AU755763B2 - Recombinant swinepox virus - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION Name of Applicant: Address of Applicant: Syntro Corporation 9669 Lackman Corporation Lenexa, Kansas 66219 United States of America COCHRAN, Mark D.

JUNKER, David E.

r r r Actual Inventor(s): r n Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete Specification for the invention entitled: Recombinant swinepox virus The following statement is a full description method of performing it known to us: of this invention, including the best Q:\OPER\MRO\2286264 div I I .doc 20/4/00 -1A- RECOMBINANT SWINEPOX VIRUS This application is related to U.S. Patent No. 6,033,904, filed June 7, 1995, U.S. Patent No. 6,251,403, filed June 7, 1995, U.S. Serial No. 08/472,679, filed June 7, 1995, and U.S. Patent No. 6,328,975, filed January 19, 1995, the contents of which are incorporated by reference into the present application.

Within this application several publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims.

The disclosures of these publications are hereby incorporated by reference into this application.

BACKGROUND OF THE INVENTION Swinepox virus (SPV) belongs to the family Poxviridae.

Viruses belonging to this group are large, doublestranded DNA viruses that characteristically develop in S 25 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 30 of tissue culture cell lines also differs dramatically from other poxviruses (24) It has also been ::demonstrated that SPV does not exhibit antigenic crossreactivity with vaccinia virus and shows no gross detectable homology at the DNA level with the ortho, 35 lepori, avi or entomopox virus groups 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. Patents 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 -3from 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 25 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 tc the use of a vaccinia virus to deliver interleukin-2 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 -4limits 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

ribozymes suppressor tRNAs interferoninducing double stranded RNA and numerous examples of protein therapeutics, from hormones, insulin, to lymphokines, 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.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of 25 integers or steps.

Ce *o P:\OPER\Fas\2286264-spcdoc-2 /l5/02 5 SUMMARY OF THE INVENTION This invention provides a recombinant swinepox virus comprising a foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within a HindIII N fragment of the swinepox virus genomic DNA and is capable of being expressed in a swinepox virus infected host cell.

The invention also provides a recombinant swinepox virus comprising a foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within an approximately 2.0 kb HindIII to BglII subfragment of the HindIII M fragment of the swinepox virus genomic DNA.

The invention further provides a homology vector for producing a recombinant swinepox virus by inserting foreign DNA into the viral genome of a swinepox virus which S 20 comprises a double-stranded DNA molecule consisting essentially of: a) double stranded foreign DNA not usually present within the swinepox virus viral genome; b) at one end the foreign DNA, double-stranded swinepox virus DNA homologous to the virus genome located at one side of the HindIII N fragment of the coding region of the swinepox virus genome; and c) at the other end of the foreign DNA, double- S3 0stranded swinepox virus DNA homologous to the viral 30 genome located at the other side of the HindIII N fragment of the coding region of the swinepox virus viral genome.

The invention further provides homology vectors, vaccines and methods of immunization.

BRIEF DESCRIPTION OF THE INVENTION Figures 1A 1B: Show a detailed diagram of SPV genomic DNA (Kasza strain) including the unique long and Terminal repeat (TR) regions. A restriction map for the enzyme HindIII is indicated 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.

Figures 2A 2B: Show the DNA sequence from homology vector 515-85.1.

The sequence of two regions of the homology vector 15 515-85.1 are shown. The first region (Figure 2A) (SEQ ID NO:1) covers a 599 base pair sequence which flanks the unique AccI site as indicated in Figures 3A-3C. 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 (Figure 2B) (SEQ ID NO:3) covers the 899 base pairs upstream of the unique HindIII site as indicated in Figures 3A-3C. The beginning (Asp) and end (Ile) of a 220 amino acid ORF is indicated by 25 the translation of amino acids below the DNA sequence.

Fiqures 3A 3C: Show the homology which exists between the 515.85.1 ORF and the Vaccinia virus 01L ORF. Figure 3A shows two maps: The first line of Figure 3A is a restriction map of the SPV HindIII M fragment and the second is a restriction map of the DNA insertion in plasmid 515-85.1. The location of the 515-85.1 [W 01L-like] ORF is also indicated on the map. The locations of the DNA sequences shown in Figures 3B and 3C are indicated below the map by heavy bars in -7- Figure 3A. Figure 3B 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. Figure 3C 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.

Ficrures 4A 4D: Show a description of the DNA insertion in Homology Vector 520-17.5. Figure 4A contains a diagram showing the orientation of DNA fragments assembled in plasmid 520-17.5 and table indicating the origin of each fragment. Figure 4B shows the sequences located at each of the junctions A and B between fragments, and Figure 4C shows the sequences located at Junctions C and D (SEQ ID NO's: 9, 10, 13, and 16). Figures 4B and 4C further describe the restriction sites used to generate each fragment as S.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 (EPI), late promoter 2 (LP2), lactose operon Z gene (lacZ), and Escherichia coli coli).

FiQures SA Show a detailed description of the DNA insertion in Homology Vector 538-46.16. Figure 5A contains a diagram showing the orientation of DNA fragments assembled in plasmid 538-46.16 and a table -8indicating the origin of each fragment. Figure shows the sequences located at Junctions A and B between fragments, Figure 5C shows sequences located at Junction C and Figure 5D shows sequences located at Junctions D and E (SEQ ID NO's: 17, 18, 21, 26, and 28). Figures 5B to 5D also describe 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 .i remnants of sites which were destroyed during construction. The following abbreviations are used, swinepox virus (SPV), pseudorabies virus (PRV), glycoprotein 63 (g63), early promoter 1 (EP1), late promoter 1 (LP1) (SEQ ID NO: 46), late promoter 2 (LP2), lactose operon Z gene (lacZ), and Escherichia coli coli).

Fiqure 6: 25 Western blot of lysates from recombinant SPV infected cells with anti-serum to PRV. Lanes (A) uninfected Vero cell lysate, S-PRV-000 (pseudorabies virus S62/26) infected cell lysate, pre-stained molecular weight markers, (D) uninfected EMSK cell lysate, S-SPV-000 infected cell lysate, 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.

Fiqure 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.

Fiqures 8A 8D: Show a detailed description of the DNA insertion in Homology Vector 538-46.26. Figure 5A contains a diagram showing the orientation of DNA fragments .i assembled in plasmid 538-46.26 and table indicating the origin of each fragment. Figure 8B shows the 15 sequences located at Junctions A and B between fragments; Figure 8C shows the sequences located at Junctions C and D, Figure 8D shows the sequences located at Junction E (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 in Figures 8B and 8D. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions 25 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 werFi destroyed during construction. The following abbreviations are used, swinepox virus (SPV), Newcastle Disease virus (NDV), hemagglutininneuraminidase early promoter 1 (EP1), late promoter 1 (LP) late promoter 2 (LP2), lactose operon Z gene (lacZ), and Escherichia coli (E.

coli).

Ficures 9A 9C: Show a detailed description of Swinepox Virus S-SPV- 010 and the DNA insertion in Homology Vector 561- 36.26. Figure 9A contains a diagram showing the orientation of DNA fragments assembled in plasmid 561-36.26 and a table indicating the origin of each fragment. Figure 9B shows the sequences located at Junctions A and B between fragments and Figure 9C show the sequences located at junction C and D (SEQ ID. NO: 47, 48, 49,50). 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 in Figures **9B and 9C. 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 coli), thymidine kinase pox synthetic late promoter 1 (LP1), base pairs (BP).

25 Fiqures 10A Show a detailed description of Swinepox Virus S-SPV- 011 and the DNA insertion in Homology Vector 570- 91.21. Figure 10A contains a diagram showing the orientation of DNA fragments assembled in plasmid 570-91.21 and a table indicating the origin of each fragment. Figure 10B show the sequences located at Junctions A and B between fragments; Figure shows the sequences located at Junction C, and Figure 10D shows the sequences located at Junctions 10D and 10E(SEQ ID NOs: 51, 52, 53, 54, 55). The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to -11join the fragments are described for each junction in Figures 10B to 10D. 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 coli), pox synthetic late promoter 1 (LP1), pox synthetic early promoter 2 (EP2) (SEQ ID NO: 45), gIII base pairs (BP).

o* Ficures 11A 11D: Show a detailed description of Swinepox Virus S-SPV- 012 and the DNA insertion in Homology Vector 570- 91.41. Figure 11A contains a diagram showing the S* orientation of DNA fragments assembled in plasmid 570-91.41 and a table indicating the origin of each fragment. Figure 11B shows the sequences located at Junctions A and B between fragments, Figure 11C shows the sequences located at Junction C, and Figure 11D shows the sequence located at Junctions D and E. (SEQ ID NOs: 56, 57, 58, 59, 60). The 25 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 in Figures 11B to 11D. 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 coli), pox synthetic late promoter 1 (LP1), pox synthetic early promoter 1 -12late promoter 2 (EP1LP2) (SEQ ID NO: 43), gIII (gC), base pairs (BP).

Ficrures 12A 12D: Show a detailed description of Swinepox Virus S-PRV- 013 and the DNA insertion in Homology Vector 570- 91.64. Figure 12A contains a diagram showing the orientation of DNA fragments assembled in plasmid 570-91.64 and a table indicating the origin of each fragment. Figure 12B shows the sequences located at Junctions A and B between fragments, Figure 12C shows the sequences located at Junction C, and 0e06 Figure 12D shows the sequences located at Junctions 4.

D and E (SEQ ID NOs: 61, 62, 63, 64, 65) The S. 15 restriction sites used to generate each fragment as e* well as synthetic linker sequences which are used to join the fragments are described for each junction in Figures 12B to 12D. 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: 25 swinepox virus (SPV), pseudorabies virus (PRV), Escherichia coli coli), pox synthetic late promoter 1 (LPl), pox synthetic late promoter 2 early promoter 2 (LP2EP2) (SEQ ID NO: 44), gIII (gC) base pairs (BP) Figures 13A 13D: Show a detailed description of Swinepox Virus S-PRV- 014 and the DNA insertion in Homology Vector 599- 65.25. Figure 13A contains a diagram showing the orientation of DNA fragments assembled in plasmid 599-65.25 and a table indicating the origin of each fragment. Figure 13B shows sequences located at -13- Junctions A and B between the fragments, Figure 13C shows sequences located at Junction C, and Figure 13D shows sequences located at Junctions D and E.

(SEQ ID NOs: 66, 67, 68, 69, and 70). 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 in Figures 13B to 13D. 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: 15 swinepox virus (SPV), infectious laryngotracheitis virus (ILT), Escherichia coli coli), pox synthetic late promoter 1 (LP1), pox synthetic early promoter 1 late promoter 2 (EP1LP2), glycoprotein G polymerase chain reaction (PCR), base pairs 20 (BP) Fiqures 14A 14D: Show a detailed description of Swinepox Virus S-SPV- 016 and the DNA insertion in Homology Vector 624- 25 20.1C. Figure 14A contains a diagram showing the orientation of DNA fragments assembled in plasmid 624-20.1C and a table indicating the origin of each fragment. Figure 14B shows the sequences located at Junctions A and B between fragments; Figure 14C shows the sequences located at Junction C, and Figure 14D shows the sequences at Junctions D and E.

(SEQ ID NOs: 71, 72, 73, 74, and 75). 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 in Figures 14B to 14D. The location of several gene coding regions and regulatory elements -14is 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 (LPI) pox synthetic late promoter 2 early promoter 2 (LP2EP2), glycoprotein I polymerase chain reaction (PCR), base pairs (BP).

Fiqures 15A Show a detailed description of Swinepox Virus S-SPV- 017 and the DNA insertion in Homology Vector 614- 83.18. Figure 15A contains a diagram showing the orientation of DNA fragments assembled in plasmid 614-83.18 and a table showing the origin of each fragment. Figure 15B shows the sequences located at 20 Junctions A and B between fragments, Figure shows the sequences at Junction C, and Figure shows the sequences located at Junctions D and E.

The restriction sites used to generate each fragment as well as synthetic linker sequences which are used 25 to join the fragments are described for each .i junction in Figures 15B to 15D. 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 (LP2EP2), glycoprotein G polymerase chain reaction (PCR), base pairs (BP).

Fiqure 16: Western blot of lysates from recombinant SPV infected cells with polyclonal goat anti-PRV gIII (gC) Lanes S-PRV-002 Patent No.

4,877,737, issued October 31, 1989) infected cell lysate, molecular weight markers, mockinfected EMSK cell lysate, S-SPV-003 infected cell lysate, S-SPV-008 infected cell lysate, (F) S-SPV-011 infected cell lysate, S-SPV-012 infected cell lysate, S-SPV-013 infected cell lysate. Cell lysates are prepared as described in 15 the PREPARATION OF INFECTED CELL LYSATES.

Approximately 1/5 of the total lysates sample is loaded in each lane.

Fiqure 17: Map showing the 5.6 kilobase pair HindIII M swinepox virus genomic DNA fragment. Open reading frames (ORF) are shown with the number of amino acids coding in each open reading frame. The swinepox virus ORFs show significant sequence identities to 25 the vaccinia virus ORFs and are labeled with the vaccinia virus nomenclature (56 and 58). The I4L ORF (SEQ ID NO: 196) shows amino acid sequence homology to ribonucleotide reductase large subunit and the 01L ORF (SEQ ID NO: 193) shows amino acid sequence homology to a leucine zipper motif characteristic of certain eukaryotic transcriptional regulatory proteins (13) The BglII site in the -I4L ORF and the AccI site in the 01L ORF are insertion sites for foreign DNA into non-essential regions of the swinepox genome. The homology vector 738-94.4 contains a deletion of SPV DNA from nucleotides 1679 to 2452 (SEQ ID NO: 189). The black bar at the -16bottom indicates regions for which the DNA sequence is known and references the SEQ ID NOs: 189 and 195.

Positions of restriction sites AccI, BglII, and HindIII are shown. I3L ORF (SEQ ID NO: 190), I2L ORF (SEQ ID NO: 191) and EO1R ORF (SEQ ID NO: 194) are shown. SEQ ID NO 221 contains the complete 5785 base pair sequence of the HindIII M fragment. Open reading frames within the SPV HindIII M fragment are the partial I4L ORF (445 AA; Nucl 2 to 1336); I3L ORF (275 AA; Nucl 1387 to 2211); I2L ORF (75 AA; Nucl 2215 to 2439); IlL ORF (313 AA; Nucl 2443 to 3381); OIL ORF (677 AA; Ncl 3520 to 5550); partial E1OR ORF (64 AA; Nucl 5787 to 5596).

Fiqures 18A 18D: Show a detailed description of Swinepox Virus S-SPV- 034 and the DNA insertion in Homology Vector 723- 59A9.22. Figure 18A contains a diagram showing the orientation of DNA fragments assembled in plasmid 20 723-59A9.22 and a table indicating the origin of each fragment. Figure 18B shows the sequences located at Junctions A and B between fragments, Figure 18C shows the sequences located at Junction C, and Figure 18D shows the sequences located at 25 Junctions D and E. 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 in Figures 18B to 18D.

The location of several gene c',ding 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), equine influenza virus (EIV), Escherichia coli (E.

coli), pox synthetic late promoter 1 (LP1) pox -17synthetic late promoter 2 early promoter 2 (LP2EP2), neuraminidase Prague polymerase chain reaction (PCR), base pairs (BP).

Fiqures 19A 19D: Show a detailed description of Swinepox Virus S-SPV- 015 and the DNA insertion in Homology Vector 727- 54.60. Figure 19A contains a diagram showing the orientation of DNA fragments assembled in plasmid 727-54.60 and a table indicating the origin of each fragment. Figure 19B shows the sequences located at Junctions A and B between fragments, Figure 19C shows the sequences located at Junction C, and Figure 19D shows the sequences located at Junctions 15 D and E. 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 in Figures 19B to 19D. The location of several gene coding regions and 20 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 25 abbreviations are used: swinepox virus (SPV), 0 pseudorabies virus (PRV), Escherichia coli (E.

coli), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2EP2), glycoprotein B base pairs (BP).

Figures 20A Show a detailed description of Swinepox Virus S-SPV- 031 and the DNA insertion in Homology Vector 727- 67.18. Figure 20A contains a diagram showing the orientation of DNA fragments assembled in plasmid 727-67.18 and a table indicating the origin of each fragment. Figure 20B shows the sequences located -18at Junctions A and B between fragments, Figure shows the sequences located at Junction C, and Figure 20D shows the sequences located at Junctions D and E. 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 in Figures 20B to 20D. 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 coli), pox synthetic late promoter 1 (LP1) pox synthetic early promoter 1 late promoter 2 (EP1LP2) antigen (Ag) base pairs

(BP)

20 Ficures 21A 21D: Show a detailed description of Swinepox Virus S-SPV- 033 and the DNA insertion in Homology Vector 732- 18.4. Figure 21A contains a diagram showing the orientation of DNA fragments assembled in plasmid 25 732-18.4 and a table indicating the origin of each fragment. Figure 21B shows the sequences located at Junctions A and B between fragments, Figure 21C shows the sequences located at Junction C, and Figure 21D shows the sequences located at Junctions D and E. 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 in Figures 21B to 21D. 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 -19brackets, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), Escherichia coli coli), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2EP2), equine influenza virus (EIV), neuraminidase Alaska polymerase chain reaction (PCR), base pairs (BP).

Figures 22A 22C: Show a detailed description of Swinepox Virus S-SPV- 036 and the DNA insertion in Homology Vector 741- 80.3. Figure 22A contains a diagram showing the S* orientation of DNA fragments assembled in plasmid 741-80.3 and a table indicating the origin of each fragment. Figure 22B shows the sequences located at Junctions A, B, and C between fragments and Figure 22C shows the sequences located at Junctions E and F. The restriction sites used to generate 20 each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction in Figures 22B and 22C. The location of several gene coding regions and regulatory elements is also given. The following two 25 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), human cytomegalovirus immediate early (HCMV IE), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2EP2), polyadenylation site (poly base pairs (BP).

Figures 23A 23D: Show a detailed description of Swinepox Virus S-SPV- 035 and the DNA insertion in Homology Vector 741- 84.14. Figure 23A contains a diagram showing the orientation of DNA fragments assembled in plasmid 741-84.14 and a table indicating the origin of each fragment. Figure 23B shows the sequences located at Junctions A and B between fragments, Figure 23C shows the sequences located at Junction C, and Figure 23D shows the sequences located at Junctions D and E. 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 in Figures 23B to 23D. The location of several gene coding regions and regulatory 15 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 20 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), interleukin-2 glycoprotein X (gX) polymerase 25 chain reaction (PCR) sequence (seq) base pairs

(BP)

Figures 24A 24D: Show a detailed description of Swinepox Virus S-SPV- 038 and the DNA insertion in Homology Vector 744-34.

Figure 24A contains a diagram showing the orientation of DNA fragments assembled in plasmid 744-34 and a table indicating the origin of each fragment. Figure 24B shows the sequences located at Junction A and B between fragments, Figure 24C shows the sequences located at Junction C, and Figure 24D shows the sequences located at Junctions D and E.

-21- 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 in Figures 24B and 24D. 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), equine herpesvirus type 1 (EHV-1), Escherichia coli coli), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2EP2), glycoprotein 15 B polymerase chain reaction (PCR), base pairs

(BP).

Figures 25A Show a detailed description of Swinepox Virus S-SPV- 20 039 and the DNA insertion in Homology Vector 744-38.

Figure 25A contains a diagram showing the orientation of DNA fragments assembled in plasmid 744-38 and a table indicating the origin of each fragment. Figure 25B shows the sequences located at Junction A and B between fragments. Figure shows the sequences located at Junction C and Figure shows the sequences located at Junctions D and E. 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 in Figures 25B to 25D. 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 -22abbreviations are used: swinepox virus (SPV), equine herpesvirus type 1 (EHV-1), Escherichia coli coli), pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LP2EP2), glycoprotein D polymerase chain reaction (PCR), base pairs (BP) Figures 26A-26D: Detailed description of Swinepox Virus S-SPV-042 and the DNA insertion in Homology Vector 751-07.A1.

Diagram showing the orientation of DNA fragments assembled in plasmid 751-07.A1. The origin of each fragment is indicated in the table. The sequence located at each of the junctions between fragments 15 is also shown. The restriction sites used to generate each fragment as well as synthetic linker S: sequences which are used to join the fragments are described for each junction. Figures 26A-26D show the sequences located at Junction A (SEQ ID NOS: 20 197), (SEQ ID NO: 198), C (SEQ ID NO: 199), D (SEQ ID NO: 200) and E (SEQ ID NO: 201) between fragments and the sequences located at the junctions. The location of several gene coding regions and regulatory elements is also given. The following 25 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), chicken interferon (cIFN), Escherichia coli (E.

coli) pox synthetic late promoter 1 (LP1) pox synthetic late promoter 2 early promoter 2 (LP2EP2), polymerase chain reaction (PCR), base pairs (BP) Figures 27A-27D: Detailed description of Swinepox Virus S-SPV-043 and the DNA insertion in Homology Vector 751-56.Al.

-23- Diagram showing the orientation of DNA fragments assembled in plasmid 751-56.A1. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 27A-27D show the sequences located at Junction A (SEQ ID NOS: 202), (SEQ ID NO: 203), C (SEQ ID NO: 204), D (SEQ ID NO: 205) and E (SEQ ID NO: 206) between fragments and the sequences located at the junctions. The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the several gene coding regions and regulatory elements is also given. The following two conventions are used: :i numbers in parentheses, refer to amino acids, 15 and restriction sites in brackets, indicate the S.remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), chicken myelomonocytic growth factor (cMGF), Escherichia coli coli), pox 20 synthetic late promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2 (LPE2EP2), polymerase chain reaction (PCR), base pairs (BP).

Figure 28A-28D: 25 Detailed description of Swinepox Virus S-SPV-043 and the DNA insertion in Homology Vector 752-22.1.

Diagram showing the orientation of DNA fragments assembled in plasmid 752-22.1. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 28A-28D show the sequences located at Junction A (SEQ ID NOS: 207), (SEQ ID NO: 208), C (SEQ ID NO: 209), and D (SEQ ID NO: 210) between fragments and the sequences located at the junctions. The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described -24for 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 restrictions sites in brackets, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: swinepox virus (SPV), Escherichia coli coli), pox synthetic late promoter 2 early promoter 2 (LP2EP2), polymerase chain reaction (PCR), base pairs (BP) Figures 29A-29B: Figure 29A: Restriction Endonuclease Map and Open 15 Reading Frames in the SPV HindIII N fragment and part of SPV HindIII M fragment. Insertions of a foreign gene into a non-essential site of the swinepox virus Hind III N and Hind III M genomic DNA include the EcoR V site (S-SPV-060), SnaB I site (S- 20 SPV-061), Bgl II site in Hind III N (S-SPV-062), and the Bgl II site in Hind III M (S-SPV-047) Insertions of a foreign gene into the I7L ORF (SEQ ID NO. 230) and I4L ORF (SEQ ID NO. 231) indicates that the sequence of the entire open reading frame 25 is non-essential for replication of the swinepox virus and suitable for insertion of foreign genes.

Additional sites for insertion of foreign genes include, but are not limited to the two Hind III sites, Ava I site, and the BamHI site.

Figure 29B: Restriction Endonuclease Map and Open Reading Frames in the SPV Hind III K fragment.

Insertion of a foreign gene into a non-essential site of the swinepox virus Hind III K genomic DNA include, but is not limited to the EcoR I site (S- SPV-059). Three open reading frames are identified within a 3.2 kB region of the SPV HindIII K fragment. Insertions of a foreign gene into the B18R ORF (SEQ ID NO. 228) indicates that the sequence of the entire open reading frame is non-essential for replication of the swinepox virus and suitable for insertion of foreign genes. Also identified is the B4R ORF (SEQ ID NO. 229) which is a site for insertion of a foreign gene. SPV B18R ORF has homology to the vaccinia virus (VV) B18R ORF. SPV B18R ORF has more homology to the 77.2 kd protein of rabbit fibroma virus (RFV). SPV B4R ORF has homology to the vaccinia virus B4R ORF. SPV B4R ORF has more homology to the T5 protein of rabbit fibroma virus (RFV). The identified open reading frames are S. within approximately 3200 base pairs of the SPV Hind 15 III K fragment. The remaining approximately 3500 base pairs of the SPV Hind III K fragment has been sequenced previously Massung, et al. Virology 197, 511-528 (1993)).

Fiqures 30A-30C: Detailed description of Swinepox Virus S-SPV-047 and the DNA insertion in Homology Vector 779-94.31.

25 Diagram showing the orientation of DNA fragments assembled in plasmid 779-94.31. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 30A-30C show the sequences located at Junction A (SEQ ID NOS: (SEQ ID NO: C (SEQ ID NO: D (SEQ ID NO: and E (SEQ ID NO: between fragments and the sequences located at the junctions. 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 -26given. The following two conventions are used: numbers in parentheses, refer to amino acids, and restrictions 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 coli), pox synthetic late promoter 2 early promoter 2 (LP2EP2), pox synthetic late promoter 1 (LP1), base pairs (BP).

Figures 31A-31D: Detailed description of Swinepox Virus S-SPV-052 and the DNA insertion in Homology Vector 789-41.7.

Diagram showing the orientation of DNA fragments assembled in plasmid 789-41.7. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 31A-31D show the sequences located at Junction A (SEQ ID NOS: (SEQ ID NO: C (SEQ ID NO: D (SEQ ID NO: E (SEQ ID NO: and F (SEQ ID NO: between fragments and the sequences located at the junctions. 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 S" 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 restrictions 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 2 early promoter 2 (LP2EP2), pox synthetic early promoter 1 late promoter 2 (EP1LP2), pox synthetic late promoter 1 (LP1), base pairs (BP).

-27- Figures 32A-32D: Detailed description of Swinepox Virus S-SPV-053 and the DNA insertion in Homology Vector 789-41.27.

Diagram showing the orientation of DNA fragments assembled in plasmid 789-41.27. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 32A-32D show the sequences located at Junction A (SEQ ID NOS: (SEQ ID NO: C (SEQ ID NO: D (SEQ ID NO: E (SEQ ID NO: F (SEQ ID NO: and G (SEQ ID NO: between fragments and the sequences located at the junctions. The restriction sites used to generate 15 each fragment as well as synthetic linker sequences which are used to join the fragments are described .i for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: 20 numbers in parentheses, refer to amino acids, and restrictions 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 coli), pox synthetic late promoter 2 early promoter 2 (LP2EP2), pox synthetic early promoter 1 late promoter 2 (EP1LP2), pox synthetic late promoter 1 (LP1), base pairs (BP).

Figures 33A-33D: Detailed description of Swinepox Virus S-SPV-054 and the DNA insertion in Homology Vector 789-41.47.

Diagram showing the orientation of DNA fragments assembled in plasmid 789-41.47. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 33A-33D show the sequences -28located at Junction A (SEQ ID NOS: (SEQ ID NO: C (SEQ ID NO: D (SEQ ID NO: E (SEQ ID NO: F (SEQ ID NO: and G (SEQ ID NO: between fragments and the sequences located at the junctions. 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 restrictions sites in brackets, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: 15 swinepox virus (SPV), pseudorabies virus (PRV), Escherichia coli coli) pox synthetic early promoter 1 late promoter 2 (EP1LP2), pox synthetic late promoter 1 (LP1), base pairs (BP).

Fiqures 34A-34E: Detailed description of Swinepox Virus S-SPV-055 and the DNA insertion in Homology Vector 789-41.73.

Diagram showing the orientation of DNA fragments assembled in plasmid 789-41.73. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 34A-34E show the sequences located at Junction A (SEQ ID NOS: (SEQ ID NO: C (SEQ ID NO: D (SEQ ID NO: E (SEQ ID NO: F (SEQ ID NO: G (SEQ ID NO: and H (SEQ ID NO: between fragments and the sequences located at the junctions. 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 -29given. The following two conventions are used: numbers in parentheses, refer to amino acids, and restrictions 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 coli), pox synthetic late promoter 2 early promoter 2 (LP2EP2), pox synthetic early promoter 1 late promoter 2 (EP1LP2), pox synthetic late promoter 1 (LP1), base pairs (BP).

e* e W DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant swinepox virus comprising a foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within a HindIII K fragment of the swinepox virus genomic DNA and is capable of being expressed in a swinepox virus infected host cell.

In one embodiment the recombinant swinepox virus contains the foreign DNA sequence is inserted into an approximately 2 kB HindIII to BamHI subfragment of the .HindIII N fragment of the swinepox virus genomic DNA. In another embodiment the foreign DNA sequence is inserted into an open reading frame within an approximately 2 kB HindIII to BamHI subfragment of the HindIII N fragment of the swinepox virus genomic DNA. In another embodiment the the open reading frame encodes a I7L gene.

In another embodiment the foreign DNA sequence is inserted within a EcoRV restriction endonuclease site within the approximately 2 kB HindIII to BamHI subfragment of the swinepox virus genomic DNA. In another embodiment the foreign DNA sequence is inserted within a SnaBI restriction endonuclease site within the approximately 2.0 kB HindIII to BamHI subfragment of the swinepox virus genomic DNA.

In another embodiment the foreign DNA sequence is inserted within an approximately 1.2 kB BamHI to HindIII subfragment of the HindIII N fragment of the swinepox virus genomic DNA. In another embodiment the foreign DNA sequence is inserted into an open reading frame within an approximately 1.2 kB BamHI to HindIII subfragment of the HindIII N fragment of the swinepox virus genomic DNA. In another embodiment the foriegn DNA sequence is inserted into an open reading frame which encodes a I4L gene. In -31another embodiment the foreign DNA sequence is inserted within a BglII restriction endonuclease site within the approximately 1.2 kB BamHI to HindIII subfragment of the swinepox virus genomic DNA.

The present invention provides a recombinant swinepox virus comprising a foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within a HindIII M fragment of the swinepox virus genomic DNA and is capable of being expressed in a swinepox virus infected host cell.

In one embodiment the recombinant swinepox virus contains the foreign DNA sequence inserted into an approximately 2 kB BglII to HindIII subfragment of the HindIII M fragment of the swinepox virus genomic DNA. In another embodiment the foreign DNA sequence is inserted into an open reading frame within an approximately 2 kB BglII to HindIII subfragment of the HindIII M fragment of the swinepox virus genomic DNA. In another embodiment the open reading frame encodes a OIL gene. In the preferred embodiment the foreign DNA sequence is inserted within a BglII restriction endonuclease site within the approximately 2 kB BglII to HindIII subfragment of the 25 swinepox virus genomic DNA.

4S In another embodiment the recombinant swinepox virus contains the foreign DNA sequence inserted within an approximately 3.6 kB larger HindIII to BglII subfragment of the HindIII M fragment of the swinepox virus genomic DNA. In another embodiment the foreign DNA sequence is inserted into an open reading frame within an approximately 3.6 kB larger HindIII to BglII subfragment of the HindIII M fragment of the swinepox virus genomic DNA. In another embodiment the open reading frame encodes a I4L gene -32- In one embodiment the foreign DNA sequence of the recombinant swinepox virus is inserted within a nonessential Open Reading Frame (ORF) of the HindIII M fragment. Example of ORF's include, but are not limited to: I4L, I2L, 01L, and In another embodiment the foreign DNA sequence of the recombinant swinepox virus is inserted within an approximately 2 Kb HindIII to BglII subfragment of the HindIII M fragment of the swinepox virus genomic DNA. In a preferred embodiment the foreign DNA sequence is inserted within a BglII site located within the approximately 2 Kb HindIII to BglII subfragment of the swinepox virus genomic DNA.

In another embodiment the foreign DNA sequence is inserted within a larger HindIII to BglII subfragment of the HindIII M fragment of the swinepox virus genomic DNA.

In a preferred embodiment the foreign DNA sequence is 20 inserted within an AccI site located within the larger HindIII to BglII subfragment of the swinepox virus genomic DNA.

In another embodiment the recombinant swinepox virus further comprises a foreign DNA sequence inserted into an open reading frame encoding swinepox virus thymidine kinase. In one embodiment the foreign DNA sequence is inserted into a NdeI site located within the open reading frame encoding the swinepox virus thymidine kinase.

This invention provides a recombinant swinepox virus comprising a -foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within a HindIII K fragment of the swinepox virus genomic DNA and is capable of being expressed in a swinepox virus infected host cell.

-33- In one embodiment the foreign DNA sequence is inserted into an approximately 3.2 kB subfragment of the HindIII K fragment of the swinepox virus genomic DNA. In another embodiment the foreign DNA sequence is inserted into an open reading frame within an approximately 3.2 kB subfragment of the HindIII K fragment of the swinepox virus genomic DNA. In another embodiment the open reading frame encodes a B18R gene. In another embodiment the open reading frame encodes a B4R gene.

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, 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.

.ee 20 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.

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.

-34- In addition, 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.

The invention further provides a foreign DNA sequence or foreign RNA which encodes a polypeptide. Preferably, the polypeptide is antigenic in the animal. Preferably, this 15 antigenic polypeptide is a linear polymer of more than 2* 'amino acids linked by peptide bonds which stimulates the animal to produce antibodies.

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 /-galactosidase or E. coli beta-glucuronidase.

Preferably, the insertion site for the foreign DNA encoding E. coli 0-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, Maryland 20852 U.S.A. under ATCC Accession No. VR 2335.

For purposes of this invention, a "polypeptide which is a detectable marker" includes the birer, trimer and tetramer form of the polypeptide. E. coli 0galactosidase 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 pseudorabies virus (PRV) gII Pseudorabies virus (PRV) gIII (gC), pseudorabies virus (PRV) glycoprotein H, pseudorabies virus (PRV) glycoprotein E, Transmissible gastroenteritis (TGE) glycoprotein 195, Transmissible gastroenteritis 15 (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 (gD).

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 Chole-.a 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 (gD) This recombinant swinepox virus can be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli 0-galactosidase. A preferred site within the -36swinepox viral genome for insertion of the foreign DNA encoding PRV g50 (gD) and E. coli 0-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, Maryland 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 15 encoding pseudorabies virus (PRV) gIII This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli 0-galactosidase. A preferred site within the swinepox viral DNA for insertion of the foreign DNA 20 encoding PRV C gene and E. coli -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 on July 16, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under AT'CC 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 This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli 0-galactosidase. A preferred site within the -37swinepox viral DNA for insertion of the foreign DNA encoding PRV gII (gB) and E. coli f-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 (ATCC Accession No. VR 2466).

The S-SPV-015 swinepox virus has been deposited on July 22, 1994 pursuant to the Budapest Treaty -on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2466.

The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) g50 (gD) and foreign DNA encoding pseudorabies virus (PRV) gIII This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such 20 as E. coli f-galactosidase. A preferred site within the swinepox viral DNA for insertion of the foreign DNA encoding PRV g50 PRV gIII (gC) and E. coli 0galactosidase 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 (gD) and foreign DNA encoding pseudorabies virus (PRV) gII (gB) This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli 3-galactosidase. A preferred site within the swinepox viral genome for insertion of foreign DNA encoding PRV g50 (gD) PRV gII (gB) and E. coli ggalactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA.

-38- The invention further provides for a recombinant swinepox virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) gIII (gC) and foreign DNA encoding pseudorabies virus (PRV) gII (gB) This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli 0-galactosidase. A preferred site within the swinepox viral genome for insertion of foreign DNA encoding PRV gIII PRV gII (gB) and E. coli 3galactosidase is the AccI site within the HindIII M fragment of the swinepox viral DNA.

The invention further provides for a recombinant swinepox i virus capable of replication which contains foreign DNA encoding pseudorabies virus (PRV) g50 foreign DNA encoding pseudorabies virus (PRV) gIII and foreign DNA encoding pseudorabies virus (PRV) gII (gB) This recombinant swinepox virus can also be further engineered to contain foreign DNA encoding a detectable marker, such as E. coli 0-galactosidase.

A preferred site within the swinepox viral genome for insertion of foreign DNA encoding PRV g50 PRV gIII PRV gII (gB) and E. coli /-galactosidase is the 25 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 -39- Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2344.

The present invention further provides a recombinant swinepox virus which comprises a foreign DNA sequence inserted into a non-essential site of the swinepox genome, wherein the foreign DNA sequence encodes an antigenic polypeptide derived from infectious bovine rhinotracheitis virus and is capable of being expressed in a host infected by the recombinant swinepox virus.

Examples of such antigenic polypeptide are infectious bovine rhinotracheitis virus glycoprotein E and glycoprotein G. Preferred embodiment of this invention **are recombinant swinepox viruses designated S-SPV-017 and S-SPV-019.

The present invention further provides a recombinant swinepox virus which comprises a foreign DNA sequence inserted into a non-essential site of the swinepox genome, wherein the foreign DNA sequence encodes an antigenic polypeptide derived from infectious laryngotracheitis virus and is capable of being expressed 25 in a host infected by the recombinant swinepox virus.

Examples of such antigenic polypeptide are infectious laryngotracheitis virus glycoprotein G and glycoprotein I. Preferred embodiment of this invention are recombinant swinepox viruses designated S-SPV-014 and S- SPV-016.

In one embodiment of the recombinant swinepox virus the foreign DNA sequence encodes a cytokine. In another embodiment the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN). Cytokines include, but are not limited to: transforming growth factor beta, epidermal growth factor family, fibroblast growth factors, hepatocyte growth factor, insulin-like growth factor, vascular endothelial growth factor, interleukin 1, IL-1 receptor antagonist, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin- 6, IL-6 soluble receptor, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, angiogenin, chemokines, colony stimulating factors, granulocyte-macrophage colony stimulating factors, erythropoietin, interferon, interferon gamma, c-kit ligand, leukemia inhibitory factor, oncostatin M, pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor, tumor necrosis factors, and soluble TNF receptors. These cytokines are from humans, bovine, equine, feline, canine, porcine or 15 avian. Preferred embodiments of such recombinant virus are designated S-SPV-042, and S-SPV-043.

The present invention further provides a recombinant swinepox virus which comprises a foreign DNA sequence 20 inserted into a non-essential site of the swinepox genome, wherein the foreign DNA sequence encodes an antigenic polypeptide derived from a human pathogen and is capable of being expressed in a host infected by the recombinant swinepox virus.

Recombinant SPV expressing cytokines is used to enhance the immune response either alone or when combined with vaccines containing cytokines or antigen genes of disease causing microorganisms.

Antigenic polypeptide of a human pathogen which are derived from human herpesvirus include, but are not limited to: hepatitis B virus and hepatitis C virus hepatitis B virus surface and core antigens, hepatitis C virus, human immunodeficiency virus, herpes simplex virus-i, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, human -41herpesvirus-6, human herpesvirus-7, human influenza, measles virus, hantaan virus, pneumonia virus, rhinovirus, poliovirus, human respiratory syncytial virus, retrovirus, human T-cell leukemia virus, rabies virus, mumps virus, malaria (Plasmodium falciparum), Bordetella pertussis, Diptheria, Rickettsia prowazekii, Borrelia berfdorferi, Tetanus toxoid, malignant tumor antigens.

In one embodiment of the invention, a recombinant swinepox virus contains the foreign DNA sequence encoding hepatitis B virus core protein. Preferably, such virus recombinant virus is designated S-SPV-031.

The present invention further provides a recombinant swinepox virus which comprises a foreign DNA sequence inserted into a non-essential site of the swinepox S[ genome, wherein the foreign DNA sequence encodes a S* cytokine capable of stimulating an immune in a host infected by the recombinant swinepox virus and is capable of being expressed in the host infected.

In one embodiment of the invention, a recombinant swinepox virus contains a foreign DNA sequence encoding 25 human interleukin-2. Preferably, such recombinant virus is designated S-SPV-035.

The present invention further provides a recombinant swinepox virus which comprises a foreign DNA sequence inserted into a non-essential site of the swinepox genome, wherein the foreign DNA sequence encodes an antigenic polypeptide derived from an equine pathogen and is capable of being expressed in a host infected by the recombinant swinepox virus.

The antigenic polypeptide of an equine pathogen can derived from equine influenza virus, or equine -42herpesvirus. In one embodiment the antigenic polypeptide is equine influenza neuraminidase or hemagglutinin.

Examples of such antigenic polypeptide are equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine influenza virus type A/Kentucky 92 neuraminidase equine herpesvirus type 1 glycoprotein B, equine herpesvirus type 1 glycoprotein D, Streptococcus equi, equine infectious anemia virus, equine encephalitis virus, equine rhinovirus and equine rotavirus. Preferred embodiments of such recombinant virus are designated S- SPV-033, S-SPV-034, S-SPV-038, S-SPV-039 and S-SPV-041.

The present invention further provides an antigenic polypeptide which includes, but is not limited to: hog cholera virus gEl, hog cholera virus gE2, swine influenza virus hemagglutinin, neurominidase, matrix and nucleoprotein, pseudorabies virus gB, gC and gD, and PRRS virus ORF7.

The present invention further provides a recombinant S" swinepox virus which comprises a foreign DNA sequence S" 25 inserted into a non-essential site of the swinepox genome, wherein the foreign DNA sequence encodes an antigenic polypeptide derived from bovine respiratory syncytial virus or bovine parainfluenza virus, and is capable of being expressed in a host infected by the recombinant swinepox virus.

For example, the antigenic polypeptide of derived from infectious bovine rhinotracheitis virus gE, bovine respiratory syncytial virus equine pathogen can derived from equine influenza virus is bovine respiratory syncytial virus attachment protein (BRSV bovine respiratory syncytial virus fusion protein (BRSV F), -43bovine respiratory syncytial virus nucleocapsid protein (BRSV bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase. In a preferred embodiment the recombinant swinepox virus is designated S-SPV-045.

Preferred embodiments of a recombinant virus containing a foreign DNA encoding an antigenic polypeptide from a bovine respiratory syncytial virus are designated S-SPV- 020, S-SPV-029, and S-SPV-030. And a preferred embodiment of a recombinant virus containing a foreign DNA encoding an antigenic polypeptide from a bovine parainfluenza virus are designated S-SPV-028.

15 The present invention further provides a recombinant swinepox virus which comprises a foreign DNA sequence inserted into a non-essential site of the swinepox genome, wherein the foreign DNA sequence encodes bovine viral diarrhea virus (BVDV) glycoprotein 48 or glycoprotein 53, and wherein the foreign DNA sequence is capable of being expressed in a host infected by the recombinant swinepox virus. Preferred embodiments of such virus are designated S-SPV-032, S-SPV-040, S-SPV- 049, and S-SPV-050.

The present invention further provides a recombinant swinepox virus which comprises a foreign DNA sequence inserted into a non-essential site of the swinepox genome, wherein the foreign DNA sequence encodes an antigenic polypeptide derived from infectious bursal disease virus and wherein the foreign DNA sequence is capable of being expressed in a host infected by the recombinant swinepox virus. Examples of such antigenic polypeptide are infectious bursal disease virus polyprotein and VP2. Preferred embodiments of such virus are designated S-SPV-026 and S-SPV-027.

-44- The present invention further provides a recombinant swinepox virus in which the foreign DNA sequence encodes an antigenic polypeptide which includes, but is not limited to: MDV gA, MDV gB, MDV gD, NDV HN, NDV F, ILT gB, ILT gI, ILT gD, IBDV VP2, IBDV VP3, IBDV VP4, IBDV polyprotein, IBV spike, IBV matrix, avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia virus, Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes, trematodes, poultry mites/lice, and poultry protozoa.

9* S. 15 The invention further provides that the inserted foreign DNA sequence is under the control of a promoter. In one embodiment the is a swinepox viral promoter. In another embodiment the foreign DNA sequence is under control of an endogenous upstream poxvirus promoter. In another embodiment the foreign DNA sequence is under control of a heterologous upstream promoter.

S

For purposes of this invention, promoters include but is not limited to: synthetic pox viral promoter, pox 25 synthetic late promoter 1, pox synthetic late promoter 2 early promoter 2, pox O1L promoter, pox I4L promoter, pox I3L promoter, pox I2L promoter, pox IlL promoter, pox promoter, PRV gX, HSV-1 alpha 4, HCMV immediate early, MDV gA, MDV gB, MDV gD, ILT gB, BHV-1.1 VP8 and ILT gD. Alternate promoters are 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.

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 sequence or (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, doublestranded 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 another embodiment of the present invention, the double-stranded swinepox viral DNA of the homology vectors described above is homologous to genomic DNA present within the HindIII M fragment. In another embodiment the double-stranded swinepox viral DNA of the homology vectors described above is homologous to genomic DNA present within an approximately 2 Kb HindIII to BglII sub-fragment. In a preferred embodiment the doublestranded swinepox viral DNA is homologous to genomic DNA present within the BglII site located in this HindIII to BglII subfragment.

In another embodiment the double-stranded swinepox viral DNA is homologous to genomic DNA present within the open reading frame contained in the larger 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 the larger HindIII to BglII subfragment.

In a preferred embodiment the homology vectors are designated 752-29.33, 751-07.Al, 751-56.Al, 751-22.1, 746-94.1, 767-67.3, 738-94.4, and 771-55.11.

-46- In one embodiment, the polypeptide is a detectable marker. Preferably, the polypeptide which is a detectable marker is E. coli 0-galactosidase.

In one embodiment, the polypeptide is antigenic in the animal. Preferably, the antigenic polypeptide is or is from pseudorabies virus (PRV) g50 pseudorabies virus (PRV) gII Pseudorabies virus (PRV) gIII (gC), Pseudorabies virus (PRV) glycoprotein H, Transmissible gastroenteritis (TGE) glycoprotein 195, Transmissible gastroenteritis (TGE) matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, .I Serpulina hydodysenteriae protective antigen, Bovine Viral Diarrhea (BVD) glycoprotein 53 and g48, 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 gEl and gE2, Swine Influenza Virus, African Swine Fever Virus or Mycoplasma hyopneumoniae, swine influenza virus hemagglutinin, neuraminidase and matrix and nucleoprotein, PRRS virus ORF7, and hepatitis B virus core protein.

25 In an embodiment of the present invention, the double stranded foreign DNA sequence in the homology vector encodes an antigenic polypeptide derived from a human pathogen.

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

Furthermore, the antigenic polypeptide of a human -47pathogen may be associated with malaria or malignant tumor from the group conisting of Plasmodium falciparum, Bordetella pertusis, and malignant tumor.

In an embodiment of the present invention, the double stranded foreign DNA sequence in the homology vector encodes a cytokine capable of stimulating human immune response. In one embodiment the cytokine is a chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN) For example, the cytokine can be, but not limited to, interleukin-2, interleukin-6, interleukin-12, interferons, granulocyte-macrophage colony stimulating factors, and interleukin receptors.

15 In an embodiment of the present invention, the double stranded foreign DNA sequence in the homology vector encodes an antigenic polypeptide derived from an equine pathogen.

The antigenic polypeptide of an equine pathogen can derived from equine influenza virus or equine herpesvirus. Examples of such antigenic polypeptide are equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, 25 equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidaseequine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein

D.

In an embodiment of the present invention, the double stranded foreign DNA sequence of the homology vector encodes an antigenic polypeptide derived from bovine respiratory syncytial virus or bovine parainfluenza virus.

For example, the antigenic polypeptide is derived from infectious bovine rhinotracheitis gE, bovine respiratory -48syncytial virus attachment protein (BRSV bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase.

In an embodiment of the present invention, the double stranded foreign DNA sequence of the homology vector encodes an antigenic polypeptide derived from infectious bursal disease virus. Examples of such antigenic polypeptide are infectious bursal disease virus polyprotein and infectious bursal disease virus VP2, VP3, or VP4.

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 of the homology vectors described above is homologous to genomic DNA present within the open reading frame encoding swinepox thymidine kinase.

Preferably, the double-stranded swinepox viral DNA is 25 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 vectors described above, the foreign DNA sequence of which is under control of a promoter located upstream of the foreign DNA sequence. The promoter can be an endogenous swinepox viral promoter or an exogenous promoter.

Promoters include, but are not limited to: synthetic pox viral promoter, pox synthetic late promoter 1, pox synthetic late promoter 2 early promoter 2, pox 01L promoter, pox I4L promoter, pox I3L promoter, pox I2L -49promoter, pox IlL promoter, pox EO1R promoter, PRV gX, HSV-1 alpha 4, HCMV immediate early, BHV-1.1 VP8, infectious laryngotracheitis virus glycoprotein B, infectious laryngotracheitis virus gD, marek's disease virus glycoprotein A, marek's disease virus glycoprotein B, and marek's disease virus glycoprotein D.

This invention provides a recombinant swinpox virus designated S-SPV-044, S-SPV-046, S-SPV-047, S-SPV-048, S- SPV-052, S-SPV-051, S-SPV-053, S-SPV-054, S-SPV-055, S- SPV-056, S-SPV-057, S-SPV-058, S-SPV-059, S-SPV-060, S- SPV-061, and S-SPV-062.

The invention further provides a vaccine which comprises 15 an effective immunizing amount of a recombinant swinepox :i virus of the present invention and a suitable carrier.

Suitable carriers for the swinepox 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.

25 For purposes of this invention, an "effective immunizing amount" of the recombinant swinepox virus of the present invention is within the range of 10 3 to 109 PFU/dose.

The present invention also provides a method of immunizing an animal, wherein the animal is a human, 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 influenza, PRRS, bovine respiratory synctial virus, bovine parainfluenza virus type 3, foot and mouth disease, hog cholera, African swine fever or Mycoplasma hyopneumoniae. For purposes of this invention, the method of immunizing also includes immunizing the animal against human pathogens, bovine pathogens, equine pathogens, avian pathogens described in the preceding part of this section.

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 15 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 25 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.

-51- The present invention provides a recombinant SPV which when inserted with a foreign DNA sequence or gene may be employed as a diagnostic assay. In one embodiment FIV env and gag genes and D. immitis p39 and 22kd are employed in a diagnostic assay to detect feline immunodeficiency caused by FIV and to detect heartworm caused by D. immnits, respectively.

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, 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 viruses described above are detailed below in Materials and Methods.

-52- EXPERIMENTAL DETAILS 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 .i or equivalent supplier, and hereafter are referred to as EMSK negative medium). Prior to infection, the cell 15 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 0 C in 5% CO, until cytopathic effect was complete. The medium and 25 cells were harvested and frozen in a 50 ml conical screw cap tube at -70C. Upon thawing at 37 0 C, the virus stock was aliquoted into 1.0 ml vials and refrozen at -70 0 C. The titers were usually about 106 PFU/ml.

PREPARATION OF SPV DNA. For swinepox virus DNA isolation, a confluent monolayer of EMSK cells in a T175 cm 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 minutes in a clinical centrifuge. The medium was decanted, and the cell pellet was gently resuspended in -53ml Phosphate Buffer Saline (PBS: 1.5g Na 2 HPO,, 0.2g

KH

2

PO

4 0.8g NaCL and 0.2g KC1 per liter H 2 0) (per T175) and subjected to two successive freeze-thaws (-700 C to 370 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 40 C. SPV virions, present in the supernatant, were then pelleted by centrifugation at 15,000 rpm for 20 minutes at 40 C in a SS34 rotor (Sorvall) and resuspended in 10 mM Tris (pH 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 15 minutes in a SW41 rotor (Beckman) at 40 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 40 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 minutes in a SW41 rotor at 40 C. The resultant pellet was then washed once with 10 mM Tris pH 7.5 to remove traces 25 of sucrose and finally resuspended in 10 mM Tris pH SPV DNA was then extracted from the purified virions by lysis (4 hours at 600 C) induced by the addition of EDTA, SDS, and proteinase K to final concentrations of 20 mM, and 0.5 mg/ml, respectively. After digestion, three phenol:chloroform extractions were conducted and the sample precipitated by the addition of two volumes of absolute ethanol and incubation at -200 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 0.01 M Tris pH 7.5, 1 mM EDTA at 40 C.

-54- 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 2 flask or a 60 mm petri dish was infected with 100 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 l of disruption buffer sodium dodecyl sulfate, 2% 0-mercapto-ethanol). The samples were sonicated for 30 seconds on ice and stored at -20 0

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: 6.61g Tris-HCl, 0.97g Tris-base, .NaC1 and 2.0g Sodium Azide per liter H 2 0) 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 3 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 and Supersee T programs from Coral Software.

CLONING WITH THE POLYMERASE CHAIN REACTION. The 15 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 S 20 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.

25 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 DNA synthesis) into the cells. The plasmid homology vector DNA is then transfected into these cells according to the INFECTION TRANSFECTION -56- 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 0 C in a humidified

CO

2 environment for 5 hours. The transfection procedure used is essentially that recommended for Lipofectin

M

Reagent (BRL). Briefly, for each 6 cm plate, 15 Ag of plasmid DNA was diluted up to 100 pl with Separately, 50 micrograms of Lipofectin Reagent was diluted to 100 1l with H 2 O. The 100 pl of diluted Lipofectin Reagent was then added dropwise to the diluted 15 plasmid DNA contained in a polystyrene 5 ml snap cap tube and mixed gently. The mixture was then incubated for 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 0 C in a humidified 5% CO 2 environment. The next day the 3 ml of EMSK negative 25 medium was removed and replaced with 5 ml EMSK complete medium. The cells were incubated at 37 0 C in 5% CO 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 9-galactosidase (BLUOGAL AND CPRG ASSAYS). When the E. coli 0galactosidase (lacZ) marker gene was incorporated into a -57recombinant virus the plaques containing the recombinants were visualized by one of two simple methods. In the first method, the chemical Bluogal T (Bethesda Research Labs) was incorporated (200 Ag/ml) into the agarose overlay during the plaque assay, and plaques expressing active 0-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 pg/ml) into the agarose overlay during the plaque assay, and plaques expressing active 0-galactosidase turned red. The red plaques were then picked onto fresh cells (EMSK) and .i purified by further red plaque isolation. In both cases viruses were typically purified with three rounds of 15 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 0 C for plaque development to occur. The agarose overlay was then removed from the dish, the cells fixed with 100% methanol for 10 minutes 25 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 (gD) 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 -58times with PBS at room temperature. The secondary antibody, either a goat anti-swine (PRV g50 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 pg/ml 4-chloro-l-naphthol, 0.003% H 2 0 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 107 PFU of S-SPV-009, -014, -016, 017, -018, or -019. Three weeks after the last vaccination, mice are injected intraperitoneally with mg of the corresponding viral glycoprotein. Spleens are S.removed from the mice three days after the last antigen dose.

25 Splenocytes are fused with mouse NS1/Ag4 plasmacytoma cells by the procedure modified from Oi and Herzenberg, Splenocytes and plasmacytoma cells are pelleted together by centrifugation at 300 x 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 (5ml) is added to the cells over three minutes. Cells are pelleted by centrifugation at 300 x 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 -59containing 100 ml of normal spleen feeder layer cells and incubated at 37 0 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 x 106 hybridoma cells into pristane-treated BALB/c mice.

Cell lysates from S-SPV-009, -014, -016, -017, -018, or 15 019 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, 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 Pre- 25 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 0 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°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 0 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 0 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 15 allowed to develop for 15 minutes. The reaction is terminated by addition of 0.1M oxalic acid. The color is read at absorbance 410nm 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. Three promoter cassettes LP1, EP1 and LP2 based on promoters that have been defined in the 25 vaccinia virus 7 and 8) were designed. 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 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 Figures 4, 5 and 8).

-61- 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 intramuscularly, intradermally or orally about 103 to 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- 15 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.

25 CLONING OF EQUINE INFLUENZA VIRUS HEMAGGLUTININ AND NEURAMINIDASE GENES. The equine influenza virus hemagglutinin (HA) and Neuraminidase (NA) genes was cloned essentially as described by Katz et al. (42) for the HA gene of human influenza virus. Viral RNA was prepared from virus grown in MDBK cells (for Influenza A/equine/Alaska/91 and Influenza A/equine/Miami/63) and MDCK cells (for Influenza A/equine/Prague/56 and Influenza A/equine/Kentucky/81) was first converted to cDNA utilizing an oligo nucleotide primer specific for the target gene. The cDNA was used as a template for PCR cloning (51) of the targeted gene region. The PCR primers were designed to incorporate restriction sites which -62permit the cloning of the amplified coding regions into vectors containing the appropriate signals for expression in EHV. One pair of oligo nucleotide primers was required for each coding region. The HA gene coding regions from the serotype 2 (H3) viruses (Influenza A/equine/Miami/63, Influenza A/equine/Kentucky/B1, and Influenza A/equine/Alaska/91) was cloned utilizing the fol 1 o w i ng p r i m e rs 5 GGAGGCCTTCATGACAGACAACCATTATTTTGATACTACTGA-3' (SEQ ID NO: 120) for cDNA priming and combined with GAAGGCCTTCTCAAATGCAAATGTTGCATCTGATGTTGCC-3' (SEQ ID NO: 121) for PCR. The HA gene coding region from the serotype 1 (H7) virus (Influenza A/equine/Prague/56) was be cloned e utilizing the following primers 5' 15 GGGATCCATGAACACTCAAATTCTAATATTAG-3' (SEQ ID NO: 122) for cDNA priming and combined with 5' GGGATCCTTATATACAAATAGTGCACCGCA-3' (SEQ ID NO: 123) for PCR. The NA gene coding regions from the serotype 2 (N8) viruses (Influenza A/equine/Miami/63, Influenza A/equine/Kentucky/81, and Influenza A/equine/Alaska/91) was cloned utilizing the following primers .GGGTCGACATGAATCCAAATCAAAAGATAA-3' (SEQ ID NO: 124) for cDNA priming and combined with GGGTCGACTTACATCTTATCGATGTCAAA-3' (SEQ ID NO: 125) for 25 PCR. The NA gene coding region from the serotype 1 (N7) virus (Influenza/A/equine/Prague/56) was cloned utilizing the following primers 5' -GGGATCCATGAATCCTAATCAAAAACTCTTT- 3' (SEQ ID NO: 118) for cDNA priming and combined with 5'-GGGATCCTTACGAAAAGTATTTAATTTGTGC-3' (SEQ ID NO: 119) for PCR. Note that this general strategy was used to clone the coding regions of HA and NA genes from other strains of equine influenza A virus. The EIV HA or NA genes were cloned as a blunt ended SalI or BamHI fragment into a blunt ended EcoRI site behind the LP2EP2 promoter of the SPV homology vector.

-63- CLONING OF PARAINFLUENZA-3 VIRUS FUSION AND HEMAGGLUTININ GENES. The parainfluenza-3 virus fusion and hemagglutinin (HN) genes were cloned by a PCR CLONING procedure essentially as described by Katz et al. (42) for the HA gene of human influenza. Viral RNA prepared from bovine PI-3 virus grown in Madin-Darby bovine kidney (MDBK) cells was first converted to cDNA utilizing an oligonucleotide primer specific for the target gene. The cDNA was then used as a template for polymerase chain reaction (PCR) cloning (15) of the targeted region. The PCR primers were designed to incorporate restriction sites which permit the cloning of the amplified coding regions into vectors containing the appropriate signals for expression in SPV. One pair of oligonucleotides were 15 required for each coding region. The F gene coding region from the PI-3 strain SF-4 (VR-281) was cloned using the following primers: 5' -TTATGGATCCTGCTGCTGTGTTGAACAACTTTGT- 3' (SEQ ID NO: 130) for cDNA priming and combined with S. 5'-CCGCGGATCCCATGACCATCACAACCATAATCATAGCC-3' (SEQ ID NO: 131) for PCR. The HN gene coding region from PI-3 strain SF-4 (VR-281) was cloned utilizing the following primers: S9. 5'-CGTCGGATCCCTTAGCTGCAGTTTTTTGGAACTTCTGTTTTGA-3' (SEQ ID NO: 132) for cDNA priming and combined with CATAGGATCCATGGAATATTGGAAACACACAAACAGCAC-3' (SEQ ID NO: 25 133) for PCR. Note that this general strategy is used to clone the coding region of F and HN genes from other strains of PI-3. The DNA fragment for PI-3 HN or F was digested with BamHI to yield an 1730 bp or 1620 bp fragment, respectively. The PI-3 HN fragment is cloned into the BamHI site next to the LP2EP2 promoter of the SPV homology vector. The PI-3 F fragment was cloned into the BamHI site next to the LP2EP2 promoter of the SPV homology vector to yield homology vector 713-55.10.

CLONING OF BOVINE VIRAL DIARRHEA VIRUS g48 and g53 GENES.

The bovine viral diarrhea g48 and g53 genes were cloned by a PCR CLONING procedure essentially as described by -64- Katz et al. (42) for the HA gene of human influenza.

Viral RNA prepared from BVD virus Singer strain grown in Madin-Darby bovine kidney (MDBK) cells was first converted to cDNA utilizing an oligonucleotide primer specific for the target gene. The cDNA was then used as a template for polymerase chain reaction (PCR) cloning of the targeted region. The PCR primers were designed to incorporate restriction sites which permit the cloning of the amplified coding regions into vectors containing the appropriate signals for expression in SPV.

One pair of oligonucleotides were required for each coding region. The g48 gene coding region from the BVDV Singer strain (49) was cloned using the following primers: 5' -ACGTCGGATCCCTTACCAAACCACGTCTTACTCTTGTTTCC-3' S. 15 (SEQ ID NO: 134) for cDNA priming and combined with ACATAGGATCCCATGGGAGAAACATAACACAGTGGAACC-3' (SEQ ID NO: 135) for PCR. The g53 gene coding region from the BVDV Singer strain (49) was cloned using the following primers: 5'-CGTGGATCCTCAATTACAAGAGGTATCGTCTAC-3' (SEQ ID NO: 136) for cDNA priming and combined with CATAGATCTTGTGGTGCTGTCCGACTTCGCA-3' (SEQ ID NO: 137) for PCR. Note that this general strategy is used to clone the coding region of g48 and g53 genes from other strains of BVDV. The DNA fragment for BVDV g 48 was digested with 25 BamHI to yield an 678 bp fragment. The DNA fragment for BVDV g53 was digested with BglII and BamHI to yield an 1187 bp fragment. The BVDV g48 or g53 DNA fragments were cloned into the BamHI site next to the LP2EP2 promoter of the SPV homology vector to yield homology vectors, 727- 78.1 and 738-96, respectively.

CLONING OF BOVINE RESPIRATORY SYNCYTIAL VIRUS FUSION, NUCLEOCAPSID AND GLYCOPROTEIN GENES. The bovine respiratory syncytial virus fusion nucleocapsid and glycoprotein genes were cloned by a PCR CLONING procedure essentially as described by Katz et al. (42) for the HA gene of human influenza. Viral RNA prepared from BRSV virus grown in bovine nasal turbinate (BT) cells was first converted to cDNA utilizing an oligonucleotide primer specific for the target gene. The cDNA was then used as a template for polymerase chain reaction (PCR) cloning (15) of the targeted region. The PCR primers were designed to incorporate restriction sites which permit the cloning of the amplified coding regions into vectors containing the appropriate signals for expression in SPV. One pair of oligonucleotides were required for each coding region. The F gene coding region from the BRSV strain 375 (VR-1339) was cloned using the Sf o 1 1 o w i n g p r i m e r s 5 TGCAGGATCCTCATTTACTAAAGGAAAGATTGTTGAT-3' (SEQ ID NO: 138) for cDNA priming and combined with 15 CTCTGGATCCTACAGCCATGAGGATGATCATCAGC-3' (SEQ ID NO: 139) for PCR. The N gene coding region from BRSV strain 375 (VR-1339) was cloned utilizing the following primers: CGTCGGATCCCTCACAGTTCCACATCATTGTCTTTGGGAT-3'(SEQ ID NO: 140) for cDNA priming and combined with CTTAGGATCCCATGGCTCTTAGCAAGGTCAAACTAAATGAC-3' (SEQ ID NO: 141) for PCR. The G gene coding region from BRSV strain 375 (VR-1339) was cloned utilizing the following primers: 5'-CGTTGGATCCCTAGATCTGTGTAGTTGATTGATTTGTGTGA-3' (SEQ ID NO: 142) for cDNA priming and combined with 25 CTCTGGATCCTCATACCCATCATCTTAAATTCAAGACATTA-3' (SEQ ID NO: 143) for PCR. Note that this general strategy is used to clone the coding region of F, N and G genes from other strains of BRSV. The DNA fragments for BRSV F, N, or G were digested with BamHI to yield 1722 bp, 1173 bp, or 771 bp fragments, respectively. The BRSV F, N, and G DNA fragments were cloned into the BamHI site next to the LP2EP2 promoter of the SPV homology vector to yield homology vectors, 727-20.10, 713-55.37 and 727-20.5, respectively.

RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. Chicken spleens were dissected from 3 week -66old SPAFAS hatched chicks, washed, and disrupted through a syringe/needle to release cells. After allowing stroma and debri to settle out, the cells were pelleted and washed twice with PBS. The cell pellet was treated with a hypotonic lysis buffer to lyse red blood cells, and splenocytes were recovered and washed twice with PBS.

Splenocytes were resuspended at 5 x 106 cells/ml in RPMI containing 5% FBS and 5 Ag/ml Concanavalin A and incubated at 39° for 48 hours. Total RNA was isolated from the cells using guanidine isothionate lysis reagents and protocols from the Promega RNA isolation kit (Promega Corporation, Madison WI). 4pg of total RNA was used in S.i! each 1st strand reaction containing the appropriate antisense primers and AMV reverse transcriptase (Promega 15 Corporation, Madison WI). cDNA synthesis was performed in *.the same tube following the reverse transcriptase reaction, using the appropriate sense primers and Vent® e* DNA polymerase (Life Technologies, Inc. Bethesda, MD).

SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. When the E.coli 0-glucuronidase (uidA) marker gene was incorporated into a recombinant virus the plaques containing recombinants were visualized by a simple assay. The enzymatic substrate was incorporated 25 (300 pg/ml) into the agarose overlay during the plaque assay. For the uidA marker gene the substrate X-Glucuro Chx (5-bromo-4-chloro-3-indolyl-3-D-glucuronic acid Cyclohexylammonium salt, Biosynth AG) was used. Plaques that expressed active marker enzyme turned blue. The blue plaques were then picked onto fresh ESK-4 cells and purified by further blue plaque isolation. In recombinant virus strategies in which the enzymatic marker gene is removed the assay involves plaque purifying white plaques from a background of parental blue plaques. In both cases viruses were typically purified with three rounds of plaque purification.

-67- 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 Figures 4A-4D. 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 3628 base pair HindIII to BglII restriction sub-fragment of the SPV HindIII restriction fragment M (23).

H. OMOLOGY VECTOR 520-17.5. The plasmid 520-17.5 was 20 constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene flanked by SPV DNA. Upstream of the marker gene is an approximately 2149 base pair fragment of SPV DNA. Downstream of the marker gene is an approximately S. 25 1484 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 3galactosidase (lacZ) marker gene is under the control of a synthetic early/late pox promoter. A detailed description of the plasmid is given in Figures 4A-4D. 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 Figures 4A-4D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega).

-68- Fragment 1 is an approximately 2149 base pair HindIII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 3006 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11) Fragment 3 is an approximately 1484 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 0-galactosidase (lacZ) marker gene and the PRV g50 (gD) gene flanked by SPV DNA.

Upstream of the foreign genes is an approximately 2149 base pair fragment of SPV DNA. Downstream of the foreign 15 genes is an approximately 1484 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 0-galactosidase 20 (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the g50 (gD) gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in Figures 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 Figures The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 2149 base pair HindIII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 3006 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 3 is an approximately 1571 base pair EcoRI to StuI restriction sub-fragment of the PRV BamHI fragment 7 Note that the EcoRI site was introduced in to this -69fragment 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

CGCGAATTCGCTCG

CAGCGCTATTGGC-3') (SEQ ID NO:41) sits down on the PRV (gD) 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 Sail to produce an approximately 509 base pair fragment. The approximately 1049 base pair Sall to Stul sub-fragment of PRV BamHI #7 may then be ligated to the approximately 509 base pair 15 EcoRI to SalI fragment to generate the approximately 1558 base pair EcoRI to StuI fragment 3. Fragment 4 is an approximately 1484 base pair AccI to BglII restriction sub-fragment of the SPV HindIII fragment M (23).

20 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 P-galactosidase (lacZ) S. marker gene and the PRV gIII (gC) gene flanked by SPV DNA. Upstream of the foreign DNA genes is an 25 approximately 1484 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 2149 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 0galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the gIII (gC) gene is under the control of a synthetic early pox promoter (EP2) A detailed description of the plasmid is given in Figures 10A-10D. It may be constructed utilizing standard recombinant DNA techniques (22 and by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 10A-10D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M 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 EcoRI linkers have replaced the NcoI and NcoI sites at the ends of this fragment. Fragment 4 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII fragment M 15 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 20 SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the PRV gIII (gC) gene flanked by SPV DNA. Upstream of the foreign DNA genes is an approximately 2149 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 1484 25 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 3galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the gIII (gC) gene is under the control of a synthetic early late pox promoter (EP1LP2). A detailed description of the plasmid is given in Figures 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 Figures 11A-11D. The plasmid vector was derived from an -71approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M 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 EcoRI linkers have replaced the NcoI and Ncol sites at the ends of this fragment. Fragment 4 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII fragment M The AccI sites in fragments 1 and 4 have been converted to PstI sites using synthetic DNA linkers.

HQMOLOGY VECTOR 570-91.64. The plasmid 570-91.64 was S.constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 3-galactosidase (lacZ) marker gene and the PRV gIII (gC) gene flanked by SPV DNA. Upstream of the foreign DNA genes is an approximately 1484 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 2149 base pair fragment of SPV DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR S. 25 GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the 3galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the gIII (gC) gene is under the control of a synthetic late early pox promoter (LP2EP2). A detailed description of the plasmid is given in Figures 12A-12D. It may be constructed utilizing standard recombinant DNA techniques (22 and by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 12A-12D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an -72approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M 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 EcoRI linkers have replaced the NcoI and NcoI sites at the ends of this fragment.

Fragment 4 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII fragment M 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 SSPV. It incorporates an E.coli 0-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 2149 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1484 base pair fragment of SPV S. DNA. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING 25 RECOMBINANT SPV a virus containing DNA coding for the foreign genes will result. Note that the -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 Figures 8A-8D. 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 Figures 8A-8D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 2149 base pair -73- HindIII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M 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 Figure 7. The cDNA clone was generated from the B1 strain of NDV using standard cDNA cloning techniques Fragment 3 is an approximately 3006 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11) Fragment 4 is an approximately 1484 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 15 SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the ILT gG gene flanked by SPV DNA.

Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 base pair fragment of SPV 20 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 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter 25 (LP1), and the ILT gG gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in Figures 13A-13D.

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 Figures 13A-13D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega).

Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 1073 base pair EcoRI to MboI fragment.

-74- 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 kb Asp718I fragment of ILT virus genome. The first primer 91.13 CCGAATTCCGGCTTCAGTAACATAGGATCG (SEQ ID NO: 81) sits down on the ILT gG 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: 82) 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 15 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 2149 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 0-galactosidase (lacZ) marker gene and the ILT gI gene flanked by SPV DNA.

Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the ILT gI gene is under the control of a synthetic late/early pox promoter (LP2EP2).

A detailed description of the plasmid is given in Figures 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 Figures 14A-14D.

The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair Bgl II to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 1090 base pair fragment with EcoRI and 15 BamHI restriction sites at the ends synthesized by PCR cloning and containing the entire amino acid coding sequence of the ILT gI gene. The ILT gI 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 7181 fragment. The first primer 103.6 -CCGGAATTCGCTACTT GGAACTCTGG-3') (SEQ ID NO: 83) sits down on the ILT gI sequence at amino acid number 2 and introduces an EcoRI site at the 5' end of the ILT gI gene. The second primer 103.3 CATTGTCCCGAGACGGACAG-3') (SEQ ID NO: 84) sits down on the ILT gI sequence at approximately amino acid 269 on the opposite strand to primer 103.6 and primes toward the 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 end of the ILT gI gene. The third primer 103.4 CGCGATCCAACTATCGGTG-3') (SEQ ID NO: 85) sits down on the ILT gI gene at approximately amino acid 153 priming toward the 3' end of the gene. The fourth primer 103.5 ACTTAATCAC-3') (SEQ ID NO: 86) sits down at the 3' end of the ILT gI gene 14 base pairs -76beyond 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 gI gene.

Fragment 2 consists of the products of the two PCR reactions ligated together to yield an ILT gI 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 2149 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 HOMOLOGY VECTOR 614-83.18. The plasmid 614-83.18 was a.

constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli -galactosidase (lacZ) Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 base pair fragment of SPV DNA. When the plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, Sa virus contai oning DNA coding for the foreign genes will result. Note that the 3-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 Figures 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 Figures 15A-15D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega).

-77- Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M 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 gG 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 ATGAATTCCCCTGCCGCCCGGACCGGCACC-3') (SEQ ID NO: 87) sits down on the IBR gG sequence at amino acid number 1 and introduces an EcoRI site at the 5' end of the IBR gG gene and two additional amino acids between amino acids 1 and 15 2 The second primer 106 .8 CATGGATCCCGCTCGAGGCGAGCGGGCTCC-3') (SEQ ID NO: 88) sits down on the IBR gG sequence at approximately amino acid 362 on the opposite strand to primer 1 and primes synthesis toward the 5' end of the IBR gG gene. Fragment 20 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 gG gene. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction 25 fragment of plasmid pJF751 (11) Fragment 4 is an approximately 2149 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 gE HOMOLOGY VECTOR): This lacZ/IBR gE homology vector is used to insert foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the IBR gE 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 -78for the foreign genes will result. Note that the 3galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter and the gE 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 The IBR gE gene is an S. approximately 1888 base pair fragment synthesized by PCR 15 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: 20 89) sits down on the IBR gE gene at amino acid number 1 and introduces an EcoRI site at the 5' end of the IBR gE gene and adds two additional amino acids at the amino S. terminus of the protein. The second primer 4/93.18DR CTCGCTCGCCCAGGATCCCTAGCGGAGGATGGACTTGAGTCG-3') (SEQ ID NO: 90) sits down on the IBR gE sequence at approximately amino acid 648 on the opposite strand to primer 1 and primes synthesis toward the 5' end of the IBR gE 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 S fragment M The AccI site in the SPV homology vector is converted to a unique XbaI site.

HOMOLOGY VECTOR FOR CONSTRUCTING S-SPV-018 (LacZ/PRV gE HOMOLOGY VECTOR): This homology vector is constructed for the purpose of inserting foreign DNA into SPV. It -79incorporates an E. coli 0-galactosidase (lacZ) marker gene and the PRV gE gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the PRV gE gene is under the control of a synthetic early/late pox promoter (EP1LP2). The homology vector is constructed utilizing standard I 'recombinant DNA techniques (22,30), by joining 15 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 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M 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 Dral to MluI sub-fragment of PRV derived from the PRV BamHI #7 DNA fragment. The Dral site is converted to an EcoRI site through the use of a synthetic DNA linker. The Dral site sits 45 base pairs upstream of the natural gE start codon and extends the open reading frame at the amino terminus of the protein for "5 amino acids. The synthetic pox promoter/EcoRI DNA linker contributes another 4 amino acids. Therefore, the engineered gE gene contains 19 additional amino acids fused to the amino terminus of gE. 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 2149 base pair AccI to HindIII sub-fragment of the SPV HindIII fragment M 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 S: 15 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) HOMOLOGY VECTOR 708-78.9. The plasmid 708-78.9 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the infectious bovine rhinotracheitis virus (IBRV) gE gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the IBRV gE gene is under the control of a synthetic late/early pox promoter (LP2EP2) It may be constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the -81following sources. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair Bgl II to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 475 base pair fragment with EcoRI and BamHI restriction sites at the ends. The EcoRI and BamHI sites are synthesized by PCR cloning. The PCR product contains the entire amino acid coding sequence of the IBRV gE 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 2/94.5DR CTGGTTCGGCCCAGAATTCGATGCAACCCACCGCGCCGCCCCG-3') (SEQ ID NO: 116) sits down on the IBR gE gene at amino acid number 1 and introduces an EcoRI site at the 5' end of the IBRV gE gene and adds two additional amino acids at the amino terminus of the protein. The second primer 4/93.18DR (SEQ ID NO: 117) sits down on the IBRV gE sequence (44) at approximately amino acid 648 on the opposite strand to the first primer and primes synthesis toward ~the 5' end of the IBRV gE gene. The PCR product was digested with EcoRI and BamHI to yield a fragment approximately 1950 base pairs in length corresponding to the IBRV gE gene. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2149 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 723-59A9.22. The plasmid 723-59A9.22 was used to insert foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the equine influenza virus NA PR/56 gene flanked by SPV DNA. When this plasmid was used according to the HOMOLOGOUS -82- RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes results. Note that the -galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the EIV PR/56 NA gene is under the control of a synthetic late/early pox promoter (LP2EP2). A detailed description of the plasmid is given in Figures 18A-18D.

The homology vector was 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 was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 1484 base pair BglII to 15 AccI restriction sub-fragment of the SPV HindIII fragment M Fragment 2 is the NA gene coding region from the equine Influenza A/Prague/56 (serotype 1 (N7) virus) cloned as an approximately 1450 base pair BamHI fragment utilizing the following primers 20 GGGATCCATGAATCCTAATCAAAAACTCTTT-3' (SEQ ID NO: 118) for cDNA priming and combined with GGGATCCTTACGAAAAGTATTTAATTTGTGC-3' (SEQ ID NO: 119) for PCR. (see CLONING OF EQUINE INFLUENZA VIRUS HEMAGGLUTININ AND NEURAMINIDASE GENES). Fragment 3 is an approximately 25 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII restriction fragment M The AccI site in the SPV homology vector was converted to a unique NotI site.

HOMOLOGY VECTOR 727-54.60. The plasmid 727-54 .60 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 3-galactosidase (lacZ) marker gene and the pseudorabies virus (PRV) gII (gB) gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA.

-83- Downstream of the foreign genes is an approximately 2149 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 3galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the PRV gB gene is under the control of a synthetic late/early pox promoter (LP2EP2). A detailed description of the plasmid is given in Figures 19A-19D. It may be constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 19A-19D. The plasmid vector was derived from an 15 approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23) Fragment 2 is an approximately 3500 base pair 20 fragment which contains the coding sequence for the PRV gB gene within the KpnI C fragment of genomic PRV DNA(21) Fragment 2 contains an approximately 53 base pair synthetic fragment containing the amino terminus of the PRV gB gene, an approximately 78 base pair SmaI to Nhe I fragment from the PRV KpnI C genomic fragment, and an approximately 3370 base pair NheI to EcoRI fragment from the PRV KpnI C genomic fragment Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2149 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 727-67.18. The plasmid 727-67.18 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) -84marker gene and the hepatitis B virus core antigen gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 2149 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 pgalactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the hepatitis

B

core antigen gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in Figures 20A-20D. It may be .i constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the *following sources with the synthetic DNA sequences indicated in Figures 20A-20D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BanHI restriction fragment of pSP64 (Promega). Fragment 20 1 is an approximately 1484 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 Fragment 3 is an approximately 589 base pair fragment with BamHI and EcoRI restriction sites at the ends. This fragment contains the hepatitis B core antigen coding sequences (amino acids 25-212) (Ref. Fragment 4 is an approximately 2149 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 732-18.4. The plasmid 732-18.4 was used to insert foreign DNA into SPV. It incorporates an E.

coli 3 -galactosidase (lacZ) marker gene and the equine influenza virus AK/91 NA gene flanked by SPV DNA. When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes results. Note that the 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the EIV AK/91 NA gene is under the control of a synthetic late/early pox promoter (LP2EP2). A detail description of the plasmid is given in Figures 21A-21D.

The homology vector was 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 was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega).

Fragment 1 is an approximately 1484 base pair BglII to 15 AccI restriction sub-fragment of the SPV HindIII fragment M Fragment 2 is the NA gene coding region from the equine Influenza A/Alaska/91 (serotype 2 (N8) virus) cloned as an approximately 1450 base pair SalI fragment utilizing the following primers GGCGACATGAATCCAAATCAAAAGATAA-3' (SEQ ID NO: 124) for cDNA priming and combined with GGGTCGACTTACATCTTATCGATGTCAAA-3' (SEQ ID NO: 125) for PCR (see CLONING OF EQUINE INFLUENZA VIRUS HEMAGGLUTININ

AND

NEURAMINIDASE GENES). Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII restriction fragment M The AccI site in the SPV homology vector was converted to a unique 0TotI site HOMOLOGY VECTOR 741-80.3 The plasmid 741-80.3 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli G-galactosidase (lacZ) marker gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 base pair fragment of SPV DNA. When the plasmid is -86used according to the HOMOLOGOUS RECOMBINATION

PROCEDURE

FOR GENERATING RECOMBINANT SPV, a virus containing

DNA

coding for the foreign genes will result. Note that the 3 -galactosidase (lacZ) marker gene is under the control of a human cytomegalovirus immediate early (HCMV

IE)

promoter. A detailed description of the plasmid is given in Figures 22A-22C. 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 Figures 22A-22C.

The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23) .Fragment 2 is a 1154 base *pair PstI to AvaII fragment derived from a HCMV 2.1 kb PstI fragment containing the HCMV IE promoter (46) Fragment 3 is a 3010 base pair BamHI to PvuII fragment derived from plasmid pJF751 (49) containing the E. coli lacZ gene. Fragment 4 is an approximately 750 base pair Ndel to SalI fragment derived from PRV BamHI #7 which contains the carboxy-terminal 19 amino acids and the polyadenylation signal of the PRV gX gene. Fragment 5 is an approximately 2149 base pair AccI to HindIII 25 subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 5 were converted to unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 741-84.14. The plasmid 741-84.14 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli P-galactosidase (lacZ) marker gene and the human interleukin-2 (IL-2) gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 2149 base pair fragment of SPV DNA. When the plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE

FOR

-87- GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the 3galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the human IL-2 gene is under the control of a synthetic late/early pox promoter (LP2EP2). The coding sequence for the human IL-2 protein is fused at the amino terminus to the PRV gX signal sequence for membrane transport. A detailed description of the plasmid is given in Figures 23A-23D.

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 Figures 23A-23D. The plasmid i* vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 475 base pair fragment with EcoRI and BglII 20 restriction sites at the ends. The EcoRI site is synthesized by PCR cloning and the BglII site is at the 3' end of the human IL-2 cDNA (43, 47). The PCR product contains the entire amino acid coding sequence of the PRV gX signal sequence-human IL-2 gene. In this procedure, the primers described below were used with a template consisting of the cDNA for PRV gX signal sequence-human IL-2 The first primer 5/94.23 CTCGAATTCGAAGTGGGCAACGTGGATCCTCGC-3') (SEQ ID NO: 126) sits down on the PRV gX signal sequence at amino acid number 1 and introduces an EcoRI site at the 5' end of the gene. The second primer 5/94.24 CAGTTAGCCTCCCCCATCTCCCCA-3') (SEQ ID NO: 127) sits down on the human IL-2 gene sequence within the 3' untranslated region on the opposite strand to primer 5/94.23 and primes toward the 5' end of the gene. The PCR product was digested with EcoRI and BglII (BglII is located approximately 3 nucleotides beyond the stop codon -88for the human IL-2 gene) to yield a fragment 475 base pairs in length corresponding to the PRV gX signal sequence-human IL-2 gene. Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2149 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 744-34. The plasmid 744-34 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli G-galactosidase (lacZ) marker gene and the equine herpesvirus type 1 gB gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 2149 S: 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 20 for the foreign genes will result. Note that the 6galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the EHV-1 gB gene is under the control of a synthetic late/early pox promoter (LP2EP2) A detailed description of the plasmid 25 is given in Figures 24A-24D. It may be constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 24A-24D The plasmid vector was derived frcm an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 1484 base pair Bgl II to AccI restriction sub-fragment of the SPV HindIII restriction fragment

M

Fragment 2 is an approximately 2941 base pair fragment with EcoRI and PmeI restriction sites at the ends. Fragment 2 is an approximately 2941 base pair EcoRI to Pmel fragment. Fragment 2 was synthesized as an -89approximately 429 base pair PCR fragment at the 5' end of the gene having a synthetic EcoRI site and a natural BamHI site within the BamHI fragment of EHV-1 genomic DNA and an approximately 2512 base pair restriction fragment at the 3' end of the gene from BanHI to Pmel within the BamHI fragment of EHV-1 genomic DNA (48).

In the procedure to produce the 5' end PCR fragment, the primers described below were used with a template consisting of the EHV-1 BamHI and fragments. The first primer 5/94.3 (5'-CGGAATTCCTCTGGTTGCCGT-3') (SEQ ID NO: 128) sits down on the EHV-1 gB sequence at amino acid number 2 and introduces an EcoRI site at the 5' end of the EHV-1 gB gene and an ATG start codon. The second primer 5/94.4 (5'-GACGGTGGATCCGGTAGGCGGT-3') (SEQ ID NO: 15 129) sits down on the EHV-1 gB sequence at approximately amino acid 144 on the opposite strand to primer 5/94.3 and primes toward the 5' end of the gene. The PCR product was digested with EcoRI and BamHI to yield a fragment 429 b ase pairs in length corresponding to the 5' end of the 20 EHV-1 gB gene. Fragment 2 consists of the products of the PCR reaction (EcoRI to BamHI) and the restriction Sfragment (BamHI to PmeI) ligated together to yield an

EHV-

1 gB gene which is an EcoRI to PmeI fragment approximately 2941 base pairs (979 amino acids) in 25 length. Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2149 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 744-38. The plasmid 744-38 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the equine herpesvirus type 1 gD gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 2149 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 Rgalactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the EHV-1 gD gene is under the control of a synthetic late/early pox promoter (LP2EP2). A detailed description of the plasmid is given in Figures 25A-25D. It may be constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in *Figures 25A-25D. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an *approximately 1484 base pair Bgl II to AccI restriction sub-fragment of the SPV HindIII restriction fragment

M

Fragment 2 is an approximately 1240 base pair HindIII fragment within the BamHI fragment of EHV-1 Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11).

Fragment 4 is an approximately 2149 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 689-50.4. The plasmid 689-50.4 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the infectious bursal disease virus (IBDV) polyprotein gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 base pair fragment of SPV DNA.

When the plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT

SPV,

-91a virus containing DNA coding for the foreign genes will result. Note that the 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the IBDV polyprotein gene is under the control of a synthetic late/early pox promoter (LP2EP2). It may be constructed utilizing standard recombinant

DNA

techniques (22, 30), by joining restriction fragments from the following sources. The plasmid vector was derived from an approximately 2972 base pair Hind III to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction subfragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 3400 base pair fragment with SmaI and HpaI restriction sites at the ends from plasmid 2-84/2-40 This fragment contains the IBDV polyprotein coding sequences. Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 0: is an approximately 2149 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.

ae HOMOLOGY VECTOR 689-50.7. The plasmid 689-50.7 was 25 constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the infectious bursal disease virus (IBDV) VP2 gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 3-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), and the IBDV VP2 gene is under the control of a -92synthetic late/early pox promoter (LP2EP2) It may be constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promegal. Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment

M

Fragment 2 is an approximately 1081 base pair fragment with BclI and BamHI restriction sites at the ends. This fragment codes for the IBDV VP2 protein and is derived from a full length IBDV cDNA clone (51) Fragment 3 is an approximately 3010 base pair BamHI to Pv-uII restriction fragment of plasmid pJF751 (11) Fragment 4 is an approximately 2149 base pair AccI to HindIII sub-fragment 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 7 51-07.A1. The plasmid 7 51-07.A1 was used to insert foreign DNA into SPV. It incorporates an E.

coli 0-galactosidase (lacZ) marker gene and the chicken interferon (cIFN) gene flanked by SPV DNA. When this plasmid was used according to the HOMOLOGOUS 25 RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes results. Note that the 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LPI) and the cIFN gene is under the control of a synthetic late/early pox promoter (LP2EP2). The homology vector was 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 was derived from an approximately 2972 base pair Hindll to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1146 base pair BglII to AccI restriction -93sub-fragment of the SPV HindIII fragment M Fragment 2 is an approximately 577 base pair EcoRI to BglII fragment coding for the cIFN gene (54) derived by reverse transcription and polymerase chain reaction

(PCR)

(Sambrook, et al., 1989) of RNA ISOLATED

FROM

CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. The antisense primer (6/94.13) used for reverse transcription and PCR was 5'-CGACGGATCCGAGGTGCGTTTGGGGCTAAGTGC-3'

(SEQ

ID NO: 211). The sense primer (6/94.12) used for PCR was 5'-CCACGGATCCAGCACAACGCGAGTCCCACCATGGCT-3' (SEQ ID NO: 212). The BamHI fragment resulting from reverse transcription and PCR was gel purified and used as a template for a second PCR reaction to introduce a unique EcoRI site at the 5' end and a unique BglII site at the 3' end. The second PCR reaction used primer 6/94.22 CCA CGAATTCGATGGCTGTGCCTGCAAGCCCACAG-3'; SEQ ID NO: 213) at the 5' end and primer 6/94.34 CGAAGATCTGAGGTGCGTTTGGGGCTAAGTGC-3'; SEQ ID NO: 214) at the 3' end to yield an approximately 577 base pair fragment. The DNA fragment contains the coding sequence from amino acid 1 to amino acid 193 of the chicken interferon protein (54) which includes a 31 amino acid signal sequence at the amino terminus and 162 amino acids of the mature protein encoding chicken interferon.

25 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 restriction sub-fragment of the SPV HindIII restriction fragment M The AccI site in the SWV homology vector was converted to a unique NotI site.

HOMQLQGY VECTOR 7 51-56.A1. The plasmid 7 5 1-56.A1 was used to insert foreign DNA into SPV. It incorporates an E.

coli 0-galactosidase (lacZ) marker gene and the chicken myelomonocytic growth factor (cMGF) gene flanked by SPV DNA. When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING -94- RECOMBINANT SPV a virus containing DNA coding for the foreign genes results. Note that the 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the cMGF gene is under the control of a synthetic late/early pox promoter (LP2EP2).

The homology vector was 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 was 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 fragment M (23) Fragment 2 is an approximately 640 base pair EcoRI to BamHI fragment coding for the cMGF derived by reverse transcription and polymerase chain reaction (PCR) (Sambrook, et al., 1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. The antisense primer (6/94.20) used for reverse transcription and PCR was 5'-CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3, (SEQ ID NO: 215). The sense primer (5/94.5) used for PCR 4. was 5'-GAGCGGATCCTGCAGGAGGAGACACAGAGCTG-3' (SEQ ID NO: 216). The BamHI fragment derived from PCR was subcloned into a plasmid and used as a template for a second

PCR

25 reaction us ing primer 6 94 16 (5 GCGCGAATTCCATGTGCTGCCTCACCCCTGTG-3'; SEQ ID NO: 217) at the 5' end and primer 6/94.20 CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3'; SEQ ID NO: 218) at the 3' end to yield an approximately 640 base pair fragment. The DNA fragment contains the coding sequence from amino acid 1 to amino acid 201 of the cMGF protein which includes a 23 amino acid signal sequence at the amino terminus and 178 amino acids of the mature protein encoding cMGF. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2156 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII restriction fragment M The AccI site in the SPV homology vector was converted to a unique NotI site.

HOMOLOGY VECTOR 752-22.1. The plasmid 752-22.1 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene flanked by SPV DNA. Upstream of the foreign gene is an approximately 855 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1113 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 15 6-galactosidase (lacZ) marker gene is under the control of a swinepox virus OIL gene promoter. The homology vector also contains the synthetic late/early promoter (LP2EP2) into which a second foreign gene is inserted into a unique BamHI or EcoRI site. A detailed description of the plasmid is given in Figures 28A-28D. It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 28A-28D. The plasmid vector was 25 derived from an approximately 2519 base pair HindIII to SphI restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 855 base pair sub-fragment of the SPV HindIII restriction fragment M (23) synthesized by polymerase chain reaction using DNA primers GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3' (SEQ ID NO: 185) and 5'-CATAAGATCTGGCATTGTGTTATTATACTAACAAAAATAAG-3' (SEQ ID NO: 186) to produce an 855 base pair fragment with SphI and EglII ends. Fragment 2 is a 3002 base pair BamHI to PvuII fragment derived from plasmid pJF751 (49) containing the E. coli lacZ gene. Fragment 3 is an approximately 1113 base pair subfragment of the SPV HindIII fragment M synthesized by polymerase chain -96reaction using DNA primers 5 CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3' (SEQ ID NO: 187) and 5'-GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3' (SEQ ID NO: 188) to produce an 1113 base pair fragment with Sail and HindIII ends.

HOMOLOGY VECTOR 752-29.33. The plasmid 759.33 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 3 -galactosidase (lac Z) marker gene and an equine herpesvirus type 1 gB gene flanked by SPV DNA. Upstream of the foreign gene is an approximately 855 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 113 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 fgalactosidase (lacZ) marker gene is under the control of 'a swinepox virus 01L gene promoter and the EHV-1 gB gene is under the control of the late/early promoter (LP2EP2) The LP2EP2 promoter-EHV-1 gB gene cassette was inserted into a NotI site of homology vector 738-94.4. Homology vector 752-29.33 was constructed utilizing standard recombinant DNA techniques (22, 30), by joining 25 restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2519 base pair HindIII to Sphl restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 855 base pair sub-fragment of the SPV HindIII restriction fragment M (23) synthesized by polymerase chain reaction using DNA primers GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3' (SEQ ID NO: 185) and 5'-CATAAGATCTGGCATTGTGTTATTATACTAACAAAAATAAG-3' (SEQ ID NO: 186) to produce an 855 base pair fragment with SphI and BglII ends. Fragment 2 is a 3002 base pair BamHI to PvuII fragment derived from plasmid pJF751 (49) containing the E. coli lacz gene. Fragment 3 is the -97product of a PCR reaction (EcoRI to BamHI) and a restriction fragment (BamHI to PmeI) ligated together to yield an EHV-1 gB gene which is an EcoRI to Pmel fragment approximately 2941 base pairs (979 amino acids) in length. The PCR fragment is an approximately 429 base pair fragment having a synthetic EcoRI site at the 5' end of the gene and a natural BamHI site at the 3' end within the BamHI fragment of EHV-1 genomic DNA. The restriction fragment is an approximately 2512 base pair fragment from BamHI to PmeI within the BamHI fragment of EHV-1 genomic DNA. In the procedure to produce the end PCR fragment, the primers described below were used with a template consisting of the EHV-1 BamHI and "i" fragments.

The first primer 5/94.3 (5'-CGGAATTCCTCTGGTTCGCCGT-3') (SEQ ID NO: 128) sits down on the EHV-1 gB sequence at amino acid number 2 and introduces an EcoRI site at the end of the EHV-1 gB gene and an ATG start codon. The second primer 5/94.4 (5'-GACGGTGGATCCGGTAGGCGGT-3')

(SEQ

ID NO: 129) sits down on the EHV-1 gB sequence at approximately amino acid 144 on the opposite strand to primer 5/94.3 and primes toward the 5' end of the gene.

S. The PCR product was digested with EcoRI and BamHI to 25 yield a fragment 429 base pairs in length corresponding to the 5' end of the EHV-1 gB gene. Fragment 3 consists of the products of the PCR reaction (EcoRI to BamHI) and the restriction fragment (BamHI to PmeI) ligated together to yield an EHV-1 gB gene which is an EcoRI to PmeI fragment approximately 2941 base pairs (979 amino acids) in length. Fragment 4 is an approximately 1113 base pair subfragment of the SPV HindIII fragment M synthesized by polymerase chain reaction using DNA primers CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3' (SEQ ID NO: 187) and 5'-GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3' (SEQ ID NO: 188) to produce an 1113 base pair fragment with SalI and HindIII ends.

-98- HOMOLOGY VECTOR 746-94.1. The plasmid 746-94.1 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli /-galactosidase (lacZ) marker gene and an infectious bovine rhinotracheitis virus glycoprotein E (gE) gene flanked by SPV DNA.

Upstream of the foreign gene is an approximately 855 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1113 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 3-galactosidase (lacZ) marker gene is under the control of a swinepox virus OIL gene promoter and the IBRV gE gene is under the control of the late/early promoter (LP2EP2) It was constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences. A 1250 base pair EcoRI to BamHI fragment coding for amino acids 1 to 417 of the IBRV gE gene (missing 158 amino acids of the carboxy terminal transmembrane region) was inserted into unique EcoRI and BamHI sites of homology vector 752-22.1 (Figures 28A-28D) The 1250 base pair EcoRI to BamHI fragment was synthesized by polymerase chain reaction S. 25 (15) using IBRV (Cooper) genomic DNA as a template and primer 10/94.23 (5'-GGGGAATTCAATGCAACCCACCGCGCCGCCCC-3'; SEQ ID NO: 219) at the 5' end of the IBRV gE gene (amino acid 1) and primer 10/94 22 GGGGGATCCTAGGGCGCGCCCGCCGGCTCGCT-3'; SEQ ID NO: 220) at amino acid 417 of the IBRV gE gene.

HOMOLOGY VECTOR 767-67.3. The plasmid 767-67.3 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and an bovine viral diarrhea virus glycoprotein 53 (BVDV gp53) gene flanked by SPV DNA.

Upstream of the foreign gene is an approximately 855 base -99pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1113 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 3-galactosidase (lacZ) marker gene is under the control of a swinepox virus OIL gene promoter and the BVDV gp53 gene is under the control of the late/early promoter (LP2EP2). It was constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences. A 1187 base .i pair BamHI fragment coding for the BVDV gp53 was inserted into the unique BamHI sites of homology vector 752-22.1 (Figures 28A-28D) The 1187 base pair BamHI fragment was synthesized by polymerase chain reaction (15) as oo described in CLONING OF BOVINE VIRAL DIARRHEA VIRUS gp48 AND gp53 GENES.

HOMOLOGY VECTOR 771-55.11. The plasmid 771-55.11 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli /-galactosidase (lacZ) marker gene and an bovine viral diarrhea virus glycoprotein 48 (BVDV gp48) gene flanked by SPV DNA.

25 Upstream of the foreign gene is an approximately 855 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1113 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 3-galactosidase (lacZ) marker gene is under the control of a swinepox virus OIL gene promoter and the BVDV gp48 gene is under the control of the late/early promoter (LP2EP2). It was constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences. A 678 base pair -100- BamHI fragment coding for the BVDV gp48 was inserted into the unique BamHI sites of homology vector 752-22.1 (Figures 28A-28D). The 678 base pair BamHI fragment was synthesized by polymerase chain reaction (15) as described in CLONING OF BOVINE VIRAL DIARRHEA VIRUS gp48 AND gp53 GENES.

PLASMID 551-47.23. The plasmid 551-47.23 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates the E. coli 0-glucuronidase (0-glu) marker gene under the control of a late/early pox promoter (LP2EP2) It is useful to insert the marker gene into sites in the SPV genome to produce a recombinant swinepox virus. It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources. The plasmid vector was derived from an approximately 3005 base pair HindIII restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 1823 base pair EcoRI to SmaI fragment of the plasmid pRAJ260 (Clonetech). Note that the EcoRI and Smal sites were introduced by PCR cloning. Plasmid 551- 47.23 was used to make recombinant swinepox viruses S- SPV-059, S-SPV-060, S-SPV-061, and S-SPV-062.

25 HOMOLOGY VECTOR 779-94.31. The plasmid 779-94.31 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the pseudorabies virus (PRV) gB (gIl) gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 538 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 1180 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 0 galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the PRV gB gene -101is under the control of a synthetic late/early pox promoter (LP2EP2). A detailed description of the plasmid is given in Figures 30A-30E. It was constructed utilizing standard recombinant DNA techniques (22, and 30), by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2986 base pair HindIII to PstI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 542 base pair HindIII to BglII restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 3500 base pair fragment which contains the coding sequence for the PRV gB gene within the KpnI C fragment of genomic PRV DNA Fragment 2 contains an approximately 53 base pair 15 synthetic fragment containing the amino terminus of the PRV gB gene, an approximately 78 base pair SmaI to Nhe I fragment from the PRV KpnI C genomic fragment, and an approximately 3370 base pair NheI to EcoRI fragment from the PRV KpnI C genomic fragment Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11) Fragment 4 is an approximately 1180 base pair BglII to PstI subfragment of the SPV HindIII fragment M. The BglII sites in fragments 1 and 4 were converted to unique HindIII sites using 25 HindIII linkers.

HOMOLOGY VECTOR 789-41.7. The plasmid 789-41.7 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene, the pseudorabies virus (PRV) gB (gIl) gene and the PRV gD (g50) gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1560 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 -102result. Note that the 3 galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), the PRV gB gene is under the control of a synthetic late/early pox promoter (LP2EP2), and the PRV gD gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in Figures 31A-31D. It was constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega).

SFragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII 15 restriction fragment M Fragment 2 is an S* approximately 1552 base pair subfragment of the PRV BamHI #7 fragment which contains the coding sequence of the PRV gD gene from amino acid 3 to amino acid 279. The EcoRI site and the ATG translation start codon are derived from a polymerase chain reaction using a 5' primer with an EcoRI site. The StuI site at the 3' end is normally within the PRV gI gene 3' to the PRV gD gene. The entire open reading frame beginning at the EcoRI site codes for S. 405 amino acids. Fragment 3 is an approximately 48 base 25 pair AccI to NdeI subfragment of the SPV HindIII M fragment. Fragment 4 is an approximately 3500 base pair fragment which contains the coding sequence for the PRV gB gene within the KpnI C fragment of genomic PRV DNA(21) Fragment 4 contains an approximately 53 base pair synthetic fragment containing the amino terminus of the PRV gB gene, an approximately 78 base pair SmaI to Nhe I fragment from the PRV KpnI C genomic fragment, and an approximately 3370 base pair NheI to EcoRI fragment from the PRV KpnI C genomic fragment Fragment 5 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 6 is an approximately 1560 base pair NdeI to HindIII -103subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 3 were converted to unique PstI sites using PstI linkers. The NdeI sites in fragments 3 and 6 were converted to unique HindIII sites using HindIII linkers. An approximately 545 base pair NdeI to NdeI subfragment (Nucleotides 1560 to 2104; SEQ ID NO. of the SPV HindIII M fragment has been deleted which would span SPV fragments 3 and 6.

HOMOLOGY VECTOR 789-41.27. The plasmid 789-41.27 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli A-galactosidase (lacZ) marker gene, the pseudorabies virus (PRV) gB (gIl) gene and the PRV gC (gIll) gene flanked by SPV DNA. Upstream 15 of the foreign genes is an approximately 1560 base pair S* fragment of SPV DNA. Downstream of the foreign genes is an approximately 1484 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 P galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), the PRV gB gene is under the control of a synthetic late/early pox promoter (LP2EP2), and the PRV 25 gC gene is under the control of a synthetic early/late pox promoter (EP1LP2) A detailed description of the plasmid is given in Figures 32A-32D. It was constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources .with the synthetic DNA sequences indicated. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1560 base pair HindIII to NdeI subfragment of the SPV HindIII fragment M. Fragment 2 is an approximately 3500 base pair fragment which contains the coding sequence for the PRV gB gene within the KpnI C fragment of genomic PRV DNA(21).

-104- Fragment 2 contains an approximately 53 base pair synthetic fragment containing the amino terminus of the PRV gB gene, an approximately 78 base pair SmaI to Nhe I fragment from the PRV KpnI C genomic fragment, and an approximately 3370 base pair NheI to EcoRI fragment from the PRV KpnI C genomic fragment Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11) Fragment 4 is an approximately 48 base pair AccI to NdeI subfragment of the SPV HindIII M fragment. Fragment 5 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 sites at the ends of the fragment. Fragment 6 is an approximately 1484 base 15 pair AccI to BglII restriction sub-fragment of the SPV HindIII restriction fragment M The NdeI sites in fragments 1 and 4 were converted to unique HindIII sites using HindIII linkers. The AccI site in fragments 4 and 6 were converted to unique PstI sites using PstI linkers.

An approximately 545 base pair NdeI to Ndel (Nucleotides 1560 to 2104; SEQ ID NO. subfragment of the SPV HindIII M fragment has been deleted which would span SPV fragments 4 and 6.

Ott.

S: 25 HOMOLOGY VECTOR 789-41.47. The plasmid 789-41.47 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli -galactosidase (lacZ) marker gene, the pseudorabies virus (PRV) gC (gIII) gene and the PRV gD (g50) gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1560 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 0 galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter -105- (LP1), the PRV gC gene is under the control of a synthetic early/late pox promoter (EP1LP2), and the PRV gD gene is under the control of a synthetic early/late pox promoter (EP1LP2). A detailed description of the plasmid is given in Figures 33A-33D. It was constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an **approximately 1552 base pair subfragment of the PRV BamHI 15 #7 fragment which contains the coding sequence of the PRV gD gene from amino acid 3 to amino acid 279. The EcoRI site and the ATG translation start codon are derived from a polymerase chain reaction using a 5' primer with an EcoRI site. The StuI site at the 3' end is normally within the PRV gI gene 3' to the PRV gD gene. The entire open reading frame beginning at the EcoRI site codes for 405 amino acids. Fragment 3 is an approximately 48 base pair AccI to NdeI subfragment of the SPV HindIII M fragment. Fragment 4 is an approximately 3010 base pair 25 BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 5 is an approximately 2378 base pair NcoI to NcoI fragment of plasmid 251-41.A, a subfragment of PRV BamHI #2 and EcoRI linkers have replaced the NcoI sites at the ends of the fragment. Fragment 6 is an approximately 1560 base pair NdeI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 3 were converted to unique PstI sites using PstI linkers. The NdeI sites in fragments 3 and 6 were converted to unique HindIII sites using HindIII linkers. An approximately 545 base pair NdeI to NdeI subfragment (Nucleotides 1560 to 2104; SEQ ID NO. of -106the SPV HindIII M fragment has been deleted which would span SPV fragments 3 and 6.

HOMOLOGY VECTOR 789-41.73. The plasmid 789-41.73 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene, the pseudorabies virus (PRV) gB (gII) gene, the PRV gC (gIII) gene and the PRV gD (g50) gene flanked by 'SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 1560 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 15 for the foreign genes will result. Note that the F galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1), the PRV gB gene is under the control of a synthetic late/early pox promoter (LP2EP2), the PRV gC gene is under the control of a synthetic early/late promoter (EP1LP2), and the PRV gD gene is under the control of a synthetic late/early pox promoter (LP2EP2) A detailed description of the plasmid is given in Figures 34A-34E. It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining 25 restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (.Promega) Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M (23) Fragment 2 is an approximately 1552 base pair subfragment of the PRV BamHI #7 fragment which contains the coding sequence of the PRV gD gene from amino acid 3 to amino acid 279. The EcoRI site and the ATG translation start codon are derived from a polymerase chain reaction using a 5' primer with an EcoRI site. The StuI site at the 3' end is normally within the PRV gI gene 3' to the -107- PRV gD gene. The entire open reading frame beginning at the EcoRI site codes for 405 amino acids. 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 sites at the ends of the fragment. Fragment 4 is an approximately 48 base pair AccI to NdeI subfragment of the SPV HindIII M fragment.

Fragment 5 is an approximately 3500 base pair fragment which contains the coding sequence for the PRV gB gene within the KpnI C fragment of genomic PRV DNA(21).

Fragment 5 contains an approximately 53 base pair synthetic fragment containing the amino terminus of the PRV gB gene, an approximately 78 base pair SmaI to Nhe I fragment from the PRV KpnI C genomic fragment, and an 15 approximately 3370 base pair NheI to EcoRI fragment from .PRV KpnI C genomic fragment (21) Fragment 6 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 7 is an approximately 1560 base pair NdeI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 3 were converted to unique PstI sites using PstI linkers. The NdeI sites in fragments 3 and 6 were converted to unique HindIII sites using HindIII .linkers. An approximately 545 base pair NdeI to NdeI 25 subfragment (Nucleotides 1560 to 2104; SEQ ID NO. of the SPV HindIII M fragment has been deleted which would span SPV fragments 3 and 6.

HOMOLOGY VECTOR 791-63.19. The plasmid 791-63.19 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli P-galactosidase (lacZ) marker gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 -108coding for the foreign genes will result. Note that the 3 galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequence. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 3010 base pair BamHI to PvuII restriction .i fragment of plasmid pJF751 (11) Fragment 3 is an approximately 2149 base pair AccI to HindIII subfragment 15 of the SPV HindIII fragment M. The AccI sites in fragments 1 and 3 were converted to unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 791-63.41. The plasmid 791-63.41 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli -galactosidase (lacZ) marker gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately S* 25 2149 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 0 galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP2) It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following souices with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII -109restriction fragment M Fragment 2 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 3 is an approximately 2149 base pair AccI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 3 were converted to unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 796-18.9. The plasmid 796-18.9 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 3-galactosidase (lacZ) marker gene flanked by SPV DNA. Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 galactosidase (lacZ) marker gene is under the control of a synthetic early pox promoter (EP1). It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 25 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment M Fragment 2 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 3 is an approximately 2149 base pair AccI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and 3 were converted to unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 783-39.2. The plasmid 783-39.2 was constructed for the purpose of inserting foreign DNA into -110- SPV. It incorporates an E. coli &-galactosidase (lacZ) marker gene and an bovine viral diarrhea virus glycoprotein 53 (BVDV gp53) gene flanked by SPV DNA.

Upstream of the foreign gene is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 0-galactosidase (lacZ) marker gene is under the control of a late promoter (LP1) and the BVDV gp53 gene is under the control of the late/early .promoter (LP2EP2). It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII restriction fragment

M

Fragment 2 is an approximately 1187 base pair BamHI fragment coding for the BVDV gp53. The 1187 base pair BamHI fragment was synthesized by polymerase chain reaction (15) as described in CLONING OF BOVINE VIRAL 25 DIARRHEA VIRUS gp48 AND gp53 GENES. Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11) Fragment 4 is an approximately 2149 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 749-75.78. The plasmid 749-75.78 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the infectious bursal disease virus (IBDV) polymerase gene flanked by SPV DNA. Upstream of -111the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 3 galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the IBDV polymerase gene is under the control of a synthetic late/early promoter (LP2EP2). It was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 15 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII orestriction fragment M (23) Fragment 2 is an approximately 2700 EcoRI to AscI restriction fragment synthesized by cDNA cloning and polymerase chain reaction (PCR) from an IBDV RNA template. cDNA and PCR primers

-CACGAATTCTGACATTTTCAACAGTCCACAGGCGC-

3 12/93.4)

(SEQ

ID NO: and 5'-GCTGTTGGACATCACGGGCCAGG-3' 9/93.28)

(SEQ

ID NO: were used to synthesize an approximately 1400 25 base pair EcoRI to BclI fragment at the 5' end of the IBDV polymerase gene. cDNA and PCR primers ACCCGGAACATATGGTCAGCTCCAT-3'; 12/93.2) (SEQ ID NO: and -GGCGCGCCAGGCGAAGGCCGGGGATACGG-3'; 12/93.3) (SEQ ID NO: were used to synthesize an approximately 1800 base pair BclI to AscI fragment at the 3' end of the IBDV polymerase gene. The two fragments were ligated at the BclI site to form the approximately 2700 base pair EcoRI to BclI fragment. Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 2149 base pair AccI to HindIII subfragment of the SPV HindIII -112fragment M. The AccI sites in fragments 1 and 4 were converted to unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 761-75.B18. The plasmid 761-75.B18 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lac Z) marker gene and a feline immunodeficiency virus (FIV) protease (gag) gene flanked by SPV DNA. Upstream of the foreign gene is an approximately 855 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1113 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 S: 15 result. Note that the 3 galactosidase (lacZ) marker gene is under the control of a swinepox virus 01L gene promoter and the FIV gag gene is under the control of C* the late/early promoter (LP2EP2) The homology vector was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2519 base pair HindIII to SphI restriction fragment of (Promega). Fragment 1 is an approximately 855 base pair 25 sub-fragment of the SPV HindIII restriction fragment

M

(23) synthesized by polymerase chain reaction using DNA primers 5' GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3' (S EQ ID NO 18 5) a n d 5 CATAAGATCTGGCATTGTGTTATTATACTAACAAGAATAAG-3' (SEQ ID NO: 186) to produce an 855 base pair fragment with SphI and BglII ends. Fragment 2 is a 3002 base pair BamHI to PvuII fragment derived from plasmid pJF751 (49) containing the E. coli lacZ gene. Fragment 3 is an approximately 1878 base pair EcoRI to BglII restriction fragment synthesized by polymerase chain reaction (PCR) using cDNA from the FIV (PPR strain) The primer GCGTGAATTCGGGGAATGGACAGGGGCGAGAT-3'; 11/94.9) (SEQ ID -113- NO: synthesizes from the 5' end of the FIV gag gene, introduces an EcoRI site at the 5' end of the gene and an ATG start codon. The primer GAGCCAGATCTGCTCTTTTTACTTTCCC-3'; 11/94.10) (SEQ ID NO: synthesizes from the 3' end of the FIV gag gene. The PCR product was digested with EcoRI and BglII to yield a fragment 1878 base pairs in length corresponding to the FIV gag gene. Fragment 4 is an approximately 1113 base pair subfragment of the SPV HindIII fragment M synthesized by polymerase chain reaction using DNA primers 5'-CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3' S E Q ID NO 1 8 7 a n d 5 S. GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3' (SEQ ID NO: 188) to produce an 1113 base pair fragment with SalI and 15 HindIII ends.

HOMOLOGY VECTOR 781-84.C11. The plasmid 781-84.C11 was used to insert foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the feline immunodeficiency virus (FIV) envelope (env) gene flanked by SPV DNA. When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes results. Note that the 0 galactosidase 25 (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the FIV env gene is under the control of a synthetic late/early pox promoter (LP2EP2) The homology vector was 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 was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega).

Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII fragment M Fragment 3 is an approximately 2564 base pair BamHI to BamHI fragment of the FIV env gene (61) -114synthesized by CLONING WITH THE POLYMERASE CHAIN REACTION. The template for the PCR reaction was FIV strain PPR genomic cDNA The upstream primer 10/93.21 (5'-GCCCGGATCCTATGGCAGAAGGGTTTGCAGC-3'; SEQ ID NO.) was synthesized corresponding to the 5' end of the FIV env gene starting at nucleotide 6263 of FIV strain PPR genomic cDNA, and the procedure introduced a BamHI site at the 5' end. The BamHI site was destroyed during the cloning of the PCR fragment. The downstream primer 10/93.20 (5'-CCGTGGATCCGGCACTCCATCATTCCTCCTC-3'; SEQ ID NO. was synthesized corresponding to the 3' end of the FIV env gene starting at nucleotide 8827 of FIV PPR genomic cDNA, and the procedure introduced a BamHI site at the 3' end. Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11) Fragment 4 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII restriction fragment M The AccI site in the SPV homology vector was converted to a unique NotI site.

-115-

EXAMPLES

Example 1 HomoloQy Vector 515-85.1. The homology vector 515-85.1 is a plasmid useful for the insertion of foreign DNA into SPy. 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 15 that the site be flanked with swinepox virus DNA appropriate for mediating homologous recombination between virus and plasmid DNAs. AccI site in homology vector 515-85.1 is 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 "555 (HindIII fragments G, J, and M see Figures IA-IB) were 25 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 -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 -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 -116greatly. In one case virus could not be purified above the level of 10i, in another case virus could not be purified above the level of 5016, 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 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 f lanks the unique AccI site (see .:Figures 2A and 2B) .The second region covers the 899 base pairs upstream of the unique HindIII site (see Figures 2A and 2B). The seqjuence of the first region codles for an open reading frame (ORE) which shows homology to amino acids 1 to 115 of the vaccinia virus (VV) O1L open reading frame identified by Goebel et al, 1990 (see Figures 3A-3C). The seqjuence of che second region codes for an open reading frame which shows homology to amino acids 568 to 666 of the same vaccinia *virus O1L open reading frame (see Figures 3A-3C) These data suggest that the AccI site interrupts the presumptive VV OiL-like ORE at approximately amino acid 41, suggesting that this ORE codes for a gene nonessential f or SPy replication. Goebel. et al. suggest that the VTV OiL ORE 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 3S replication.

-117- The DNA sequence located upstream of the VVW OiL-like ORF (see Figure 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 -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 15 promoter (EPILP2).

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 20 HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING -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 -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 rof 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.

-118- Example 3 S-SPV-008 S-SPV-008 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli. G-galactosidase (lacZ gene) and the gene for pseudorabies virus (PRV) (gD) (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 (gD) 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 SHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING -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 /3-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 25 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 -119monolayers 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 (gD) gene product, cells were infected with SPV and samples of infected cell lysates were subjected to SDSpolyacrylamide 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 Figure 6, the lysate from S-SPV-008 infected cells exhibits a specific band of approximately 48 kd, the reported size of PRV g50 (gD) PRV g50 (gD) is the g50 (gD) homologue of HSV-1 (26) Several investigators have shown that W expressing HSV-1 (gD) will protect mice against challenge with HSV-1 (6 and 34). Therefore the S-SPV-008 should be valuable 20 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 gII gIII and gH (19) The PRV gIII 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 f;ur 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 gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals. S-SPV-008 has been deposited with the ATCC under Accession No. VR 2339.

-120- Example 4 S-SPV-011 S-SPV-011 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for pseudorabies virus gill (gC) 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 (gC) gene is under the control of the synthetic early promoter (EP2).

15 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 20 the SCREEN FOR RECOMBINANT SPV EXPRESSING P-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 /-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 (gC) 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 -121expressing 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 (gC) gene product, cells were infected with SPV and samples of infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.

Polyclonal goat anti-PRV gIII (gC) antibody was used to detect expression of PRV specific proteins. As shown in Figure 16, the lysate from S-SPV-011 infected cells exhibits a specific band of approximately 92 kd, the s: reported size of PRV gIII (gC) (37).

Recombinant-expressed PRV gIII (gC) has been shown to elicit a significant immune response in mice and swine (37, 38). Furthermore, when gIII (gC) is coexpressed 20 with gII (gB) or g50 significant protection from challenge with virulent PRV is obtained Therefore S-SPV-011 should be valuable as a vaccine to protect ~swine against PRV disease. Since the PRV vaccines described here do not express PRV gX or gI, they would be 25 compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

-122- Example S-SPV-012 S-SPV-012 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for pseudorabies virus gill (gC) 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 gill (gC) gene is under the control of the synthetic early late promoter (EP1LP2) 15 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 20 the SCREEN FOR RECOMBINANT SPV EXPRESSING 0-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 -galactosidase expression, purity, and insert stability by multiple 25 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 gill (gC) 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 -123stably 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 (gC) gene product, cells were infected with S-SPV-012 and samples of infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.

Polyclonal goat anti-PRV gIII (gC) antibody was used to detect expression of PRV specific proteins. As shown in Figure 16, the lysate from S-SPV-012 infected cells Si* exhibits two specific bands which are the reported size 15 of PRV gIII (gC) (37) a 92 kd mature form and a 74 kd *pre-golgi form.

Recombinant-expressed PRV gIII (gC) has been shown to elicit a significant immune response in mice and swine (37, 38). Furthermore, when gIII (gC) is coexpressed with gII (gB) or g50 significant protection from challenge with virulent PRV is obtained 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 gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

-124- Example 6 S-SPV-013 S-SPV-013 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli '-galactosidase (lacZ) and the gene for pseudorabies virus gill (gC) 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 (gC) gene is under the control of the synthetic late early promoter (LP2EP2) 15 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 20 the SCREEN FOR RECOMBINANT SPV EXPRESSING 0-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 3 -galactosidase expression, purity, and insert stability by multiple 25 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 gill (gC) 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 -125expressing 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 (gC) gene product, cells were infected with SPV and samples of infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING

PROCEDURE.

Polyclonal goat anti-PRV gIII (gC) antibody was used to detect expression of PRV specific proteins. As shown in :i Figure 16, the lysate from S-SPV-013 infected cells .i exhibits two specific bands which are the reported size of PRV gIII (gC) (37)-a 92 kd mature form and a 74 kd pre-Golgi form.

Recombinant-expressed PRV gill (gC) has been shown to Selicit a significant immune response in mice and swine 20 (37, 38). Furthermore, when gIII (gC) is coexpressed with gII (gB) or g50 significant protection from challenge with virulent PRV is obtained. (39) Therefore S* S-SPV-013 is valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described 25 here do not express PRV gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

S-SPV-013 has been deposited with the ATCC under Accession No. 2418.

Protection against Aujeszky's disease using recombinant swinepox virus vaccines S-SPV-008 and S-SPV-013.

A vaccine containing S-SPV-008 and S-SPV-013 (1 x 6 PFU/ml) (2ml of a 1:1 mixture of the two viruses) was given to two groups of pigs (5 pigs per group) by -126intradermal inoculation or by oral/pharyngeal spray.

A

control group of 5 pigs received S-SPV-001 by both intradermal and oral/pharyngeal inoculation. Pigs were challenged three weeks post-vaccination with virulent PRV, strain 4892, by intranasal inoculation. The table presents a summary of clinical responses. The data support an increase in protection against Aujeszky's disease in the S-SPV-008/S-SPV-013 vaccinates compared to the S-SPV-001 vaccinate controls.

Vaccine Route of Post- Post- Post-challenge inoculation challenge challenge Group average: Respiratory CNS signs: (Days of Signs: with clinical with signs/ signs) signs/ total number) total number) S-SPV-008 Intradermal 3/5 0/5 2.6 S-SPV-013 S-SPV-008 Oral/ 3/5 0/5 2.2 S-SPV-013 pharyngeal S-SPV-001 Intradermal 5/5 2/5 7.8 (Control) Oral/ Pharyngeal Example 7 S-SPV-015 S-SPV-015 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for pseudorabies virus (PRV) gII (gB) 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 (LPl), and the PRV gB gene is under the control of the synthetic late/early promoter (LP2EP2).

-127- S-SPV-015 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 727-54.60 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated

S-SPV-

015. This virus was assayed for 0-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 15 expressing the foreign gene.

S-SPV-015 was assayed for expression of PRV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN

GENE

EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti-PRV 20 serum was shown to react specifically with S-SPV-015 plaques and not with S-SPV-001 negative control plaques.

All S-SPV-015 observed plaques reacted with the antiserum indicating that the virus was stably expressing the PRV foreign gene. The assays described here were carried out 25 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 PRV gII gene product, cells were infected with SPV-015 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 swine anti-PRV serum was used to detect expression of PRV specific proteins. The lysate from S-SPV-015 infected cells exhibited bands corresponding to 120 kd, 67 kd and 58 kd, which are the expected size of the PRV gII glycoprotein.

-128- S-SPV-015 is useful as a vaccine in swine against pseudorabies virus. A superior vaccine is formulated by combining S-SPV-008 (PRV g50), S-SPV-013 (PRV gIII), and S-SPV-015 for protection against pseudorabies in swine.

Therefore S-SPV-015 should be valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

S-SPV-015 has been deposited with the ATCC under Accession No. 2466.

Example 8 Recombinant swinepox virus expressing more than one pseudorabies virus (PRV) glycoproteins, which can elicit S. production of neutralizing antibodies against 20 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 (gD) and gIII a recombinant swinepox virus expressing

PRV

g50 (gD) and gII a recombinant swinepox virus expressing PRV gII (gB) and gill and a recombinant swinepox virus expressing PRV g50 gill (gC) and gII Each of the viruses cited above is also engineered to contain and express E. coli 0-galactosidase (lac Z) gene, which will facilitate the cloning of the recombinant swinepox virus.

-129- Listed below are three examples of a recombinant swinepox virus expressing PRV g50 PRV gIIl PRV gI (gB) and E. coli 0-galactosidase (lacZ): a) Recombinant swinepox virus containing and expressing PRV g50 (gD) gene, PRV gIII (gC) gene, PRV gII (gB) 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 (gD) gene is under the control of a synthetic early/late promoter (EP1LP2), PRV gIII (gC) gene is under the control of o,00 a synthetic early promoter (EP2), PRV gII (gB) gene .00 1 is under the control of a synthetic late/early 15pand 'oo.00 promoter (LP2EP2) and lacZ gene is under the control 00o of-a synthetic late promoter (LP1). o0 0 o 0 0 b) Recombinant swinepox virus containing and expressing PRV g50 (gD) gene, PRV gIII (gC) gene, o20 PRV gII (gB) gene and lacZ gene. All four genes are inserted into the unique AccI restriction o. endonuclease site within the HindIII M fragment of o. the swinepox virus genome. PRV g50 (gD) gene is under the control of a synthetic early/late promoter 25 (EP1LP2) PRV gIII (gC) gene is under the control of a synthetic early/late promoter (EP1LP2), PRV gII (gB) gene is under the control of a synthetic late/early promoter (LP2EP2) and lacZ gene is under the control of a synthetic late promoter

(LP).

c) Recombinant swinepox virus containing and expressing PRV g50 (gD) gene, PRV gIII (gC) gene, PRV gII (gB) gene and lacZ gene. All four genes are inserted into the unique AccI restriction endonuclease site within the Hindll M fragment of the swinepox virus genome. PRV g50 (gD) gene is under the control of a synthetic early/late promoter -131- S-SPV-013 Intramuscular 1/5 0/5 0.4 S-SPV-015 Intramuscular 3/5 0/5 S-SPV-008 Intramuscular 0/5 0/5 0.0 S-SPV-013 S-SPv-015 S-SPV-001 Intramuscular 5/5 2/5 3.6 (Control) Example 9 S-SPV-009 S-SPV-009 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 3galactosidase (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 (LPl) and the HN gene is under the control of an synthetic early/late promoter (EPlLP2).

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 3-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 1-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.

-132- 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 Accession No. VR 2344).

:i 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 15 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 S. exhibited a specific band of approximately 74 kd, the reported size of NDV HN (29).

Example S-SPV-014 S-SPV-014 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 3galactosidase (lacZ) and the gene for infectious laryngotracheitis virus glycoprotein G (ILT gG) 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 gG 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 -133- 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 G-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 ?-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, 15 indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant vaccines.

To confirm the expression of the ILT gG gene product, cells were infected with SPV-014 and samples of infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN

BLOTTING

PROCEDURE. Peptide antisera to ILT gG was used to 25 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 gG protein and additional bands of higher molecular weight which represent glycosylated forms of the protein which are absent in deletion mutants for ILT gG.

This virus is used as an expression vector for expressing ILT glycoprotein G Such ILT gG is used as an antigen to identify antibodies directed against the wild-type ILT virus as opposed to antibodies directed against gG deleted ILT viruses.

This virus is also used as an antigen for the -134production of ILT gG specific monoclonal antibodies.

Such antibodies are useful in the development of diagnostic tests specific for the ILT gG 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 3galactosidase (lacZ) and the gene for infectious laryngotracheitis virus glycoprotein I (ILT gI) 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 gI gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-016 was derived from S-SPV-001 (Kasza Strain) 25 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 -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 0-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 -135plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.

S-SPV-016 was assayed for expression of ILT gI- and 3galactosidase-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 15 recombinant vaccines.

To confirm the expression of the ILT gI gene product, cells were infected with SPV-016 and samples of infected cell lysates were subjected to SDSpolyacrylamide 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 25 range of bands reactive to the anti-ILT antibody from to 200 kd indicating that the ILT gI may be heavily modified.

This virus is used as an expression vector for expressing ILT glycoprotein I Such ILT gI is used as an antigen to identify antibodies directed against the wild-type ILT virus as opposed to antibodies directed against gI deleted ILT viruses.

This virus is also used as an antigen for the production of ILT gI specific monoclonal antibodies.

Such antibodies are useful in the development of diagnostic tests specific for the ILT gI protein.

-136- 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 3galactosidase (lacZ) and the gene for infectious bovine *i rhinotracheitis virus glycoprotein G (IBR gG) were inserted into the SPV 617-48.1 ORF (a unique NotI 15 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 gG 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 25 GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING f-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 0-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, ail plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.

-137- 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 gG 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 gG gene product, cells were infected with SPV-017 and samples of infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Antisera to IBR gG was used to detect expression of IBR specific proteins. The lysate from SS-SPV-017 infected cells exhibited a band at 43 kd which is the expected size of the IBR gG protein and additional bands of higher molecular weight which S..i 25 represent glycosylated forms of the protein which are absent in deletion mutants for IBR gG.

This virus is used as an expression vector for expressing IBR glycoprotein G Such IBR gG is used as an antigen to identify antibodies directed against the wild-type IBR virus as opposed to antibodies directed against gG deleted IBR viruses.

This virus is also used as an antigen for the production of IBR gG specific monoclonal antibodies.

Such antibodies are useful in the development of diagnostic tests specific for the IBR gG protein.

Monoclonal antibodies are generated in mice utilizing -138this 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 ?-galactosidase (lacZ) and the gene for infectious bovine S. rhinotracheitis virus (IBRV) gE 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 IBRV gE gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-019 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 708-78.9 (see Materials and Methods) and virus S-SPV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR \GENERATING RECOMBINANT SPV. The transfection stock was 25 screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-019. This virus was assayed for P-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.

This virus is used as an expression vector for expressing IBR glycoprotein E Such IBR gE is -139used as an antigen to identify antibodies directed against the wild-type IBR virus as opposed to antibodies directed against gE deleted IBR viruses.

This virus is also used as an antigen for the production of IBR gE specific monoclonal antibodies.

Such antibodies are useful in the development of diagnostic tests specific for the IBR gE 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 15 S-SPV-018 S-SPV-018 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli Pgalactosidase (lacZ) and the gene for pseudorabies virus glycoprotein E (PRV gE) 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 gE gene is under the control of the synthetic early/late 25 promoter (EPLP2) 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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). Red plaque purification of the recombinant virus is designated S-SPV-018. This virus is assayed for -galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay described in Materials and Methods.

-140- 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 Such PRV gE is used as an antigen to identify antibodies directed against the wild-type PRV virus as opposed to antibodies directed against gE deleted PRV viruses.

This virus is also used as an antigen for the production of PRV gE specific monoclonal antibodies.

Such antibodies are useful in the development of diagnostic tests specific for the PRV gE protein.

Monoclonal antibodies are generated in mice utilizing 15 this virus according to the PROCEDURE FOR PURIFICATION .OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials Methods) Example Homoloqv 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 25 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 Therefore, thymidine kinase gene of swinepox virus was shown to be -141non-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 A-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.

.Thus, swinepox virus thymidine kinase gene was shown to be 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 S* into the Asc I site within the thymidine kinase gene.

25 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 9-galactosidase were selected by SCREEN FOR RECOMBINANT SPV EXPRESSING 3galactosidase (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 0-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 -142that 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 15 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.

Serial No. 078,519, filed July 27, 1987. It is contemplated that the procedures that have been used to express PRV g50 (gD) in SPV and are disclosed herein are applicable to TGE.

PORCINE PARVOVIRUS The major capsid protein of the porcine (swine) 25 parvovirus (PPV) was cloned for use in the swinepox virus vector. The clone of the capsid protein is disclosed in U.S. Patent No. 5,068,192 issued November 26, 1991. It is contemplated that the procedures that have been used to express PRV g50 (gD) in SP and are disclosed herein are applicable to PPV.

SWINE ROTAVIRUS The major neutralizing antigen of the swine rotavirus, glycoprotein 38, was cloned for use in the swinepox virus vector. The clone of glycoprotein 38 is disclosed in U.S. Patent No. 5,068,192 issued November 26, 1991.

It is contemplated that the procedures that have been -143used to express PRV g50 (gD) 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.

Serial No. 225,032, filed July 27, 1988. Since the BVD and hog cholera viruses are cross protective 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 (gD) in SPV and are disclosed herein are applicable to BVD virus.

S**

C

15 SERPULINA HYODYSENTERIAE A protective antigen of Serpulina hyodysenteriae for use in the swinepox virus vector has been cloned.

It is contemplated that the procedures that have been used to express PRV g50 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 25 fever virus, hog cholera virus, Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome/swine infertility and respiratory syndrome

(PRRS/SIRS)

Antigens from the following microorganisms may also be utilized for animal vaccines: 1) canine herpesvirus, canine distemper, canine adenovirus type 1 (hepatitis), adenovirus type 2 (respiratory disease), parainfluenza, Leptospira canicola, icterohemorragia, parvovirus, coronavirus, Borrelia burgdorferi, canine herpesvirus, Bordetella bronchiseptica, Dirofilaria immitis (heartworm) and rabies virus. 2) Feline Fiv gag and -144env, feline leukemia virus, feline immunodeficiency virus, feline herpesvirus, feline infectious peritonitis virus, canine herpesvirus, canine coronavirus, canine parvovirus, parasitic diseases in animals (including Dirofilaria immitis in dogs and cats), equine infectious anemia, Streptococcus equi, coccidia, emeria, chicken anemia virus, Borrelia bergdorferi, bovine coronavirus, Pasteurella haemolytica.

Example 17A Vaccines containing recombinant swinepox virus expressing antigens from hog cholera virus, swine 15 influenza virus and (porcine reproducting and respiratory syndrome) PRRS virus.

Recombinant swinepox virus expressing genes for neutralizing antigens to hog cholera virus, swine influenza virus and PRRS virus is useful to prevent disease in swine. The genes expressed in the recombinant SPV include, but are not limited to hog cholera virus gEl and gE2 genes, swine influenza virus hemagglutinin, neuraminidase, matrix and nucleoprotein, and PRRS virus ORF7.

Example 18 Recombinant swinepox viruses express equine influenza virus type A/Alaska 91, equine influenza virus type A/Prague 56, equine herpesvirus type 1 gB, or equine herpesvirus type 1 gD genes. S-SPV-033 and S-SPV-034 are useful as vaccines against equine influenza infection, and S-SPV-038 and S-SPV-039 are useful as a vaccine against equine herpesvirus infection which causes equine rhinotracheitis and equine abortion.

These equine influenza and equine herpesvirus antigens -145are key to raising a protective immune response in the animal. The recombinant viruses are useful alone or in combination as an effective vaccine. The swinepox virus is useful for cloning other subtypes of equine influenza virus (including equine influenza virus type A/Miami/63 and equine influenza virus type A/Kentucky/81) to protect against rapidly evolving variants in this disease. S-SPV-033, S-SPV-034, S-SPV- 038, and S-SPV-039 are also useful as an expression vector for expressing equine influenza or equine herpesvirus antigens. Such equine influenza or equine herpesvirus antigens are useful to identify antibodies directed against the wild-type equine influenza virus or equine herpesvirus. The viruses are also useful to in producing antigens for the production of monospecific polyclonal or monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the viral proteins. Monoclonal or polyclonal antibodies are generated in mice utilizing these viruses according to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials and Methods).

Example 18A S-SPV-033: S-SPV-033 is a recombinant swinepox virus that expresses at least two foreign genes. The gene for E.

coli 0-galactosidase (lacZ) and the gene for equine influenza virus type A/Alaska 91 neuraminidase 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 EIV AK/91 NA gene is under the control of the synthetic late/early promoter (LP2EP2).

-146- S-SPV-033 was derived from S-SPV-001 (Kasza Strain).

This was accomplished utilizing the homology vector 732-18.4 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-033. This virus was assayed for 0-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 i plaques observed were blue indicating that the virus S* 15 was pure, stable, and expressing the foreign gene.

Example 18B S-SPV-034: S-SPV-034 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for equine influenza virus type A/Prague 56 neuraminidase 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 EIV PR/56 NA gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-034 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 723-59A9.22 (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 -galactosidase (BLUOGAL AND CPRG ASSAYS). The final -147result of red plaque purification was the recombinant virus designated S-SPV-034. This virus was assayed for 0-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-034 was assayed for expression of EIV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Monospecific polyclonal antibodies to EIV PR/56 NA were shown to react specifically with S-SPV-034 plaques and not with S-SPV- 001 negative control plaques. All S-SPV-034 observed plaques reacted with the antiserum indicating that the virus was stably expressing the EIV PR/56 NA gene. The assays described here were carried out in ESK-4 cells, indicating that ESK-4 cells would be a suitable 20 substrate for the production of SPV recombinant vaccines.

Example 18C S-SPV-038: S-SPV-038 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for equine herpesvirus type 1 glycoprotein B are 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 t-he control of the synthetic late promoter (LP1), and the EHV-1 gB gene is under the control of the synthetic late/early promoter (LP2EP2).

-148- S-SPV-038 is derived from S-SPV-001 (Kasza Strain).

This is accomplished utilizing the homology vector 744- 34 (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 3galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification is the recombinant virus designated S-SPV-038. This virus is assayed for 0-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 are blue indicating that the virus is 15 pure, stable, and expressing the foreign gene.

Example 18D S-SPV-039: S-SPV-039 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli P-galactosidase (lacZ) and the gene for equine herpesvirus type 1 glycoprotein D are inserted into the SPV 617-48.1 ORF 25 (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 EHV-1 gD gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-039 is derived from S-SPV-001 (Kasza Strain) This is accomplished utilizing the homology vector 744- 38 (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 0galactosidase (BLUOGAL AND CPRG ASSAYS) The final -149result of red plaque purification is the recombinant virus designated S-SPV-039. This virus is assayed for -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 are blue indicating that the virus is pure, stable, and expressing the foreign gene.

4. :i e -150- Example 19 Recombinant swinepox viruses express bovine respiratory syncytial virus attachment protein (BRSV BRSV Fusion protein (BRSV BRSV nucleocapsid protein (BRSV bovine viral diarrhea virus (BVDV) g48, BVDV g53, bovine parainfluenza virus type 3 (BPI-3) F, or BPI-3 HN. S-SPV-020, S-SPV-029, S-SPV-030, and S-SPV- 032, S-SPV-028 are useful as vaccines against bovine disease. These BRSV, BVDV, and BPI-3 antigens are key to raising a protective immune response in the animal.

The recombinant viruses are useful alone or in combination as an effective vaccine. The swinepox virus is useful for cloning other subtypes of BRSV, BVDV, and 15 BPI-3 to protect against rapidly evolving variants in this disease. S-SPV-020, S-SPV-029, S-SPV-030, and S- SPV-032, S-SPV-028 are also useful as an expression vector for expressing BRSV, BVDV, and BPI-3 antigens.

Such BRSV, BVDV, and BPI-3 antigens are useful to identify antibodies directed against the wild-type BRSV, BVDV, and BPI-3. The viruses are also useful as antigens for the production of monospecific polyclonal or monoclonal antibodies. Such antibodies are useful in S* the development of diagnostic tests specific for the 25 viral proteins. Monoclonal or polyclonal antibodies are generated in mice utilizing these viruses according to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials and Methods).

Example 19A S-SPV-020: S-SPV-020 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for bovine respiratory syncytial virus (BRSV) G were inserted into the SPV 617-48.1 ORF -151- (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 BRSV G gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-020 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 727-20.5 (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 -galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-020. This virus was assayed for /-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-020 was assayed for expression of BRSV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Bovine anti-BRSV FITC (Accurate Chemicals) was shown to react specifically with S-SPV-020 plaques and not with S-SPV-003 negative control plaques. All S-SPV-020 observed plaques reacted with the antiserum indicating that the virus was stably expressing the BRSV 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 BRSV G gene product, cells were infected with S-SPV-020 and samples of -152infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.

Bovine anti-BRSV FITC (Accurate Chemicals) was used to detect expression of BRSV specific proteins. The lysate from S-SPV-020 infected cells exhibited a band at 36 kd which is the expected size of the non-glycosylated form of BRSV G protein and bands at 43 to 45 kd and 80 to kd which are the expected size of glycosylated forms of the BRSV G protein.

Example 19B S-SPV-029: a S-SPV-029 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for bovine respiratory syncytial virus (BRSV) F 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 BRSV F gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-029 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 727-20.10 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS) The final result of red plaque purification was the recombinant virus designated S-SPV-029. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods.

-153- 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-029 was assayed for expression of BRSV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Bovine anti-BRSV FITC (Accurate Chemicals) was shown to react specifically with S-SPV-029 plaques and not with S-SPV-003 negative control plaques. All S-SPV-029 observed plaques reacted with the antiserum indicating that the virus was stably expressing the BRSV 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 15 production of SPV recombinant vaccines.

Example 19C S-SPV-030: S-SPV-030 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for bovine respiratory syncytial virus (BRSV) N were inserted into the SPV 617-48.1 ORF 25 (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 BRSV N gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-030 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 713-55.37 (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 3-galactosidase (BLUOGAL AND CPRG ASSAYS). The final -154result of red plaque purification was the recombinant virus designated S-SPV-030. This virus was assayed for 0-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-030 was assayed for expression of BRSV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Bovine anti-BRSV FITC (Accurate Chemicals) was shown to react specifically with S-SPV-030 plaques and not with S-SPV-003 negative 15 control plaques. All S-SPV-030 observed plaques reacted with the antiserum indicating that the virus was stably expressing the BRSV 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 BRSV N gene product, cells were infected with SPV-030 and samples of infected cell lysates were subjected to SDS- 25 polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.

Bovine anti-BRSV FITC (Accurate Chemicals) was used to detect expression of BRSV specific proteins. The lysate from S-SPV-030 infected cells exhibited a band at 43 kd which is the expected size of the BRSV N protein.

Example 19D S-SPV-028: S-SPV-028 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 9-galactosidase -155- (lacZ) and the gene for bovine parainfluenza virus type 3 (BPI-3) F 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 BPI-3 F gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-028 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 713-55.10 (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 15 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-028. This virus was assayed for -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.

o* 25 S-SPV-028 was assayed for expression of BPI-3-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN

GENE

EXPRESSION IN RECOMBINANT SPV. Bovine anti-BPI-3

FITC

(Accurate Chemicals) was shown to react specifically with S-SPV-028 plaques and not with S-SPV-003 negative control plaques. All S-SPV-028 observed plaques reacted with the antiserum indicating that the virus was stably expressing the BPI-3 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.

-156- To confirm the expression of the BPI-3 F gene product, cells were infected with SPV-028 and samples of infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.

Bovine anti-BPI-3 FITC (Accurate Chemicals) was used to detect expression of BPI-3 specific proteins. The lysate from S-SPV-028 infected cells exhibited bands at 43, and 70 kd which is the expected size of the BPI-3 F protein.

Example 19E S-SPV-032: S' S-SPV-032 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for bovine viral diarrhea virus (BVDV) g48 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 *o of the synthetic late promoter (LP1), and the BVDV g48 gene is under the control of the synthetic late/early .o promoter (LP2EP2).

S-SPV-032 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 727-78.1 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-032. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods.

-157- After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.

Example 19F S-SPV-040: S-SPV-040 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 9-galactosidase (lacZ) and the gene for bovine viral diarrhea virus (BVDV) g53 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 15 of the synthetic late promoter (LP1), and the BVDV g53 gene is under the control of the synthetic late/early promoter (LP2EP2) S-SPV-040 is derived from S-SPV-001 (Kasza Strain) S20 This is accomplished utilizing the homology vector 738- 96 (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 3galactosidase (BLUOGAL AND CPRG ASSAYS) The final result of red plaque purification is the recombinant virus designated S-SPV-040. This virus is assayed for 0-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 are blue indicating that the virus is pure, stable, and expressing the foreign gene.

-158- Example 19G Shipping Fever Vaccine Shipping fever or bovine respiratory disease (BRD) complex is manifested as the result of a combination of infectious diseases of cattle and additional stress related factors Respiratory virus infections augmented by pathophysiological effects of stress, alter the susceptibility of cattle to Pasteurella organisms by a number of mechanisms. Control of the S: viral infections that initiate BRD is essential to preventing the disease syndrome (53) The major infectious disease pathogens that contribute to BRD include but are not limited to infectious bovine rhinotracheitis virus (IBRV), parainfluenza virus type 3 bovine respiratory syncytial virus (BRSV), 20 and Pasteurella haemolytica Recombinant swinepox virus expressing protective antigens to organisms causing BRD is useful as a vaccine. S-SPV-020, S-SPV- 029, S-SPV-030, S-SPV-032, and S-SPV-028 are useful components of such a vaccine.

Example Recombinant swinepox viruses S-SPV-031 and S-SPV-035 are useful as a vaccine against human disease. S-SPV- 031 expresses the core antigen of hepatitis B virus. S- SPV-031 is useful against hepatitis B infection in humans. S-SPV-035 expresses the cytokine, interleukin- 2, and is useful as an immune modulator to enhance an immune response in humans. When S-SPV-031 and S-SPV-035 are combined, a superior vaccine against hepatitis B is produced.

-159- Example S-SPV-031: S-SPV-031 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for Hepatitis B Core antigen 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 Hepatitis B Core antigen gene is under the control of the synthetic early/late promoter (EP1LP2).

15 S-SPV-031 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 727-67.18 (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 -galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-031. This virus was assayed for t0-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-031 was assayed for expression of Hepatitis B Core antigen-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.

Rabbit antisera to Hepatitis B Core antigen was shown to react specifically with S-SPV-031 plaques and not with S-SPV-001 negative control plaques. All S-SPV-031 -160observed plaques reacted with the antiserum indicating that the virus was stably expressing the Hepatitis B Core antigen 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 Hepatitis B Core antigen gene product, cells were infected with SPV-031 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Rabbit antisera to Hepatitis B Core antigen was used to detect expression of Hepatitis B specific 15 proteins. The lysate from S-SPV-031 infected cells exhibited a band at 21 kd which is the expected size of the Hepatitis B Core antigen.

Example e S-SPV-035: S-SPV-035 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for human IL-2 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 human IL-2 gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-035 was derived from S-SPV-001 (Kasza Strain).

This was accomplished utilizing the homology vector 741-84.14 (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 -161- 3-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-035. This virus was assayed for 3-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.

-162- Example 21 Human vaccines using recombinant swinepox virus as a vector Recombinant swinepox virus is useful as a vaccine against human diseases. For example, human influenza virus is a rapidly evolving virus whose neutralizing viral epitopes rapidly change. A useful recombinant swinepox vaccine is one in which the influenza virus neutralizing epitopes are quickly adapted by recombinant DNA techniques to protect against new strains of influenza virus. Human influenza virus 15 hemagglutinin (HN) and neuraminidase (NA) genes are cloned into the swinepox virus as described in CLONING OF EQUINE INFLUENZA VIRUS HEMAGGLUTININ AND NEURAMINIDASE GENES (See Materials and Methods and Example 17) S. Recombinant swinepox virus is useful as a vaccine against other human diseases when foreign antigens from the following diseases or disease organisms are expressed in the swinepox virus vector: hepatitis B virus surface and core antigens, hepatitis C virus, human immunodeficiency virus, human herpesviruses, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, measles virus, hantaan virus, pneumonia virus, rhinovirs, poliovirus, human respiratory syncytial virus, retrovirus, human T-cell leukemia virus, rabies virus, mumps virus, malaria (Plasmodium falciparum), Bordetelia pertussis, Diptheria, Rickettsia prowazekii, Borrelia bergdorferi, Tetanus toxoid, malignant tumor antigens.

-163- Furthermore, S-SPV-035 (Example 20), when combined with swinepox virus interleukin-2 is useful in enhancing immune response in humans. Additional cytokines, including but not limited to, interleukin-2, interleukin-6, interleukin-12, interferons, granulocyte-macrophage colony stimulating factors, interleukin receptors from human and other animals when vectored into a non-essential site in the swinepox viral genome, and subsequently expressed, have immune stimulating effects.

Recombinant swinepox virus express foreign genes in a human cell line. S-SPV-003 (EP1LP2 promoter expressing the lacZ gene) expressed the lacZ gene in THP human 15 monocyte cell lines by measuring 0-galactosidase activity. Cytopathic effect of swinepox virus was observed on the THP human monocyte cells, indicating that recombinant swinepox virus can express foreign genes in a human cell line, but will not productively 20 infect or replicated in the human cell line. Swinepox virus was demonstrated to replicate well in ESK-4 cells (embryonic swine kidney) indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant vaccines.

Example 22 Avian vaccines using recombinant swinepox virus as a vector.

Example 22A S-SPV-026 S-SPV-026 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for infectious bursal disease virus -164- (IBDV) polyprotein 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 IBDV polyprotein gene is under the control of the synthetic early/late promoter (EP1LP2).

S-SPV-026 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 689-50.4 (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 -galactosidase (BLUOGAL AND CPRG ASSAYS). The final 15 result of red plaque purification was the recombinant virus designated S-SPV-026. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods.

20 After the initial three rounds of purification, all plaques observed were blue indication that the virus was pure, stable, and expressing the foreign gene.

S-SPV-026 was assayed for expression of IBDV polyprotein-specific antigens using the BLACK

PLAQUE

SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT

SPV.

Rat antisera to IBDV polyprotein were shown to react specifically with S-SPV-026 plaques and not with

S-SPV-

001 negative control plaques. All S-SPV-026 observed plaques reacted with the antiserum indicating that the virus was stably expressing the IBDV polyprotein 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.

-165- To confirm the expression of the IBDV polyprotein gene product, cells were infected with SPV-026 and samples of infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN

BLOTTING

PROCEDURE. Rat antisera to IBDV proteins VP2, VP3, and VP4 and monoclonal antibody R63 to IBDV VP2 were used to detect expression of IBDV proteins. The lysate from S-SPV-026 infected cells exhibited bands at 32 to 40 kd which is the expected size of the IBDV proteins.

Example 22B S-SPV-027 S-SPV-027 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for infectious bursal disease virus (IBDV) VP2 (40kd) were inserted into the SPV 617-48.1 20 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 IBDV VP2 gene is under the control of the synthetic early/late promoter (EP1LP2).

S-SPV-027 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 689-50.7 (see Materials and Methods) and virus S-SPV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE

FOC.

GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING 1-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-027. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods.

-166- 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-027 was assayed for expression of IBDV VP2specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Rat antisera to IBDV protein was shown to react specifically with S-SPV-027 plaques and not with S-SPV- 001 negative control plaques. All S-SPV-027 observed plaques reacted with the antiserum indicating that the virus was stably expressing the IBDV VP2 gene. The assays described here were carried out in ESK-4 cells, indicating that ESK-4 cells would be a suitable 15 substrate for the production of SPV recombinant vaccines.

To confirm the expression of the IBDV VP2 gene product, cells were infected with S-SPV-027 and samples of 20 infected cell lysates were subjected to SDSpolyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN

BLOTTING

PROCEDURE. Rat antisera to IBDV protein and monoclonal antibody R63 to IBDV VP2 were used to detect expression of IBDV VP2 protein. The lysate from S-SPV-027 infected cells exhibited a band at 40 kd which is the expected size of the IBDV VP2 protein.

S-SPV-026 and S-SPV-027 are useful as vaccines against infectious bursal disease in chickens and also as expression vectors for IBDV proteins. Recombinant swinepox virus is useful as a vaccine against other avian disease when foreign antigens from the following diseases or disease organisms are expressed in the swinepox virus vector: Marek's disease virus, infectious laryngotracheitis virus, Newcastle disease virus, infectious bronchitis virus, and chicken anemia -167virus, Chick anemia virus, Avian encephalomyelitis virus, Avian reovirus, Avian paramyxoviruses, Avian influenza virus, Avian adenovirus, Fowl pox virus, Avian coronavirus, Avian rotavirus, Salmonella spp E coli, Pasteurella spp, Haemophilus spp, Chlamydia spp, Mycoplasma spp, Campylobacter spp, Bordetella spp, Poultry nematodes, cestodes, trematodes, Poultry mites/lice, Poultry protozoa (Eimeria spp, Histomonas spp, Trichomonas spp).

Example 23 SPV-036: 15 S-SPV-036 is a swinepox virus that expresses at one foreign gene. The gene for E. coli 0-galactosidase (lacZ) was 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 20 of the human cytomegalovirus immediate early (HCMV IE) promoter.

S-SPV-036 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 741-80.3 (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 /-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-036. This virus is assayed for /-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 are blue indicating that the virus is pure, stable, and expressing the foreign gene.

-168- The expression of lacZ from the HCMV IE promoter provides a strong promoter for expression of foreign genes in swinepox. S-SPV-036 is a novel and unexpected demonstration of a herpesvirus promoter driving expression of a foreign gene in a poxvirus. S-SPV-036 is useful in formulating human vaccines, and recombinant swinepox virus is useful for the expression of neutralizing antigens from human pathogens.

Recombinant swinepox virus expressed foreign genes in a human cell line as demonstrated by S-SPV-003 (EP1LP2) promoter expressing the lacZ gene) expressed 3galactosidase in THP human monocyte cell lines.

Cytopathic effects of swinepox virus on the THP human monocyte cells were not observed, indicating that recombinant swinepox virus can express foreign genes in a human cell line, but will not productively infect or ~replicated in the human cell line Example 24 Homology Vector 738-94.4 Homology Vector 738-94.4 is a swinepox virus vector that expresses one foreign gene. The gene for E. coli /-galactosidase (lacZ) was inserted into the the OIL open reading frame (SEQ ID NO: 115). The lacZ gene is under the control of the OIL promoter. The homology vector 738-94.4 contains a deletion of SPV DNA from nucleotides 1679 to 2452 (SEQ ID NO: 189; Figure 17) which deletes part of the OIL ORF.

The upstream SPV sequences were synthesized by polymerase chain reaction using DNA primers GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3' (SEQ ID NO: 185) and 3' (SEQ ID NO: 186) to produce an 855 base pair fragment with BglII and SphI ends. The 01L promoter is -169present on this fragment. The downstream SPV sequences were synthesized by polymerase chain reaction using DNA primers 5'-CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3' S E Q ID NO 1 8 7 a n d 5 GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3' (SEQ ID NO: 188) to produce an 1113 base pair fragment with SalI and HindIII ends. A recombinant swinepox virus was derived utilizing homology vector 738-94.4 and S-SPV- 001 (Kasza strain) in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING 3-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification is the recombinant virus. This virus is 15 assayed for 0-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 are blue indicating that the virus is pure, stable, and expressing the foreign gene. Recombinant swinepox viruses derived from *homology vector 738-94.4 are utilized as an expression vector to express foreign antigens and as a vaccine to raise a protective immune response in animals to 25 foreign genes expressed by the recombinant swinepox virus. Other promoters in addition to the OIL promoter are inserted into the deleted region including LP1, EP1LP2, LP2EP2, HCMV immediate early, and one or more foreign genes are expressed from these promoters.

Example 24B Homology Vector 752-22.1 is a swinepox virus vector that is utilized to express two foreign genes. The gene for E. coli 0-galactosidase (lacZ) was inserted into the the OIL open reading frame (SEQ ID NO: 115). The lacZ gene is under the control of the OIL promoter. A -170second foreign gene is expressed from the LP2EP2 promoter inserted into an EcoRI or BamHI site following the LP2EP2 promoter sequence. The homology vector 752- 22.1 contains a deletion of SPV DNA from nucleotides 1679 to 2452 (SEQ ID NO: 189; Figure 17) which deletes part of the OIL ORF. The homology vector 752-22.1 was derived from homology vector 738-94.4 by insertion of the LP2EP2 promoter fragment (see Materials and Methods). The homology vector 752-22.1 is further improved by placing the lacZ gene under the control of the synthetic LP1 promoter. The LP1 promoter results in higher levels of lacZ expression compared to the SPV OIL promoter Example S-SPV-041: S-SPV-041 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for equine herpesvirus type 1 glycoprotein B (gB) were inserted into the 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1679 to 2452, SEQ ID NO: 189). The lacZ 25 gene is under the control of the swinepox OIL promoter, and the EHV-1 gB gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-041 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 752-29.33 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS) The final result of red plaque purification was the recombinant virus designated S-SPV-041. This virus was assayed for -171- 3-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-041 is useful as a vaccine in horses against EHV- 1 infection and is useful for expression of EHV-1 glycoprotein B.

S-SPV-045: S-SPV-045 is a swinepox virus that expresses at least 15 two foreign genes. The gene for E. coli 3-galactosidase (lacZ) and the gene for infectious bovine rhinotracheitis virus glycoprotein E (gE) were inserted into the 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1679 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of the swinepox OIL promoter, and the IBRV gE gene is under the control of the synthetic late/early promoter (LP2EP2).

25 S-SPV-045 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 746-94.1 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-045. This virus was assayed for 0-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 -172plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.

S-SPV-045 is useful for expression of IBRV glycoprotein

E.

S-SPV-049: S-SPV-049 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for bovine viral diarrhea virus glycoprotein 48 (gp48) were inserted into the 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1679 to 2452, SEQ ID NO: 189) 15 The lacZ gene is under the control of the swinepox OIL promoter, and the BVDV gp48 gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-049 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 771-55.11 (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 25 3-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-049. This virus was assayed for 0-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-049 is useful as a vaccine in cattle against BVDV infection and is useful for expression of BVDV glycoprotein 48.

-173- S-SPV-050: S-SPV-050 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 3-galactosidase (lacZ) and the gene for the bovine viral diarrhea virus glycoprotein 53 (gp53) were inserted into the 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1679 to 2452, SEQ ID NO: 189).

The lacZ gene is under the control of the swinepox OIL promoter, and the IBRV gE gene is under the control of the synthetic late/early promoter (LP2EP2).

"i S-SPV-050 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 767-67.3 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-050. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue 25 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-050 is useful as a vaccine in cattle against BVDV infection and is useful for expression of BVDV glycoprotein 53.

Example 26 Recombinant swinepox virus, S-SPV-042 or S-SPV-043, expressing chicken interferon (cIFN) or chicken -174myelomonocytic growth factor (cMGF), respectively, are useful to enhance the immune response when added to vaccines against diseases of poultry. Chicken myelomonocytic growth factor (cMGF) is homologous to mammalian interleukin-6 protein, and chicken interferon (cIFN) is homologous to mammalian interferon. When used in combination with vaccines against specific avian diseases, S-SPV-042 and S-SPV-043 provide enhanced mucosal, humoral, or cell mediated immunity against avian disease-causing viruses including, but not limited to, Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus.

Example 26A S-SPV-042: S-SPV-042 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 3-galactosidase (lacZ) and the gene for chicken interferon (cIFN) 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 25 synthetic late promoter (LP1) and the cIFN gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-042 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 751-07.Al (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 -galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-042. This virus was assayed for -175- 0-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-042 has interferon activity in cell culture.

Addition of S-SPV-042 conditioned media to chicken embryo fibroblast (CEF) cell culture inhibits infection of the CEF cells by vesicular stomatitis virus or by herpesvirus of turkeys. S-SPV-042 is useful to enhance the immune response when added to vaccines against diseases of poultry.

Example 26B S-SPV-043: S-SPV-043 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli P-galactosidase (lacZ) and the gene for chicken myelomonocytic growth factor (cMGF) were inserted into the SPV 617-48.1 ORF (a unique NotI restriction site has replaced a unique 25 AccI restriction site) The lacZ gene is under the control of the synthetic late promoter (LP1), and the cMGF gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-043 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 751-56.Al (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant -176virus designated S-SPV-043. This virus was assayed for 0-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-043 is useful to enhance the immune response when added to vaccines against diseases of poultry.

Example 27 *Insertion into a non-essential site in the 2.0 kb HindIII to BqlII region of the swinepox virus HindIII M fragment.

A 2.0 kb HindIII to BglII region of the swinepox virus HindIII M fragment is useful for the insertion of foreign DNA into SPV. The foreign DNA is inserted into a unique BglII restriction site in the region (Figure 17; Nucleotide 540 of SEQ ID NOs: 195). A plasmid containing a foreign DNA insert is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING S: 25 RECOMBINANT SPV to generate an SPV containing the foreign DNA. For this procedure to be successful, it is important that the insertion site be in a region nonessential 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 BglII restriction site in the kb HindIII to BglII region of the swinepox virus HindIII M fragment is located within the coding region of the SPV I4L open reading frame. The I4L ORF has sequence similarity to the vaccinia virus and smallpox virus ribonucleotide reductase (large subunit) gene (56-58). The ribonucleotide reductase (large subunit) -177gene is non-essential for DNA replication of vaccinia virus and is an appropriate insertion site in swinepox virus.

Example 28 S-SPV-047 S-SPV-047 is a swinepox virus that expresses two foreign genes. The gene for E. coli 3-galactosidase (lacZ) and the gene for pseudorabies virus gB (gIl) were inserted into a unique HindIII site (HindIII linker inserted into the BglII restriction endonuclease S. site within the 2.0 kb BglII to HindIII subfragment of the HindIII M fragment.) The BglII insertion site is within the SPV I4L open reading frame which has significant homology to the vaccinia virus ribonucleoside-diphosphate reductase gene. The lacZ gene is under the control of the synthetic late promoter (LP1), and the PRV gB (gIl) gene is under the control of the synthetic late/early promoter (LP2EP2) S-SPV-047 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 779-94.31 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-047. This virus was assayed for P-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.

-178- S-SPV-047 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- PRV serum was shown to react specifically with S-SPV- 047 plaques and not with S-SPV-001 negative control plaques. All S-SPV-047 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 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 PRV gB gene product, cells were infected with S-SPV-047 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis.

The gel was blotted and analyzed using the WESTERN ~BLOTTING PROCEDURE. A polyclonal swine anti-PRV serum was used to detect expression of PRV specific proteins.

The cell lysate and supernatants from S-SPV-047 infected cells exhibited bands corresponding to 120 kD, 67 kD and 58 kD, which are the expected size of the PRV glycoprotein B.

2 SPV recombinant-expressed PRV gB has been shown to elicit a significant immune response in swine (37, 38; See example Furthermore, PRV gB is expressed in reconLoinant SPV, significant protection from challenge with virulent PRV is obtained. (See Examples 6 and 8) Therefore S-SPV-047 is valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

-179- S-SPV-052 S-SPV-052 is a swinepox virus that expresses three foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for pseudorabies virus gB (gIl) were inserted into the unique HindIII restriction site (HindIII linkers inserted into a unique NdeI site in the SPV OIL open reading frame; An approximately 545 base pair NdeI to NdeI subfragment (Nucleotides 1560 to 2104; SEQ ID NO. of the SPV HindIII M fragment has been deleted). The gene for PRV gD (g50) was inserted into the unique PstI restriction site (PstI linkers inserted into a unique AccI site in the SPV 01L open S. 15 reading frame). The lacZ gene is under the control of the synthetic late promoter (LP1), the PRV gB (gIl) gene is under the control of the synthetic late/early promoter (LP2EP2), and the PRV gD (g50) gene is under the control of the synthetic early/late promoter 20 (EP1LP2).

4*.

S-SPV-052 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 789-41.7 (see Materials and Methods) and virus S-SPV- 25 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 052. This virus was assayed for 0-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.

-180- S-SPV-052 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- PRV serum was shown to react specifically with S-SPV- 052 plaques and not with S-SPV-001 negative control plaques. All S-SPV-052 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 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 PRV gB and gD gene 15 products, cells were infected with S-SPV-052 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. A polyclonal swine anti-PRV serum was used to detect expression of PRV specific proteins. The cell lysate and 1. supernatants from S-SPV-052 infected cells exhibited bands corresponding to 120 kD, 67 kD and 58 kD, which are the expected size of the PRV glycoprotein B; and a 25 48 kD which is the expected size of the PRV glycoprotein

D.

SPV recombinant-expressed PRV gB and gD has been shown to elicit a significant immune response in swine (37, 38; See example Furthermore, PRV gB and gD are expressed in recombinant SPV, significant protection from challenge with virulent PRV is obtained. (See Examples 6 and 8) Therefore S-SPV-052 is valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gX or gI, they would be compatible with current

PRV

diagnostic tests (gX HerdChek®, gI HerdChek® and V- *Wl^f^ -181- ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

S-SPV-053 S-SPV-053 is a swinepox virus that expresses three foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for pseudorabies virus gB (gIl) were inserted into the unique HindIII restriction site (HindIII linkers inserted into a unique NdeI site in the SPV OIL open reading frame; An approximately 545 base pair NdeI to NdeI subfragment (Nucleotides 1560 to 2104; SEQ ID NO. of the SPV HindIII M fragment has been deleted). The gene for PRV gC (gIII) was inserted into the unique PstI restriction site (PstI linkers *inserted into a unique AccI site in the SPV 01L open reading frame). The lacZ gene is under the control of ~the synthetic late promoter (LP1), the PRV gB (gIl) gene is under the control of the synthetic late/early promoter (LP2EP2), and the PRV gC (gIII) gene is under Sthe control of the synthetic early/late promoter (EP1LP2).

S-SPV-053 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 789-41.27 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 053. This virus was assayed for 0-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 -182plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.

S-SPV-053 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- PRV serum was shown to react specifically with S-SPV- 053 plaques and not with S-SPV-001 negative control plaques. All S-SPV-053 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 ESK-4 cells, indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant 15 vaccines.

To confirm the expression of the PRV gB and gC gene products, cells were infected with S-SPV-053 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. A polyclonal swine anti-PRV serum was used to detect expression of PRV specific proteins. The cell lysate and supernatants from S-SPV-053 infected cells exhibited bands corresponding to 120 kD, 67 kD and 58 kD, which are the expected size of the PRV glycoprotein B; and a 92 kD which is the expected size of the PRV glycoprotein C.

SPV recombinant-expressed PRV gB and gC has been shown to elicit a significant immune response in swine (37, 38; See example Furthermore, PRV gB and gC are expressed in recombinant SPV, significant protection from challenge with virulent PRV is obtained. (See Examples 6 and 8) Therefore S-SPV-053 is valuable as a vaccine to protect swine against PRV disease. Since -183the PRV vaccines described here do not express PRV gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

S-SPV-054 S-SPV-054 is a swinepox virus that expresses three foreign genes. The gene for E. coli 3-galactosidase (lacZ) and the gene for pseudorabies virus gC (gIII) were inserted into the unique HindIII restriction site (HindIII linkers inserted into a unique NdeI site in :the SPV OL open reading frame; An approximately 545 base pair NdeI to NdeI subfragment (Nucleotides 1560 to 2104; SEQ ID NO. of the SPV HindIII M fragment has been deleted). The gene for PRV gD (g50) was inserted into the unique PstI restriction site (PstI linkers inserted into a unique AccI site in the SPV 01L open reading frame). The lacZ gene is under the control of ~the synthetic late promoter (LP1), the PRV gC (gIII) gene is under the control of the synthetic early/late promoter (EP1LP2), and the PRV gD (g50) gene is under ~the control of the synthetic early/late promoter 25 (EP1LP2).

S-SPV-054 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 789-41.47 (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 3-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 054. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue -184plaque 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-054 was assayed for expression Qf PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- PRV serum was shown to react specifically with S-SPV- 054 plaques and not with S-SPV-001 negative control plaques. All S-SPV-054 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 ESK-4 cells, 15 indicating that ESK-4 cells would be a suitable substrate for the production of SPV recombinant vaccines.

To confirm the expression of the PRV gC and gD gene products, cells were infected with S-SPV-054 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. A polyclonal swine anti-PRV serum was used to detect expression of PRV specific proteins. The cell lysate and supernatants from S-SPV-054 infected cells exhibited a band corresponding to 92 kD which is the expected size of the PRV glycoprotein C and a 48 kD which is the expected size of the PRV glycoprotein D.

SPV recombinant-expressed PRV gC and gD has been shown to elicit a significant immune response in swine (37, 38; See example Furthermore, PRV gC and gD are expressed in recombinant SPV, significant protection from challenge with virulent PRV is obtained. (See Examples 6 and 8) Therefore S-SPV-054 is valuable as -185a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

S-SPV-055 S-SPV-055 is a swinepox virus that expresses four foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for pseudorabies virus gB (gIl) were inserted into the unique HindIII restriction site (HindIII linkers inserted into a unique NdeI site in 15 the SPV OIL open reading frame; An approximately 545 base pair NdeI to NdeI subfragment (Nucleotides 1560 to 2104; SEQ ID NO. of the SPV HindIII M fragment has been deleted). The gene for PRV gD (g50) and PRV gC (gIII) were inserted into the unique PstI restriction site (PstI linkers inserted into a unique AccI site in the SPV 01L open reading frame). The lacZ gene is under the control of the synthetic late promoter (LP1), the PRV gB (gIl) gene is under the control of the synthetic late/early promoter (LP2EP2), the PRV gD (g50) gene is under the control of the synthetic late/early promoter (LP2EP2) and the PRV gC (gIII) gene is under the control of the synthetic early/late promoter (EP1LP2).

S-SPV-055 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 789-41.73 (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 3-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 055. This virus was assayed for -186- P-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-055 was assayed for expression of PRV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- PRV serum was shown to react specifically with S-SPV- 055 plaques and not with S-SPV-001 negative control plaques. All S-SPV-055 observed plaques reacted with the swine anti-PRV serum indicating that the virus was 15 stably expressing the PRV 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 PRV gB, gC and gD gene products, cells were infected with S-SPV-055 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. A polyclonal swine anti-PRV serum was used to detect expression of PRV specific proteins. The cell lysate and supernatants from S-SPV-055 infected cells exhibited a bands corresponding to 120 kD, 67 kD, and 58 kD which is the expected size of the PRV glycoprotein B; a 92 kD which is the expected size of the PRV glycoprotein C; and a 48 kD which is the expected size of the PRV glycoprotein D SPV recombinant-expressed PRV gB, gC and gD has been shown to elicit a significant immune response in swine -187- (37, 38; See example Furthermore, PRV gB, gC and gD are expressed in recombinant SPV, significant protection from challenge with virulent PRV is obtained. (See Examples 6 and 8) Therefore S-SPV-055 is valuable as a vaccine to protect swine against PRV disease. Since the PRV vaccines described here do not express PRV gX or gI, they would be compatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguish vaccinated animals from infected animals.

Example 29 SPV-059 *S-SPV-059 is a swinepox virus that expresses one foreign gene. The gene for E. coli 0-glucuronidase ~(uidA) was inserted into the unique EcoRI restriction 9 site in the SPV B18R open reading frame within the SPV HindIII K genomic fragment. The uidA gene is under the control of the synthetic late/early promoter (LP2EP2).

Partial sequence from a 3.2 kb region of the SPV 6.5 kb HindIII K fragment (SEQ ID NO. indicates three potential open reading frames. The SPV B18R ORF shows sequence homology to the vaccinia virus B18R gene, 77.2K protein from rabbit fibroma virus, vaccinia virus C19L/B25R ORF and an ankyrin repeat region from a human brain variant. The B18R gene codes for a soluble interferon receptor with high affinity and broad specificty. The SPV B4R open reading frame shows sequence homology to the T5 protein of rabbit fibroma virus.

S-SPV-059 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 796-50.31 and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV.

-188- Homology vector 796-50.31 was generated by insertion of a blunt ended NotI fragment containing the LP2EP2 promoter uidA cassette from plasmid 551-47.23 (see Materials and Methods) into a unique EcoRI site (blunt ended) in the SPV 6.5 kb HindIII K fragment, (Figure 29B) The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S- SPV-059. This virus was assayed for 0-glucuronidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as I* described in Materials and Methods. After the initial *three rounds of purification, plaques observed were blue indicating that the virus was pure, stable, and expressing the foreign gene.

S-SPV-059 has been purified and expresses the foreign gene, E. coli uidA, indicating that the EcoRI site within the 6.5 kb HindIII K fragment is a stable insertion site for foreign genes. Recombinant swinepox virus utilizing this insertion site is useful for expression of foreign antigen genes, as a vaccine against disease or as an expression vector to raise antibodies to the expressed foreign gene.

SPV-060 S-SPV-060 is a swinepox virus that expresses one foreign gene. The gene for E. coli -glucuronidase (uidA) was inserted into the unique EcoRV restriction site within the SPV HindIII N genomic fragment. The uidA gene is under the control of the synthetic late/early promoter (LP2EP2). Partial sequence of the SPV 3.2 kb HindIII N fragment (SEQ ID NO.

indicates two potential open reading frames. The SPV I7L ORF shows sequence homology to protein 17 of -189vaccinia virus. The SPV I4L open reading frame shows sequence homology to the ribonucleoside diphosphate reductase gene of vaccinia virus. Two potential open reading frames I5L and I6L, between I4L ORF and I7L ORF are of unknown function.

S-SPV-060 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 796-71.31 and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. Homology vector 796-71.31 was generated by insertion of a blunt ended NotI fragment containing the 'LP2EP2 promoter uidA cassette from plasmid 551-47.23 (see Materials and Methods) into a unique EcoRV site in 15 the SPV 3.2 kb HindIII N fragment (Figure 29A) The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification is the recombinant virus designated S-SPV-060. This virus is assayed for 0-glucuronidase 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, plaques observed are blue indicating that the virus is pure, stable, and expressing the foreign gene.

S-SPV-060 is purified and expresses the foreign gene, E. coli uidA, indicating that the EcoRI site within the 3.2 kb HindIII N fragment is a stable insertion site for foreign genes. Recombinant swinepox virus utilizing this insertion site is useful for expression of foreign antigen genes, as a vaccine against disease or as an expression vector to raise antibodies to the expressed foreign gene.

S-SPV-061 -190- S-SPV-061 is a swinepox virus that expresses one foreign gene. The gene for E. coli -glucuronidase (uidA) was inserted into the unique SnaBI restriction site within the SPV HindIII N genomic fragment. The uidA gene is under the control of the synthetic late/early promoter (LP2EP2). Partial sequence of the SPV 3.2 kb HindIII N fragment (SEQ ID NO. indicates two potential open reading frames. The SPV I7L ORF shows sequence homology to protein 17 of vaccinia virus. The SPV I4L open reading frame shows sequence homology to the ribonucleoside diphosphate reductase gene of vaccinia virus. Two potential open reading frames I5L and I6L, between I4L ORF and I7L ORF are of unknown function.

S-SPV-061 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 796-71.41 and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV.

Homology vector 796-71.41 was generated by insertion of a blunt ended NotI fragment containing the LP2EP2 promoter uidA cassette from plasmid 551-47.23 (see Materials and Methods) into a unique SnaBI site in the 4** SPV 3.2 kb HindIII N fragment. The transfection stock 25 was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification is the recombinant virus designated S-SPV-061. This virus is assayed for 0glucuronidase 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, plaques observed are blue indicating that the virus is pure, stable, and expressing the foreign gene.

S-SPV-061 is purified and expresses the foreign gene, E. coli uidA, indicating that the SnaBI site within the -191- 3.2 kb- HindIII N fragment is a stable insertion site for foreign genes. Recombinant swinepox virus utilizing this insertion site is useful for expression of foreign antigen genes, as a vaccine against disease or as an expression vector to raise antibodies to the expressed foreign gene.

S-SPV-062 S-SPV-062 is a swinepox virus that expresses one foreign gene. The gene for E. coli 0-glucuronidase ~(uidA) was inserted into the unique BglII restriction o.

site within the SPV HindIII N genomic fragment (Figure 29A). The uidA gene is under the control of the synthetic late/early promoter (LP2EP2). Partial sequence of the SPV 3.2 kb HindIII N fragment (SEQ ID NO. indicates two potential open reading frames. The SPV I7L ORF shows sequence homology to protein 17 of ~vaccinia virus. The SPV I4L open reading frame shows sequence homology to the ribonucleoside diphosphate reductase gene of vaccinia virus. Two potential open reading frames I5L and I6L, between I4L ORF and I7L ORF are of unknown function.

'25 S-SPV-062 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 796-71.51 and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV.

Homology vector 796-71.51 was generated by insertion of a blunt ended NotI fragment containing the LP2EP2 promoter uidA cassette from plasmid 551-47.23 (see Materials and Methods) into a unique BglII site in the SPV 3.2 kb HindIII N fragment. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification is the recombinant virus designated S-SPV-062. This virus is assayed for 3- -192glucuronidase 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, plaques observed are blue indicating that the virus is pure, stable, and expressing the foreign gene.

S-SPV-062 is purified and expresses the foreign gene, E. coli uidA, indicating that the BglII site within the 3.2 kb HindIII N fragment is a stable insertion site for foreign genes. Recombinant swinepox virus utilizing this insertion site is useful for expression of foreign i antigen genes, as a vaccine against disease or as an expression vector to raise antibodies to the expressed 15 foreign gene.

Example Recombinant swinepox virus expressing E coli 3galactosidase (lacZ) under the control of a synthetic early or synthetic late pox promoter.

Three recombinant swinepox viruses, S-SPV-056, S-SPV- 057, and S-SPV-058 expressing E coli 0-galactosidase 25 (lacZ) under the control of a synthetic pox promoter, LP1, LP2, and EP1, respectively, have been constructed.

S-SPV-056 was derived from S-SPV-001 (Kasza Strain).

This was accomplished utilizing the homology vector 791-63.19 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). S-SPV-057 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 791-63.41 (see Materials and Methods) and virus S-SPV-001 in the -193- HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING 3galactosidase (BLUOGAL AND CPRG ASSAYS). S-SPV-058 was derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing the homology vector 796-18.9 (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 3galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification were the recombinant viruses designated S-SPV-056, S-SPV-057 and S-SPV-058.

The viruses were assayed for 0-galactosidase 15 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.

*o* Recombinant swinepox virus expresses a foreign gene such as E. coli 0-galactosidase in a human cell line but does not replicate in the human cell line. To 25 optimize expression of the foreign gene, S-SPV-056, S- SPV-057 and S-SPV-058 are used to compare optimal expression levels of E. coli g-galactosidase under the control of early or late synthetic pox viral promoters.

The human cell lines in which expression of recombinant swinepox virus has been detected include, but are not limited to 143B (osteosarcoma), A431 (epidermoid carcinoma), A549 (lung carcinoma), Capan-1 (liver carcinoma), CF500 (foreskin fibroblasts), Chang Liver (liver), Detroit (down's foreskin fibroblasts), HEL-199 (embryonic lung) HeLa (cervical carcinoma), HEp-2 (epidermal larynx carcinoma), HISM (intestinal smooth muscle), HNK (neonatal kidney), MRC-5 (embryonic lung), VV JF -194- NCI-H292 (pulmonary mucoepidermoid carcinoma), OVCAR-3 (ovarian carcinoma), RD (rhabdosarcoma), THP (monocyte leukemia), WIL2-NS (B lymphocyte line, non-secreting), WISH (amnion).

Example 31: S-SPV-051 S-SPV-051 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli P-galactosidase ;(lacZ) and the gene for the bovine viral diarrhea virus glycoprotein 53 (g53) were inserted into the SPV 617- 48.1 ORF (a unique NotI restriction site has replaced 15 a unique AccI restriction site). The lacZ gene is under the control of the synthetic late promoter (LP1), and .the BVDV g53 gene is under the control of the synthetic late/early promoter (LP2EP2) 20 S-SPV-051 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 783-39.2 (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 SF7V EXPRESSING -galactosidase (BLUOGAL AND CPRG ASSAYS) The final result of red plaque purification was the recombinant virus designated S-SPV 051. This virus was assayed for 0-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-051 was assayed for expression of BVDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN

GENE

-195- EXPRESSION IN RECOMBINANT SPV. A mouse monoclonal antibody to BVDV g53 was shown to react specifically with S-SPV-051 plaques and not with S-SPV-001 negative control plaques. All S-SPV-051 observed plaques reacted with the monoclonal antibody to BVDV g53 indicating that the virus was stably expressing the BVDV 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 BVDV g53 gene product, cells were infected with S-SPV-051 and samples of infected cell lysates and culture supern 15 atants were subjected to SDS polyacrylamide gel electrophoresis. The gel was blotted and analyzed using S* the WESTERN BLOTTING PROCEDURE. A mouse monoclonal antibody to BVDV g53 was used to detect expression of BVDV specific proteins. The cell lysate and supernatant 20 from S-SPV-051 infected cells exhibited bands at 53 kd and higher indicating glycosylated and unglycosylated forms of the BVDV g53 protein.

S-SPV-051 is useful as a vaccine in cattle against

BVDV

infection and is useful for expression of BVDV glycoprotein 53.

Example 32: S-SPV-044: S-SPV-044 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for the infectious bursal disease virus (IBDV) polymerase protein were inserted into the 617-48.1 ORF (a unique NotI site has replaced a unique AccI restriction site) The lacZ gene is under the -196control of the synthetic late promoter (LP1), and the IBDV polymerase gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-044 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 749-75.78 (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 /-galactosidase (BLUOGAL AND CPRG ASSAYS) The final result of red plaque purification was the recombinant .I virus designated S-SPV-044. This virus was assayed for S0/-galactosidase expression, purity, and insert 15 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-044 is useful for expression of IBDV polymerase protein. S-SPV-044 is useful in an in vitro approach to a recombinant IBDV attenuated vaccine. RNA strands from the attenuated IBDV strain are synthesized in a bacterial expression system using T3 or T7 promoters (pBlueScript plasmid; Stratagene, Inc.) to synthesize double stranded short and long segments of the IBDV genome. The IBDV double stranded RNA segments and S- SPV-044 are transfected into CEF cells. The swinepox virus expresses the IBDV polymerase but does not replicate in CEF cells. The IBDV polymerase produced from S-SPV-044 synthesizes infectious attenuated

IBDV

virus from the double stranded RNA genomic templates.

The resulting attenuated IBDV virus is useful as a vaccine against infectious bursal disease in chickens.

Example 33: -197- S-SPV-046: S-SPV-046 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 9-galactosidase (lacZ) and the gene for the feline immunodeficiency virus (FIV) gag protease (gag) were inserted into the 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189) The lacZ gene is under the control of the swinepox OIL promoter, and the FIV gag gene is under the control of the synthetic late/early promoter (LP2EP2).

S" S-SPV-046 was derived from S-SPV-001 (Kasza Strain) 15 This was accomplished utilizing the homology vector 761-75.B18 (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 20 3-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 046. This virus was assayed for f-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.

To confirm the expression of the FIV gag gene product, cells were infected with S-SPV-046 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis.

The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Feline anti-FIV (PPR strain) sera was used to detect expression of FIV specific proteins.

The cell lysate and supernatant from S-SPV-046 infected -198cells exhibited bands at 26 kd and 17 kd which are the expected sizes of the processed form of the FIV gag protein. The recombinant swinepox virus expressed FIV gag protein is processed properly and secreted into the culture media.

S-SPV-048 S-SPV-048 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for feline immunodeficiency virus .(FIV) envelope (env) were inserted into the SPV 617 S" 48.1 ORF (a unique NotI restriction site has replaced a unique AccI restriction site). The lacZ gene is under [15 the control of the synthetic late promoter (LP1), and the FIV env gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-048 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 781-84.C11 (see Materials and Methods) and virus S-SPV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock was S: screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING 0-galactosidase (BLUOGAL AND CPRG ASSAYS) The final result of red plaque purification was the recombinant virus designated S-SPV 048. This virus was assayed for 3-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-046 and S-SPV-048 are useful alone or in combination as a vaccine in cats against FIV infection and are useful for expression of the FIV env and gag -199proteins. A recombinant swinepox virus expressing both the FIV env and gag proteins is useful as a vaccine in cats against FIV infection.

Recombinant swinepox virus expressing human respiratory synctial virus F and G proteins is useful as a vaccine against the human disease.

Example 34 In Vitro Properties of Chicken IFN Expressed in Recombinant Pox viruses.

Growth properties of recombinant viruses in cell culture. Gowth properties of recombinant S-SPV-042 were not effected in embryonic swine kidney cells (ESK- 4) compared to wild-type swinpox virus.

Western blot analysis was performed on supernatants 20 from cells infected with SPV/cIFN recombinant virus.

Rabbit and mouse antisera were raised against cIFN from concentrated SPV/cIFN infected supernatants and precleared against ESK-4 cells infected with wild-type SPV in preparation for western analysis. Rabbit and mouse anti-cIFN antisera were reacted with denatured proteins on nitrocellulose from recombinant SPV/cIFN and SPV wild type virus infected supernatants. A reactive band with an estimated molecular weight size range of 17-20 kilodaltons was present in the SPV/cIFN lanes, and absent in the SPV wild type control lanes.

Effect of cIFN expressed in supernatants from SPV/cIFN (S-SPV-042), FPV/cIFN, and FPV/cIFN/NDV infected cells on the growth of Vesicular Stomatis Virus.

-200- Virion cleared supernatants from SPV/cIFN, FPV/cIFN and FPV/cIFN/NDV infected cells were tested for the presence of viral inhibitory activity, results shown in Table 1. Briefly, CEF cells were incubated with serially diluted viral supernatants. Subsequently, 40,000 plaque forming units (pfu)/well of vesicular stomatitis virus (VSV) were added and 48 hours later, wells were scored for the presence of VSV cytopathic effect (CPE). Recombinant viral supernatants containing cIFN were shown to inhibit VSV induced CPE, whereas, control viral supernatants did not. VSV induced cytopathic effect could be reversed in the I presence of rabbit anti-cIFN sera.

15 Table 1.

Recombinant Viral cIFN Activity (units/ml)a Supernatants.

SPV/IFN 2,500 000 SPV <100 20 FPV/IFN 250,000 FPV/cIFN/NDV 250,000 FPV <100 One unit of cIFN activity is defined as the dilution of pox virus supernatant at which 100% VSV CPE was inhibited.

Effect of clFN exDressed from supernatants or SPV/cIFN infected cells on heroes virus of turkeys.

Supernatant containing recombinant cIFN from ESK-4 cells infected with SPV/cIFN virus, was tested for its ability to inhibit the growth of herpes virus of -201turkeys (HVT) in CEF cells, results shown in Table 2.

Briefly, serially diluted supernatants were incubated with CEF cells, and then subsequently infected with 100 pfu/well of wild-type HVT. Plaques were counted in all wells after 48 hours. It was shown that 10-100 units of cIFN activity inhibited plaque formation of HVT(100 pfu/well). Supernatants from wild type SPV did not inhibit HVT plaque formation.

Table 2.

SPV/cIFN Supernatant Number of HVT plaques (units/mla) 9 0 99 1000 0 100 0 10 a- One unit of cIFN activity is defined as the 20 dilution of pox virus supernatant at which 100% VSV CPE was inhibited.

Induction of NO by chicken macrophages after treatment with cIFN expressed in supernatants from SPV/cIFN infected cells.

HD 11 cells or bone marrow adherent cells were incubated with 1000unit/ml of cIFN from SPV/cIFN supernatants, lipopolysaccharide (LPS) (6ng/ml) or with both cIFN and LPS, results shown in Table 3. After 24 hours, supernatant fluids were collected and nitrite levels were measured. These data demonstrate that cIFN expressed from SPV/cIFN supernatants has the ability to activate chicken macrophages in the presence of LPS.

-202- Table 3.

Nitrite (micro/mol) levels following stimulation with Cell source LPS SPV/cIFN LPS SPV/cIFN HD11 10.76 6.4 35.29 BMAC 13.1 5.8 35.10 Conclusions: 1. Recombinant swinepox viruses express biologically active chicken interferon into the supernatants of infected cells, as measured by protection of CEF cells from VSV infection.

2. Chicken interferon expressed in supernatants from recombinant SPV/cIFN infected cells has been shown to protect CEF cells against infection with HVT in a dose dependent manner.

3. Chicken interferon expressed from SPV/cIFN acted synergistically with LPS to activate chicken macrophages as detected by nitric oxide induction.

4. The foregoing data indicate that recombinant swinepox viruses expressing chicken IFN may have beneficial applications as immune modulating agents in vitro, in vivo and in ovo.

Example -203- As an alternative to the construction of a IBD vaccine using a viral vectored delivery system and/or subunit approaches, IBD virus RNA is directly manipulated reconstructing the virus using full length RNA derived from cDNA clones representing both the large (segment A) and small (segment B) double-stranded RNA subunits.

Generation of IBD virus is this manner offers several advantages over the first two approaches. First, if IBD virus is re-generated using RNA templates, one is able to manipulate the cloned cDNA copies of the viral genome prior to transcription (generation of RNA).

~Using this approach, it is possible to either attenuate a virulent IBD strain or replace the VP2 variable region of the attenuated vaccine backbone with that of virulent strains. In doing so, the present invention S"provides protection against the virulent IBDV strain while providing the safety and efficacy of the vaccine strain. Furthermore, using this approach, the present invention constructs and tests temperature sensitive 20 IBD viruses generated using the RNA polymerase derived from the related birnavirus infectious pancreatic necrosis virus (IPNV) and the polyprotein derived from IBDV. The IPNV polymerase has optimum activity at a temperature lower than that of IBDV. If the IPNV polymerase recognizes the regulatory signals present on IBDV, the hybrid virus is expected to be attenuated at the elevated temperature present in chickens.

Alternatively, it is possible to construct and test IBD viruses generated using the RNA polymerase derived from IBDV serotype 2 viruse and the polyprotein derived from IBDVserotype 1 virus.

cDNA clones representing the complete genome of IBDV (double stranded RNA segments A and B) is constructed, initially using the BursaVac vaccine strain (Sterwin Labs). Once cDNA clones representing full length copies of segment A and B are constructed, template RNA is -204prepared. Since IBDV exists as a bisegmented doublestranded RNA virus, both the sense and anti-sense

RNA

strands of each segment are produced using the pBlueScript plasmid; Stratagene, Inc.). These vectors utilize the highly specific phage promoters SP6 or T7 to produce substrate amounts of RNA in vitro. A unique restriction endonuclease site is engineered into the 3' PCR primer to linearize the DNA for the generation of run-off transcripts during transcription.

The purified RNA transcripts (4 strands) are 'transfected into chick embryo fibroblasts (CEF) cells to determine whether the RNA is infectious. If IBD .t 0 virus is generated, as determined by black plaque assays using IBDV specific Mabs, no further manipulations are required and engineering of the vaccine strain can commence. The advantage of this method is that engineered IBD viruses generated in this manner will be pure and require little/no purification, 20 greatly decreasing the time required to generate new vaccines. If negative results are obtained using the purified RNA's, functional viral RNA polymerase is required by use of a helper virus. Birnaviruses replicate their nucleic acid by a strand displacement (semi-conservative) mechanism, with the RNA polymerase binding to the ends of the double-stranded

RNA

molecules forming circularized ring structures (Muller Nitschke, Virology 159, 174-177, 1987).

RNA

polymerase open reading frame of about 878 amino acids in swinepox virus is expressed and this recombinant virus (S-SPV-044) is used to provide functional

IBDV

RNA polymerase in trans. Swinpox virus expressed immunologically recognizable foreign antigens in avian cells (CEF cells), where there are no signs of productive replication of the viral vector. In the present invention the IBDV polymerase protein is expressed in the same cells as the transfected

RNA

-205using the swinepox vector without contaminating the cells with SPV replication.

With the demonstration that IBD virus is generated in vitro using genomic RNA, an improved live attenuated virus vaccines against infectious bursal disease is developed. Using recombinant DNA technology along with the newly defined system of generating IBD virus, specific deletions within the viral genome, facilitating the construction of attenuated viruses are made. Using this technology, the region of IBDV responsible for virulence and generate attenuated, *e g immunogenic IBDV vaccines are identified. The present invention provides a virulent IBD strain or replacement 15 of the VP2 variable region of the attenuated vaccine backbone with that of a virulent strain, thus protecting against the virulent strain while providing the safety and efficacy of the vaccine strain.

Example 36 Effects of Rabbit anti-chicken interferon (cIFN) antibody on the growth of Herpes Virus of Turkeys.

Supernatants from SPV/cIFN (SPV 042) infected ESK-4 cells were harvested 48 hours after infection and then concentrated 5-10 times, by Centricon 10 columns (Amicon). One ml of concentrated supernatant was injected into a rabbit 3 times, at 3 week intervals, and then bled. This rabbit antisera was then used in culture to study the effect of interferon on the growth of HVT. It was shown that anti-cIFN reverses the block to HVT (1:200) and VSV(1:80) growth induced by the addition of cIFN in plaque assays. Furthermore, it was shown that the addition of anti-cIFN (1:100) in the media of CEFs transiently transfected with sub- -206plaqueing levels of HVT viral DNA, enhances the formation of HVT plaques (200 plaques/well). CEFs transfected with HVT DNA in the absence of anti-cIFN did not yield plaques.

HVT is highly susceptible to interferon produced from CEFs and that when cIFN is blocked, HVT growth is enhanced.

Applications include: Use antibody to cIFN as an additive to increase HVT titers in vaccine stocks; (2) Use antibody to cIFN as an additive to facilitate the formation of new recombinant HVT viruses via cosmid o o reconstructions.

00 Example 37 S-SPV-063 20 S-SPV-063 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for swine influenza virus (SIV) NP (HIN1) 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 SIV NP gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-063 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 807-41.3 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS) The final result of red plaque purification was the recombinant -207virus designated S-SPV 063. This virus was assayed for 0-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-063 was assayed for expression of SIV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- SIV serum or a polyclonal goat anti-NP serum was shown to react specifically with S-SPV-063 plaques and not with S-SPV-001 negative control plaques. All S-SPV-063 15 observed plaques reacted with the swine anti-SIV serum or goat anti-NP serum indicating that the virus was stably expressing the SIV 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 SIV NP gene products, cells were infected with S-SPV-063 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis, The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. A polyclonal swine anti-SIV serum or a polyclonal goat anti-NP serum was used to detect expression of SIV specific proteins. The cell lysate and supernatant from S-SPV-063 infected cells exhibited bands corresponding to 56 kd, which is the expected size of the SIV NP protein.

S-SPV-063 is useful as a vaccine in swine against SIV infection and is useful for expression of SIV NP. S- SPV-063 is useful as a vaccine in combination with S- -208- SPV-066 which expresses NA and S-SPV-065 which expresses SIV HA.

S-SPV-064 S-SPV-064 is a swinepox virus that expresses one foreign gene. The gene for E. coli (uidA) was inserted into the unique XhoI restriction site within the 6.9 kb SPV HindIII J genomic fragment.

The uidA gene is under the control of the synthetic late/early promoter (LP2EP2). The HindIII J genomic fragment contains part of the A50R ORF (aa 227 to 552) The unique XhoI site is not within the A50R ORF. The XhoI site is 25 kb from the 3'end of the swinepox virus genome (62).

S-SPV-064 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 807-42.28 and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV.

Homology vector 807-42.28 was generated by insertion of a NotI fragment containing the LP2EP2 promoter uidA gene cassette from plasmid 551-47.23 (see Materials and Methods) into a NotI site (unique XhoI site converted 25 to NotI by a DNA linker) in the SPV 6.9 kb HindIII J fragment. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification is the recombinant virus designated S-SPV- 064. This virus is assayed for -glucuronidase 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, plaques observed are blue indicating that the virus is pure, stable, and expressing the foreign gene.

-209- S-SPV-064 is purified and expresses the foreign gene, E. coli uidA, indicating that the XhoI site within the 6.9 kb HindIII J fragment is a site non-essential for virus growth and a stable insertion site for foreign genes. Recombinant swinepox virus utilizing this insertion site is useful for expression of foreign antigen genes, as a vaccine against disease or as an expression vector to raise antibodies to the expressed foreign gene.

S-SPV-065 S-SPV-065 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase 15 (lacZ) and the gene for swine influenza virus (SIV) HA (H1N1) 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 SIV HA gene is under the control of the synthetic late/early promoter (LP2EP2) e S-SPV-065 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 25 807-84.8 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 065. This virus was assayed for 3-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.

-210- S-SPV-065 was assayed for expression of SIV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- SIV serum or a Polyclonal goat anti-HA serum was shown to react specifically with S-SPV-065 plaques and not with S-SPV-001 negative control plaques.. All S-SPV-065 observed plaques reacted with the swine anti-SIV serum or the SIV 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 SIV NP gene products, cells were infected with S-SPV-065 and samples of 15 infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis.

The gel was blotted and analyzed using the WESTERN

*C*

BLOTTING PROCEDURE. A Polyclonal swine anti-SIV serum or a Polyclonal goat anti-HA serum was used to detect expression of SIV specific proteins. The cell lysate and supernatant from S-SPV-065 infected cells exhibited bands corresponding to 64 kd, which is the expected size of the SIV HA protein.

S-SPV-065 is useful as a vaccine in swine against SIV infection and is useful for expression of SIV HA. S- SPV-065 is useful as a vaccine in combination with S- SPV-066 which expresses NA and S-SPV-063 which expresses SIV NP.

S-SPV-066 S-SPV-066 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for swine influenza virus (SIV) NA (H1N1) were inserted into the SPV 617 48.1 ORF (a unique NotI restriction site has replaced a unique AccI -211restriction site). The lacZ gene is under the control of the synthetic late promoter (LP1), and the SIV NA gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-066 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 807-84.35 (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 -galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 066. This virus was assayed for 0-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.

To confirm the expression of the SIV NA gene products, cells were infected with S-SPV-066 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis.

The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. A Polyclonal swine anti-SIV serum or a Polyclonal goat anti-NA serum was used to detect expression of SIV specific proteins. The cell lysate and supernatant from S-SPV-066 infected cells exhibited bands corresponding to 64 kd, which is the expected size of the SIV HA protein.

S-SPV-066 is useful as a vaccine in swine against SIV infection and is useful for expression of SIV NA. S- SPV-066 is useful as a vaccine in combination with S- -212- SPV-065 which expresses HA and S-SPV-063 which expresses SIV NP.

S-SPV-071 S-SPV-071 is a swinepox virus that expresses at least four foreign genes. The gene for E. coli 3galactosidase (lacZ) and the genes for swine influenza virus (SIV) HA (HIN1) and NA (H1N1) 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 SIV HA, and NA genes are under the control of the synthetic late/early promoter 15 (LP2EP2) S-SPV-071 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector817- 86.35 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 071. This virus was assayed for /-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-071 was assayed for expression of SIV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN

GENE

EXPRESSION IN RECOMBINANT SPV. Polyclonal goat anti-HA serum was shown to react specifically with S-SPV-071 plaques and not with S-SPV-001 negative control -213plaques. All S-SPV-071 observed plaques reacted with the goat anti-HA serum indicating that the virus was stably expressing the SIV 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 SIV HA and NA gene products, cells were infected with S-SPV-071 and samples of infected cell lysates and culture supernatants were subjected to SDS polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. A Polyclonal 15 swine anti-SIV serum or a Polyclonal goat anti-HA serum was used to detect expression of SIV specific proteins.

The cell lysate and supernatant from S-SPV-071 infected cells exhibited bands corresponding to 64 kd and 52 kd, which is the expected size of the SIV HA and NA protein.

S-SPV-071 is useful as a vaccine in swine against SIV infection and is useful for expression of SIV HA and NA. S-SPV-071 is useful as a vaccine in combination 25 with S-SPV-063 which expresses SIV NP.

S-SPV-074 S-SPV-074 is a swinepox virus that expresses at least four foreign genes. The gene for E. coli 3glucuronidase (uidA) and the genes for swine influenza virus (SIV) HA (H1N1) and NA (H1N1) were inserted into the SPV 617 48.1 ORF (a unique NotI restriction site has replaced a unique AccI restriction site). The uidA gene is under the control of the synthetic late/early promoter (LP2EP2), and the SIV HA and NA genes are -214under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-074 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 817-14.2 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV 074. This virus was assayed for G. 3-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue 15 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-074 was assayed for expression of SIV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal swine anti- SIV serum was shown to react specifically with S-SPV- 074 plaques and not with S-SPV-001 negative control plaques. All S-SPV-074 observed plaques reacted with the goat anti-HA serum indicating that the virus was stably expressing the SIV 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.

S-SPV-074 is useful as a vaccine in a swine against SIV infection and is useful for expression of SIV HA and Na. S-SPV-074 is useful as a vaccine in combination with S-SPV-063 which expresses SIV NP.

-215- S-SPV-063, -065, -066, -071, and -074, are useful alone or in combination as a vaccine in swine against swine influenza infection and are useful for expression of the SIV NP, HA, and NA proteins.

S-SPV-069 S-SPV-069 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for human respiratory syncytial virus (HRSV) fusion protein were inserted into the SPV 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of the 15 swinepox PoIL promoter and the HRSV F gene is under the control of the synthetic late/early promoter (LP2EP2) S-SPV-069 was derived from S-SPV-001 (Kasza Strain) This was accomplished utilizing the homology vector 810-29.A2 (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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant 25 virus designated S-SPV 069. This virus was assayed for 0-galacrosidase 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-069 was assayed for expression of HRSV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Monoclonal antibody 621 (Biodesign, Inc.) against HRSV F was shown to react specifically with S-SPV-069 plaques and not with S-SPV- 001 negative control plaques. All S-SPV-069 observed -216plaques reacted with the monoclonal antibody 621 indicating that the virus was stably expressing the PRV 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.

S-SPV-078 S-SPV-078 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for human respiratory syncytial virus (HRSV) attachment protein were inserted into the SPV 617 48.1 ORF (a unique NotI restriction site 15 has replaced a unique AccI restriction site). The lacZ gene is under the control of the synthetic late/early promoter (LP2EP2), and the HRSV G gene is under the control of the synthetic late/early promoter (LP2EP2) S-SPV-078 was derived from S-SPV-001 (Kasza Strain).

This was accomplished utilizing the homology vector 822-52G.7 (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 25 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification is the recombinant virus designated S-SPV-078. This virus is assayed for 0-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 are blue indicating that the virus is pure, stable, and expressing the foreign gene.

S-SPV-069 and S-SPV-078 are useful individually or in combination as a vaccine in swine against human -217respiratory syncytial virus infection and are useful for expression of HRSV F and G genes.

HOMOLOGY VECTOR 810-29.A2. The plasmid 810-29.A2 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli P-galactosidase (lac Z) marker gene and a human respiratory syncytial virus (HRSV) fusion gene flanked by SPV DNA.

Upstream of the foreign gene is an approximately 855 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 1113 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 15 coding for the foreign genes will result. Note that the 0 galactosidase (lacZ) marker gene is under the control of a swinepox virus 01L gene promoter and the 0* HRSV F gene is under the control of the late/early 00 promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA techniques (22, by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2519 base pair S' HindIII to SphI restriction fragment of 25 (Promega). Fragment 1 is an approximately 855 base pair sub-fragment of the SPV HindIII restriction fragment M (23) synthesized by polymerase chain reaction using DNA primers 5 GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAATA-3' (SEQ ID NO: 185) and 5' -CATAAGATCTGGCATTGTGTTATTATACTAACAAAAATAAG- 3' (SEQ ID NO: 186) to produce an 855 base pair fragment with SphI and BglII ends. Fragment 2 is a 3002 base pair BamHI to PvuII fragment derived from plasmid pJF751 (49) containing the E. coli lacZ gene.

Fragment 3 is an approximately 1728 base pair EcoRI restriction fragment synthesized by reverse transcriptase and polymerase chain reaction (PCR) -218- 42) using RNA from the HRSV Strain A2 (ATCC VR-1302) The primer GCCGAATTCGCTAATCCTCAAAGCAAATGCAAT- 3';4/95.23) (SEQ ID NO:) synthesizes from the 5' end of the HRSV F gene, introduces an EcoRI site at the 5' end of the gene and an ATG start codon. The primer GGTGAATTCTTTATTTAGTTACTAAATGCAATATTATTT-3'; 4/95.24) (SEQ ID NO:) synthesizes from the 3' end of the HRSV F gene and was used for reverse transcription and polymerase chain reaction. The PCR product was digested with EcoRI to yield a fragment 1728 base pairs in length corresponding to the HRSV F gene. Fragment 4 is an approximately 1113 base pair subfragment of the SPV HindIII fragment M synthesized by polymerase chain reaction using DNA primers 5 15 CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3' (SEQ ID N O 1 8 7 a n d GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3' (SEQ ID NO: 188) to produce an 1113 base pair fragment with SalI and HindIII ends.

HOMOLOGY VECTOR 822-52G.7. The plasmid 822-52G.7 was constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E. coli 0-galactosidase (lacZ) marker gene and the human respiratory syncytial virus (HRSV attachment gene flanked by SPV DNA.

Upstream of the foreign genes is an approximately 1484 base pair fragment of SPV DNA. Downstream of the foreign genes is an approximately 2149 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 5-galactosidase (lacZ) marker gene is under the control of a synthetic late/early pox promoter (LP2EP2) and the HRSV G gene is under the control of a synthetic late/early pox promoter (LP2EP2) .It was constructed utilizing standard recombinant DNA techniques (22 and -219by joining restriction fragments from the following sources. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP65 (Promega) Fragment 1 is an approximately 1484 base pair AccI to BglII restriction sub-fragment of the SPV HindIII fragment M Fragment 2 is an approximately 3006 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11) Fragment 3 is an approximately 899 base pair EcoRI restriction fragment synthesized by reverse transcriptase and polymerase chain reaction (PCR) 42) using RNA from the HRSV Strain A2 (ATCC VR-1302) The primer GCCGAATTCCAAAAACAAGGACCAACGCAC- 3';4/95.25) (SEQ ID NO:) synthesizes from the 5' end of I 15 the HRSV F gene, introduces an EcoRI site at the 5' end of the gene and an ATG start codon. The primer C* GCCGAATTCACTACTGGCGTGGTGTGTTG-3'; 4/95.26) (SEQ ID NO:) synthesizes from the 3' end of the HRSV G gene and was used for reverse transcription and polymerase chain 20 reaction. The PCR product was digested with EcoRI to yield a fragment 899 base pairs in length corresponding to the HRSV G gene. Fragment 4 is an approximately 2149 base pair HindIII to AccI restriction sub-fragment of .the SPV HindIII restriction fragment M (23) HOMOLOGY VECTOR 807-41.3. The plasmid 807-41.3 was used to insert froeign DNA into SPV. It incorporates an E.

coli B- galactosidase (lacZ) marker gene and the swine influenza virus (SIV) nucleoprotein (NP) gene flanked by SPV DNA. When this plasmid was used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing 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 SIV NP gene is under the control of a synthetic late/early pox prometer (LP2EP2) The homology vector was constructed utilizing -220standard recombinant DNA techniques (22 and 30), by joining restriction fragments from the following sources with the appropriate synthetic DNA sequences.

The plasmid vector was derived from an approximately 2972 base pair Hindiii to BAm HI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 1484 base Bglii to AccI restriction sub-fragment of the SPV HindIII fragment M Fragment 2 is an approximately 1501 base pair EcoRI to EcoRI fragment of the SIV NP gene synthesized by reverse :i transcription (RT) and polymerase chain reaction (PCR) (15,42) using RNA from the SIV H1N1 strain (NVSL). The .primer (5'CATGAATTCTCAAGGCACCAAACGATCATATGAAC-3'; 6/95.13) (SEQ ID NO:) synthesizes from the 5' end of .15 the SIV NP gene and introduces an EcoRI site at the end of the gene. The primer ATTTGAATTCAATTGTCATACTCCTCTGCATTGTCT-3';6/95.14) (SEQ ID NO:) synthesizes from the 3' end of the SIV NP gene, introduces an EcoRI site st the 3' end of the gene, and 20 was used for reverse transcription and polymarase chain reaction. The PCR product was digested with EcoRI to yield a fragment 1501 base pairs in length corresponding to the SIV NP gene. Fragment 3 is Sapproximately 3010 base pair BamHI ro PuvII restriction fragmebt of plsmid pJF751 (11) Fragent 4 is approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV Hind III restriction fragment M (23) HOMOLOGY VECTOR 807-84.8. THe plasmid 807-84.8 was used to insert foreign DNA into SPV. It incorporates an E.

coli B- galactosidase (lacZ) marker gene and the swine influenza virus (SIV) hemmagglutinin (HA) gene flanked by SPV DNA. When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GANERATING RECOMBINANT SPV a virus containing DNA coding for the -221foreign genes results. Note that the B galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the SIV HA gene is under the control of a synthetic late/early promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA techniques (22 and 30), by joining restricting fragments from the following sources with the appropriate synthetic DNA sequences.

The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega) Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII fragment M (23) Fragment 2 is an approximately 1721 base pair BamHI to BamHI fragment of 15 the SIV HA gene synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) (15,42) using RNA from the SIV H1N1 strain (NVSL). The primer CGAGGATCCGGCAATACTATTAGTCTTGCTATGTACAT-3'; 6/95.5) (SEQ ID NO:) synthesizes from the 5' end of the SIV HA 20 gene and introduces an BamHI site at the 5; end of the g e n e T h e p r i m e r 5

CTCTGGATCCTAATTTAAATACATATTCTGCACTGTS-

3 6/95.6) (SEQ SID NO:) synthesizes from the 3' end of the SIV HA gene, introduces a BAm HI site at the 3' end of the gene, and was used for the reverse transcription and polymerase chain reaction. The PCR product was digested with EcoRI to yield a fragment 1721 base pairs in length corresponding to the SIV HA gene. Fragment 3 is an approximately 3010 base pair BamHI ot PvuII restriction fragment of plasmid pJF75 (11) Fragment 4 is an approximately 2149 base pair AccI to fragmeny M (23) HOMOLOGY VACTOR 807-84.35. The plasmid 807-84.35 was used to insert foreign DNA into SPV> It incorporates an E> coli B-galactosidase (lacZO marker gene and the swine influenza virus (SIV) neuraminidase (NA) gene flanked by SPV DNA. When this PROCEDURE FRO GENERATING -222- RECOMBINANT SPV a virus containing DNA coding for the foreign genes results. Note that the B galactosidase (lacZ) marker gene is under the comtrol of a synthetic late pox promoter (LP1) and the SIV NA gene is under the comtrol of a synthetic late/early pox promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA techniques (22 and by joinung restricting fragments from the following sources with the appropriate synthetic DNA sequences The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriciton fragment of pSP64 (Promega). Fragment 1 is an approximately 1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIII fragment M 15 Fragment 2 is an approximately 1414 base pair EcoRI ti BglII fragment of the SIV NA gene synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) (15,42) using RNA from the SIV H1N1 strain (NVSL) THe primer 5 20 AATGAATTCAAATCAAAAAATAATAACCATTGGGTCAAT 6/95.12) (SEQ ID NO:) synthesizes from the 5' end of the SIV NA gene and introduces an EcoRI site at the 5' end of the g e ne T h r e pr i m e r 5

GGAAGATCTACTTGTCAATHHTHAATGGCAGATCAG-

3 6/95.13) (SEQ ID NO:) synthesizes from the 3' end of the SIV NA gene, introduces an BglII site at the 3' end of the gene, and was used for reverse transcription and polymerase chain recation. The PCR product was digested with EcoRI to yield a fragment 1414 base pairs in length corresponding to the SIV NA gene. Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriciton fragment of plasmid pJF751 Fragment 4 is an approximately 2149 base pair AccI to HIndIII restriciton sub-fragment of the SPV HindIII restriction fragment M (23).

-223- HOMOLOGY VECTOR 807-86.35. The plasmid 807-86.35 was used to insert foreign DNA into SPV. It incorporates an E> coli B- galactosidase (lacZ) marker gene and the swine influenza virus (SIV) hemagglutinin (HA) and neuraminidase (NA) gene flanked by SPV DNA> When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing 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 SIV NA and HA genes are each under the control of a synthetic late/early pox ."promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA techniques (22 and 15 30), by joining restriction fragments from the following sources with the appropriate synthetic DNA sequences. The plasmid vector was derived from an approximately 2972 base pair HIndIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is 20 approximately 1484 base pair BglII to AccI restriction sub-fragmentof the SPV HindIII fragment M (23) Fragment 2 is an approximately 1721 base pair BamHI to BamHI fragment of the SIV HA gene synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) (15,42) using RNA from the SIV H1N1 strain (NVSL) The primer (5 CCGAGGATCCGGCAATACTATTAGTCTTGCTATGTACAT-3';6/95.5) (SEQ ID NO:) synthesizes from the 5' end of the SIV HA gene and introduces an Bam HI site at the 5' end of the g e n e. T h e p r i m e r (5 CTCTGGGATCCTAATTTTAAATACATATTCTGCACTGTA-3'; 6/95.6) (SEQ ID NO:) synthesizes from the 3' end of the SIV HA gene, introduces an BamHI site at the 3' end of the gene, and was used for reverse transcription and polymerase chain reaction. The PCR product was digested with EcoRI to yield a fragment 1721 base pairs in length corresponding to the SIV HA gene. Fragment 3 is -224an approximately 1414 base pair EcoRI to BglII fragment of the SIV NA gene synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) (15,42) using RNA from the SIV H1N1 strain (NVSL). The primer

AATGAATTCAAATCAAAAATAATAACCATTGGGTCAAT-

3 ;6/95.12) (SEQ ID NO:) synthesizes from the 5' end of the SIV NA gene and introduces an EcoRI site at the 5' end of the g e n e. T h e pr i me r 5 GGAAGATCTACTTGTCAATGGTGAATGGCAGATCAG-3'; 6/95.13) (SEQ ID NO:) synthesizes from the 3' end of the SIV NA gene, introduces an BglII site at the 3' end of the gene, and was used for reverse transcription and plymerase chain .reaction. The PCR product was digested with EcoRI to yield a fragment 1414 base pairs in length 15 corresponding to the SIV NA gene. Fragment 4 is an approximately 3010 base pair BamHI to PvuII restriction fagment of plasmid pJF751 (11) Fragment 5 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII restriction 20 fragment M (23).

HOMOLOGY VECTOR 817-14.2. The plasmid 817-14.2 was used to insert foreign DNA into SPV. It incorporates an E.

coli B- glucuronidase (uidA) marker gene and the swine influenza virus (SIV) henagglutinin (HA) and neuraminidase (NA) gene flanked by SPV DNA, When theis poasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA coding for the foreign genes results. Note that the B glucuronidase (uidA) marker gene is under the control of a synthetic late/early pox pomoter (LP2EP2) and the SIV NA and HA genes are each under the control of a synthetic late/early pox promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA techniques (22 and by joining restriction fragments from the following sources with the appropriate synthetic DNA -225sequences. The plasmid vector was derived from an approximately 2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 is an approximatelyl484 base pair BglII to AccI restriction subfragment fo the SPV HindIII fragment M Fragment 2 is an approximately 1721 base pair BamHI to BamHI fragment of the SIV HA gene synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) (15,42) using RNA from the SIV H1N1 strain (NVSL) The primer (5 CCGAGGATCCGGCAATACTATTAGTCTTGCTATGTACAT-3';6/95.5) (SEQ ID NO:) synthesizes from the 5' end of the SIV HA gene and introduces an BamHI site at the 5' end of the gene.

The primer -CTCTGGGATCCTAATTTTAAATACATATTCTGCACTGTA- 15 6/95.6) (SEQ ID NO:) synthesizes from the 3' end of the SIV HA gene, introduces an BamHI site at the 3' end of the gene, and was used for reverse transcription and polymerase chain reaction. The PCR product was digested with EcoRI to yield a fragment 1721 base pairs in 20 length corresponding to the SIV HA gene. Fragment 3 is an approximately 1414 base pair EcoRI to BglII fragment of the SIV NA gene synthesized by reverse transcription S(RT) and polymerase chain reaction (PCR) (15,42) using RNA fro the SIV H1N1 strain (NVSL). The primer AATGAATTCAAATCAAAAAATAATAACATTGGGTCAAT-3';6/95.12)

(SEQ

ID NO:) synthesizes from the 5' end of the SIV NA gene and introduces an EcoRI site at the 5' end of the gene.

The primer -GGAAGATCTACTTGTCAATGGTGAATGGCAGATCAG-3'; 6/95.13) (SEQ ID NO:) synthesizes from the end of the SIV NA gene, introduces an BglII site at the 3' end of the gene, and was used for reverse transcription and polymerase chainreaction. The PCR product was digested with EcoRI to yield a fragment 1414 base pairs in length corresponding to the SIV NA gene. Fragment 4 is an approximately 1823 vase pair NotI restriction fragment of plasmid pRAJ260 (Clonetech). Fragment 5 is an approximately 2149 base pair AccI to HindIII restriction sub-fragment of the SPV HindIII restriction fragment M (23).

PRRS HOMOLOGY VECTORS CONTAINING SINGLE OR MULTIPLE PRRS GENES (ORF2, ORF3, ORF4, ORF5, ORF6, or ORF7: The PRRS homology vector is constructed for the purpose of inserting foreign DNA into SPV. It incorporates an E.

coli 0-galactosidase (lac Z) marker gene and a porcine reproductive and respiratory syndrome virus (PRRS) ORF2, ORF3, ORF4, ORF5, ORF6, or ORF7 gene flanked by SPV DNA. Upstream of the foreign gene is an approximately 855 base pair fragment of SPV DNA.

Downstream of the foreign genes is an approximately 1113 base pair fragment of SPV DNA. When the plasmid 15 is used according to the HOMOLOGOUS RECOMBINATION g PROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA coding for the foreign genes will result. Note that the 0 galactosidase (lacZ) marker gene is under the control of a swinepox virus 01L gene 20 promoter and the PRRS gene is under the control of the late/early promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA techniques (22, 30), by joining restriction fragments from the following sources with the synthetic DNA sequences. The plasmid vector was derived from an approximately 2519 base pair HINDIII to SphI restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 855 base pair sub-fragment of the SPV HindIII restriction fragment M (23) synthesized by polymerase chain reaction using DNA primers GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3' (SEQ ID NO: 185) and 3' (SEQ ID NO: 186) to produce an 855 base pair fragment with SphI and BglII ends. Fragment 2 is a 3002 base pair BamHI to PvuII fragment derived from plasmid pJF751 (49) containing the E. coli lacZ gene.

Fragment 3 is an EcoRI to BamHI restriction fragment -227synthesized by reverse transcription and polymerase chain reaction (PCR) using genomic RNA from a U.S.

Isolate of PRRS obtained from the NVSL (Reference strain). Each homology vector contains one or multiple of the PRRS virus ORF2 through 7. To synthesize PRRS O R F 2 t h e p r i m e r (5 AATGAATTCGAAATGGGGTCCATGCAAAGCCTTTTTG-3';1/96.15)

(SEQ

ID NO:) synthesizes from the 5' end of the PRRS ORF2 gene, introduces an EcoRI site at the 5' end of the g e n e. T h e p r i m e r 5 CAAGGATCCCACACCGTGTAATTCACTGTGAGTTCG-3'; 1/96.16) (SEQ ID NO.) is used for reverse transcription and PCR and synthesizes from the 3' end of the PRRS ORF2 gene. The PCR product was digested with EcoRI and BamHI to yield 15 a fragment 771 base pairs in length corresponding to the PRRS ORF2 gene. To synthesize PRRS ORF3, the S. primer TTCGAATTCGGCTAATAGCTGTACATTCCTCCATATTT-3'; 1/96.7) (SEQ ID NO:) synthesizes from the 5' end of the PRRS ORF3 gene, introduces an EcoRI site at the 5' end 20 of the gene The primer (5 GGGGATCCTATCGCCGTACGGCACTGAGGG-3'; 1/96.8) (SEQ ID NO:) is used for reverse transcription and PCR and S. synthesizes from the 3' end of the PRRS ORF3 gene. To .synthesize PRRS ORF4, the primer (5'CCGAATTCGGCTGCGTCCCTTCTTTTCCTCATGG-3' 1/96.11) (SEQ ID NO:) synthesizes from the 5' end of the PRRS ORF4 gene, introduces an EcoRI site at the

CTGGATCCTTCAAATTGCCAACAGAATGGCAAAAAGAC-

3 1/96.12) (SEQ ID NO.) is used for reverse transcription and PCR and synthesizes from the 3' end of the PRRS ORF4 gene.

The PCR product was digested with EcoRI and BamHI to yield a fragment 537 base pairs in length corresponding to the PRRS ORF4 gene. To synthesize PRRS ORF5, the primer TTGAATTCGTTGGAGAAATGCTTGACCGCGGGC-3'; 1/96.13) (SEQ ID NO:) synthesizes from the 5' end of the PRRS ORF5 gene, introduces an EcoRI site at the end of the gene. The primer -228-

GAAGGATCCTAAGGACGACCCCATTGTTCCGCTG-

3 1/96.14) (SEQ ID NO:) is used for reverse transcription and PCR and synthesizes from the 3' end of the PRRS ORF5 gene. The PCR product was digested with EcoRI and BamHI to yield a fragment 603 base pairs in length corresponding to the PRRS ORF5 gene. To synthesize PRRS ORF6, the primer CGGGAATTCGGGGTCGTCCTTAGATGACTTCTGCC-3'; 1/96.17) (SEQ ID NO:) synthesizes from the 5' end of the PRRS ORF6 gene, introduces an EcoRI site at the end of the gene. The primer

GCGGATCCTTGTTATGTGGCATATTTGACAAGGTTTAC-

3 1/96.18) (SEQ ID NO:) is used for reverse transcription and PCR and synthesizes from the 3' end of the PRRS ORF6 gene.

.The PCR product was digested with EcoRI and BamHI to 15 yield a fragment 525 base pairs in length corresponding to the PRRS ORF6 gene. To synthesize PRRS ORF7, the primer GTCGAATTCGCCAAATAACAACGGCAAGCAGCAGAAG-3' 1/96.19) (SEQ ID NO:) synthesizes from the 5' end of the PRRS ORF7 gene, introduces an EcoRI site at the 20 end of the gene. The primer se*

CAAGGATCCCAGCCCATCATGCTGAGGGTGATG-

3 1/96.20) (SEQ ID NO:) is used for reverse transcription and PCR and synthesizes from the 3' end of the PRRS ORF7 gene.

Fragment 4 is an approximately 1113 base pair subfragment of the SPV HindIII fragment M synthesized by polymerase chain reaction using DNA primers CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3' (SEQ ID NO: 187) and 5' GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAT- 3' )SEQ ID NO: 188) to produce an 1113 base pair fragment with SalI and HindIII ends.

Recombinant swineDox virus expressing pseudorabies genes S-SPV-076 is a swinepox virus that expresses at least three foreign genes. The gene for E. coli 3galactosidase (lacZ) and the genes for pseudorabies -229virus (PRV) gD and gI 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 PRV gD and gI genes are under the control of the synthetic late/early promoter (LP2EP2) S-SPV-077 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli galactosidase (lacZ) and the gene for pseudorabies virus (PRV) gI 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 PRV gI gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-079 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0- 20 galactosidase (lacZ) and the genes for pseudorabies virus (PRV) gB 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 PRV gB gene are under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-076, S-SPV-077, and S-SPV-079 were derived from S-SPV-001 (Kasza Strain). This was accomplished utilizing a homology vector and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock were screened by the SCREEN FOR RECOMBINANT SPV EXPRESSING 3galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-076, S-SPV-077, and S-SPV-079.

The viruses were assayed for p-galactosidase -230expression, 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-076, S-SPV-077, S-SPV-079 have been tested by BLACK PLAQUE ASSAY and WESTERN BLOT for expression of the PRV glycoproteins.

S-SPV-076, S-SPV-077, and S-SPV-079 are useful as a vaccine in swine against PRV infection and is useful for expression of PRV gD, gI or gB. S-SPV-071 is useful as a vaccine in combination with a recombinant swinepox virus which expresses PRV gC, such as S-SPV- 011, S-SPV-012, or S-SPV-013.

9 4o -231-

Z

0* e.

a 0*0.

0*0* *0 143B osteosarcona* A431 epidermoid carcinomna* A549 lung carcinoma* Capari-l liver carcinoma* CF500 foreskin fibroblasts Chang Liver liver Detroit Downs'foreskin fibroblasts HEL-199 embryonic lung HeLa cervical carcinoma* Hep-2 epidermal larynx carcinoma* HISM intestinal smooth muscle HNK neonatal kidney MRC-5 embryonic lung NCI-H292 pulmonary mucoepidermoid carc inoma* OVCAR-3 ovarian carcinoma* RD rhabdosarcoma* THP monocyte (leukemia)* WIL2-NS B lymphocyte line, non-secreting WISH amnion PEL peripheral blood lymphocytes -232- ExamDle 38 Recombinant swinepox virus expressing PRRS genes ORF2, ORF3, ORF4, ORF5, and ORF6 S-SPV-080 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for porcine reproductive and respiratory syndrome virus (PRRS) ORF2 were inserted into the SPV 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of the swinepox POIL promoter and the PRRS ORF2 gene is under the control of the synthetic late/early promoter (LP2EP2).

s S-SPV-081 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase 20 (lacZ) and the gene for porcine reproductive and respiratory syndrome virus (PRRS) ORF3 were inserted into the SPV 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of the swinepox POIL promoter and the PRRS ORF3 gene is under the control of the synthetic late/early promoter (LP2EP2) S-SPV-082 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 0-galactosidase (lacZ) and the gene for porcine reproductive and respiratory syndrome virus (PRRS) ORF4 were inserted into the SPV 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of the swinepox PoIL promoter and the PRRS ORF4 gene is -233under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-083 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli P-galactosidase (lacZ) and the gene for porcine reproductive and respiratory syndrome virus (PRRS) ORF5 were inserted into the SPV 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of S. the swinepox POIL promoter and the PRRS ORF5 gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-084 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli 3-galactosidase (lacZ) and the gene for porcine reproductive and respiratory syndrome virus (PRRS) ORF6 were inserted into the SPV 738-94.4 ORF (a 773 base pair deletion of S. 20 the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of the swinepox POIL promoter and the PRRS ORF6 gene is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-085 is a swinepox virus that expresses at least two foreign genes. The gene for E. coli -galactosidase (lacZ) and the gene for porcine reproductive and respiratory syndrome virus (PRRS) ORF7 were inserted into the SPV 738-94.4 ORF (a 773 base pair deletion of the SPV OIL ORF; Deletion of nucleotides 1669 to 2452, SEQ ID NO: 189). The lacZ gene is under the control of the swinepox POIL promoter and the PRRS ORF6 gene is under the control of the synthetic late/early promoter (LP2EP2).

-234- S-SPV-080, S-SPV-081, S-SPV-082, S-SPV-083, S-SPV-084, S-SPV-085 were derived from S-SPV-001 (Kasza Strain).

This was accomplished utilizing the homology vector Materials and Methods (PRRS HOMOLOGY VECTORS) 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 0-galactosidase (BLUOGAL AND CPRG ASSAYS). The final result of red plaque purification was the recombinant virus designated S-SPV-080, S-SPV-081, S-SPV-082, S- SPV-083, S-SPV-084, S-SPV-085. This virus was assayed for /-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-080, S-SPV-081, S-SPV-082, S-SPV-083, S-SPV-084, 20 S-SPV-085 are useful individually or in combination as vaccines in swine against PRRS infection and are ,useful for expression of PRRS ORF2, ORF3, ORF4, ORF6 and ORF7.

Example 39 The following experiment was performed to determine the ability of swinepox virus to infect human cells in culture and express a foreign DNA such as lacZ.

S-SPV-003 was adsorbed to the human cell lines listed in the Table below at an MOI=0.1 for 2 to 3 hours.

Cells were rinsed three times with PBS, growth media was added, and cells were incubated at 37 for four days. Cells were harvested and a lysate prepared in 200 microliters of PBS by freeze/thaw three times.

-235- Cell debris was pelleted, and 10 microliters of supernatant was assayed for -galactosidase activity by ONPG assay at 37 for 1 1/2 hours. The table shows the results of infection of various human cell lines with S-SPV-003 and the relative levels of cytopathetic effect and expression of lacZ.

The results show that various human cell lines vary in the ability to take up S-SPV-003 and express lacZ. CPE was minimal in all cases and did not result in viral replication. One exception A549 cells which did show some rounding of cells and lifting off the plate in one instance, and another instance of a ten-fold increase in titer during passage suggesting limited viral replication. Several cell lines how significant lacZ activity with no cytopathic effect.

Different pox promoters express lac Z from recombinant swinepox virus in a number of human cell lines. Six different swinepox viruses were constructed which expressed lac Z from EP1, LP1, LP2, EP1LP2, LP2EP2, or the SPV POlL promoter. Viruses were each used to infect A549, Chang liver, or 143B cells at 0.1 moi, and cells were rinsed between 2 and 3 hours later and then incubated for 4 days at 37 C. Each cell line maintained a different hierarchy of promoter activity, which was reproducible in following experiments.

For example, the EP1, LP2EP2, and POlL promoters gave the most expression in 143B cells, while the LP2 was strongest in Chang liver cells, and the EP1LP2 in A549.

In the Chang liver and A549 cells, expression form the POlL promoter was poorest, whereas in 143B, expression from LP2 was poorest. Therefore different human cell lines utilize pox promoters in dissimilar ways. This may reflect how far the swinepox virus can proceed -236r o r r along the replication pathway in different cell lines.

These early and late promoters exhibited lower or higher lacZ activity depending on the human cell type infected by the recombinant swinepox virus. By choosing different promoters for different target tissues, one is able to regulate the amounts of foreign gene product delivered by the swinepox virus to target tissues.

Recombinant swinepox virus is useful as a vaccine for human infectious disease and to deliver therapeutic agents to humans. Recombinant swinepox virus is useful as a vaccine against viral or bacterial infection in humans, and as a therapeutic for cancer or genetic disease to deliver antibodies, tumor antigens, cell surface ligands and receptors, immune modulating molecules such as cytokines.

ExamDle S-SPV-003 Expression of lacZ in human cell lines Measurement of cytopathic effect and lacZ expression Cell Type Cytopathetic Effect" LacZ Expression" A431 epidermoid carcinoma A549 lung carcinoma Capan-1 3 0 liver carcinoma CF500 foreskin fibroblasts Chang Liver +-r -237- Detroit Down's foreskin fibroblasts HEL- 199+1 embryonic lung HEp-2 epiderrmal larynx carcinoma' HISM intestinal smooth muscle H-NK neonatal kidne-y embryonic lung NCI-H292 pulmonary mucoepidermnoid carcinoma' OVCAR-3 ovarian carcinoma' RD rhabdosarcoma' THP monocvte (leukemia)' WIL2-NS B lymphocyte line, nonsecreting WISH amnion HeLa WO 96/22363 PCT/US96/01485 -238-

PBL

peripheral blood lymphocytes When human cells are infected with SPV, a cytopathic effect is sometimes seen. In most cell lines, this cytopathic effect is evidenced by a change in the appearance of the cells, with cells becoming thinner and more ragged along the edges; cells look stressed. This phenomenon was assessed as follow: indicates no difference between infected uninfected cells; indicates that the monolayer is visibly different from uninfected, though most cells appear normal; indicates that the monolayer is obviously *9* 20 affected, with most cells looking stressed. It should be noted that in certain cell lines (HeLa, CF500, 143B), in which titers were obtained after serial passage, there was no evidence for replication of SPV, with one exception.

A549 was given a for cytopathic effect in one instance, when cells appeared to round up and come off the plate during infection, though this observation was not repeated. A549 also showed evidence ii another case of a ten-fold increase in titer during passage, suggesting that it might support limited viral replication.

B-galactosidase activity in A 260 units per cell lysate from 1/20 of a 35 mm dish: No Activity 0.2-0.9 A 260 unit 0.9-1,6 A 260 unit -239-

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S

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-243- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Cochran, Mark D.

Junker, David E.

(ii) TITLE OF INVENTION: Recombinant Swinepox Virus (iii) NUMBER OF SEQUENCES: 231 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: John P. White STREET: 1185 Avenue of the Americas CITY: New York STATE: New York COUNTRY: USA ZIP: 10036 S(v) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: Not Yet Known FILING DATE: 19-JAN-1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: White, John P REGISTRATION NO: 28,678 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 278-0400 TELEFAX: (212) 391-0525 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 599 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS -244- LOCATION: 202..597 OTHER INFORMATION: /partial /codon start= 202 /function= "Potential eukaryotic transcriptional regulatory protein" /standard name= "515-85.1 ORF" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AATGTATCCA GAGTTGTTGA ATGCCTTATC GTACCTAATA TTAATATAGA GTTATTAACT GAATAAGTAT ATATAAATGA TTGTTTTTAT AATGTTTGTT ATCGCATTTA GTTTTGCTGT ATGGTTATCA TATACATTTT TAAGGCCGTA TATGATAAAT GAAAATATAT AAGCACTTAT 120 180 TTTTGTTAGT ATAATAACAC A ATG CCG TCG TAT ATG TAT CCG AAG AAC GCA Met Pro Ser Tyr Met Tyr Pro Lys Asn Ala

C

0 AGA AAA GTA ATT Arg Lys Val Ile AAG ATT ATA TCA Lys Ile Ile Ser

TTA

Leu CAA CTT GAT ATT Gln Leu Asp Ile AAA AAA Lys Lys 279 CTT CCT AAA Leu Pro Lys

AAA

Lys 30 TAT ATA AAT ACC Tyr Ile Asn Thr TTA GAA TTT GGT Leu Glu Phe Gly CTA CAT GGA Leu His Gly GAT ATA AAT Asp Ile Asn AAT CTA CCA GCT TGT ATG TAT Asn Leu Pro Ala Cys Met Tyr

AAA

Lys GAT GCC GTA TCA Asp Ala Val Ser AAT ATA Asn Ile AGA TTT TTA CCT Arg Phe Leu Pro

TAT

Tyr 65 AAT TGT GTT ATG Asn Cys Val Met

GTT

Val AAA GAT TTA ATA Lys Asp Leu Ile

AAT

Asn GTT ATA AAA TCA Val Ile Lys Ser

TCA

Ser 80 TCT GTA ATA GAT Ser Val Ile Asp AGA TTA CAT CAA Arg Leu His Gln

TCT

Ser *0 0 GTA TTA AAA CAT Val Leu Lys His

CGT

Arg AGA GCG TTA ATA Arg Ala Leu Ile GAT TAC GGC GAT CAA GAC ATT Asp Tvr Gly Asp Gln Asp Ile 100 105 ATC ACT TTA ATG ATC ATT AAT AAG Ile Thr Leu Met Ile Ile Asn Lys TAT ATA TTA Tyr Ile Leu 125 110

GAT

Asp TTA CTA TCG ATA GAT Leu Leu Ser Ile Asp 115 CAT GTA AC His Val GAT ATA TCC Asp Ile Ser 120 AAA AAA ATA Lys Lys Ile INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 132 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Pro Ser Tyr Met Tyr Pro Lys Asn Ala Arg Lys Val Ile Ser Lys 1 5 10 -245- Ile Ile Ser Asn Thr Met Leu Gin Leu Asp Ile Lys Leu Pro Lys Lys Tyr Ile Ala Cys Met Leu Glu Phe Gly Leu His Gly Asn Leu Tyr Lys Asp Ala Val Ser Asp Ile Asn Asn Ile Arg Phe Leu Pro Tvr Asn Cys Val Met Val Lys Asp Leu Ile Val Ile Lys Ser Ser Val Ile Asp Thr Arg Leu His Gin Ser 90 Val Leu Lys His Arg Arg Ala Leu Ile Asn Lys Leu 115 Asp 100 Tyr Gly Asp Gin Ile Ile Thr Leu Met Ile Ile 110 Leu Ser Ile Asp Asp 120 Ile Ser Tyr Ile Leu Asp Lys Lys 125 0 5* *5

S

OSOO

S. S 0@ Ile Ile His Val 130 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 899 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..662 OTHER INFORMATION: /partial /codon start= 3 /function= "Potential eukaryotic transcriptional regulatory protein" /standardname= "515-85.1 ORF" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GA GAT ATT AAA TCA TGT AAA TGC TCG ATA TGT TCC GAC TCT ATA ACA Asp Ile Lys Ser Cys Lys Cys Ser Ile Cys Ser Asp Ser Ile Thr 1 5 10 -246- CAT CAT ATA TAT GAA ACA ACA TCA TGT ATA AAT TAT AAA TCT ACC GAT His His Ile Tyr Glu Thr Thr Ser Cys Ile Asn Tyr Lys Ser Thr Asp 25 AAT GAT CTT ATG ATA GTA TTG TTC AAT CTA ACT AGA TAT TTA ATG CAT 143 Asn Asp Leu Met Ile Val Leu Phe Asn Leu Thr Arg Tyr Leu Met His 40 GGG ATG ATA CAT CCT AAT CTT ATA AGC GTA AAA GGA TGG GGT CCC CTT 191 Gly Met Ile His Pro Asn Leu Ile Ser Val Lys Gly Trp Gly Pro Leu 55 ATT GGA TTA TTA ACG GGT GAT ATA GGT ATT AAT TTA AAA CTA TAT TCC 239 Ile Gly Leu Leu Thr Gly Asp Ile Gly Ile.Asn Leu Lys Leu Tyr Ser 70 ACC ATG AAT ATA AAT GGG CTA CGG TAT GGA GAT ATT ACG TTA TCT TCA 287 Thr Met Asn Ile Asn Gly Leu Arg Tyr Gly Asp Ile Thr Leu Ser Ser 85 90 TAC GAT ATG AGT AAT AAA TTA GTC TCT ATT ATT AAT ACA CCC ATA TAT 335 Tyr Asp Met Ser Asn Lys Leu Val Ser Ile Ile Asn Thr Pro Ile Tyr 100 105 110 GAG TTA ATA CCG TTT ACT ACA TGT TGT TCA CTC AAT GAA TAT TAT TCA 383 Glu Leu Ile Pro Phe Thr Thr Cys Cys Ser Leu Asn Glu Tyr Tyr Ser S115 120 125 AAA ATT GTG ATT TTA ATA AAT GTT ATT TTA GAA TAT ATG ATA TCT ATT 431 Lys Ile Val Ile Leu Ile Asn Val Ile Leu Glu Tyr Met Ile Ser Ile 130 135 140 ATA TTA TAT AGA ATA TTG ATC GTA AAA AGA TTT AAT AAC ATT AAA GAA 479 Ile Leu Tyr Arg Ile Leu Ile Val Lys Arg Phe Asn Asn Ile Lys Glu 145 150 155 TTT ATT TCA AAA GTC GTA AAT ACT GTA CTA GAA TCA TCA GGC ATA TAT 527 Phe Ile Ser Lys Val Val Asn Thr Val Leu Glu Ser Ser Gly Ile Tyr 160 165 170 175 TTT TGT CAG ATG CGT GTA CAT GAA CAA ATT GAA TTG GAA ATA GAT GAG 575 Phe Cys Gin Met Arg Val His Glu Gin Ile Glu Leu Glu Ile Asp Glu 180 185 190 CTC ATT ATT AAT GGA TCT ATG CCT GTA CAG CTT ATG CAT TTA CTT CTA 623 Leu Ile Ile Asn Gly Ser Met Pro Val Gin Leu Met His Leu Leu Leu 195 200 205 AAG GTA GCT ACC ATA ATA TTA GAG GAA ATC AAA GAA ATA TAACGTATTT 672 Lys Val Ala Thr Ile Ile Leu Glu Glu Ile Lys Glu Ile 210 215 220 TTTCTTTTAA ATAAATAAAA ATACTTTTTT TTTTAAACAA GGGGTGCTAC CTTGTCTAAT 732 TGTATCTTGT ATTTTGGATC TGATGCAAGA TTATTAAATA ATCGTATGAA AAAGTAGTAG 792 ATATAGTTTA TATCGTTACT GGACATGATA TTATGTTTAG TTAATTCTTC TTTGGCATGA 852 ATTCTACACG TCGGANAAGG TAATGTATCT ATAATGGTAT AAAGCTT 899 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 220 amino acids TYPE: amino acid TOPOLOGY: linear -247- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEC Asp Ile Lys Ser Cys Lys Cys Ser Ile r e r His Asp Met Gly Met Asp Leu Ile Leu 145 Ile Cys Ile Val Ile Leu Ile Leu Asn Met Ile Val 130 Tyr Ser Gin Ile Ala 210 Tyr Met His Leu Ile Ser Pro 115 Ile Arg Lys Met Asn 195 Thr Glu Ile Pro Thr Asn Asn 100 Phe Leu Ile Val Arg 180 Gly Ile Thr Val Asn Gly Gly Lys Thr Ile Leu Val 165 Val Ser Ile Thr Leu Leu Asp 70 Leu Leu Thr Asn Ile 150 Asn His Met Leu Ser Phe Ile 55 Ile Arg Val Cys Val 135 Val Thr Glu Pro Glu 215 Cys Asn 40 Ser Gly Tyr Ser Cys 120 Ile Lys Val Gin Val 200 Glu Ile 25 Leu Val Ile Gly Ile 105 Ser Leu Arg Leu Ile 185 Gin Ile ID NO:4: Cys Ser Asp Asn Tyr Lys Thr Arg Tyr Lys Gly Trp Asn Leu Lys 75 Asp Ile Thr Ile Asn Thr Leu Asn Glu Glu Tyr Met 140 Phe Asn Asn 155 Glu Ser Ser 170 Glu Leu Glu Leu Met His Lys Glu Ile 220 Ser Ser Leu Gly Leu Leu Pro Tyr 125 lle lie Gly lie Leu 205 Ile Thr Met Pro Tyr Ser Ile 110 Tyr Ser Lys Ile Asp 190 Leu His Asn Gly Ile Thr Tyr Glu Lys Ile Phe 160 Phe Leu Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 129 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Vaccinia virus STRAIN: Copenhagen -248- (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID Met Phe Met Tyr Pro Glu Phe Ala Arg Lys Ala Leu Ser Lys Leu Ile 1 5 10 Ser Lys Lys Leu Asn Ile Glu Lys Val Ser Ser Lys His Gin Leu Val 25 Leu Leu Asp Tyr Gly Leu His Gly Leu Leu Pro Lys Ser Leu Tyr Leu 40 Glu Ala Ile Asn Ser Asp Ile Leu Asn Val Arg Phe Phe Pro Pro Glu 55 Ile Ile Asn Val Thr Asp Ile Val Lys Ala Leu Gin Asn Ser Cys Arg 65 70 75 e Val Asp Glu Tyr Leu Lys Ala Val Ser Leu Tyr His Lys Asn Ser Leu 85 90 Met Val Ser Gly Pro Asn Val Val Lys Leu Met Ile Glu Tyr Asn Leu 100 105 110 Leu Thr His Ser Asp Leu Glu Trp Leu Ile Asn Glu Asn Val Val Lys 115 120 125 Ala INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 132 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Pro Ser Tyr Met Tyr Pro Lys Asn Ala Arg Lys Val Ile Ser Lys 1 5 10 -249- Ile Ile Ser Leu Gin Leu Asp Ile Lys Lys Leu Pro Lys Lys Tyr Ile 25 Asn Thr Met Leu Glu Phe Gly Leu His Gly Asn Leu Pro Ala Cys Met 40 Tyr Lys Asp Ala Val Ser Tyr Asp Ile Asn Asn Ile Arg Phe Leu Pro 55 Tyr Asn Cys Val Met Val Lys Asp Leu Ile Asn Val Ile Lys Ser Ser 70 75 Ser Val Ile Asp Thr Arg Leu His Gin Ser Val Leu Lys His Arg Arg 90 Ala Leu Ile Asp Tyr Gly Asp Gin Asp Ile Ile Thr Leu Met Ile Ile 100 105 110 Asn Lys Leu Leu Ser Ile Asp Asp Ile Ser Tyr Ile Leu Asp Lys Lys 115 120 125 Ile Ile His Val 130 S* INFORMATION FOR SEQ ID NO:7: o SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: ORGANISM: Vaccinia virus STRAIN: Copenhagen (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi' SEQUENCE DESCRIPTION: SEQ ID NO:7: .'al Leu Asn Asp Gin Tyr Ala Lys Ile Val Ile Phe Phe Asn Thr Ile 1 5 10 Ile Glu Tyr Ile Ile Ala Thr Ile Tyr Tyr Arg Leu Thr Val Leu Asn 25 Asn Tyr Thr Asn Val Lys His Phe Val Ser Lys Val Leu His Thr Val 40 Met Glu Ala Cys Gly Val Leu Phe Ser Tyr Ile Lys Val Asn Asp Lys 55 Ile Glu His Glu Leu Glu Glu Met Val Asp Lys Gly Thr Val Pro Ser 70 75 -250- Tyr Leu Tyr His Leu Ser Ile Asn Val Ile Ser Ile Ile Leu Asp Asp 90 Ile Asn Gly Thr Arg 100 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 100 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ser Leu Asn Glu Tyr Tyr Ser Lys Ile Val Ile Leu Ile Asn Val Ile 1 5 10 Leu Glu Tyr Met Ile Ser Ile Ile Leu Tyr Arg Ile Leu Ile Val Lys 25 Arg Phe Asn Asn Ile Lys Glu Phe Ile Ser Lys Val Val Asn Thr Val 35 40 Leu Glu Ser Ser Gly Ile Tyr Phe Cys Gin Met Arg Val His Glu Gin 55 Ile Glu Leu Glu Ile Asp Glu Leu Ile Ile Asn Gly Ser Met Pro Val 70 75 Gin Leu Met His Leu Leu Leu Lys Val Ala Thr Ile Ile Leu Glu Glu 90 Ile Lys Glu Ile 100 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 102 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO -251- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 520-17.5 (Junction A) PUBLICATION INFORMATION: AUTHORS: Ferrari, Franco A Trach, Kathleen Hoch, James A TITLE: Sequence Analysis of the spoOB Locus Revels a Polycistronic Transcription Unit JOURNAL: J. Bacteriol.

VOLUME: 161 ISSUE: 2 PAGES: 556-562 DATE: Feb.-1985 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CACATACGAT TTAGGTGACA CTATAGAATA CAAGCTTTAT ACCATTATAG ATACATTACC TTGTCCGACG TGTAGAATTC ATGCCAAAGA AGAATTAACT AA 102 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 102 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 520-17.5 (Junction B) (ix) FEATURE: NAME/KEY: CDS LOCATION: 85..99 OTHER INFORMATION: /codon start= /function= "Translational start of hybrid protein" /product= "N-terminal peptide" /number= 1 /standard_name= "Translation of synthetic DNA sequence" (ix) FEATURE: NAME/KEY: CDS LOCATION: 100..102 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /partial /codon start= 100 /function= "marker enzyme" /product= "Beta-Galactosidase" /evidence= EXPERIMENTAL -252- /gene= "lacZ" /number= 2 /citation= PUBLICATION INFORMATION: AUTHORS: Ferrari, Franco A Trach, Kathleen Hoch, James A TITLE: Seqquence Analysis of the spoOB Locus Reveals a Polycistronic Transcription Unit JOURNAL: J. Bacteriol.

VOLUME: 161 ISSUE: 2 PAGES: 556-562 DATE: Feb.-1985 (xi) SEQUENCE DESCRIPTION: SEQ ID GTAGTCGACT CTAGAAAAAA TTGAAAAACT ATTCTAATTT ATTGCACGGA GATCTTTTTT TTTTTTTTTT TTTTTGGCAT ATAA ATG AAT TCG GAT CCC GTC 102 Met Asn Ser Asp Pro Val SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Asn Ser Asp Pro 1 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 1 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Val 1 5 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 103 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (iv) ANTI-SENSE: NO -253- (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 520-17.5 (Junction C) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..72 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /partial /codon start= 1 /function= "marker enzyme" /product= "Beta-galactosidase" /evidence= EXPERIMENTAL /gene= "lacZ" /number= 1 /citation= (ix) FEATURE: NAME/KEY: CDS LOCATION: 73..78 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /codon start= 73 /function= "Translational finish of hybrid protein" /product= "C-terminal peptide" /evidence= EXPERIMENTAL /number= 2 S /standardname= "Translation of synthetic DNA sequence" PUBLICATION INFORMATION: AUTHORS: Ferrari, Franco A Trach, Kathleen °Hoch, James A TITLE: Seqquence Analysis of the spoOB Locus Reveals a Polycistronic Transcription Unit JOURNAL: J. Bacteriol.

VOLUME: 161 S(E) ISSUE: 2 PAGES: 556-562 DATE: Feb.-1985 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AGC CCG TCA GTA TCG GCG GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT 48 Ser Pro Ser Val Ser Ala Glu Ile Gln Leu Ser Ala Gly Arg Tyr His 1 5 10 TAC CAG TTG GTC TGG TGT CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG 98 Tyr Gin Leu Val Trp Cys Gln Lys Asp Pro AAGAC 103 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: -254- Ser Pro Ser Val Ser Ala Glu Ile Gin Leu Ser Ala Gly Arg Tyr His 1 5 10 Tyr Gin Leu Val Trp Cys Gin Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Pro 1 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 520-17.5 (Junction D) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AGATCCCCGG GCGAGCTCGA ATTCGTAATC ATGGTCATAG CTGTTTCC 48 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.26 (Junction A) -255- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CACATACGAT TTAGGTGACA CTATAGAATA CAAGCTTTAT ACCATTATAG ATACATT 57 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 102 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.16 (Junction B) (ix) FEATURE: NAME/KEY: CDS LOCATION: 91..102 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /partial /codon start= 91 /function= "marker enzyme" /product= "Beta-Galactosidase" /evidence=

EXPERIMENTAL

/gene= "lacZ" /number= 2 /citation= (ix) FEATURE: NAME/KEY: CDS LOCATION: 76..90 OTHER INFORMATION: /partial /codon start= 76 /function= "Translational start of hybrid protein" /product= "N-terminal peptide" /number= 1 /standard_name= "Translation of synthetic DNA sequence" PUBLICATION

INFORMATION:

AUTHORS: Ferrari, Franco A Trach, Kathleen Hoch, James A TITLE: Seqquence Analysis of the spoOB Locus Reveals a Polycistronic Transcription Unit JOURNAL: J. Bacteriol.

VOLUME: 161 ISSUE: 2 PAGES: 556-562 DATE: Feb.-1985 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: AAGCTGGTAG ATTTCCATGT AGGGCCGCCT GCAGGTCGAC TCTAGAATTT CATTTTGTTT TTTTCTATGC TATAA ATG AAT TCG GAT CCC GTC GTT TTA CAA 102 Met Asn Ser Asp Pro Val Val Leu Gln 1 -256- INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met Asn Ser Asp Pro 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid o TOPOLOGY: linear 94 (ii) MOLECULE TYPE: peptide *8 9 (xi) SEQUENCE DESCRIPTION: SEQ ID Val Val Leu Gin 1 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 206 base pairs TYPE: nucleic acid o STRANDEDNESS: double TOPOLOGY: circular *see (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.16 (Junction C) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /partial /codon start= 1 /function= "marker enzyme" /product= "Beta-galactosidase" /evidence= EXPERIMENTAL /number= 1 /citation= (ix) FEATURE: NAME/KEY: CDS LOCATION: 64..69 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /codon start= 64 /function= "Translational finish of hybrid protein" -257- /product= "C-terminal peptide" /evidence= EXPERIMENTAL /standard_name= "Translation of synthetic DNA sequence" (ix) FEATURE: NAME/KEY: CDS LOCATION: 177..185 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /codonstart= 177 /function= "Translational start of hybrid protein" /product= "N-terminal peptide" /evidence= EXPERIMENTAL /standardname= "Translation of synthetic DNA sequence" (ix) FEATURE: NAME/KEY: CDS LOCATION: 186..206 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /partial /codon_start= 186 **/function= "glycoprotein" /product= "PRV /evidence= EXPERIMENTAL /gene= /number= 3 /citation= PUBLICATION INFORMATION: AUTHORS: Ferrari, Franco A Trach, Kathleen Hoch, James A TITLE: Seqquence Analysis of the spoOB Locus Reveals a Polycistronic Transcription Unit JOURNAL: J. Bacteriol.

VOLUME: 161 "oo ISSUE: 2 PAGES: 556-562 DATE: Feb.-1985 PUBLICATION INFORMATION: AUTHORS: Petrovskis, Erik A Timmins, James G Armentrout, Marty A Marchioli, Carmine C Jr. Yancy, Robert J Post, Leonard E TITLE: DNA Sequence of the Gene for Pseudorabies Virus gp50, a Glycoprotein without N-Linked Glycosylation JOURNAL: J. Virol.

VOLUME: 59 ISSUE: 2 PAGES: 216-223 DATE: Aug.-1986 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GTA TCG GCG GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG 48 Val Ser Ala Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu 1 5 10 GTC TGG TGT CAA AAA GAT CCA TAATTAATTA ACCCGGCCGC CTGCAGGTCG 99 Val Trp Cys Gln Lys Asp Pro ACTCTAGAAA AAATTGAAAA ACTATTCTAA TTTATTGCAC GGAGATCTTT TTTTTTTTTT -258- TTTTTTTTGG CATATAA ATG AAT TCG CTC GCA GCG CTA TTG GCG GCG Met Asn Ser Leu Ala Ala Leu Leu Ala Ala 1 1 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Val Ser Ala Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu 1 5 10 Val Trp Cys Gin Lys 206

I

r r r

J

INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 2 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Asp Pro 1 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 3 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Met Asn Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Ala Ala Leu Leu Ala Ala 1 -259- INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 101 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.16 (Junction D) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..15 OTHER INFORMATION: /partial *./codon start= 1 /function= "glycoprotein" /product= "PRV gp63" /gene= "gp63" /number= 1 /citation= PUBLICATION INFORMATION: AUTHORS: Petrovskis, Erik A Timmins, James G Post, Lenoard E TITLE: Use of Lambda-gtll To Isolate Genes for two Pseudorabies Virus Glycoproteins with homology to Herpes Simplex Virus and Varicella-Zoster Virus Glycoproteins JOURNAL: J. Virol.

VOLUME: ISSUE: 1 PAGES: 185-193 DATE: Oct.-1986 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CGC GTG CAC CAC GAG GGACTCTAGA GGATCCATAA TTAATTAATT AATTTTTATC Arg Val His His Glu 1 CCGGGTCGAC CTGCAGGCGG CCGGGTCGAC CTGCAGGCGG CCAGAC 101 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Arg Val His His Glu -260- INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.16 (Junction E) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ***AGATCCCCGG GCGAGCTCGA ATTCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAA 57 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 1907 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Newcastle disease virus STRAIN: Bl (vii) IMMEDIATE SOURCE: CLONE: 137-23.803 (PSY1142) (viii) POSITION IN GENOME: MAP POSITION: UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 92..1822 OTHER INFORMATION: /codon start= 92 /product= "NDV heamagglutinin-Neuraminidase" /gene= "HN" /number= 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ACGGGTAGAA CGGTAAGAGA GGCCGCCCCT CAATTGCGAG CCAGACTTCA CAACCTCCGT TCTACCGCTT CACCGACAAC AGTCCTCAAT C ATG GAC CGC GCC GTT AGC CAA 112 Met Asp Arg Ala Val Ser Gln 1 -261- GTT GCG TTA Val Ala Leu GAG AAT GAT GAA Glu Asn Asp Glu AGA GAG GCA AAA AAT ACA TGG CGC TTG Arg Glu Ala Lys Asn Thr Trp Arg Leu 15 TTC TTA ACA GTA GTG ACC TTG GCT ATA Phe Leu Thr Val Val Thr Leu Ala Ile ATA TTC Ile Phe CGG ATT GCA ATC Arg Ile Ala Ile

TTA

Leu 30

TCT

Ser GTA GCC TCC CTT Val Ala Ser Leu TAT AGC ATG GGG Tyr Ser Met Gly

GCT

Ala AGC ACA CCT AGC Ser Thr Pro Ser CTT GTA GGC ATA Leu Val Gly Ile

CCG

Pro ACT AGG ATT TCC Thr Arg Ile Ser GCA GAA GAA AAG Ala Glu Giu Lys ATT ACA Ile Thr TCT ACA CTT Ser Thr Leu GTG GCC CTT Val Ala Leu GGT TCC AAT CAA GAT Gly Ser Asn Gin Asp GAG TCT CCA TTG GCA Glu Ser Pro Leu Ala

GTA

Val 80 GTA GAT AGG ATA Val Asp Arg Ile TAT AAG CAA Tyr Lys Gin ACC ACA ATT Thr Thr Ile TTG TTA AAT ACT Leu Leu Asn Thr

GAG

Glu 100 ATG AAC Met Asn 105 GCA ATA ACA TCT Ala Ile Thr Ser

CTC

Leu 110 TCT TAT CAG ATT Ser Tyr Gin Ile

AAT

Asn 115 GGA GCT GCA AAC Gly Ala Ala Asn

AAC

Asn 120 AGC GGG TGG GGG Ser Gly Trp Gly CCT ATT CAT GAC Pro Ile His Asp

CCA

Pro 130 GAT TAT ATA GGG Asp Tyr Ile Gly

GGG

Gly 135 ATA GGC AAA GAA Ile Gly Lys Glu

CTC

Leu 140 ATT GTA GAT GAT Ile Val Asp Asp

GCT

Ala 145 AGT GAT GTC ACA Ser Asp Val Thr TCA TTC Ser Phe 150 544 TAT CCC TCT Tyr Pro Ser

GCA

Ala 155 TTT CAA GAA CAT Phe Gin Glu His

CTG

Leu 160 AAT TTT ATC CCG Asn Phe Ile Pro GCG CCT ACT Ala Pro Thr 165 AGT GCT ACC Ser Ala Thr r h ACA GGA TCA GGT TGC ACT CGA Thr Gly Ser Giy Cys Thr Arg 170 CCC TCA TTT GAC Pro Ser Phe Asp

ATG

Met 180 640 CAT TAC His Tyr 185 TGC TAC ACC CAT Cys Tyr Thr His

AAT

Asn 190 GTA ATA TTG TCT Val Ile Leu Ser

GGA

Gly 195 TGC AGA GAT CAC Cys Arg Asp His

TCA

Ser 200 CAC TCA CAT CAG His Ser His Gin

TAT

Tyr 205 TTA GCA CTT GGT Leu Ala Leu Gly

GTG

Val 210 CTC CGG ACA TCT Leu Arg Thr Ser ACA GGG AGG GTA Thr Gly Arg Val TTT TCT ACT CTG Phe Ser Thr Leu

CGT

Arg 225 TCC ATC AAC CTG Ser Ile Asn Leu GAC GAC Asp Asp 230 ACC CAA AAT Thr Gin Asn

CGG

Arg 235 AAG TCT TGC AGT Lys Ser Cys Ser AGT GCA ACT CCC Ser Ala Thr Pro CTG GGT TGT Leu Gly Cys 245 GAT TAT AAC Asp Tyr Asn 832 880 GAT ATG CTG TGC TCG AAA GCC ACG Asp Met Leu Cys Ser Lys Ala Thr 250 255 GAG ACA GAG GAA Glu Thr Glu Glu

GAA

Glu 260 -262- TCA GCT GTC CCT ACG CGG ATG GTA

S

Sex

CAA

Gin 280

GTG

Val2

GTG

Val

ACT

Thr

TGC

Cys

AAG

Lys 360

ATC

Ile

CCC

Pro

GGG

Gly

GCG

Al a

AGT

Ser 440 CAr, G-'-n

GAT

Asp

TTC

Phe Al a 265

TAT

Tyr

GCC

Al a

TGG

Trp

GTA

Val

CCA

Pro 345

CCT

Pro

AAA

Lys

AAC

Asn

ACA

Thr

TTA

Leu 425

CCT

Pro

GCT

Al a

CCA

Pro

GGG

Gly Val

CAC

His

AAC

Asn

TTC

Phe

CAG

Gin 330

GAT

Asp

GGA

Gly

GTG

Val

ACA

Thr

TCC

Ser 410

TTA

Leu

TAT

Tyr

TCA

Ser

TAT

Tyr

ACA

Thr 490 Pro

GAA

Giu

TAC

Tyr

TCA

Ser 315

GAA

Giu

GAG

Giu

CGG

Arg

TCA

Ser

GTC

Val 395

CAT

His

TAT

Tyr

ACA

Thr

GCA

Ala

CCC

Pro 475

ATG

Met Thr

AAG

Lys

CCA

Pro 300

GTC

Val

GGG

Gly

CAA

Gin

TTT

Phe

ACA

Thr 380

ACA

Thr

TTC

Phe

CCT

Pro

TTC

Phe

AGA

Arg 460

CTA

Leu

CTT

Leu Arg

GAC

Asp 285

GGA

Gly

TAC

Tyr

AAA

Lys

GAC

Asp

GGT

Gly 365

TCC

Ser

CTC

Leu

TTG

Leu

ATG

Met

AAT

Asn 445

TGC

Cys

ATC

Ile

GAT

Asp Met 270

CTA

Leu

GTA

Val

GGA

Gly

TAT

Tyr

TAC

Tyr 350

GGG

Gly

TTA

Leu

ATG

Met

TAT

Tyr

ACA

Thr 430

GCC

Al a

CCC

Pro

TTC

Phe

GGT

Gly Val

GAT

Asp

GGG

Gly

GGG

Gly

GTG

Val 335

CAG

Gin

AAA

Lys

GGC

Gly

GGG

Gly

CAG

Gin 415

GTC

Val

TTC

Phe

AAC

Asn

TAT

Tyr

GAA

Giu 495

CAT

His

GTC

Val

GGT

Gly

TTA

Leu 320

ATA

Ile

ATT

Ile

CGC

Arg

GAA

Glu

GCC

Ala 400

CGA

Arg

AGC

Ser

ACT

Thr

TCA

Sex

AGA

Arg 480

CAA

Gin Gly Arg Leu 275 ACA ACA TTA Thr Thr Leu 290 GGA TCT TTT Giy Ser Phe 305 AAA CCC AAT Lys Pro Asn TAC AAG CGA Tyr Lys Arg CGA ATG GCC Arg Met Ala Gly Phe Asp Giy

ATA

Ile

GAC

Asp 385

GAA

Giu

GGG

Giy

AAC

Asn

CGG

Arg

TGT

Cys 465

AAC

Asn

GCA

Ala

CAG

Gin 370

CCG

Pro

GGC

Giy

TCA

Ser

AAA

Lys

CCA

Pro 450

GTT

Val

CAC

His

AGA

Arg 355

CAG

Gin

GTA

Val1

AGA

Arg

TCA

Sex

ACA

Thr 435

GGT

Gly

ACT

Thr

ACC

Thr

CTT

Leu

TTC

Phe Ile

ACA

Thr

TAC

Tyr 340

AAG

Lys

GCT

Ala

CTG

Leu

ATT

Ile

TAC

Tyr 420

GCC

Ala

AGT

Ser

GGA

Giy

TTG

Leu

AAC

Asn 500 GGG AGG TTA GGG TTC GAC GGC

GGG

Gly

GAC

Asp

CCC

Pro 325

AAT

Asn

TCT

Ser

ATC

Ile

ACT

Thr

CTC

Leu 405

TTC

Phe

ACT

Thr

ATC

Ile

GTC

Vai

CGA

Arg 485

CCT

Pro

GAC

Asp

AGC

Sex 310

AGT

Sex

GAC

Asp

TCG

Sex

TTA

Leu

GTA

Val 390

ACA

Thr

TCT

Sex

CTT

Leu

CCT

Pro

TAT

Tyr 470

GGG

Gly

GCG

Al a

TGG

Trp 295

CGC

Arg

GAC

Asp

ACA

Thr

TAT

Tyr

TCT

Ser 375

CCG

Pro

GTA

Vai

CCC

Pro

CAT

His

TGC

Cys 455

ACA

Thx

GTA

Val

TCT

Ser 928 976 1024 1072 1120 1168 1216 1264 1312 1360 1408 1456 1504 1552 1600 GCA GTA TTC GAT AGC ACA TCC CGC AGT CGC ATA ACT CGA GTG AGT TCA 1648 Ala Val 505 Phe Asp Ser Thx Ser Arg Ser Axg Ile Thr 515 Arg Val Ser Ser -263- AGC AGC ATC AAA GCA GCA TAC ACA ACA TCA ACT TGT TTT AAA GTG GTC 1696 Ser Ser Ile Lys Ala Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val Val 520 525 530 535 AAG ACC AAT AAG ACC TAT TGT CTC AGC ATT GCT GAA ATA TCT AAT ACT 1744 Lys Thr Asn Lys Thr Tyr Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr 540 545 550 CTC TTC GGA GAA TTC AGA ATC GTC CCG TTA CTA GTT GAG ATC CTC AAA 1792 Leu Phe Gly Glu Phe Arg Ile Val Pro Leu Leu Val Glu Ile Leu Lys 555 560 565 GAT GAC GGG GTT AGA GAA GCC AGG TCT GGC TAGTTGAGTC AACTATGAAA 1842 Asp Asp Gly Val Arg Glu Ala Arg Ser Gly 570 575 GAGTTGGAAA GATGGCATTG TATCACCTAT CTTCTGCGAC ATCAAGAATC AAACCGAATG 1902 CCGGC 1907 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 577 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asp Arg Ala Val Ser Gin Val Ala Leu Glu Asn Asp Glu Arg Glu 1 5 10 Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu 25 Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser Met 35 40 Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser 55 Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn Gin Asp Val 70 75 Val Asp Arg Ile Tyr Lys Gin Val Ala Leu Glu Ser Pro Leu Ala Leu 90 9C Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser Leu £er Tyr 100 105 110 Gin Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro Ile His 115 120 125 Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp 130 135 140 Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gln Glu His Leu 145 150 155 160 Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro 165 170 175 -264- Ser Phe Asp Met Ser Ala Thr His r r r r r Leu Gly Arg 225 Ser Thr Gly Thr Gly 305 Lys Tyr Arg Ile Asp 385 Glu Gly Asn Arg Cys 465 Asn Ala Ser Gly 195 Val Leu 210 Ser Ile Ala Thr Glu Glu Arg Leu 275 Thr Leu 290 Ser Phe Pro Asn Lys Arg Met Ala 355 Gin Gin 370 Pro Val Gly Arg Ser Ser Lys Thr 435 Pro Gly 450 Val Thr His Thr Arg Leu Cys Arg Asn Pro Glu 260 Gly Phe Ile Thr Tyr 340 Lys Ala Leu Ile Tyr 420 Ala Ser Gly Leu Asn 500 Arg Asp Thr Ser Leu Asp 230 Leu Gly 245 Asp Tyr Phe Asp Gly Asp Asp Ser 310 Pro Ser 325 Asn Asp Ser Ser Ile Leu Thr Val 390 Leu Thr 405 Phe Ser Thr Leu Ile Pro Val Tyr 470 Arg Gly 485 Pro Ala His Ala 215 Asp Cys Asn Gly Trp 295 Arg Asp Thr Tyr Ser 3.75 Pro Val Pro His Cys 455 Thr Val Ser Ser 200 Thr Thr Asp Ser Gin 280 Val Val Thr Cys Lys 360 Ile Pro Gly Ala Ser 440 Gin Asp Phe Ala Tyr 185 His Gly Gin Met Ala 265 Tyr Ala Trp Val Pro 345 Pro Lys Asn Thr Leu 425 Pro Ala Pro Gly Val 505 Cys Tyr Thr His Asn Val Ile 190 Ser Arg Asn Leu 250 Val His Asn Phe Gin 330 Asp Gly Val Thr Ser 410 Leu Tyr Ser Tyr Thr 490 Phe His Val Arg 235 Cys Pro Glu Tyr Ser 315 Glu Glu Arg Ser Val 395 His Tyr Thr Ala Pro 475 Met Asp Gin Phe 220 Lys Ser Thr Lys Pro 300 Val Gly Gin Phe Thr 380 Thr Phe Pro Phe Arg 460 Leu Leu Ser Tyr 205 Phe Ser Lys Arg Asp 285 Gly Tyr Lys Asp Gly 365 Ser Leu Leu Met Asn 445 Cys Ile Asp Thr Leu Ala Ser Thr Cys Ser Ala Thr 255 Met Val 270 Leu Asp Val Gly Gly Gly Tyr Val 335 Tyr Gin 350 Gly Lys Leu Gly Met Gly Tyr Gin 415 Thr Val 430 Ala Phe Pro Asn Phe Tyr Gly Glu 495 Ser Arg 510 Leu Leu Val 240 Glu His Val Gly Leu 320 Ile Ile Arg Glu Ala 400 Arg Ser Thr Ser Arg 480 Gin Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala Tyr Thr Thr 515 520 -265- Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser 530 535 540 Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro 545 550 555 560 Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg Glu Ala Arg Ser 565 570 575 Gly INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.26 (Junction A) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CACATACGAT TTAGGTGACA CTATAGAATA CAAGCTTTAT ACCATTATAG ATACATT 57 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 108 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.26 (Junction B) (ix) FEATURE: NAME/KEY: exon LOCATION: 88..102 OTHER INFORMATION: /codon start= 88 /function= "Translational start of hybrid protein" /product= "N-terminal peptide" /number= 1 /standardname= "Translation of synthetic DNA sequence" -266- (ix) FEATURE: NAME/KEY: CDS LOCATION: 103..108 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /partial /codon start= 103 /product= "NDV Heamagglutinin-Neuraminidase" /evidence= EXPERIMENTAL /gene= "HN" /number= 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CATGTAGTCG ACTCTAGAAA AAATTGAAAA ACTATTCTAA TTTATTGCAC GGAGATCTTT TTTTTTTTTT TTTTTTTTGG CATATAAATG AATTCGGATC CG GAC CGC 108 Asp Arg 1 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 2 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Asp Arg 1 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 108 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.26 (Junction C) (ix) FEATURE: NAME/KEY: CDS LOCATION: 70..84 OTHER INFORMATION: /codon start= /function= "Translational start of hybrid protein" /product= "N-terminal peptide" /number= 1 /standard name= "Translation of synthetic DNA sequence" -267- (ix) FEATURE: NAME/KEY: CDS LOCATION: 85..108 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /partial /codonstart= /function= "marker enzyme" /product= "Beta-galactosidase" /evidence= EXPERIMENTAL /gene= "lacZ" /number= 2 /citation= PUBLICATION INFORMATION: AUTHORS: Ferrari, Franco A Trach, Kathleen Hoch, James A TITLE: Sequence Analysis of the spoOB Locus Reveals a Polycistronic Transcription Unit JOURNAL: J. Bacteriol.

VOLUME: 161 ISSUE: 2 PAGES: 556-562 DATE: Feb.-1985 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: TGCGACATCA AGAATCAAAC CGAATGCCCT CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAA ATG AAT TCG GAT CCC GTC GTT TTA CAA CGT CGT GAC TGG 108 Met Asn Ser Asp Pro Val Val Leu Gln Arg Arg Asp Trp 1 5 INFORMATION FOR SEQ ID S(i) SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asn Ser Asp Pro 1 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Val Val Leu Gln Arg Arg Asp Trp 1 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 108 base pairs -268- TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.26 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..54 IDENTIFICATION METHOD: experimental S* OTHER INFORMATION: /partial /codon_start= 1 /function= "marker enzyme" /product= "Beta-galactosidase" /evidence= EXPERIMENTAL /gene= "lacZ" /number= 1 /citation= (ix) FEATURE: NAME/KEY: CDS LOCATION: 55..63 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /codon start= /function= "Translational finish of hybrid protein" /product= "C-terminal peptide" /evidence= EXPERIMENTAL /number= 2 /standard_name= "Translation of synthetic DNA sequence" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG AGGGTCGAAG ACCAAATTCT 100 Gin Lys Asp Pro AACATGGT 108 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: -269- Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 Gin Lys INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 2 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Asp Pro 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular ses (ii) MOLECULE TYPE: DNA (genomic)

S

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Plasmid (vii) IMMEDIATE SOURCE: CLONE: 538-46.26 (Junction E) e (xi) SEQUENCE DESCRIPTION: SEQ ID AGATCCCCGG GCGAGCTCGA ATTCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAA 57 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (vi) ORIGINAL SOURCE: ORGANISM: Pseudorabies virus Synthetic oligonucleotide primer -270- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: CGCGAATTCG CTCGCAGCGC TATTGGC 27 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (vi) ORIGINAL SOURCE: ORGANISM: Pseudorabies virus Synthetic oligonucleotide primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GTAGGAGTGG CTGCTGAAG 19 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: AAAAATTGAA AAACTATTCT AATTTATTGC ACGGAGATCT TTTTTTTTTT TTTTTTTTTG GCATATAAAT INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 74 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -271- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: TTTTTTTTTT TTTTTTTTTT GGCATATAAA TAGATCTGTA TCCTAAAATT GAATTGTAAT S* TATCGATAAT AAAT 74 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

S*

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID GTATCCTAAA ATTGAATTGT AATTATCGAT AATAAAT 37 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -272- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAAA T 41 INFORMATION FOR SEQ ID NO:47: S* SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO D* (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CACATACGAT TTAGGTGACA CTATAGAATA CAAGCTTTGA GTCTATTGGT TATTTATACG INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 123 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO -273- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 100..123 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: TGAATATATA GCAAATAAAG GAAAAATTGT TATCGTTGCT GCATTAGATG GAACATAGGT **CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAA ATG AAT TCG GAT CCC 114 Met Asn Ser Asp Pro 1 GTC GTT TTA 123 Val Val Leu INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 132 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 -274- (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (xi) SEQUENCE DESCRIPTION: SEQ ID GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG ACCTATGAAC GTAAACCATT 100 Gin Lys Asp Pro TGGTAATATT CTTAATCTTA TACCATTATC GG 132 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids S* TYPE: amino acid S(D) TOPOLOGY: linear (ii) MOLECULE"TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 Gin Lys Asp Pro INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G -275- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: TCTACTATTG TATATATAGG ATdCCCGGGC GAGCTCGAAT TCGTAATCAT GGTCATAGCT GTTTCC 66 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 104 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS -276- LOCATION: 81..104 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: AAATATATAA ATACCATGTT AGAATTTGGT CTGCTGCAGG TCGACTCTAG AATTTCATTT TGTTTTTTTC TATGCTATAA ATG AAT TCG GAT CCC GTC GTT TTA 104 Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 150 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (ix) FEATURE: NAME/KEY: CDS LOCATION: 130..150 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 -277- CAA AAA GAT CCA TAATTAATTA ACCCGGTCGA CTCTAGAAAG ATCTGTATCC 100 Gin Lys Asp Pro TAAAATTGAA TTGTAATTAT CGATAATAA ATG AAT TCC GGC ATG GCC TCG 150 Met Asn Ser Gly Met Ala Ser 1 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 10 Gin Lys Asp Pro S20 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Met Asn Ser Gly Met Ala Ser 1 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 109 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G -278- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CCATGCTCTA GAGGATCCCC GGGCGAGCTC GAATTCGGAT CCATAATTAA TTAATTAATT TTTATCCCGG GTCGACCGGG TCGACCTGCA GCCTACATGG AAATCTACC 109 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza e* INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: -279- ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 104 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 81..104 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: AAATATATAA ATACCATGTT AGAATTTGGT CTGCTGCAGG TCGACTCTAG AATTTCATTT TGTTTTTTTC TATGCTATAA ATG AAT TCG GAT CCC GTC GTT TTA 104 Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 182 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -280- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (ix) FEATURE: NAME/KEY: CDS LOCATION: 156..182

C

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 10 CAA AAA GAT CCA TAATTAATTA ACCCGGTCGA CTCTAGAAAA AATTGAAAAA 100 t Gin Lys Asp Pro CTATTCTAAT TTATTGCACG GAGATCTTTT TTTTTTTTTT TTTTTTGGCA TATAA ATG 158 Met *ge 1 AAT TCC GGC ATG GCC TCG CTC GCG Asn Ser Gly Met Ala Ser Leu Ala 182 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 Gin Lys Asp Pro INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: -281- LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Met Asn Ser Gly Met Ala Ser Leu Ala 1 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 109 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CCATGCTCTA GAGGATCCCC GGGCGAGCTC GAATTCGGAT CCATAATTAA TTAATTAATT TTTATCCCGG GTCGACCGGG TCGACCTGCA GCCTACATGG AAATCTACC 109 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE

SOURCE:

-282- CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 104 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G -283- (ix) FEATURE: NAME/KEY: CDS LOCATION: 81..104 (xi) SEQUENCE DESCRIPTION: SEQ ID AAATATATAA ATACCATGTT AGAATTTGGT CTGCTGCAGG TCGACTCTAG AATTTCATTT TGTTTTTTTC TATGCTATAA ATG AAT TCG GAT CCC GTC GTT TTA 104 Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 180 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (ix) FEATURE: NAME/KEY: CDS LOCATION: 160..180 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: -284- GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 CAA AAA GAT CCA TAATTAATTA ACCCGGTCGA CTCTAGATTT TTTTTTTTTT 100 Gin Lys Asp Pro TTTTTTTGGC ATATAAATAG ATCTGTATCC TAAAATTGAA TTGTAATTAT CGATAATAA 159 ATG AAT TCC GGC ATG GCC TCG 180 Met Asn Ser Gly Met Ala Ser 1 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys S1 5 10 Gin Lys Asp Pro INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: Met Asn Ser Gly Met Ala Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 109 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 -285- (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID CCATGCTCTA GAGGATCCCC GGGCGAGCTC GAATTCGGAT CCATAATTAA TTAATTAATT TTTATCCCGG GTCGACCGGG TCGACCTGCA GCCTACATGG AAATCTACC 109 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL

SOURCE:

ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE

SOURCE:

CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS:

%G

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 -286- (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 117 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO S(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 94..117 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: GGTCTGCTGC AGGTCGACTC TAGAAAAAAT TGAAAAACTA TTCTAATTTA TTGCACGGAG ATCTTTTTTT TTTTTTTTTT TTTTGGCATA TAA ATG AAT TCC GGC TTC AGT AAC ATA 117 Met Asn Ser Gly Phe Ser Asn Ile 1 5 8 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: Met Asn Ser Gly Phe Ser Asn Ile 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: -287- LENGTH: 126 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 103..126 (xi) SEQUENCE DESCRIPTION: SEQ ID CGCAACATAC CTAACTGCTT CATTTCTGAT CCATAATTAA TTAATTTTTA TCCCGGCGCG CCTCGACTCT AGAATTTCAT TTTGTTTTTT TCTATGCTAT AA ATG AAT TCG GAT 114 Met Asn Ser Asp CCC GTC GTT TTA Pro Val Val Leu 126 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

-288- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 *Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG ACCTGCAGCC TACATG 96 Gin Lys Asp Pro INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 Gin Lys Asp Pro INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 -289- (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 124 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear Uii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 -290- UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 104..124 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAA ATG AAT TCG CTA CTT 113 Met Asn Ser Leu Leu 1 GGA ACT 124 Gly Thr *0ee INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: Met Asn Ser Leu Leu Gly Thr 1 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 126 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..12 (ix) FEATURE: NAME/KEY: CDS LOCATION: 103..126 -291- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: ATA AAA ATG TGATTAAGTC TGAATGTGGA TCCATAATTA ATTAATTTTT 49 Ile Lys Met 1 ATCCCGGCGC GCCTCGACTC TAGAATTTCA TTTTGTTTTT TTCTATGCTA TAA ATG 105 Met 1 AAT TCG GAT CCC GTC GTT TTA 126 Asn Ser Asp Pro Val Val Leu INFORMATION FOR SEQ ID NO:89: SEQUENCE CHARACTERISTICS: LENGTH: 3 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: Ile Lys Met 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asn Ser Asp Pro Val Val Leu *1 INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 -292- (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG 100 Gin Lys Asp Pro CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 Gin Lys Asp Pro INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs S(B) TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: -293- TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 "i (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 124 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 104..124 (xi) SEQUENCE DESCRIPTION: SEQ ID GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA 6 -294- ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAA ATG AAT TCC CCT GCC 113 Met Asn Ser Pro Ala 1 GCC CGG 124 Ala Arg INFORMATION FOR SEQ ID NO:96: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: Met Asn Ser Pro Ala Ala Arg S(i) SEQUENCE CHARACTERISTICS: LENGTH: 126 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 "(vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NRAE/KEY: CDS LOCATION: 1..36 (ix) FEATURE: NAME/KEY: CDS LOCATION: 103..126 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: CTC CAG GAG CCC GCT CGC CTC GAG CGG GAT CCA TAATTAATTA ATTTTTATCC 53 Leu Gln Glu Pro Ala Arg Leu Glu Arg Asp Pro 1 5 -295- CGGCGCGCCT CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAA ATG AAT 108 Met Asn 1 TCG GAT CCC GTC GTT TTA 126 Ser Asp Pro Val Val Leu INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: Leu Gin Glu Pro Ala Arg Leu Glu Arg Asp Pro 1 5 INFORMATION FOR SEQ ID NO:99: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: "Met Asn Ser Asp Pro Val Val Leu 1 INFORMATION FOR SEQ ID NO:100: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL; NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G -296- (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG 100 Gin Lys Asp Pro CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:101: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: Glu Ile Gin Leu Ser Ala Gly Arg Tyr His Tyr Gin Leu Val Trp Cys 1 5 10 Gin Lys Asp Pro INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double *oo TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 -297- INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: CCGAATTCCG GCTTCAGTAA CATAGGATCG INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: GTACCCATAC TGGTCGTGGC INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs -298- TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: S. CCGGAATTCG CTACTTGGAA CTCTGG 26 INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) o* (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: CATTGTCCCG AGACGGACAG INFORMATION FOR SEQ ID NO:107: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -299- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: CGCGATCCAA CTATCGGTG 19 INFORMATION FOR SEQ ID NO:108: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: GCGGATCCAC ATTCAGACTT AATCAC 26 INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO -300- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: ATGAATTCCC CTGCCGCCCG GACCGGCACC INFORMATION FOR SEQ ID NO:110: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO o« (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 9*00 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: CATGGATCCC GCTCGAGGCG AGCGGGCTCC INFORMATION FOR SEQ ID NO:111: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza -301- INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: CTGGTTCGGC CCAGAATTCT ATGGGTCTCG CGCGGCTCGT GG 42 INFORMATION FOR SEQ ID NO:112: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) :(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: CTCGCTCGCC CAGGATCCCT AGCGGAGGAT GGACTTGAGT CG 42 INFORMATION FOR SEQ ID NO:113: SEQUENCE CHARACTERISTICS: LENGTH: 3628 base pairs TYPF: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 -302- (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (ix) FEATURE: NAME/KEY: CDS LOCATION: 57. .1226 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1362. .3395 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: TTGAAGATGA ATGCATAGAG GAAGATGATG TCGANACGTC ATTATTTAAT GTATAAATGG

ATAAATTGTA

ACTTTATAGA

CGTCCAATAA

TATTAGTTTT

ACCNACTCGT

NTANTGATAT

TAAAACGCAT

TTAAACTAGA

ATAATTACTT

CAGACATAGA

TCAGTGACGT

ATGATCATGA

GAAACCATGA

CTGATGATCT

ATACTATTTC

CGAAAATATA

TAACGATAGG

ATGTTGATAG

TAAAGGATAA

TTAATATAGA

ATCGCATTTA

GAAAATATAT

CGAAGAACGC

TTCCTAAAAA

GTATGTATAA

TGCGGCAATA

AGTTGTAAAA

TAATACATGG

TANTTTGTAT

TNAAATAGT

GGTCGNTGAA

AGATACACGA

AA.ATTATAAA

TGAACAAGAT

GAAAAAGATT

TAAAGTTCTC

ATATATTATA

TATTATACTA

TTTGAATATA

TAGAACACGT

CGTTATATTG

ATCATTTGTA

ATATTCAAAA

TGAAAAAATT

GTTATTAACT

GTTTTGCTGT

AAGCACTTAT

AAGAAAAGTA

ATATATAAAT

AGATGCCGTA

TTCGGCGTTT

TCTGTATTAA

ATATATATAT

GTAGAAGTTC

ATTAGTGCTA

NTAGTTNCCC

ATTCCTATTG

AATGGAATTT

GGTAAACTTA

TTATCGTTGA

GCATCTAGGT

CAATCTGATA

GATGAAAAAA

CTTCGTGAAA

CTATATGATT

AAAGATTTAA

TATACGAAAT

AGGTTCCATG

AATGTATCCA

GAATAAGTAT

ATGGTTATCA

TTTTGTTAGT

ATTTCAAAGA

ACCATGTTAG

TCATATGATA

TTATGACATC

CAGATACATC

TTCTAGCGAT

CTAAACCNAC

GAGCATTGGN

AAAAATNTC C

ATCTTATTAA

TATCCGTTCT

ATAGCAGTGA

TTCCTAGATG

TAANNAAAAG

AAATAGAGGA

GTTCTATTAA

GATTATTTAG

ATTTTACTAG

AGATTGCTGA

ATAGCATGTT

ACTCTTTTTA

GAGTTGTTGA

ATATAAATGA

TATACATTTT

ATAATAACAC

TTATATCATT

AATTTGGTCT

TAAATAATAT

TAAAGATGAT

ANCTAATCAT

ATTATTTGGT

TTANATGGAG

GGCATTTTTT

NCCAAAGAAG

TCAACAATTC

TATCAATAGT

TATTGATGAA

TTCTCCTCTT

TGCTAAATCA

ATTAATAAAT

AGACAGCATA

ATGTCCACAG

AGTGTCAAAG

TATACTCGGT

GATTAATTCA

TGAAGATATT

ATGCCTTATC

TTGTTTTTAT

TAAGGCCGTA

AATGCCGTCG

ACAACTTGAT

ACATGGAAAT

AAGATTTTTA

GATTTTAATA

ACAATATCGT

GTTATGGNAT

GAAGCAGATA

GTATCTAAAA

ANATCACAAA

GTTAAAAGAT

TTAGTCGAA

TTAGTGCTCA

TATATAGATA

TTTACGTTTA

AGTTTATCTA

TATATACTAT

GTTAAAGATA

AAAGAAGAAG

ATCGAAACAG

ATTTCGTCTA

GCGGAATTTA

GTACCTAATA

AATGTTTGTT

TATGATAAAT

TATATGTATC

ATTAAAAAAC

CTACCAGCTT

CCTTATAATT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 -303-

GTGTTATGGT

TACATCA.ATC

TCACTTTAAT

AAAAAATAAT

TGAAACTGAA

CATATTTATA

TCCACATACC

AATTGCTTAA

CAAATAGTTC

TCCCTAGTGT

ATGGGATTAA

TAGATGACTC

GAGATTTTGC

CGAAATGTGA

AAAATAATAA

TATCACTGTC

GAAATATATT

AAAACTGGAA

TCGTAATGGA

ATGATAAAAT

GTTCCGACTC

CCGATAATGA

TACATCCTAA

ATATAGGTAT

ATATTACGTT

TATATGAGTT

TGATTTTAAT

TCGTAAAAAG

AATCATCAGG

ATGAGCTCAT

CTACCATAAT

AAATACTTTT

TCTGATGCAA

TAAAGATTTA

TGTATTAAAA

GATCATTAAT

TCATGTAACA

TAAGATAGAG

TAATAATATG

CCCCACACAT

AAAATATCCG

ATTCATTCAC

AAACGATTTT

GTGTGTTGCT

AGATTACATA

GAATATGATT

AGATATTATA

GGTGGAGGAG

TGATATATCA

ATTATCTTCA

ATGTTATTCA

TATGGTTGAT

GAGTACGTTT

TATAACACAT

TCTTATGATA

TCTTATAAGC

TAATTTAAAA

ATCTTCATAC

AATACCGTTT

AAATGTTATT

ATTTAATAAC

CATATATTTT

TATTAATGGA

ATTAGAGGA-A

TTTTTTAAAC

GATTATTAAA

ATAAATGTTA

CATCGTAGAG

AAGTTACTAT

AAAATATTAA

CTTGTAGATG

ATCATTGATC

ATATCATTAC

AATAATAATA

ATACTTCATA

ATATCTACCG

ATGTTTTCGT

TTTATAGAAA

AGAGATAAGG

AGATATATAA

GTGTTGATAC

TCTTTAATGG

GCAACTATAA

TTGACAAATG

TATATATCTA

GAATATAAAC

CATATATATG

GTATTGTTCA

GTAA.AAGGAT

CTATATTCCA

GATATGAGTA

ACTA. .ATGTT

TTAGAATATA

ATTAAAGAAT

TGTCAGATGC

TCTATGCCTG

ATCAAAGAAA

AAGGGGTGCT

TAATCGTATG

TAAAATCATC

CGTTAATAGA

CGATAGATGA

AAATAGACCC

TAATAACATC

TCGATACATT

GTTCACTTAG

TTATTGATTA

TGATAATATC

TAGTTGATAA

ACGATATAAA

AAAATATATC

TTAAAAGAGA

AATTATTCAG

ATATTGATAA

ATCAATTTCG

AATCAAAACT

TATCAATGTA

CTAACATTCT

GAGATATTAA

AAACAACATC

ATCTAACTAG

GGGGTCCCCT

CCATGAATAT

ATAAATTAGT

GTTCACTCAA

TGATATCTAT

TTATTTCAAA

GTGTACATGA

TACAGCTTAT

TATAACGTAT

ACCTTGTCTA

AAAAAGTAGT

ATCTGTAATA

TTACGGCGAT

TATATCCTAT

TACAGTAGCC

AATACCTAAG

ATTATATTTA

AGATATAAAC

TATATCCGAT

AAATATGTTT

AGATCGACTT

CATGATCGAT

TATATACGAC

AAAGAATAGA

TAAAAATAGA

TGTATCTAAA

TTTAAATCCA

ATTAGCGTTA

TAAAAAAATA

TAAATACCAT

ATCATGTAAA

ATGTATAAAT

ATATTTAATG

TATTGGATTA

AAATGGGCTA

CTCTATTATT

TGAATATTAT

TATATTATAT

AGTCGTAAAT

ACAAATTGAA

GCATTTACTT

TTTTTCTTTT

ATTGTATCTT

AGATATAGTT

GATACTAGAT

CAAGACATTA

ATATTAGATA

AATTCAAACA

TCTTCCTATA

TCCGATGCAT

AGGATTATTG

AGCATAAAAT

CCTGCTATAA

ATTAATATGT

TTAGAGTCAT

GTTAAATGTA

ATATTAACTA

ATAAACGATG

AATAATAAAT

TGTACCATAA

CGGGCAGT;A

AAGGGTGTTA

AAACAATTAT

TGCTCGATAT

TATAALATCTA

CATGGGATGA

TTAACGGGTG

CGGTATGGAG

AATACACCCA

TCAAAAATTG

AGAATATTGA

ACTGTACTAG

TTGGAAATAG

CTAAAGGTAG

AAATAAATAA

GTATTTTGGA

TATATCGTTA

1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 4.

CTGGACATGA TATTATGTTT AGTTAATTCT TCTTTGGCAT GAATTCTACA CGTCGGANA -304- GGTAATGTAT CTATAATGGT ATAAAGCT 3628 INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: LENGTH: 389 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus S. STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: Met Asp Lys Leu Tyr Ala Ala Ile Phe Gly Val Phe Met Thr Ser Lys 1 5 10 Asp Asp Asp Phe Asn Asn Phe Ile Glu Val Val Lys Ser Val Leu Thr 25 Asp Thr Ser Xaa Asn His Thr Ile Ser Ser Ser Asn Asn Asn Thr Trp 35 40 Ile Tyr Ile Phe Leu Ala Ile Leu Phe Gly Val Met Xaa Leu Leu Val 50 55 Phe Xaa Leu Tyr Val Glu Val Pro Lys Pro Thr Xaa Met Glu Glu Ala 70 75 Asp Asn Xaa Leu Val Xaa Asn Ser Ile Ser Ala Arg Ala Leu Xaa Ala 90 Phe Phe Val Ser Lys Xaa Xaa Asp Met Val Xaa Glu Xaa Val Xaa Gin 100 105 110 Lys Xaa Pro Pro Lys Lys Xaa Ser Gin Iie Lys Arg Ile Asp Thr Arg 115 120 125 Ile Pro Ile Asp Leu Ile Asn Gin Gin Phe Val Lys Arg Phe Lys Leu 130 135 140 Glu Asn Tyr Lys Asn Gly Ile Leu Ser Val Leu Ile Asn Ser Leu Val 145 150 155 160 Glu Asn Asn Tyr Phe Glu Gin Asp Gly Lys Leu Asn Ser Ser Asp Ile 165 170 175 Asp Glu Leu Val Leu Thr Asp Ile Glu Lys Lys Ile Leu Ser Leu Ile 180 185 190 -305- Pro Arg Cys 195 Ser Pro Leu Tyr Ile Asp Ile Ser Asp Val Lys Val Leu >nn 205 Ala Ser 210 Glu Tyr 225 Ser Arg Ser Ile Leu Phe Leu Tyr 290 Tyr Val 305 Thr Val Asn Ser Ser Phe Asn Val 370 Glu Leu Arg Ile Asr Tyr Arg 275 Asp Ile Thr Ile Tyr 355 Ser Leu Leu Xaa Ile Gin His Asp 245 Ile Leu 260 Cys Pro Tyr Phe Leu Lys Ile Gly 325 Ser Ser 340 Glu Asp Arg Val Thr Glu Lys Ser Ala Lys Ser Phe 215 Ser Asp Lys Ile Glu Glu 230 235 Ile Ile Leu Asp Glu Lys 250 Ser Asp Asp Leu Leu Asn 265 Gin Val Lys Asp Asn Thr 280 Thr Arg Val Ser Lys Lys 295 Asp Leu Lys Ile Ala Asp 310 315 Ser Phe Val Tyr Thr Lys 330 Asn Val Asp Arg Tyr Ser 345 Ile Ala Glu Phe Ile Lys 360 Thr Phe Asn Asp His 220 Leu Ile Asn Ser Leu 240 Ser Ser Ile Lys Asp 255 Ile Leu Arg Glu Arg 270 Ile Ser Arg Thr Arg 285 Glu Glu Ala Lys Ile 300 Ile Leu Gly Ile Glu 320 Tyr Ser Met Leu Ile 335 Lys Arg Phe His Asp 350 Asp Asn Glu Lys Ile 365 Val Glu Cys Leu Ile Val Pro Asn Ile Asn Ile INFORMATION FOR SEQ ID NO:115: SEQUENCE CHARACTERISTICS: LENGTH: 677 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (vii) IMMEDIATE SOURCE: CLONE: 515-85.1 (viii) POSITION IN GENOME: MAP POSITION: -23.2 UNITS: %G -306- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: Met Pro Ser Tyr Met Tyr Pro Lys 1 5 Ile Ile Ser Leu Gin Leu Asp Asn Thr Met Leu Glu Phe Gly Tyr Lys Asp Ala Val Ser Tyr 55 Tvr Asn Cys Val Met Val Lys 70 Ser Val Ile Asp Thr Arg Leu Ala Leu Ile Asp Tyr Gly Asp 100 Asn Lys Leu Leu Ser Ile Asp 115 Ile Ile His Val Thr Lys Ile 130 135 Ser Asn Met Lys Leu Asn Lys 145 150 Ile Pro Lys Ser Ser Tyr Thr 165 Leu Asp Thr Leu Leu Tyr Leu 180 His Ile Ser Leu Arg Ser Leu 195 Leu Lys Lys Tyr Pro Asn Asn 210 215 Ile Lys Ser Asn Ser Ser Phe 225 230 Asn Met Phe Pro Ala Ile Ile 245 Val Val Asp Lys Asp Arg Leu 260 Ala Met Phe Ser Tyr Asp Ile 275 Asp Ser Asp Tyr Ile Phe Ile 290 295 Lys Cys Arg Asp Phe Ala Asn 305 310 Lys Asn Arg Ile Leu Thr Thr 325 Ile Leu 40 Asp Asp His Gln Asp 120 Leu Ile Tyr Ser Arg 200 Asn Ile Pro Ile Asn 280 Glu Met Lys Asn Ala Arg 10 Lys Lys Leu 25 His Gly Asn Ile Asn Asn Leu Ile Asn 75 SGin Ser Val 90 Asp Ile Ile 105 Ile Ser Tyr Lys Ile Asp Glu Leu Val 155 Leu Tyr Asn 170 Asp Ala Phe 185 Asp Ile Asn Ile Ile Asp His Ile Leu 235 Ser Val Asn 250 Asn Met Tyr 265 Met Ile Asp Lys Asn Ile Ile Arg Asp 315 Cys Glu Asp 330 Lys Pro Leu Ile Val Leu Thr Ile Pro 140 Asp Asn His Arg Tyr 220 His Asp Gly Leu Ser 300 Lys Ile Val Ile Ser Lys Lys Lys Tyr Ile Pro Ala Cys Met Arg Phe Leu Pro Ile Lys Ser Ser 1 Lys His Arg Arg Leu Met Ile Ile 110 Leu Asp Lys Lys 125 Thr Val Ala Asn Val Ile Thr Ser 160 Met Ile Ile Asp 175 Ile Pro Pro Thr 190 Ile Ile Glu Leu 205 Ile Ser Asp Ser Met Ile Ile Ser 240 Phe Ile Ser Thr 255 Ile Lys Cys Val 270 Glu Ser Leu Asp 285 Ile Tyr Asp Val Val Lys Arg Glu 320 Ile Arg Tyr Ile 335 Lys Leu Phe Lys Asn Arg Ile Asn Asp Glu Asn Asn Lys Val Glu 345 350 -307- Glu Val Leu Ile His Ile Asp Asn Val Ser Lys Asn Asn Lys Leu Ser 355 r r r r r Leu Ser Asp Ile Ser 370 Thr Ile Arg Asn Ile 385 Leu Ala Leu Arg Ala 405 Val Ser Met Tyr Lys 420 Asp Tyr Ile Ser Thr 435 Lys Met Ser Thr Phe 450 Ser Ile Cys Ser Asp 465 Cys Ile Asn Tyr Lys 485 Asn Leu Thr.Arg Tyr 500 Ser Val Lys Gly Trp 515 Gly Ile Asn Leu Lys 530 Tyr Gly Asp Ile Thr 545 Ser Ile Ile Asn Thr 565 Cys Ser Leu Asn Glu 580 Ile Leu Glu Tyr Met 595 Lys Arg Phe Asn Asn 610 Val Leu Glu Ser Ser 625 Gin Ile Glu Leu Glu 645 Val Gin Leu Met His 660 Glu Ile Lys Glu Ile 675 Ser Leu Met Asp Gin I 375 Leu Leu Ser Ser Ala 390 Val Lys Asn Trp Lys 410 Lys Ile Lys Gly Val 425 Asn Ile Leu Lys Tyr I 440 Glu Tyr Lys Arg Asp 455 Ser Ile Thr His His 470 Ser Thr Asp Asn Asp I 490 Leu Met His Gly Met 505 Gly Pro Leu Ile Gly I 520 Leu Tyr Ser Thr Met 1 535 Leu Ser Ser Tyr Asp 550 Pro Ile Tyr Glu Leu 570 Tyr Tyr Ser Lys Ile 585 Ile Ser Ile Ile Leu 600 Ile Lys Glu Phe Ile 615 Gly Ile Tyr Phe Cys 630 Ile Asp Glu Leu Ile 650 ?he rhr 395 :ys Ile His Ile Ile 475 .eu Ile Leu Asn Met 555 Ile Val Tyr Ser Gin 635 Ile Arg 380 Ile Tyr Val Lys Lys 460 Tyr Met His Leu Ile 540 Ser Pro Ile Arg Lys 620 Met Asn 365 Leu Asn Pro Lys Ser Lys Ser Leu Thr 415 Met Asp Met 430 Gin Leu Tyr 445 Ser Cys Lys Glu Thr Thr Ile Val Leu 495 Pro Asn Leu 510 Thr Gly Asp 525 Asn Gly Leu Asn Lys Leu Phe Thr Thr 575 e Leu Ile Asn 590 Ile Leu Ile 605 Val Val Asn Arg Val His Gly Ser Met Cys Leu 400 Asn Jal Asp Cys Ser 480 Phe lie Ile Arg Val 560 Cys Val Val Thr Glu 640 Pro 655 Leu Leu Leu Val Ala Thr Ile Ile Leu Glu 670 INFORMATION FOR SEQ ID NO:116: SEQUENCE CHARACTERISTICS: -308- LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Infectious bovine rhinotracheitis virus STRAIN: Cooper Strain (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: CTGGTTCGGC CCAGAATTCG ATGCAACCCA CCGCGCCGCC CCG 43 INFORMATION FOR SEQ ID NO:117: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Infectious bovine rhinotracheitis virus STRAIN: Cooper Strain (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: CTCGCTCGCC CAGGATCCCT AGCGGAGGAT GGACTTGAGT CG 42 INFORMATION FOR SEQ ID NO:118: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine Influenza A neuraminidase STRAIN: Prague/56 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: GGGATCCATG AATCCTAATC AAAAACTCTT T 31 -309- INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine Influenza A neuraminidase STRAIN: Prague/56 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: GGGATCCTTA CGAAAAGTAT TTAATTTGTG C 31 INFORMATION FOR SEQ ID NO:120: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine influenza A hemagglutinin (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: GGAGGCCTTC ATGACAGACA ACCATTATTT TGATACTACT GA 42 INFORMATION FOR SEQ ID NO:121: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine influenza A hemagglutinin (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: GAAGGCCTTC TCAAATGCAA ATGTTGCATC TGATGTTGCC -310- INFORMATION FOR SEQ ID NO:122: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine Influenza A hemagglutinin STRAIN: Prague/56 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: GGGATCCATG AACACTCAAA TTCTAATATT AG 32 INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine Influenza A hemagglutinin STRAIN: Prague/56 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: GGGATCCTTA TATACAAATA GTGCACCGCA INFORMATION FOR SEQ ID NO:124: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine Influenza A neuraminidase (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: -311- GGGTCGACAT GAATCCAAAT CAAAAGATAA INFORMATION FOR SEQ ID NO:125: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Equine Influenza A neuraminidase (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125: GGGTCGACTT ACATCTTATC GATGTCAAA 29 INFORMATION FOR SEQ ID NO:126: SEQUENCE CHARACTERISTICS: eeoc LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO S* (vi) ORIGINAL SOURCE: ORGANISM: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: CTCGAATTCG AAGTGGGCAA CGTGGATCCT CGC 33 INFORMATION FOR SEQ ID NO:127: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127: -312- CAGTTAGCCT CCCCCATCTC CCCA 24 INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO *a (vi) ORIGINAL SOURCE: ORGANISM: Equine herpesvirus type 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: o.

CGGAATTCCT CTGGTTGCCG T 21 INFORMATION FOR SEQ ID NO:129: eb SEQUENCE CHARACTERISTICS: *0o LENGTH: 22 base pairs S(B) TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO S* 0 (vi) ORIGINAL SOURCE: ORGANISM: Equine herpesvirus type 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: GACGGTGGAT CCGGTAGGCG GT 22 INFORMATION FOR SEQ ID NO:130: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine parainfluenza-3 virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130: -313- TTATGGATCC TGCTGCTGTG TTGAACAACT TTGT 34 INFORMATION FOR SEQ ID NO:131: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine parainfluenza-3 virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: CCGCGGATCC CATGACCATC ACAACCATAA TCATAGCC 38 INFORMATION FOR SEQ ID NO:132: SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine parainfluenza-3 virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: CGTCGGATCC CTTAGCTGCA GTTTTTTGGA ACTTCTGTTT TGA 43 INFORMATION FOR SEQ ID NO:133: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine parainfluenza-3 virus -314- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: CATAGGATCC CATGGAATAT TGGAAACACA CAAACAGCAC INFORMATION FOR SEQ ID NO:134: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: Singer Strain (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134: ACGTCGGATC CCTTACCAAA CCACGTCTTA CTCTTGTTTT CC 42 INFORMATION FOR SEQ ID NO:135: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: double I* TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: Singer Strain (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135: ACATAGGATC CCATGGGAGA AAACATAACA CAGTGGAACC INFORMATION FOR SEQ ID NO:136: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO -315- (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: Singer Strain (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: CGTGGATCCT CAATTACAAG AGGTATCGTC TAC 33 INFORMATION FOR SEQ ID NO:137: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: Singer Strain (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137: CATAGATCTT GTGGTGCTGT CCGACTTCGC A 31 INFORMATION FOR SEQ ID NO:138: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine respiratory syncytial virus STRAIN: Strain 375 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: TGCAGGATCC TCATTTACTA AAGGAAAGAT TGTTGAT 37 INFORMATION FOR SEQ ID NO:139: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -316- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine respiratory syncytial virus STRAIN: Strain 375 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139: CTCTGGATCC TACAGCCATG AGGATGATCA TCAGC INFORMATION FOR SEQ ID NO:140: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine respiratory syncytial virus STRAIN: Strain 375 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140: CGTCGGATCC CTCACAGTTC CACATCATTG TCTTTGGGAT INFORMATION FOR SEQ ID NO:141: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine respiratory syncytial virus STRAIN: Strain 375 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141: CTTAGGATCC CATGGCTCTT AGCAAGGTCA AACTAAATGA C 41 INFORMATION FOR SEQ ID NO:142: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid -317- STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine respiratory syncytial virus STRAIN: Strain 375 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142: CGTTGGATCC CTAGATCTGT GTAGTTGATT GATTTGTGTG A 41 INFORMATION FOR SEQ ID NO:143: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: double S(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine respiratory syncytial virus STRAIN: Strain 375 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143: CTCTGGATCC TCATACCCAT CATCTTAAAT TCAAGACATT A 41 9* INFORMATION FOR SEQ ID NO:144: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:145: SEQUENCE CHARACTERISTICS: LENGTH: 128 base pairs -318- TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145: GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT TGATCCATGA 120 ATCCTAAT 128 INFORMATION FOR SEQ ID NO:146: SEQUENCE CHARACTERISTICS: LENGTH: 120 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146: CTTTTCGTAA GGATCAATTC GGATCCATAA TTAATTAATT TTTATCCCGG CGCGCCTCGA CTCTAGAATT TCATTTTGTT TTTTTCTATG CTATAAATGA ATTCGGATCC CGTCGTTTTA 120 INFORMATION FOR SEQ ID NO:147: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:148: SEQUENCE CHARACTERISTICS: -319- LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:149: S. SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:150: SEQUENCE CHARACTERISTICS: LENGTH: 168 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150: GTATTGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT CACCCGCTGG 120 TGGCGGTCTT TGGCGCGGGC CCCGTGGGCA TCGGCCCGGG CACCACGG 168 INFORMATION FOR SEQ ID NO:151: SEQUENCE CHARACTERISTICS: LENGTH: 112 base pairs -320- TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151: GAGCTCGAAT TCGGATCCAT AATTAATTAA TTTTTATCCC GGCGCGCCTC GACTCTAGAA TTTCATTTTG TTTTTTTCTA TGCTATAAAT GAATTCGGAT CCCGTCGTTT TA 112 INFORMATION FOR SEQ ID NO:152: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:152: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:153: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:154: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid -321- STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:155: SEQUENCE CHARACTERISTICS: LENGTH: 104 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155: AAATATATAA ATACCATGTT AGAATTTGGT CTGCTGCAGG TCGACTCTAG AATTTCATTT TGTTTTTTTC TATGCTATAA ATGAATTCGG ATCCCGTCGT TTTA 104 INFORMATION FOR SEQ ID NO:156: SEQUENCE CHARACTERISTICS: LENGTH: 185 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGTCGA CTCTAGAAAA AATTGAAAAA CTATTCTAAT TTATTGCACG 120 GAGATCTTTT TTTTTTTTTT TTTTTTGGCA TATAAATGAA TTCGGATCCC CGGTGGCTTT 180 GGGGG 185 INFORMATION FOR SEQ ID NO:157: SEQUENCE CHARACTERISTICS: -322- LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157: CTCAATGTTA GGGTACCGAG CTCGAATTGG GTCGACCGGG TCGACCTGCA GCCTACATGG AAATCT 66 INFORMATION FOR SEQ ID NO:158: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:158: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:159: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:160: SEQUENCE CHARACTERISTICS: LENGTH: 127 base pairs TYPE: nucleic acid STRANDEDNESS: double -323- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160: GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT TCGACATGAA 120 TCCAAAT 127 INFORMATION FOR SEQ ID NO:161: 4* SEQUENCE CHARACTERISTICS: LENGTH: 122 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161: GATAAGATGT AAGTCGAAAT TCGGATCCAT AATTAATTAA TTTTTATCCC GGCGCGCCTC GACTCTAGAA TTTCATTTTG TTTTTTTCTA TGCTATAAAT GAATTCGGAT CCCGTCGTTT 120 *TA 122 INFORMATION FOR SEQ ID NO:162: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 -324- INFORMATION FOR SEQ ID NO:163: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:164: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:165: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165: GTATAGCGGC CGCCTGCAGG TCGACCTGCA GTGAATAATA AAATGTGTGT TTGTCCGAAA T 61 INFORMATION FOR SEQ ID NO:166: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs -325- TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166: CTCCATAGAA GACACCGGGA CCATGGATCC CGTCGTTTTA CAACG INFORMATION FOR SEQ ID NO:167: SEQUENCE CHARACTERISTICS: LENGTH: 105 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167: TCGGCGGAAA TCCAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA .I GATCTAGAAT AAGCTAGAGG ATCGATCCCC TATGGCGATC ATCAG 105 INFORMATION FOR SEQ ID NO:168: S. SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168: CTGCAGGTCG ACCTGCAGGC GGCCGCTATA C 31 INFORMATION FOR SEQ ID NO:169: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -326- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:170: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170: **ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:171: SEQUENCE CHARACTERISTICS: LENGTH: 193 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171: GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT CCGAAGTGGG 120 CAACGTGGAT CCTCGCCCTC GGGCTCCTCG TGGTCCGCAC CGTCGTGGCC AGAAGTGCTC 180 CTACTAGCTC GAG 193 INFORMATION FOR SEQ ID NO:172: SEQUENCE CHARACTERISTICS: LENGTH: 123 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -327- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172: ATCATTAGCA CGTTAACTTA ATAAGATCCA TAATTAATTA ATTTTTATCC CGGCGCGCCT CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAAA TGAATTCGGA TCCCGTCGTT 120 TTA 123 INFORMATION FOR SEQ ID NO:173: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear oo e S(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:174: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:175: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -328- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:176: SEQUENCE CHARACTERISTICS: LENGTH: 133 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 0$ (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176: oGTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT CCTCTGGTTG 120 CCGTTCTGTC GGC 133 INFORMATION FOR SEQ ID NO:177: SEQUENCE CHARACTERISTICS: *et LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177: GAAAATGAAA AAATGGTTTA AACCGGGGGC GCGCCTCGAC TCTAGAATTT CATTTTGTTT TTTTCTATGC TATAAATGAA TTCGGATCCC GTCGTTTTA 99 INFORMATION FOR SEQ ID NO:178: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -329- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:179: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO o (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:180: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRTITION: SEQ ID NO:180: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEO ID NO:181: SEQUENCE CHARACTERISTICS: LENGTH: 140 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO -330- (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181: GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT CGGATCAGCT 120 TATGATGGAT GGACGTTTGG 140 INFORMATION FOR SEQ ID NO:182: SEQUENCE CHARACTERISTICS: LENGTH: 123 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182: GGAGGTGTCC ACGGCCTTAA AGCTGATCCA TAATTAATTA ATTTTTATCC CGGCGCGCCT CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAAA TGAATTCGGA TCCCGTCGTT 120 TTA 123 o INFORMATION FOR SEQ ID NO:183: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:184: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -331- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:185: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185: GAAGCATGCC CGTTCTTATC AATAGTTTAG TCGAAAATA 39 o•* o* *oo INFORMATION FOR SEQ ID NO:186: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186: CATAAGATCT GGCATTGTGT TATTATACTA ACAAAAATAA G 41 INFORMATION FOR SEQ ID NO:187: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO -332r r r (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187: CCGTAGTCGA CAAAGATCGA CTTATTAATA TGTATGGGAT T INFORMATION FOR SEQ ID NO:188: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188: GCCTGAAGCT TCTAGTACAG TATTTACGAC TTTTGAAAT INFORMATION FOR SEQ ID NO:189: SEQUENCE CHARACTERISTICS: LENGTH: 3942 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..369 (ix) FEATURE: NAME/KEY: CDS LOCATION: 370..597 (ix) FEATURE: NAME/KEY: CDS LOCATION: 598..1539 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1675..3708 (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (3748..3942) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189: -333- TGT TTG TTC ATT AAT AAG ATG GGT GGA GCT ATT ATA GAA TAC AAG ATA 48 Cys Leu Phe Ile Asn Lys Met Gly Gly Ala Ile Ile Glu Tyr Lys Ile 1 5 10 CCT GGT TCC AAA TCT ATA ACC AAA TCT ATT TCC GAA GAA CTA GAA AAT 96 Pro Gly Ser Lys Ser Ile Thr Lys Ser Ile Ser Glu Glu Leu Glu Asn 25 TTA ACA AAG CGA GAT AAA CCA ATA TCT AAA ATT ATA GTT ATT CCT ATT 144 Leu Thr Lys Arg Asp Lys Pro Ile Ser Lys Ile Ile Val Ile Pro Ile 40 GTA TGT TAC AGA AAT GCA AAT AGT ATA AAG GTT ACA TTT GCA CTA AAA 192 Val Cys Tyr Arg Asn Ala Asn Ser Ile Lys Val Thr Phe Ala Leu Lys 55 AAG TTT ATC ATA GAT AAG GAG TTT AGT ACA AAT GTA ATA GAC GTA GAT 240 Lys Phe Ile Ile Asp Lys Glu Phe Ser Thr Asn Val Ile Asp Val Asp 70 75 GGT AAA CAT GAA AAA ATG TCC ATG AAT GAA ACA TGC GAA GAG GAT GTT 288 Gly Lys His Glu Lys Met Ser Met Asn Glu Thr Cys Glu Glu Asp Val 90 GCT AGA GGA TTG GGA ATT ATA GAT CTT GAA GAT GAA TGC ATA GAG GAA 336 Ala Arg Gly Leu Gly Ile Ile Asp Leu Glu Asp Glu Cys Ile Glu Glu 100 105 110 GAT GAT GTC GAT ACG TCA TTA TTT AAT GTA TAAATG GAT AAA TTG TAT 384 Asp Asp Val Asp Thr Ser Leu Phe Asn Val Met Asp Lys Leu Tyr 115 120 1 GCG GCA ATA TTC GGC GTT TTT ATG ACA TCT AAA GAT GAT GAT TTT AAT 432 ,,Ala Ala Ile Phe Gly Val Phe Met Thr Ser Lys Asp Asp Asp Phe Asn 15 AAC TTT ATA GAA GTT GTA AAA TCT GTA TTA ACA GAT ACA TCA TCT AAT 480 Asn Phe Ile Glu Val Val Lys Ser Val Leu Thr Asp Thr Ser Ser Asn 25 30 CAT ACA ATA TCG TCG TCC AAT AAT AAT ACA TGG ATA TAT ATA TTT CTA 528 His Thr Ile Ser Ser Ser Asn Asn Asn Thr Trp Ile Tyr Ile Phe Leu 45 GCG ATA TTA TTT GGT GTT ATG GTA TTA TTA GTT TTT ATT TTG TAT TTA 576 Ala Ile Leu Phe Gly Val Met Val Leu Leu Val Phe Ile Leu Tyr Leu 60 AAA GTT ACT AAA CCA ACT TAAATG GAG GAA GCA GAT AAC CAA CTC GTT 624 Lys Val Thr Lys Pro Thr Met Glu Glu Ala Asp Asn Gin Leu Val 75 1 TTA AAT AGT ATT AGT GCT AGA GCA TTA AAG GCA TTT TTT GTA TCT AAA 672 Leu Asn Ser Ile Ser Ala Arg Ala Leu Lys Ala Phe Phe Val Ser Lys 15 20 ATT AAT GAT ATG GTC GAT GAA TTA GTT ACC AAA AAA TAT CCA CCA AAG 720 Ile Asn Asp Met Val Asp Glu Leu Val Thr Lys Lys Tyr Pro Pro Lys 35 AAG AAA TCA CAA ATA AAA CTC ATA GAT ACA CGA ATT CCT ATT GAT CTT 768 Lys Lys Ser Gin Ile Lys Leu Ile Asp Thr Arg Ile Pro Ile Asp Leu 50 ATT AAT CAA CAA TTC GTT AAA AGA TTT AAA CTA GAA AAT TAT AAA AAT 816 Ile Asn Gin Gin Phe Val Lys Arg Phe Lys Leu Glu Asn Tyr Lys Asn 65 -334- GGA ATT TTA TCC GTT CTT ATC AAT AGT TTA GTC CAA AAT PAT TAC TTT Gly Ile Leu Ser Val Leu Ile Asn Ser Leu Val Giu Asn Asn Tyr Phe 80 a a a a a.

GAA

Glu

ACA

Thr

CTT

Leu

AAA

Lys

TCT

Ser

ATT

Ile 170

TCT

Ser

CAG

Gin

ACT

Thr

GAT

Asp

TCA

Ser 250

PAT

Asn

ATT

Ile

GTT

CAA

Gin

GAC

Asp

TAT

Tyr

AGT

Ser

GAT

Asp 155

ATA

Ile

GAT

Asp

GTT

Val

AGA

Arg

TTA

Leu 235

TTT

Phe

GTT

Vai

GCG

Ala

GAA

CAT

Asp

ATA

Ile

ATA

Ile

GCT

Ala 140

AA

Lys

CTA

Leu

GAT

Asp

AAA

Lys

GTG

Val 220

AAG

Lys

GTA

Val

CAT

Asp

GAA

Clu

TGC

GGT

Cly

GAG

Glu

CAT

Asp 125

AAA

Lys

ATA

Ile

CAT

Asp

CTT

Leu

CAT

Asp 205

TCA

Ser

ATT

Ile

TAT

Tyr

AGA

Arg

TTT

Phe 285

CTT

AAA

Lys

AAA

Lys 110

ATC

Ile

TCA

Ser

GAC

Clu

GAA

Glu

TTG

Leu 190

PAT

Asn

AAG

Lys

CCT

Ala

ACC

Thr

TAT

Tyr 270

ATA

Ile

ATC

CTT

Leu 95

AAG

Lys

AGT

Ser

TTT

Phe

GAA

Clu

AAA

Lys 175

PAT

Asn

ACT

Thr

AAA

Lys

CAT

Asp

AAA

Lys 255

TCA

Ser

AAG

Lys

CTA

PAT AGC Asn Ser ATT TTA Ile Leu GAC CTT Asp Val ACC TTT Thr Phe 145 TTA ATA Leu Ile 160 AGT TCT Ser Ser ATA CTT Ile Leu ATT TCT Ile Ser GAA GAA Glu Glu 225 ATA CTC Ile Leu 240 TAT AGC Tyr Ser AAA AGG Lys Arg CAT PAT Asp Asn CCT PAT

AGT

Ser

TCC

Ser

AAA

Lys 130

PAT

Asn

PAT

Asn

ATT

Ile

CGT

Arg

AGA

Arg 210

GCG

Ala

GGT

Cly

ATC

Met

TTC

Phe

GAA

Clu 290

ATT

CAT

Asp

TTG

Leu 115

CTT

Val

CAT

Asp

AGT

Ser

AAA

Lys

GAA

Clu 195

ACA

Thr

AAA

Lys

ATC

Ile

TTC

Leu

CAT

His 275

AAA

Lys

PAT

ATT

Ile 100

ATT

Ile

CTC

Leu

CAT

His

TTA

Leu

CAC

Asp 180

AGA

Arg

CGT

Arg

ATA

Ile

GAA

Glu

ATT

Ile 260

CAC

Asp

ATT

Ile

ATA

CAT

Asp

CCT

Pro

CCA

Ala

GAA

Glu

TCT

Ser 165

AGC

Ser

TTA

Leu

CTA

Leu

TAC

Tyr

ACA

Thr 245

PAT

Asn

TCT

Ser

PAT

Asn

GAG

GAA

Clu

AGA

Arg

TCT

Ser

TAT

Tyr 150

AGA

Arg

ATA

Ile

TTT

Phe

TAT

Tyr

CTT

Val 230

CTA

Val

TCA

Ser

TTT

Phe

CTA

Va1

TTA

TTA

Leu

TGT

Cys

AGG

Arg 135

ATT

Ile

PAC

Asn

TAT

Tyr

AGA

Arg

CAT

Asp 215

ATA

Ile

ACC

Thr

ATT

Ile

TAT

Tyr

TCC

Ser 295

TTA

GTG

Va1

TCT

Ser 120

TTA

Leu

ATA

Ile

CAT

His

ATA

Ile

TGT

Cys 200

TAT

Tyr

TTC

Leu

ATA

Ile

TCC

Ser

GAA

Glu 280

AGA

Arg

ACT

CTC

Leu 105

CCT

Pro

AAA

Lys

CAA

Gin

CAT

Asp

CTA

Leu 185

CCA

Pro

TTT

Phe

AA

Lys

GGA

Cly

TCT

Ser 265

CAT

Asp

GTT

Val

GAA

912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1596 1656 1707 Val Giu Cys 300 Leu Ile Val Pro Asn Ile Asn Ile Giu Leu Leu Thr Glu TAAGTATATA TPAATCATTC TTTTTATPAT GTTTCTTATC CTTATCATAT ACATTTTTPA GGCCGTATAT GATAPATCAA TGTTACTATA ATPACACA ATC CCC TCC TAT ATC TAT Met Pro Ser Tyr Met Tyr 1 5 310 CCATTTACTT TTGCTGTATC PATATATPAG CACTTATTTT CCC AAG PAC GCA AGA Pro Lys Asn Ala Arg -333- AAA GTA ATT Lys Val Ilie TCA AAG ATT ATA TCA TTA CAA CTT GAT ATT AAA AAA CTT Ser Lys Ile Ile Ser Leu Gin Leu Asp Ile Lys Lys Leu 0*

CCT

Pro

CTA

Leu

ATA

Ile

GTT

Val

TTA

Leu

ACT

Thr

ATA

Ile

CCT

Pro 140

GAT

Asp

AAT

Asn

CAC

His

AGG

Arg

TAT

Tyr 220

CAT

His

GAT

Asp

AAA

Lys

CCA

Pro

AGA

Arg

ATA

Ile

AAA

Lys

TTA

Leu

TTA

Leu 125

ACA

Thr

GTA

Val1

ATG

Met

ATA

Ile

ATT

Ile 205

ATA

Ile

ATG

Met

TTT

Phe AAA TAT Lys Tyr CCT TGT Ala Cys TTT TTA Phe Leu AAA TCA Lys Ser CAT CGT His Arg 95 ATG ATC Met Ile 110 GAT AAA Asp Lys GTA GCC Val Ala ATA ACA Ile Thr ATC ATT Ile Ile 175 CCC CCC Pro Pro 190 ATT GAA Ile Clu TCC GAT Ser Asp ATA ATA Ile Ile ATA TCT Ile Ser 255

ATA

Ile

ATC

Met

CCT

Pro

TCA

Ser 80

AGA

Arg

ATT

Ile

AAA

Lys

AAT

Asn

TCA

Ser 160

CAT

Asp

ACA

Thr

TTC

Leu

AC

Ser

TCA

Ser 240

ACC

Thr

AAT

Asn

TAT

Tyr

TAT

Tyr 65

TCT

Ser

CC

Ala

AAT

Asn

ATA

Ile

TCA

Ser 145

ATA

Ile

CTC

Leu

CAT

His

CTT

Leu

ATA

Ile 225

AAT

Asn

GTA

Val1

ACC

Thr

AAA

Lys 50

AAT

Asn

CTA

Val1

TTA

Leu

AAC

Lys

ATT

Ile 130

AAC

Asn

CCT

Pro

CAT

Asp

ATA

Ile

AAA

Lys 210

AAA

Lys

ATC

Met

CTT

Val

ATC

Met 35

CAT

Asp

TGT

Cys

ATA

Ile

ATA

Ile

TTA

Leu 115

CAT

His

ATC

Met

AAC

Lys

ACA

Thr

TCA

Ser 195

AAA

Lys Ser

TTT

Phe

CAT

Asp 20 TTA CAA Leu Ciu CCC CTA Aia Val CTT ATC Val Met CAT ACT Asp Thr 85 CAT TAC Asp Tyr 100 CTA TCG Leu Ser CTA ACA Val Thr AAA CTG Lys Leu TCT TCC Ser Ser 165 TTA TTA Leu Leu 180 TTA CGT Leu Arg TAT CCC Tyr Pro A.AT ACT Asn Ser CCT GCT Pro Ala 245 AAA CAT Lys Asp 260

TTT

Phe

TCA

Ser

CTT

Vali 70

ACA

Arg

GCC

Cly

ATA

Ile

AAA

Lys

AAT

Asn 150

TAT

Tyr

TAT

Tyr

TCA

Ser

AAT

Asn

TCA

Ser 230

ATA

Ile

CCA

Arg CCT CTA Gly Leu TAT CAT Tyr Asp AAA CAT Lys Asp TTA CAT Leu His CAT CAA Asp Cmn GAT CAT Asp Asp 120 ATA TTA Ile Leu 135 AAC ATA Lys Ile ACA TAT Thr Tyr TTA TCC Leu Ser CTT ACA Leu Arg 200 AAT AAT Asn Asn 215 Phe Ile ATC CCT Ile Pro CTT ATT Leu Ile

CAT

His

ATA

Ile

TTA

Leu

CAA

Cmn

CAC

Asp 105

ATA

Ile

AAA

Lys

GAG

Ciu

TTA

Leu

CAT

Asp 185

CAT

Asp

ATT

Ile

CAC

His

ACT

Ser

AAT

Asn 265

GCA

Gly

AAT

Asn

ATA

Ile

TCT

Se r

ATT

Ile

TCC

Ser

ATA

Ile

CTT

Leu

TAT

Tyr 170

CCA

Ala

ATA

Ile

ATT

Ile

ATA

Ile

CTA

Va I 250

ATC

Met

AAT

Asn

AAT

Asn

AAT

Asn

CTA

Val

ATC

Ile

TAT

Tyr

CAC

Asp

CTA

Val 155

AAT

Asn

TTC

Phe

AAC

Asn

CAT

Asp

CTT

Leu 235

AAC

Asn

TAT

Tyr 1755 1803 1851 1899 1947 1995 2043 2091 2139 2187 2235 2283 2331 2379 2427 2475 2523 CCC ATT AAG TCT CTT CCT ATC TTT TCG TAC CAT ATA AAC Cly Ile Lys Cys Vai Ala Met Phe Ser Tyr Asp Ile ATC ATC CAT Met Ile Asp Asn 280 336- TTA GAG TCA TTA GAT GAC TCA GAT TAC ATA TTT ATA GAA AAA AAT ATA Leu Glu Ser Leu Asp Asp 285 Ser Asp Tyr Ile Phe Ile Glu Lys Asn Ile 295 2 -1 9 0

S

0* d 0** 'Os.

O n S. 0e *5

TCT

Ser 300

AAG

Lys

ATT

Ile

AAT

Asn

AAT

Asn

CGT

Arg 380

ATA

Ile

TAT

Tyr

GTA

Val

AAA

Lys

AAA

Lys 460

TAT

Tyr

ATG

Met

CAT

His

TTA

Leu

ATA

Ile

GTT

Val

ATA

Ile

AAT

Asn

AAT

Asn 365

TTA

Leu

AAA

Lys

TCA

Ser

ATG

Met

CAA

Gin 445

TCA

Ser

GAA

Glu

ATA

Ile

CCT

Pro

ACG

Thr 525

TAC

Tyr

AAA

Lys

AGA

Arg

AAG

Lys 350

AAA

Lys

AAT

Asn

TCA

Ser

TTG

Leu

GAT

Asp 430

TTA

Leu

TGT

Cys

ACA

Thr

GTA

Val

AAT

Asn 510

GGT

Gly GAC GTT Asp Val AGA GAA Arg Glu 320 TAT ATA Tyr Ile 335 GTG GAG Val Glu TTA TCA Leu Ser CCA TGT Pro Cys AAA CTA Lys Leu 400 ACA AAT Thr Asn 415 ATG GTT Met Val TAT GAT Tyr Asp AAA TGC Lys Cys ACA TCA Thr Ser 480 TTG TTC Leu Phe 495 CTT ATA Leu Ile GAT ATA Asp Ile

AAA

Lys 305

AAG

Lys

AAA

Lys

GAG

Glu

CTG

Leu

ACC

Thr 385

TTA

Leu

GTA

Val

GAT

Asp

AAA

Lys

TCG

Ser 465

TGT

Cys

AAT

Asn

AGC

Ser

GGT

Gly

TGT

Cys

AAT

Asn

TTA

Leu

GTG

Val

TCT

Ser 370

ATA

Ile

GCG

Ala

TCA

Ser

TAT

Tyr

ATG

Met 450

ATA

Ile

ATA

Ile

CTA

Leu

GTA

Val

ATT

Ile 530

AGA

Arg

AGA

Arg

TTC

Phe

TTG

Leu 355

GAT

Asp

AGA

Arg

TTA

Leu

ATG

Met

ATA

Ile 435

AGT

Ser

TGT

Cys

AAT

Asn

ACT

Thr

AAA

Lys 515

AAT

Asn GAT TTT Asp Phe ATA TTA Ile Leu 325 AGT AAA Ser Lys 340 ATA CAT Ile His ATA TCA Ile Ser AAT ATA Asn Ile CGG GCA Arg Ala 405 TAT AAA Tyr Lys 420 TCT ACT Ser Thr ACG TTT Thr Phe TCC GAC Ser Asp TAT AAA Tyr Lys 485 AGA TAT Arg Tyr 500 GGA TGG Gly Trp TTA AAA Leu Lys

GCG

Ala 310

ACT

Thr

AAT

Asn

ATT

Ile

TCT

Ser

TTA

Leu 390

GTA

Val

AAA

Lys

AAC

Asn

GAA

Glu

TCT

Ser 470

TCT

Ser

TTA

Leu

GGT

Gly

CTA

Leu

AAT

Asn

ACG

Thr

AGA

Arg

GAT

Asp

TTA

Leu 375

TTA

Leu

AAA

Lys

ATA

Ile

ATT

Ile

TAT

Tyr 455

ATA

Ile

ACC

Thr

ATG

Met

CCC

Pro

TAT

Tyr 535

ATG

Met

AAA

Lys

ATA

Ile

AAT

Asn 360

ATG

Met

TCT

Ser

AAC

Asn

AAG

Lys

CTT

Leu 440

AAA

Lys

ACA

Thr

GAT

Asp

CAT

His

CTT

Leu 520

TCC

Ser ATT AGA Ile Arg TGT GAA Cys Glu 330 AAC GAT Asn Asp 345 GTA TCT Val Ser GAT CAA Asp Gin TCA GCA Ser Ala TGG AAA Trp Lys 410 GGT GTT Gly Val 425 AAA TAC Lys Tyr CGA GAT Arg Asp CAT CAT His His AAT GAT Asn Asp 490 GGG ATG Gly Met 505 ATT GGA Ile Gly ACC ATG Thr Met

GAT

Asp 315

GAT

Asp

GAA

Glu

AAA

Lys

TTT

Phe

ACT

Thr 395

TGT

Cys

ATC

Ile

CAT

His

ATT

Ile

ATA

Ile 475

CTT

Leu

ATA

Ile

TTA

Leu

AAT

Asn 2571 2619 2667 2715 2763 2811 2859 2907 2955 3003 3051 3099 3147 3195 3243 3291 3339 ATA AAT GGG CTA CGG TAT GGA GAT ATT ACG TTA TCT TCA TAC GAT ATG Ile Asn 540 Gly Leu Arg Tyr Gly Asp Ile Thr Leu Ser Ser Tyr Asp 545 550 Met 555 -337- AGT AAT AAA TTA GTC TCT ATT ATT AAT ACA CCC Ser Asn Lys Leu Val Ser Ile Ile Asn Thr Pro ATA TAT GAG TTA ATA Ile Tyr Glu Leu Ile 570 CCG TTT Pro Phe ATT TTA Ile Leu AGA ATA Arg Ile 605 AAA GTC Lys Val 620 ATG CGT Met Arg AAT GGA Asn Gly ACC ATA Thr Ile

ACT

Thr

ATA

Ile 590

TTG

Leu

GTA

Val

GTA

Val

TCT

Ser

ATA

ACA

Thr 575

AAT

Asn

ATC

Ile

AAT

Asn

CAT

His

ATG

Met 655

TTA

TGT

Cys

GTT

Val

GTA

Val

ACT

Thr

GAA

Glu 640

CCT

Pro

GAG

TGT

Cys

ATT

Ile

AAA

Lys

GTA

Val 625

CAA

Gin

GTA

Val

GAA

TCA CTC AAT GAA Ser

TTA

Leu

AGA

Arg 610

CTA

Leu

ATT

Ile

CAG

Gin

ATC

Ile Leu

GAA

Glu 595

TTT

Phe

GAA

Glu

GAA

Glu

CTT

Leu

AAA

Asn 580

TAT

Tyr

AAT

Asn

TCA

Ser

TTG

Leu

ATG

Met 660

GAA

Glu

ATG

Met

AAC

Asn

TCA

Ser

GAA

Glu 645

CAT

His

ATA

TAT

Tyr

ATA

Ile

ATT

Ile

GGC

Gly 630

ATA

Ile

TTA

Leu

TAA(

00 .:00 ::*00 TAT TCA AAA ATT GTG Tyr Ser Lys Ile Val 585 TCT ATT ATA TTA TAT Ser Ile Ile Leu Tyr 600 AAA GAA TTT ATT TCA Lys Glu Phe Ile Ser 615 ATA TAT TTT TGT CAG Ile Tyr Phe Cys Gin 635 GAT GAG CTC ATT ATT Asp Glu Leu Ile Ile 650 CTT CTA AAG GTA GCT Leu Leu Lys Val Ala 665 CGTATTT TTTCTTTTAA CTTGTCTAAT TGTATCTTGT AAAGTAGTAG ATATAGTTTA TTTGGCATGA ATTCTACACG 3387 3435 3483 3531 3579 3627 3675 3725 3785 3845 3905 3942 Ile Leu Glu Glu 670 Lys Glu Ile 675 ATAAATAAAA ATACTTTTTT TTTTAAACAA GGGGTGCTAC ATTTTGGATC TGATGCAAGA TTATTAAATA ATCGTATGAA TATCGTTACT GGACATGATA TTATGTTTAG TTAATTCTTC TCGGACAAGG TAATGTATCT ATAATGGTAT AAAGCTT INFORMATION FOR SEQ ID NO:190: SEQUENCE CHARACTERISTICS: LENGTH: 122 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 Cys Leu Phe Ile Asn Lys Met Gly Gly Ala Ile 1 5 10 Pro Gly Ser Lys Ser Ile Thr Lys Ser Ile Ser 25 Leu Thr Lys Arg Asp Lys Pro Ile Ser Lys Ile 40 Val Cys Tyr Arg Asn Ala Asn Ser Ile Lys Val 55 Lys Phe Ile Ile Asp Lys Glu Phe Ser Thr Asn 70 75 Ile Glu Ile Thr Val Glu Tyr Lys Ile Glu Leu Glu Asn Val Ile Pro Ile Phe Ala Leu Lys Ile Asp Val Asp -338- Gly Lys His Glu Lys Met Ser Met Asn Glu Thr Cys Glu Glu Asp Val 90 Ala Arg Gly Leu Gly Ile Ile Asp Leu Glu Asp Glu Cys Ile Glu Glu 100 105 110 Asp Asp Val Asp Thr Ser Leu Phe Asn Val 115 120 S..o .o.

08..

00.0 0 0 4* INFORMATION FOR SEQ ID NO:191: SEQUENCE CHARACTERISTICS: LENGTH: 75 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asp Lys Leu Tyr Ala Ala Ile Phe Gly 1 5 10 Asp Asp Asp Phe Asn Asn Phe Ile Glu Val 25 Asp Thr Ser Ser Asn His Thr Ile Ser Ser 35 40 Ile Tyr Ile Phe Leu Ala Ile Leu Phe Gly 50 55 Phe Ile Leu Tyr Leu Lys Val Thr Lys Pro 70 NO:191: Val Phe Met Thr Ser Lys Val Lys Ser Val Leu Thr Ser Asn Asn Asn Thr Trp Val Met Val Leu Leu Val Thr INFORMATION FOR SEQ ID NO:192: SEQUENCE CHARACTERISTICS: LENGTH: 313 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192: Met Glu Glu Ala Asp Asn Gin Leu Val Leu Asn Ser 1 5 10 Ala Leu Lys Ala Phe Phe Val Ser Lys Ile Asn Asp 25 Leu Val Thr Lys Lys Tyr Pro Pro Lys Lys Lys Ser 40 Ile Asp Thr Arg Ile Pro Ile Asp Leu Ile Asn Gin 55 Arg Phe Lys Leu Glu Asn Tyr Lys Asn Gly Ile Leu 70 75 Asn Ser Leu Val Glu Asn Asn Tyr Phe Glu Gin Asp 90 Ile Met Gin Gin Ser Gly Ser Val Ile Phe Val Lys Ala Asp Lys Val Leu Leu Arg Glu Leu Lys Ile Asn -339- Ser Ser Asp Ile Asp Glu Leu Val Leu Thr Asp Ile Glu Lys Lys Ile 100 cc c r nr Leu Val Phe 145 Ile Ser Leu Ser Glu 225 Leu Ser Arg Asn Asn 305 Ser Lys 130 Asn Asn Ile Arg Arg 2.10 Ala Gly Met Phe Glu 290 Ile Leu 115 Val Asp Ser Lys Glu 195 Thr Lys Ile Leu His 275 Lys Asn Ile Leu His Leu Asp 180 Arg Arg Ile Glu Ile 260 Asp Ile Ile Pro Arg Ala Ser Glu Tyr 150 Ser Arg 165 Ser Ile Leu Phe Leu Tyr Tyr Val 230 Thr Val 245 Asn Ser Ser Phe Asn Val Glu Leu 310 Cys Arg 135 Ile Asn Tyr Arg Asp 215 Ile Thr Ile Tyr Ser 295 Leu Ser 120 Leu Ile His Ile Cys 200 Tyr Leu Ile Ser Glu 280 Arg Thr 105 Pro Lys Gin Asp Leu 185 Pro Phe Lys Gly Ser 265 Asp Val Glu Leu Tyr Lys Ser Ser Asp 155 Ile Ile 170 Ser Asp Gin Val Thr Arg Asp Leu 235 Ser Phe 250 Asn Val Ile Ala Val Glu Ile Ala 140 Lys Leu Asp Lys Val 220 Lys Val Asp Glu Cys 300 Asp 125 Lys Ile Asp Leu Asp 205 Ser Ile Tyr Arg Phe 285 Leu Ile Ser Glu Glu Leu 190 Asn Lys Ala Thr Tyr 270 Ile Ile Ser Asp Phe Thr Glu Leu 160 Lys Ser 175 Asn Ile Thr Ile Lys Glu Asp Ile 240 Lys Tyr 255 Ser Lys Lys Asp Val Pro INFORMATION FOR SEQ ID NO:193: SEQUENCE CHARACTERISTICS: LENGTH: 677 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193: Met Pro Ser Tyr Met Tyr Pro Lys Asn Ala Arg Lys Val Ile Ser Lys 1 5 10 Ile Ile Ser Leu Gin Leu Asp Ile Lys Lys Leu Pro Lys Lys Tyr Ile 25 Asn Thr Met Leu Glu Phe Gly Leu His Gly Asn Leu Pro Ala Cys Met 40 Tyr Lys Asp Ala Val Ser Tyr Asp Ile Asn Asn Ile Arg Phe Leu Pro 55 -340- Tyr Asn Cys Val Met Val 70 Lys Asp Leu Ile Asn Val Ile Lys Ser Ser r r r r Ser Ala Asn Ile Ser 145 Ile Leu His Leu Ile 225 Asn Val Ala Asp Lys 305 Lys Lys Glu Leu Thr 385 Leu Val Leu Lys Ile 130 Asn Pro Asp Ile Lys 210 Lys Met Val Met Ser 290 Cys Asn Leu Val Ser 370 Ile Ala Ile Ile Leu 115 His Met Lys Thr Ser 195 Lys Ser Phe Asp Phe 275 Asp Arg Arg Phe Leu 355 Asp Arg Leu Asp Asp 100 Leu Val Lys Ser Leu 180 Leu Tyr Asn Pro Lys 260 Ser Tyr Asp Ile Ser 340 Ile Ile Asn Arg Thr Arg Tyr Gly Ser Ile Thr Lys Leu Asn 150 Ser Tyr 165 Leu Tyr Arg Ser Pro Asn Ser Ser 230 Ala Ile 245 Asp Arg Tyr Asp Ile Phe Phe Ala 310 Leu Thr 325 Lys Asn His Ile Ser Ser Ile Leu 390 Ala Val 405 Leu Asp Asp Ile 135 Lys Thr Leu Leu Asn 215 Phe Ile Leu Ile Ile 295 Asn Thr Arg Asp Leu 375 Leu Lys His Gin Asp 120 Leu Ile Tyr Ser Arg 200 Asn Ile Pro Ile Asn 280 Glu Met Lys Ile Asn 360 Met Ser Asn Gin Asp 105 Ile Lys Glu Leu Asp 185 Asp Ile His Ser Asn 265 Met Lys Ile Cys Asn 345 Val Asp Ser Trp Ser Val 90 Ile Ile Ser Tyr Ile Asp Leu Val 155 Tyr Asn 170 Ala Phe Ile Asn Ile Asp Ile Leu 235 Val Asn 250 Met Tyr Ile Asp Asn Ile Arg Asp 315 Glu Asp 330 Asp Glu Ser Lys Gin Phe Ala Thr 395 Lys Cys 410 Leu Thr Ile Pro 140 Asp Asn His Arg Tyr 220 His Asp Gly Leu Ser 300 Lys Ile Asn Asn Arg 380 Ile Tyr Lys Leu Leu 125 Thr Val Met Ile Ile 205 Ile Met Phe Ile Glu 285 Ile Val Ile Asn Asn 365 Leu Lys Ser His Met 110 Asp Val Ile Ile Pro 190 Ile Ser Ile Ile Lys 270 Ser Tyr Lys Arg Lys 350 Lys Asn Ser Leu Arg Arg Ile Ile Lys Lys Ala Asn Thr Ser 160 Ile Asp 175 Pro Thr Glu Leu Asp Ser Ile Ser 240 Ser Thr 255 Cys Val Leu Asp Asp Val Arg Glu 320 Tyr Ile 335 Val Glu Leu Ser Pro Cys Lys Leu 400 Thr Asn 415 -341- Val Ser Met Tyr Lys Lys Ile Lys Gly Val Ile Val Met Asp Met Val 420 425 430 Asp Tyr Ile Ser Thr Asn Ile Leu Lys Tyr His Lys Gin Leu Tyr Asp 435 440 445 Lys Met Ser Thr Phe Glu Tyr Lys Arg Asp Ile Lys Ser Cys Lys Cys 450 455 460 Ser Ile Cys Ser Asp Ser Ile Thr His His Ile Tyr Glu Thr Thr Ser 465 470 475 480 Cys Ile Asn Tyr Lys Ser Thr Asp Asn Asp Leu Met Ile Val Leu Phe 485 490 495 Asn Leu Thr Arg Tyr Leu Met His Gly Met Ile His Pro Asn Leu Ile 500 505 510 Ser Val Lys Gly Trp Gly Pro Leu Ile Gly Leu Leu Thr Gly Asp Ile 515 520 525 Gly Ile Asn Leu Lys Leu Tyr Ser Thr Met Asn Ile Asn Gly Leu Arg 530 535 540 Tyr G l y Asp Ile Thr Leu Ser Ser Tyr Asp Met Ser Asn Lys Leu Val S545 550 555 560 Ser Ile Ile Asn Thr Pro Ile Tyr Glu Leu Ile Pro Phe Thr Thr Cys 565 570 575 SCys Ser Leu Asn Glu Tyr Tyr Ser Lys Ile Val Ile Leu Ile Asn Val 580 585 590 Ile Leu Glu Tyr Met Ile Ser Ile Ile Leu Tyr Arg Ile Leu Ile Val 595 600 605 Lys Arg Phe Asn Asn Ile Lys Glu Phe Ile Ser Lys Val Val Asn Thr 610 615 620 Val Leu Glu Ser Ser Gly Ile Tyr Phe Cys Gln Met Arg Val His Glu *o 625 630 635 640 Gin Ile Glu Leu Glu Ile Asp Glu Leu Ile Ile Asn Gly Ser Met Pro 645 650 655 Val Gin Leu Met His Leu Leu Leu Lys Val Ala Thr Ile Ile Leu Glu 660 665 670 Glu Ile Lys Glu Ile 675 INFORMATION FOR SEQ ID NO:194: SEQUENCE CHARACTERISTICS: LENGTH: 64 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194: Lys Leu Tyr Thr Ile Ile Asp Thr Leu Pro Cys Pro Thr Cys Arg Ile 1 5 10 -342- His Ala Lys Glu Glu Leu Thr Lys His Asn 25 Ile Met Ser Ser Asn Asp Asn Leu Ala Ile Asn Tyr Ile Tyr Tyr Phe Phe Ile Arg Leu Phe Ser Asp Pro Lys Tyr Lys Ile Gin Leu Asp Lys Val 55 Ala Pro Leu Val rr r r r r r r INFORMATION FOR SEQ ID NO:195: SEQUENCE CHARACTERISTICS: LENGTH: 583 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Swinepox virus STRAIN: Kasza INDIVIDUAL ISOLATE: S-SPV-001 (ix) FEATURE: NAME/KEY: CDS LOCATION: 2..583 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195: A AGC TTA AGA AAG AAT GTA GGG AAC GAA GAA TAT AGA ACC AAA GAT Ser Leu Arg Lys Asn Val Gly Asn Glu Glu Tyr Arg Thr Lys Asp 1 r TTA TTT ACT GCA Leu Phe Thr Ala

TTA

Leu 20 TGG GTA CCT GAT Trp Val Pro Asp

TTA

Leu 25 TTT ATG GAA CGC Phe Met Glu Arg GTA GAA Val Glu AAA GAT GAA Lys Asp Glu TGC GAT GTA Cys Asp Val

GAA

Glu TGG TCT CTA ATG Trp Ser Leu Met CCA TGC GAA TGT Pro Cys Glu Cys CCA GGA TTA Pro Gly Leu GAA TAC GAA Glu Tyr Glu 142 190 TGG GGG AAT GAT Trp Gly Asn Asp

TTT

Phe 55 AAC AAA TTA TAT Asn Lys Leu Tyr

ATA

Ile ACA AAG Thr Lys AAA AAA ATT AAA Lys Lys Ile Lys

GCG

Ala 70 ATC GCT AAA GCA Ile Ala Lys Ala

AGA

Arg AGT TTA TGG AAA Ser Leu Trp Lys

TCT

Ser ATT ATC GAG GCT Ile Ile Glu Ala

CAA

Gin ATA GAA CAA GGA Ile Glu Gin Gly CCG TAT ATA CTA Pro Tyr Ile Leu AAA GAT TCT TGT Lys Asp Ser Cys AGA TCG AGT AAT Arg Ser Ser Asn 115

AAT

Asn 100 AAA AAA TCC AAT Lys Lys Ser Asn

CAA

Gin 105 AGC AAT TTG GGA Ser Asn Leu Gly ACA ATT Thr Ile 110 CTC TGT ACA GAG Leu Cys Thr Glu

ATT

Ile 120 ATA CAA TTT AGT Ile Gin Phe Ser AAC GAG GAT Asn Glu Asp 125 382

GAA

Giu

AAT

Asn.

CTA

Leu 160

ATA

Ile

GTT

Val1

GTT

Val1

AAT

Asn 145

GTT

Val1

GAA

Glu

CAG

Gin

GCT

Al a 130

AAC

Asn

AAA

Lys

TCG

Ser

GGT

Gly

GTA

Val

GTA

Val

AAT

Asn

TCT

Ser -343- TGT AAT CTA GGA TCT ATT TCG TGG AGT AAA Cys Asn Leu Gly Ser Ile Ser Trp Ser Lys 135 140 TTT ATG TTC GAC AAG TTG AGA ATA ATT ACG Phe Met Phe Asp Lys Leu Arg Ile Ile Thr 150 155 CTA.AAT AAA ATA ATA GAT ATC AAT TAT TAT Leu Asn Lys Ile Ile Asp Ile Asn Tyr Tyr 165 170 AGA TCT AAT AAG AAA CAT AGA CCC ATA GGT Arg Ser Asn Lys Lys His Arg Pro Ile Gly 180 185

TTT

Phe

AAA

Lys

CCA

Pro

ATC

Ile 190

GTT

Val

ATA

Ile

GTG

Val1 175

GGG

Gly 430 478 526 574 INFORMATION FOR SEQ ID NO: 196: SEQUENCE CHARACTERISTICS: LENGTH: 194 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196: Ser Leu Arg Lys Asn Val Gly Asn Giu Glu Tyr a. a a a.

Phe Asp Asp Lys Ile Asp Ser Val Asn 145 Val1 Thr Glu Val1 50 Lys Ile Se r Ser Al a 130 Asn Lys Ala Leu Giu Trp, Trp Gly Lys Ile Glu Ala Cys Asn 100 Asn Leu 115 Val Cys Val Phe Asn Leu Trp Ser Asn Lys Gin Lys Cys Asn Met Asn 165 Val Leu Asp Al a 70 Ile Lys Thr Leu Phe 150 Lys Pro Met Phe 55 Ile Glu Ser Giu Gly 135 Asp Ile Asp Leu 25 Cys Pro 40 Asn Lys Ala Lys Gin Gly Asn Gin 105 Ile Ile 120 Ser Ile Lys Leu Ile Asp Phe Met Cys Giu Leu Tyr Ala Arg 75 Thr Pro 90 Ser Asn Gin Phe Ser Trp Arg Ile 155 Ile Asn 170 Arg Glu Cys Ile Ser Tyr Leu Ser Ser 140 Ile Tyr Thr Lys Asp Arg Val Glu Pro Gly Leu Glu Tyr Glu Leu Trp Lys Ile Leu Tyr Gly Thr Ile 110 Asn Glu Asp 125 Lys Phe Vai Thr Lys Ile Tyr Pro Val 175 Leu Lys Cys Thr Ser Lys Arg Glu Asn Leu 160 Ile Giu Ser Ser Arg 180 gSer Asn Lys Lys His Arg Pro Ile Giy Ile Gly Val 80185 r -344- Gln Gly INFORMATION FOR SEQ ID NO:197: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197: S* ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 INFORMATION FOR SEQ ID NO:198: SEQUENCE CHARACTERISTICS: LENGTH: 138 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO e* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198: GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT CGATGGCTGT 120 GCCTGCAAGC CCACAGCA 138 INFORMATION FOR SEQ ID NO:199: SEQUENCE CHARACTERISTICS: LENGTH: 120 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:199: CTTAGCCCCA AACGCACCTC AGATCCATAA TTAATTAATT TTTATCCCGG CGCGCCTCGA -345- CTCTAGAATT TCATTTTGTT TTTTTCTATG CTATAAATGA ATTCGGATCC CGTCGTTTTA 120 INFORMATION FOR SEQ ID NO:200: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 INFORMATION FOR SEQ ID NO:201: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:202: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:202: ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 -346- INFORMATION FOR SEQ ID NO:203: SEQUENCE CHARACTERISTICS: LENGTH: 141 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:203: GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA Sa ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT CCATGTGCTG 120 CCTCACCCCT GTGCTGGCGC T 141 *a S' INFORMATION FOR SEQ ID NO:204: SEQUENCE

CHARACTERISTICS:

LENGTH: 120 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO no (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:204: TCGCCCGCCT CTGACGCCCC GGATCCATAA TTAATTAATT TTTATCCCGG CGCGCCTCGA CTCTAGAATT TCATTTTGTT TTTTTCTATG CTATAAATGA ATTCGGATCC CGTCGTTTTA 120 INFORMATION FOR SEQ ID NO:205: SEQUENCE CHARACTERISTICS: LENGTH: 116 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205: GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 -347- INFORMATION FOR SEQ ID NO:206: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:206: TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 INFORMATION FOR SEQ ID NO:207: SEQUENCE

CHARACTERISTICS:

LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) S(iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:207: S.CAAGGAATGG TGCATGCCCG TTCTTATCAA TAGTTTAGTC GAAAA INFORMATION FOR SEQ ID NO:208: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:208: TATATAAGCA CTTATTTTTG TTAGTATAAT AACACAATGC CAGATCCCGT CGTTTTA 57 INFORMATION FOR SEQ ID NO:209: -348- SEQUENCE CHARACTERISTICS: LENGTH: 249 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209: TCCAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA GATCCATAAT TAATTAACCA GCGGCCGCCT GCAGGTCGAC TCTAGATTTT TTTTTTTTTT TTTTTTGGCA 120 TATAAATAGA TCTGTATCCT AAAATTGAAT TGTAATTATC GATAATAAAT GAATTCGGAT 180 o^ CCATAATTAA TTAATTTTTA TCCCGGCGCG CCGGGTCGAC CTGCAGGCGG CCGCTGGGTC 240 •GACAAAGAT 249 INFORMATION FOR SEQ ID NO:210: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

S

xi) SEQUENCE DESCRIPTION: SEQ ID NO:210: CAAAAGTCGT AAATACTGTA CTAGAAGCTT GGCGTAATCA TGGTC INFORMATION FOR SEQ ID NO:211: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211: CGACGGATCC GAGGTGCGTT TGGGGCTAAG TGC 33 INFORMATION FOR SEQ ID NO:212: -349- SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212: CCACGGATCC AGCACAACGC GAGTCCCACC ATGGCT 36 INFORMATION FOR SEQ ID NO:213: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:213: CCACGAATTC GATGGCTGTG CCTGCAAGCC CACAG INFORMATION FOR SEQ ID NO:214: SEQUENCE CHARACTERISTICS: e LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214: CGAAGATCTG AGGTGCGTTT GG3GCTAAGT GC 32 INFORMATION FOR SEQ ID NO:215: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear -350- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215: CGCAGGATCC GGGGCGTCAG AGGCGGGCGA GGTG 34 INFORMATION FOR SEQ ID NO:216: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear S, (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO e e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216: **GAGCGGATCC TGCAGGAGGA GACACAGAGC TG 32 INFORMATION FOR SEQ ID NO:217: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:217: GCGCGAATTC CATGTGCTGC CTCACCCCTG TG 32 INFORMATION FOR SEQ ID NO:218: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO -351- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218: CGCAGGATCC GGGGCGTCAG AGGCGGGCGA GGTG 34 INFORMATION FOR SEQ ID NO:219: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219: GGGGAATTCA ATGCAACCCA CCGCGCCGCC CC 32 INFORMATION FOR SEQ ID NO:220: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220: GGGGATCCT AGGGCGCGCC CGCCGGCTCG CT 32 INFORMATION FOR SEQ ID NO:221: SEQUENCE CHARACTERISTICS: LENGTH: 5785 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221: AAGCTTAAGA AAGAATGTAG GGAACGAAGA ATATAGAACC AAAGATTTAT TTACTGCATT ATGGGTACCT GATTTATTTA TGGAACGCGT AGAAAAAGAT GAAGAATGGT CTCTAATGTG 120 -352-

TCCATGCGAA

AGAATACGAA

TATTATCGAG

TAAAAAATCC

TATACAATTT

TAAATTTGTT

AGTTAAAAAT

ATCTAATAAG

ATTATTGGGC

AACAATATAT

TGAGACATAT

GAACCCAACA

GAGAAATAGC

TAATGAGTCC

TTTTCAGGTT

TGACATAAAG

TAATATAAAA

GGCAGCCGAC

TCCTACATAC

TGGGATGTAT

TAGTAATTGT

TTAAAAATGC

ACTGATAATA

TCACTAAGTA

CCTTCTTGTT

ACATATATAG

TCGTTAGATC

TTATTAGATI

AATATATCT;

AAGATGGGTC

ATTTCCGAAC

ATTCCTATTC

TTTATCATAC

ATGTCCATGj

TGTCCAGGAT

ACAAAGAAAA

GCTCAAATAG

AATCAAAGCA

AGTAACGAGG

AATAATAACG

CTAAATAAA

AAACATAGAC

TATGCATTCG

TATGCCGCAC

AACGATTCTC

GATTTATGGG

CTTCTAATAG

ACCGAACCAT

GTAAATCCAC

AATACAATTG

CCAATATATA

AGAGGTGCGT

GCAAAACTGA

TATATGAGAA

TCTGCATAAT

GTAAAAGTGG

ATAAAAAACA

AGAGCACCGA

CATCGATAAC

CATTAGAAGG

*AATATTTTT-T

*GTATATATTC

TATCGGACTI

GAGCTATTA71 AACTAGAAA-7

TATGTTACAC

3 ATAAGGAGT'.

ATGAAACAT(

TATGCGATGT

AAATTAAAGC

AACAAGGAAC

ATTTGGGAAC

ATGAAGTTGC

TATTTATGTT

TAATAGATAT

CCATAGGTAT

ATAGCGAAGA

TAGAATCTAG

CAGCGAGTAA

ATTGGAATGA

CACCAATGCC

ATACTAGCAA

ACCTATTGAG

TGTTACATAA

AAACGGTTTG

TTATAGATCC

CCAGTATGCA

CAAAATCGGC

TTTTATAAAA

CATTCCAAAA

ACAAATAACA

AAAATGTATA

TATTAATTTPA

TGAGTCTAAP

*AAAAATACG;

TAATATTAG;

*AGAAGAGAAM

AGAATACAAC

TTTAACA-AAC

AAATGCAAA'

C TAGTACAAX! 3 CGAAGAGGA'

ATGGGGGAAT

GATCGCTAAA

GCCGTATATA

AATTAGATCG

TGTATGTAAT

CGACAAGTTG

CAATTATTAT

CGGTGTTCAG

AGCAAAAATA

TTGCGAACTA

AGGTATTCTA

ACTAAAAAAG

TACTGCATCT

TATATATACA

AGAACTAATA

TGGTTCTATT

GGAGATATCT

AAGTCAATCA

TTTTTACGGA

ATCAAATCCT

ATGAAATACT

CAACCCGTTC

ACGTGTTCAG

GAAAATGTAA

ACAGACTATC

LATATACAAGA

CTITACTGCAG

GATAATAAAC

k ACGCTTAACC 3ATACCTGGTI 3 CGAGATAAAC r AGTATAAAGC r' GTAATAGACC r GTTGCTAGAC

GATTTTAACA

GCAAGAAGTT T CTATATAAAG A AGTAATCTCT G CTAGGATCTA T AGAATAATTA C CCAGTGATAG GGTTTGGCTG P

TTAAATATAC

GCTAAAATTT P CAATATGATA I1 AGAATTAATA C ACATCTCAAA I AGAAGAGTAT I AGTAGAAATA I

CAACATTTAG

CCAAAATGTA

ATGACAATAT

TGGAGATTGG

ATAAAATTCA

ATCTCATGTA

CCAAAAAAGA

AAGTCGTTGA

TATTAACGCC

TATCATCTAA

ATAAAAAGAA

CAAGTCCTA-T

ATATACCCCC

AGGGGTGTTT

CCAAATCTAT

AAATATCTAA

TTACATTTGC

TAGATGGTAA

GATTGGGAAT

ATTATATkT

ATGGAAATC

*TTTTGTIA

TACAGAGAT

TTCGTGGAG

~GAAAATACT

ATCGTCTAG

LTGTGTTTAT

LAATTTCCGA

~CGGACCTTA

'GTGGTTAAA

ACATGGATT

ATTAAGTA-A

~ATCTGGAGA

~GTGGAATAA

kTTTACCAGA

PTTTAGAAAT

kTATAGATAA 3

GCTAAAAAC

CAGTTGAGTG

rCTTAATATA

TTATGTTCAA

GTATCTTAAA

TTCTCAATAT

AATGACATCT

TGAAAGTAGA

AATGTATCAA

TTCCTTATCA

GTTCATTAAT

AACAAAATCT

AATTATAGTT

ACTAAAAAAG

ACATGAAAAA

TATAGATCTT

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1-740) 1800 1860 1920 1980 2040 2100 2160 -353-

GAAGATGAAT

AAATTGTATG

TTTATAGAAG

TCCAATAATA

TTAGTTTTTA

CAACTCGTTT

AATGATATGG

AAACTCATAG

AAACTAGAAA

AATTACTTTG

GACATAGAGA

AGTGACGTTA

TCATGAATAT

CCATGATATT

TGATCTTTTG

TATTTCTAGA

AATATACGTT

GATAGGATCA

TGATAGATAT

GGATAATGAA

TATAGAGTTA

CATTTAGTTT

ATATATAAGC

GAACGCAAGA

TAAAAAATAT

GTATAAAGAT

TATGGTTAAA

TCAATCTGTA

TTTAATGATC

AATAATTCAT

ACTGAATAAG

TTTATATAAT

CATACCCCCC

GCTTAAAAA.P

GCATAGAGGA

CGGCAATATT

TTGTAAAATC

ATACATGGAT

TTTTGTATTT

TAAATAGTAT

TCGATGAATT

ATACACGAAT

ATTATAAAAA

AACAAGATGG

AAAAGATTTT

AAGTTCTCGC

ATTATACAAT

ATACTAGATG

AATATACTTC

ACACGTCTAT

ATATTGAAAG

TTTGTATATA

TCAAAAAGGT

A.AAATTAATG

TTAACTGAAT

TGCTGTATGG

ACTTATTTTT

AAAGTAATTT

ATAAATACCA

GCCGTATCAT

GATTTAATAA

TTAAAACATC

ATTAATAAGT

GTAACAAAAA

ATAGAGCTTG

AATATGATCA

ACACATATAT

TATCCGAATP

AGATGATGTC

CGGCGTTT

TGTATTAACA

ATATATATTT

AAAAGTTACT

TAGTGCTAGA

AGTTACCAAA

TCCTATTGAT

TGGAATTTTA

TAAACTTAAT

ATCGTTGATT

ATCTAGGTTA

CTGATAAAAT

AAAAAAGTTC

GTGAAAGATT

ATGATTATTT

ATTTAAAGAT

CGAAATATAG

TCCATGACTC

TATCCAGAGT

AAGTATATAT

TTATCATATA

GTTAGTATAA

CAAAGATTAT

TGTTAGAATT

ATGATATAAA

ATGTTATAAA

GTAGAGCGTT

TACTATCGAT

TATTAAAAAT

TAGATGTAAT

TTGATCTCGA

CATTACGTTC

ATAATATTAXI

GATACGTCAT

ATGACATCTA

GATACATCAT

CTAGCGATAT

AAACCAACTT

GCATTAAAGG

AAATATCCAC

CTTATTAATC

TCCGTTCTTA

AGCAGTGATA

CCTAGATGTT

AAAAAGTGCT

AGAGGAATTA

TATTA.AAGAC

ATTTAGATGT

TACTAGAGTG

TGCTGATATA

CATGTTGATT

TTTTTATGAA

TGTTGAATGC

AAATGATTGT

CATTTTTAAG

TAACACAATG

ATCATTACAA

TGGTCTACAT

A;ATATAAGA

ATCATCATCT

AATAGATTAC

AGATGATATA

AGACCCTACA

AACATCAATA

TACATTATTA

ACTTAGAGAT

TGATTATATA

TATTTAATGT

AAGATGATGA

CTALATCATAC

TATTTGGTGT

AAATGGAGGA

CATTTTTTGT

CAAAGAAGAA

AACAATTCGT

TCAATAGTTT

TTGATGAATT

CTCCTCTTTA

AAATCATTTA

ATAAATAGTT

AGCATATATA

CCACAGGTTA

TCAAAGAAAG

CTCGGTATCG

AATTCAATTT

GATATTGCGG

CTTATCGTAC

TTTTATAATG

GCCGTATATG

CCGTCGTATA

CTTGATATTA

GGAAATCTAC

TTTTTACCTT

GTAATAGATA

GGCGATCAAG

TCCTATATAT

GTAGCCAATT

CCTAAGTCTT

*TATTTATCCG

*ATAAACAGGA

*TCCGATAGCA

ATAAATGGAT

TTTTAATAAC

AATATCGTCG

TATGGTATTA

P.GCAGATAAC

A.TCTAAAATT

P.TCACAA.ATA

TAAAAGATTT

AGTCGAAAAT

AGTGCTCACA

TATAGATATC

CGTTTAATGA

TATCTAGAAA

TACTATCTGA

AAGATAATAC

AAGAAGCGAA

AAACAGTAAC

CGTCTAATGT

AATTTATAAA

CTAATATTAA

TTTGTTATCG

ATAAATGAAA

TGTATCCGAA

AAAAACTTCC

CAGCTTGTAT

ATAATTGTGT

CTAGATTACA

ACATTATCAC

TAGATAAAAA

CAAACATGAA

CCTATACATA

ATGCATTCCA

TTATTGAATT

TAAAALTCAAA

2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200

S

S.

TAGTTCATTC

TAGTGTAAAC

GATTAAGTGT

TGACTCAGAT

TTTTGCGAAT

ATGTGAAGAT

TAATAAGGTG

ACTGTCTGAT

TATATTATTA

CTGGAA.ATGT

AATGGATATG

TAAAATGAGT

CGACTCTATA

TAATGATCTT

TCCTA.ATCTT

AGGTATTAAT

TACGTTATCT

TGAGTTAATA

TTTAATAAAT

AAAAAGATTT

ATCAGGCATA

GCTCATTATT

CATAATATTA

ACTTTTTTTT

ATGCAAGATT

ACATGATATT

ATGTATCTAT

ATTCACATAC

GATTTTATAT

GTTGCTATGT

TACATATTTA

ATGATTAGAG

ATTATAAGAT

GAGGAGGTGT

ATATCATCTT

TCTTCAGCAA

TATTCATTGA

GTTGATTATA

ACGTTTGAAT

ACACATCATA

ATGATAGTAT

ATAAGCGTAA

TTAAAACTAT

TCATACGATA

CCGTTTACTA

GTTATTTTAG

AATAACATTA

T.ATTTTTGTC

AATGGATCTA

GAGGAAATCA

TTAAACAAGG

ATTAAATAAT

ATGTTTAGTT

AATGGTATAA

TTCATATGAT

CTACCGTAGT

TTTCGTACGA.

TAGAAAAA

ATAAGGTTAA

ATATAAAATT

TGATACATAT

TAATGGATCA

CTATAAAATC

CAAATGTATC

TATCTACTAA

ATAAACGAGA

TATATGAAAC

TGTTCAATCT

AAGGATGGGG

ATTCCACCAT

TGAGTAATAA

CATGTTGTTC

AATATATGAT

AAGAATTTAT

AGATGCGTGT

TGCCTGTACA

AAGAAATATA

GGTGCTACCT

CGTATGAAAA

AATTCTTCTT

AGCTT

-354-

AATATCAAAT

TGATAAAGAT

TATAAACATG

TATATCTATA

AAGAGAAAAG,

ATTCAGTPAA

TGATAATGTA

ATTTCGTTTA

AAAACTATTA

AATGTATAAA

CATTCTTAAA

TATTAAATCA

AACATCATGT

AACTAGATAT

TCCCCTTATT

GAATATAAAT

ATTAGTCTCT

ACTCAATGAA

ATCTATTATA

TTCAAAAGTC

ACATGAACAA

GCTTATGCAT

ACGTATTTTT

TGTCTA.ATTG

AGTAGTAGAT

TGGCATGAAT

ATGTTTCCTG

CGACTTATTA

ATCGATTTAG

TACGACGTTA

AATAGAATAT

AATAGAATAA

TCTAAAAATA

AATCCATGTA

GCGTTACGGG

AAAATAAAGG

TACCATAAAC

TGTAAATGCT

ATAAATTATA

TTAATGCATG

GGATTATTAA

GGGCTACGGT

ATTATTAATA

TATTATTCAA

TTATATAGAA

GTAAATACTG

ATTGAATTGG

TTACTTCTAA

TCTTTTAA.AT

TATCTTGTAT

ATAGTTTATA

TCTACACGTC

CTATAATCCC

ATATGTATGG

AGTCATTAGA

AATGTAGAGA

TAACTACGAA

ACGATGAAAA

ATAAATTATC

CCATAAGAAA

CAGTAAAAA

GTGTTATCGT

AATTATATGA

CGATATGTTC

AATCTACCGA

GGATGATACA

CGGGTGATAT

ATGGAGATAT

CACCCATATA

AAATTGTGAT

TATTGATCGT

TACTAGA-ATC

AAATAGATGA

AGGTAGCTAC

AAATAAAAAT

TTTGGATCTG

TCGTTACTGG

GGACAAGGTA

4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 57 INFORMATION FOR SEQ ID NO:222: SEQUENCE CHARACTERISTICS: LENGTH: 722 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N -3 (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:222: TTTTGATTTT ACGCCATTAT ACTGTTCTGT AGATGCAAAT AATGAAGATG TGTTCTTATT

TACTAGAGAG

TTCAATAATC

TATCCAAAAT

CTATTACAAT

TAAGAGGTAT

TTTCGTGA.A

GGTAAACGGA

CGTGTAAAAA

TAATAAAATC

CTATCTGTTC

TTATTAATAA

AA

ATGCAGACCC

ATAATCCCCC

ACATTATTTA

AATCTTGTTA

GAAAAAAATA

TTTATAGATA

TATATATTCT

GCTAAAGAAA

TTCAAAGATT

GGAAGAAACT

TATAAACAAT

TATATTATCA

ATATTATATA

AACTGGAACA

AAAAAGAACA

TAGGAATATT

CGTGTATGAT

ATTATATTTA

TACGAATCCT

AGATATGCGA

AGTTTATGGA

CATGATATGT

CAGTATTTGG

ACTATTAAAT

AGATATTATT

TTTTATAAAA

ATTTCTTGAT

GGAAATATTT

CGAATAAGTA

TTGTTGTATA

GTGTTATA-AA

ACAGCATCCC

ATATATTATA

TGAACGTGTA TACTAACAGC TATGATATAG ATATAAATAC

AACTCTACCA

TTATTTCTAG

TATCCCACTC

GAGATGAAAT

TATTCCTATC

TGGAACCGAA

AGTAACGGAG

AATTGAGATA

TAAAAATAGA

TAGATACTTA

CCTACATAGT

TTGGTGAATA

CAGATAAGGT

CCATATATAA

ATAAAATCAA

TATATCACAT

AAACATAAGA

AAAAAAAAAT

120 180 240 300 360 420 480 540 600 660 720 INFORMATION FOR SEQ ID NO:223: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 234 base pairs TYPE: nucleic acid STRANflEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223: AAACAATGCG CTTTAATATC AAACATGCAG GTGGAATAGG ATTGTCGATA AGTAATATAC GAGCTAAGGG TACTTATATA TCCGGTATAA ACGGCAAATC TATGGTATAG TACCTATGTT AAGAATATAT AATAACACAG TTAGATATAT TAATCAGGGA GGTGATAAAA GACCAGGAGC AATGTCGATT TATATAGAAC CATGGCACGC TGATATATTC GATTTTCTAA. GCTT INFORMATION FOR SEQ ID NO:224: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 1025 base pairs TYPE: nucleic acid STRANDEDNESS: single (Di) TOPOLOGY: linear 120 180 2 34 -356- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224: GGTTGCTCCT AACTTAATAA GATAATCCAC CAAGATAGTT *0

CACAACAGGA

TGGGACCGCC

CGACATCATT

TATATTTAAA

ACTATTATTT

ATCTATATTC

GTTA.ATGTCA

AAATGTAAAA

ACCTTTATTA

CGATGAACTT

TGATATTATT

AATGTATTCA

TTATGACATT

TATCGTTGTA

ATTACATAAA

TCTATATGTG

AGCTT

GAATATCCTA

GAAAAACATG

TCATCATTTT

TTAGTGTTAT

AGTTTACATA

TCATTACCAA

CCACCATGAT

TAACAATGAA

AGCAGTAATA

AAAAATGTAG

CTTCTTATAT

TGGGCTACTA

ATTACTTAAG

TTTCTATAGG

TCGCTTTATC

ATCCATACAT

ATTTATCTCT

TATAATCGTC

TATTATATTC

TGATGTGTCT

CATCAAATCC

GGTATTTATG

AAATAAGAAA

ATGGTGTTTT

ATACAATTTC

CTAACATATC

TATCCATTTC

ATAATAGCCA

TATTATTGAT

AATCCTCATC

AATGTTAGAA

TACTAAATTT

ATAGTTTATG

GTGACAATAG

ATGTTTTATA

AATTGTAAAT

TTTTCTTATT

CAATATGGTG

CTTTATTACT

ATCATATATA

TTTCAACTCT

AGTGGCTATA

CTTCTTACAA

CATTACTAGA

AAGTCCTTCA

CAGTAACTAT

TAATACATAT

TTTAATCTTA

TTATCCGTGG

GTTGTGATAT

TTTAACATCG

TGCGAACAAA

ATATGAATAG

AAAAACTCAA

TCTCCACATC

TTAATTGTAA

GATATCCCAT

TTTAATTTAA

TTATCATCCT

ACTATTTAA

AAAAAAATTT

TTGTTAA.ATG

GTTTCTTGCA

ATGTATTTTT

AAAAATTATC

TAGATGCATA

CTATAGTATA

TGTTTAATAT

GCAAATTCAA

GATTCTTCAG

CAACTTTATA

TATGTACACT

CATTCGTATT

TTAAATTAGC

ATACGTGCAA

GTTTTATATT

CGATAACCAA

TTTTTCAATA

TAATAATATA

GTGCTTCATA

GATAATATTT

ATAATTGAGA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1025 INFORMATION FOR SEQ ID NO:22S: SEQUENCE CHARACTERISTICS: LENGTH: 305 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:225: AAGCTTGGAT GAGCAATAAG AGTATACAAA ATTTAGTGTT TCAATTCGCT CATGGATCAG AAGTAGAATA TATAGGTCAA TACGATATGA GATTTTTAAA TAATATACCT ATTCATGATA -357- AGTTTGATGT GTTTTTAAAT AAGCACATAC TATCGTATGT ACTTAGAGAT AAAATAAAGA 180 AATCAGACCA CAGATATGTA ATGTTTGGAT TTTGGTTATT TATCTCATTG GAAATGTGTT 240 ATATTCGATA AGGAACATCA TATGTCTGTT TCTATGATTC AGGAGGAATT ACCAAACGAA 300 TTCCA 305 INFORMATION FOR SEQ ID NO:226: SEQUENCE CHARACTERISTICS: LENGTH: 1721 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N 9* (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1721 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226: ATG AAT TCG GAT CCG GCA ATA CTA TTA GTC TTG CTA TGT ACA TTT ACA 48 Met Asn Ser Asp Pro Ala Ile Leu Leu Val Leu Leu Cys Thr Phe Thr 1 5 10 ACC GCA AAT GCA GAC ACA TTA TGT ATA GGT TAC CAT GCA AAT AAT TCA 96 Thr Ala Asn Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 25 ACT GAC ACT GTT GAC ACA GTA CTA GAA AAG AAT GTA ACA GTA ACA CAC 144 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 TCT GTT AAC CTT CTA GAA GAC AGA CAT AAC GGG AAA CTA TGT AAA CTA 192 Ser Val Asn Leu Leu Glu Asp Arg His Asn Gly Lys Leu Cys Lys Leu 55 AGA GGG GTA GCC CCA TTG CAT TTG GGT AAA TGT AAC ATT GCT GGA TGG 240 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 70 75 CTC CTG GGA AAC CCA GAG TGT GAA TTA CTA TTC ACA GCA AGC TCA TGG 288 Leu Leu Gly Asn Pro Glu Cys Glu Leu Leu Phe Thr Ala Ser Ser Trp 90 TCT TAC ATT GTG GAA ACA TCT AAT TCA GAC AAT GGG ACA TGT TAC CCA 336 Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 GGA GAT TTC ATC AAT TAT GAA GAG CTA AGA GAG CAG TTG AGC TCA GTG 384 Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 TCA TCA TTT GAA AGA TTT GAG ATA TTC CCC AAG GCA AGT TCA TGG CCC 432 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Ala Ser Ser Trp Pro 130 135 140 -358-

AAT

Asn 145 CAT GAA ACG AAC His Giu Thr Asn

ATA

Ile 150 GGT GTG ACG GCA Gly Val Thr Ala TGT CCT TAT GCT Cys Pro Tyr Ala 480 GCA A-AC AGC TTC Ala Asn Ser Phe

TAC

Tyr 165 AGA AAC TTA ATA Arg Asn Leu Ile

TGG

Trp, 170 CTG GTA AAA AAA Leu Val Lys Lys GGA AAT Gly Asn 175 TCA TAC CCA Ser Tyr Pro GTC CTC GTG Val Leu Val 195 CTC AGC AAA TCC Leu Ser Lys Ser ATT AAC AAT AAG Ile Asn Asn Lys GAG AAG GAA Giu Lys Glu 190 CTA TGG GGC ATT Leu Trp Gly Ile

CAC

His 200 CAT CCA CCT ACC AGT ACT GAC CAP.

His Pro Pro Thr Ser Thr Asp Gin 205 CAA AGT Gin Ser 210 CTC TAC CAG AAT Leu Tyr Gin Asn GAT GCC TAT GTT Asp Ala Tyr Val

TTT

Phe 220 GTG GGG TCA TCA Val Gly Ser Ser

AAA

Lys 225 TAC AAC AAG AAA Tyr Asn Lys Lys

TTC

Phe 230 AAG CCA GAA ATA Lys Pro Giu Ilie ACA AGA CCC AAG Thr Arg Pro Lys AGA GGT CAA GCA Arg Gly Gin Ala

GGG

Gly 245 AGA ATG AAC TAT Arg Met Asn Tyr

TAC

Tyr 250 TGG ACG CTA GTA Trp Thr Leu Val AAG CCT Lys Pro 255 GGA GAC ACA Gly Asp Thr TAT GCC TTC Tyr Ala Phe 275

ATA

Ile 260 ACA TTC GAA GCA Thr Phe Giu Ala GGA AAT CTA GTG Gly Asn Leu Val GTA CCA AGA Val Pro Arg 270 ATC ATT TCA Ile Ile Ser GCA ATG AAA AGA Ala Met Lys Arg

GGT

Gly 280 TCT GGA TCT GGT Ser Gly Ser Gly GAT ACA Asp Thr 290 CCA GTC CAC GAT Pro Val His Asp

TGT

Cys 295 AAT ACG ACT TGT Asn Thr Thr Cys

CAA

Gin 300 ACA CCC AAA GGT Thr Pro Lys Gly

GCT

Ala 305 ATA AAC ACC AGC Ile Asn Thr Ser CCA TTT CAG AAT Pro Phe Gin Asn

ATA

Ile 315 CAT CCA GTC ACA His Pro Val Thr

ATT

Ile 320 GGA GAA TGT CCA Gly Giu Cys Pro

AAA

Lys 325 TAT GTC AAA AGC Tyr Val Lys Ser

ACA

Thr 330 AAA TTG AGA ATG Lys Leu Arg Met GCT ACA Ala Thr 335 GGA TTA AGG Gly Leu Arg ATT GCT GGC Ilie Ala Gly 355

AAT

Asn ATC CCG TCT ATT Ile Pro Ser Ile TCT AGA GGC CTG Ser Arg Gly Leu TTT GGA GCC Phe Gly Ala 350 TTT ATT GAG GGG Phe Ile Giu Gly

GGA

Gly 360 TGG ACA GGA ATG ATA GAT GGC TGG Trp Thr Gly Met Ilie Asp Gly Trp 365 1008 1056 S10j4 1152 1200 1248 TAC GGT Tyr Gly 370 TAT CAC CAT CAG Tyr His His Gin

AAT

Asn 375 GAG CAG GGA TCA Giu Gin Gly Ser

GGA

Gly 380 TAT GCA GCC GAC Tyr Ala Ala Asp

CGA

Arg 385 AAG AGC ACA CAG Lys Ser Thr Gin GCC ATT GAC GGG ATC ACT AAC AAA GTA Ala Ile Asp Gly Ile Thr Asn Lys Val

AAC

As n 400 TCT GTT ATT GAA Ser Val Ile Giu

AAG

Lys 405 ATG AAC ACA CAA TTC ACA GCA GTG GGT Met Asn Thr Gin Phe Thr Ala Vai Gly 410 AAA GAA Lys Giu 41S -359- TTC AAC CAC CTG GAA AAA AGA ATA GAG AAT TTA AAC AAA AAG GTT GAT Phe Asn His Leu Glu Lys Arg Ile Glu Asn 420

GAT

Asp

TTG

Leu

CTA

Leu 465

GGA

Gly

GAG

Glu

GCA

Ala

AGG

Arg

GTA

Val 545

GGG

Gly

GGT

Gly

GAA

Glu 450

TAT

Tyr

AAT

Asn

AGC

Ser

AAA

Lys

ATT

Ile 530

CTG

Leu

TCT

Ser

TTT

Phe 435

AAT

Asn

GAG

Glu

GGC

Gly

GTC

Val

CTA

Leu 515

TAC

Tyr

TTA

Leu

TTA

Leu

CTG

Leu

GAA

Glu

AAA

Lys

TGC

Cys

AAA

Lys 500

AAC

Asn

CAG

Gin

GTC

Val

CAG

Gin

GAT

Asp

AGA

Arg

GTA

Val

TTT

Phe 485

AAT

Asn

AGA

Arg

ATT

Ile

TCC

Ser

TGC

Cys 565

GTT

Val

ACT

Thr

AGA

Arg 470

GAA

Glu

GGG

Gly

GAG

Glu

TTG

Leu

CTG

Leu 550

AGA

Arg

TGG

Trp

TTG

Leu 455

AGC

Ser

TTT

Phe

ACT

Thr

GAG

Glu

GCG

Ala 535

GGG

Gly

ATA

Ile

ACT

Thr 440

GAT

Asp

CAG

Gin

TAC

Tyr

TAT

Tyr

ATA

Ile 520

ATC

Ile

GCA

Ala

TGT

Cys 425

TAC

Tyr

TAT

Tyr

CTA

Leu

CAC

His

GAT

Asp 505

GAT

Asp

TAT

Tyr

ATC

Ile

ATT

Ile

AAT

Asn

CAC

His

AAA

Lys

AAA

Lys 490

TAC

Tyr

GGG

Gly

TCA

Ser

AGT

Ser

TAA

570 Leu

GCC

Ala

GAT

Asp

AAC

Asn 475

TGT

Cys

CCA

Pro

GTA

Val

ACT

Thr

TTC

Phe 555

AAT

Asn Asn

GAA

Glu

TCA

Ser 460

AAT

Asn

GAT

Asp

AAA

Lys

AAG

Lys

GTC

Val 540

TGG

Trp

TAG

Lys

CTG

Leu 445

AAT

Asn

GCC

Ala

GAC

Asp

TAC

Tyr

CTG

Leu 525

GCC

Ala

ATG

Met

GAT

Asp Lys 430

TTG

Leu

GTG

Val

AAG

Lys

ACG

Thr

TCA

Ser 510

GAA

Glu

AGT

Ser

TGC

Cys

CC

Val

GTT

Val

AAG

Lys

GAA

Glu

TGC

Cys 495

GAG

Glu

TCA

Ser

TCA

Ser

TCC

Ser Asp

CTA

Leu

AAC

Asn

ATT

Ile 480

ATG

Met

GAA

Glu

ACA

Thr

TTG

Leu

AAT

Asn 560 1296 1344 1392 1440 1488 1536 1584 1632 1680 1721 r o u r n INFORMATION FOR SEQ ID NO:227: SEQUENCE CHARACTERISTICS: LENGTH: 573 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asn Ser Asp Pro Ala Ile Leu Leu Val 1 5 10 Thr Ala Asn Ala Asp Thr Leu Cys Ile Gly 25 Thr Asp Thr Val Asp Thr Val Leu Glu Lys 40 Ser Val Asn Leu Leu Glu Asp Arg His Asn 55 Arg Gly Val Ala Pro Leu His Leu Gly Lys 70 NO:227: Leu Leu Cys Thr Phe Thr Tyr His Ala Asn Asn Ser Asn Val Thr Val Thr His Gly Lys Leu Cys Lys Leu Cys Asn Ile Ala Gly Trp 75 -360- Leu Leu Gly Asn Pro Giu Cys Giu Leu Leu Phe Thr Ala Ser Ser Trp 90 Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asn Tyr Giu Giu Leu Arg Giu Gin Leu Ser Ser Vai 115 120 125 Ser Ser Phe Glu Arg Phe Giu Ile Phe Pro Lys Ala Ser Ser Trp Pro 130 135 140 Asn His Giu Thr Asn Ile Giy Val Thr Ala Ala Cys Pro Tyr Ala Gly 145 150 155 160 Ala Asn Ser Phe Tyr Arg Asri Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asn Lys Glu Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Pro Thr Ser Thr Asp Gin 195 200 205 Gin Ser Leu Tyr Gin Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser *210 215 220 *Lys Tyr Asn Lys. Lys Phe Lys Pro Giu Ile Ala Thr Arg Pro Lys Val :225 230 235 240 Arg Gly Gin Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Lys Pro 245 250 255 Gly Asp Thr Ile Thr Phe Glu Ala Thr Giy Asn Leu Val Val Pro Arg a..260 265 270 Tyr Ala Phe Ala Met Lys Arg Gly Ser Gly Ser Gly Ile Ile Ile Ser 275 280 285 *Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gin Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gin Asn Ile His Pro Val Thr Ile 305 310 315 320 .Gly Giu Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Met Ala Thr 325 330 .335 Gly Leu Arg Asn Ile Pro Ser Ile Gin Ser Arg Gly Leu Phe Gly Ala 340 345 350 Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met I~e Asp Gly Trp 355 360 365 Tyr Gly Tyr His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp 370 375 380 Ang Lys Sen Thr Gin Asn Ala Ile Asp Gly Ile Thr Asn Lys Val Asn 385 390 395 400 Ser Val Ile Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Giu 405 410 415 Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp 420 425 430 Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Giu Leu Leu Val Leu -361- 435 440 445 Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn 450 455 460 Leu Tyr Glu Lys Val Arg Ser Gin Leu Lys Asn Asn Ala Lys Glu Ile 465 470 475 480 Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asp Thr Cys Met 485 490 495 Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu 500 505 510 Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr 515 520 525 Arg Ile Tyr Gin Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu 530 535 540 Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn 545 550 555 560 Gly Ser Leu Gin Cys Arg Ile Cys Ile Asn .Asp 565 570 INFORMATION FOR SEQ ID NO:228: SEQUENCE CHARACTERISTICS: LENGTH: 1414 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1414 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID N0:228: ATG AAT TCA AAT CAA AAA ATA ATA ACC ATT GGG TCA ATC TGT CTG ATA 48 Met Asn Ser Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Cys Leu Ile 1 5 10 GTT GGA ATA GTT AGT CTA TTA TTG CAG ATA GGA AAT ATA GTC TCG TTA 96 Val Gly Ile Val Ser Leu Leu Leu Gin Ile Gly Asn Ile Val Ser Leu 25 TGG ATA AGC CAT TCA ATT CAG ACT GGA GAA AAA AAC CAC TCT GAG ATA 144 Trp Ile Ser His Ser Ile Gin Thr Gly Glu Lys Asn His Ser Glu Ile 40 TGC AAC CAA AAT ATC ATT ACA TAT GAA AAC AAC ACA TGG GTG AAC CAA 192 Cys Asn Gin Asn Ile Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gin 55 ACT TAT GTA AAC ATT AGC AAT ACC AAC ATT GCT GAT GGA CAG GGC GTG 240 Thr Tyr Val Asn Ile Ser Asn Thr Asn Ile Ala Asp Gly Gin Gly Val -362- 70 75 ACT TCA ATA ATA CTA GCC GGC AAT CCC CCT CTT TGC CCA ATA ATT GGG 288 Thr Ser Ile Ile Leu Ala Gly Asn Pro Pro Leu Cys Pro Ile Ile Gly 90 TGG GCT ATA TAC AGC AAA AAC AAT AGC ATA AGG ATT GGT CCC AAA GGA 336 Trp Ala Ile Tyr Ser Lys Asn Asn Ser Ile Arg Ile Gly Pro Lys Gly 100 105 110 AAC ATT TTT GTC ATA AAA AAA CCA TCC ATT TCA TGC TCT CAC TTG GAG 384 Asn Ile Phe Val Ile Lys Lys Pro Ser Ile Ser Cys Ser His Leu Glu 115 120 125 TGC AAA ACC TTT TTC CTG ACC CAA GGT GCT TTG CTA AAT GAC AGG CAT 432 Cys Lys Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Arg His 130 135 140 CCT AAT GGA ACC GTC AAG GAC AGG AGC CCT TAC CGA ACC TTA ATG AGC 480 Pro Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 ,**TGC CCG ATC GGT GAA GCT CCA TCT CCG TAT AAT TCA AGA TTC GAA TCA 528 Cys Pro Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 GTT GCT TGG TCA GCA AGT GCA TGC CAT GAT GGA ATG GGA TGG CTA ACA 576 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 ATC GGG ATT TCC GGT CCA GAT AAT GGA GCA GTG GCT GTT TTG AAA TAC 624 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 AAT GGT ATA ATA ACA GAT ACA ATA AAA AGT TGG AGA AAC AAA ATA CTA 672 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Lys Ile Leu 210 215 220 AGA ACA CAA GAG TCA GAA TGT GTT TGT ATA AAC GGT TCA TGT TTT ACT 720 Arg Thr Gin Glu Ser Glu Cys Val Cys Ile Asn Gly Ser Cys Phe Thr 225 230 235 240 ATA ATG ACT GAT GGC CCA AGC AAT GGG CAA GCC TCG TAC AAA ATA TTC 768 Ile Met Thr Asp Gly Pro Ser Asn Gly Gin Ala Ser Tyr Lys Ile Phe 245 250 255 AAA ATG GAG AAA GGG AAG ATT ATT AAG TCA GTT GAG CTG GAT GCA CCT 816 Lys Met Glu Lys Gly Lys Ile Ile Lys Ser Val Glu Leu Asp Ala Pro 260 265 270 AAT TAC CAC TAT GAG GAA TGC TCC TGT TAC CCT GAT ACA GGC AAA GTG 864 Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 GTG TGT GTG TGC AGA GAC AAT TGG CAT GCT TCA AAT CGA CCG TGG GTC 912 Val Cys Val Cys Arg Asp Asn Trp His Ala Ser Asn Arg Pro Trp Val 290 295 300 TCT TTC GAT CAG AAT CTT GAT TAT CAG ATA GGG TAC ATA TGC AGT GGG 960 Ser Phe Asp Gin Asn Leu Asp Tyr Gin Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 GTT TTC GGT GAT AAT CCG CGT TCT AAT GAT GGG AAA GGC AAT TGT GGC 1008 Val Phe Gly Asp Asn Pro Arg Ser Asn Asp Gly Lys Gly Asn Cys Gly 325 330 335 CCA GTA CTT TCT AAT GGA GCA AAT GGA GTG AAA GGA TTC TCA TTT AGA 1056 -363- Pro Val Leu Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 TAT GGC AAT GGT GTT TGG ATA GGA AGA ACT AAA AGT ATC AGC TCT AGA 1104 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg 355 360 365 AGT GGA TTT GAG ATG ATT TGG GAT CCA AAT GGA TGG ACG GAA ACT GAT 1152 Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 AGT AGT TTC TCT ATA AAG CAG GAT ATT ATA GCA TTA ACT GAT TGG TCA 1200 Ser Ser Phe Ser Ile Lys Gin Asp Ile Ile Ala Leu Thr Asp Trp Ser 385 390 395 400 GGA TAC AGT GGA AGT TTT GTC CAA CAT CCT GAA TTA ACA GGA ATG AAC 1248 Gly Tyr Ser Gly Ser Phe Val Gin His Pro Glu Leu Thr Gly Met Asn 405 410 415 TGC ATA AGG CCT TGT TTT TGG GTA GAG TTA ATC AGA GGA CAA CCC AAG 1296 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Gin Pro Lys 420 425 430 SGAG AGC ACA ATC TGG ACT AGT GGA AGC AGC ATT TCT TTC TGT GGC GTG 1344 t Glu Ser Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 GAC AAT GAA ACC.GCA AGC TGG TCA TGG CCA GAC GGA GCT GAT CTG CCA 1392 Asp Asn Glu Thr Ala Ser Trp Ser Trp Pro Asp Gly Ala Asp Leu Pro 450 455 460 TTC ACC ATT GAC AAG TAG ATC T 1414 Phe Thr Ile Asp Lys Ile 465 470 INFORMATION FOR SEQ ID NO:229: SEQUENCE CHARACTERISTICS: LENGTH: 471 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229: Met Asn Ser Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Cys Leu Ile 1 5 10 Val Gly Ile Val Ser Leu Leu Leu Gin Ile Gly Asn Ile Val Ser Leu 25 Trp Ile Ser His Ser Ile Gin Thr Gly Glu Lys Asn His Ser Glu Ile 40 Cys Asn Gin Asn Ile Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gin 55 Thr Tyr Val Asn Ile Ser Asn Thr Asn Ile Ala Asp Gly Gin Gly Val 70 75 Thr Ser Ile Ile Leu Ala Gly Asn Pro Pro Leu Cys Pro Ile Ile Gly 90 Trp Ala Ile Tyr Ser Lys Asn Asn Ser Ile Arg Ile Gly Pro Lys Gly 100 105 110 -364- Asn Ile Phe Val Ile Lys Lys Pro Ser Ile Ser Cys Ser His Leu Glu r r r

C

115 Cys Lys Thr 130 Pro Asn Gly 145 Cys Pro Ile Val Ala Trp Ile Gly Ile 195 Asn Gly Ile 210 Arg Thr Gin 225 Ile Met Thr Lys Met Glu Asn Tyr His 275 Val Cys Val 290 Ser Phe Asp 305 ial Phe Gly Pro Val Leu ryr Gly Asn 355 Ser Gly Phe 370 3er Ser Phe 385 3iy Tyr Ser 'ys Ile Arf ,iu Ser Thr 435 sp Asn Glu Phe Thr Gly Ser 180 Ser Ile Glu Asp Lys 260 Tyr Cys Gin Asp Ser 340 Gly Glu Ser Gly Pro 420 Ile Thr Phe Val Glu 165 Ala Gly Thr Ser Gly 245 Gly Glu Arg Asn Asn 325 Asn Val Met Ile Ser 405 Cys Trp Ala Leu Lys 150 Ala Ser Pro Asp Glu 230 Pro Lys Glu Asp Leu 310 Pro Gly Trp Ile Lys 390 Phe Phe Thr Ser Thr 135 Asp Pro Ala Asp Thr 215 Cys Ser Ile Cys Asn 295 Asp Arg Ala Ile Trp 375 Gin Val Trp Ser Trp 455 Ile 120 Gin Arg Ser Cys Asn 200 Ile Va1 Asn Ile Ser 280 Trp Tyr Ser Asn Gly 360 Asp Asp Gin Va1 Gly 440 Gly Ser Pro His 185 Gly Lys Cys Gly Lys 265 Cys His Gin Asn Gly 345 Arg Pro Ile His Glu 425 Ser Ala Pro Tyr 170 Asp Ala Ser Ile Gin 250 Ser Tyr Ala Ile Asp 330 Va1 Thr Asn Ile Pro 410 Leu Ser Leu Tyr 155 Asn Gly Vai Trp Asn 235 Ala Val Pro Ser Gly 315 Gly Lys Lys Gly Ala 395 Glu Ile Ile Leu 140 Arg Ser Met Ala Arg 220 Gly Ser Glu Asp Asn 300 Tyr Lys Gly Ser Trp 380 Leu Leu Arg Ser Gly 460 125 Asn Thr Arg Gly Va1 205 Asn Ser Tyr Leu Thr 285 Arg Ile Gly Phe Ile 365 Thr Thr Thr Gly Phe 445 Asp Leu Phe Trp 190 Leu Lys Cvs Lys Asp 270 Gly Pro Cys Asn Ser 350 Ser Glu Asp Gly Gin 430 Cys Arg Met Glu 175 Leu Lys Ile Phe Ile 255 Ala Lys Trp Ser Cys 335 Phe Ser Thr Trp Met 415 Pro Gly His Ser 160 Ser Thr Tyr Leu Thr 240 Phe Pro Va1 Va1 Gly 320 Gly Arg Arg Asp Ser 400 Asn Lys Val Ser Trp Pro Asp Ala Asp Leu Pro 450 Phe Thr Ile Asp Lys -365- 465 470 INFORMATION FOR SEQ ID NO:230: SEQUENCE CHARACTERISTICS: LENGTH: 1501 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

N

(iv) ANTI-SENSE: N (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 1..1501 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230: ATG AAT TCT CAA GGC ACC AAA CGA TCA TAT GAA CAA ATG GAG ACT r Met Asn Ser Gln 1

GGG

Gly

ATT

Ile

CTC

Leu

AGA

Arg

GAG

Glu

TAT

Tyr

AAA

Lys

GCA

Ala

GAT

Asp 145 GAA CGC CAG Glu

GGT

Gly

AGT

Ser

ATG

Met

CAT

His

AGA

Arg

GAA

Glu

ACA

Thr 130

GCC

Ala Arg

GGA

Gly

GAC

Asp

GTG

Val

CCC

Pro

AGG

A-g

GAA

Glu 115

GCC

Ala

ACC

Thr Gln

ATC

Ile

TAT

Tyr

CTC

Leu

AGT

Ser

GTA

Va1 100

ATA

Ile

GGT

Gly

TAT

Tyr Gl 5

GAT

Asp

GGA

Gly

GAG

Glu

TCT

Ser

GCT

Ala

GAC

Asp

AGG

Arg

CTT

Leu

CAG

Gin Thr Lys Arg

GCC

Ala

AGA

Arg

GGA

Gly

GCT

Ala 70

GGG

Gly

GGA

Gly

AGA

Arg

ACT

Thr

AGA

Arg 150 ACA GAA Thr Glu TTC TAC Phe Tyr 40 CGA CTA Arg Leu 55 TTT GAT Phe Asp AAG GAT Lys Asp AAA TGG Lys Trp GTT TGG Val Trp 120 CAC ATC His Ile 135 ACA AGA Thr Arg

ATC

Ile 25

ATC

Ile

ATT

Ile

GAG

Glu

CCT

Pro

ATG

Met 105

CGC

Arg

ATG

Met

GCG

Ala

AGA

Arg

CAA

Gin

CAA

Gin

AGA

Arg

AAG

Lys 90

AGA

Arg

CAA

Gln

ATT

Ile

CTT

Leu

GCA

Ala

ATG

Met

AAT

Asn

AGG

Arg 75

AAA

Lys

GAA

Glu

GCA

Ala

TGG

Trp

GTT

Val 155

TCT

Ser

TGC

Cys

AGC

Ser

AAT

Asn

ACT

Thr

CTC

Leu

AAC

Asn

CAC

His 140

CGC

Arg

GTC

Val

ACT

Thr

ATA

Ile

AAA

Lys

GGA

Gly

ATC

Ile

AAT

Asn 125

TCC

Ser

ACT

Thr

GGA

Gly

GAA

AGA

Arg

CTC

ATG

Met

AAAA

Ser Tyr 10 Glu Gin Met Glu Thr Gly u u~u ul Glu Leu Lys

ACA

Thr

TAC

Tyr

GGA

Gly

CTT

Leu 110

GGT

Gly

A.AT

Asn

GGA

Gly

ATA

Ile

CTA

Leu

CCC

Pro

TAT

Tyr

GAG

Glu

CTT

Leu

ATG

Met

GAG

Glu

GAA

Glu

ATA

Ile

GAC

Asp

GAT

Asp

AAT

Asn

GAT

Asp 160

TCT

Ser 144 192 240 288 336 384 432 480 CCC AGA ATG TGC TCC Pro Arg Met Cys Ser CTA ATG CAA GGT TCA ACA CTT CCC AGA AGG Leu Met Gin Gly Ser Thr Leu Pro Arg Arg

GGG

Gly

TTA

Leu

GGT

Gly

ATT

Ile 225

CAA

Gin

ATT

Ile

AAG

Lys

CAT

His

AAA

Lys 305

AAC

Asn

GCA

Aia

GTT

Vai

GAG

Giu

TAT

Tyr 385

GCA

Aia

AAT

Asn

GCC

Al

ATC

Il

GAZ

G11 210

CTC

Leu

GTA

Val

TTC

Phe

TCC

Ser

GAC

Asp 290

TTA

Leu

CCA

Pro

~TTT

Phe

CCA

Pro %sn 370 rGG rrp

['CT

3er

'TC

e u C GCA a Aia

-AGA

Arg 195

AAT

Asn

.AA

1Lys

AGA

Arg

CTG

Leu

TGC

Cys 275

TTT

Phe

CTT

Leu

GCT

Aia

GAG

Giu

AGA

Arg 355

GTG

Val

CC

Aia

GCG

Aia

CCT

Pro

GGT

Giy 180

ATG

Met

GGA

Ci y

GGA

Giy

GAA

Giu

GCA

Al a 260

CTG

Leu

GAA

Giu

CAA

Gin

CAC

H{is

GAT

A~sp 340

GGA

3AA C riTA; Ile; 'GC C fly C E'TT C ?he C 165 GC'3 Al a

ATC

Ile

CGA

Arg

AAA

Lys

AGT

Ser 245

CGG

Arg

CCT

Pro

A.GA

Arg

P.AC

Asn

%AG

Lys 325

['TA

eu ys

CT

la

.GG

~rg

AG

;in 0OS

;AA

;iu GCA GTG AAV Ala Val Lys 366- 170 GTT GGA ACA Val Gly Thr

AAA

Lys

AGG

Arg

*TTT

Phe 230

CGA

Arg

TCA

Ser

GCT

Ala

GAA

Giu

AGT

Ser 310

AGT

Ser

AGA

Arg

CTT

Leu

ATG

Met

ACC

Thr2 390 ATC2 Ile

AGAC

Arg2

CGI

Arg

ACA

Thr 215

CAG

Gin

AAC

Asn

GCA

Al a

TGT

Cys

GGA

Giy 295

CAA

Gin

CAA

Gin

PATA

Ile rCC Ser

'AC

ksp 375 k.GA krg k.GT 3er 3CA kla

GGA

Giy 200

AGG

Arg

ACA

Thr

CCA

Pro

CTT

Leu

GTG

Val1 280

TAT

Tyr

GTG

Val

TTG

Leu

TCA

Ser

ACA

Thr 360

TCT

Ser

AGT

Ser

GTG

Val

ACC

Thr

GA

Gly 185

ATC

Ile

ATT

Ile

GCT

Ala

GGA

Gly

ATT

Ile 265

TAT

Tyr

TCA

Ser

TTC

Phe

GTG

Val

AGT

Ser 345 AGA C Arg C AGT Ser GCA C Gly C CAA C Gin 1 GTT Val M~ 425 AA1 Asr

GC;

Ala

GCC

Ala

AAC

Asn 250

CTA

Leu

GGG

Giy

CTG

Ueu kGC Ser

['GG

['rp 330

['TC

?he

GG

fly .cc 'hr

GA

;iy

CT

>ro ~10

LTG

e t

GAC

Asp

TAT

Tyr

*CAG

*Gin 235

GCT

Ala

AGG

Arg

CTT

Leu

GTC

Val

CTG

Leu 315

ATG

Met

ATA

Ile

GTT

Val

CTA

Leu

AAT

Asn 395

ACA

Thr

GCA

Ala2

CG;

Arg

GAA

Clu 220

AGG

Arg

GAA

Giu

GGG

Gly

GCA

Ala

GGG

Gly 300

ATC

Ile

GCA

Al a

A.GA

Arg

CAG

Gin Lys 380

%CC

rhr

['TC

Phe 3CT kla

~AAC

Asi 201

AGZ

Arc

GC;

Ala

ATT

Ile

TCA

Ser

GTA

Val 285

ATA

Ile

AGA

Arg

TGC

Cys

GGG

Gly

ATT

Ile 365

CTA

Leu

AAC

Asn

TCA

Ser

TTC

Phe

TTC

IPhe

SATG

Met

ATG

Met

GAA

Giu

GTT

Val 270

GCA

Ala

GAC

Asp

CCA

Pro

CAT

His

AAG

Lys 350

GCT

Ala

AGA

Arg

CAA

Gin

GTG

Val

AGC

Ser

TG(

Tr

TGC

Cyl

ATC

Met

GATI

Asp 255

GCA

Ala

AGT

Ser

CCC

Pro

AAT

Asn

TCT

Ser 335

AAA

Lys

TCA

Ser

AGC

Sen

CAG

Gin

CAA

Gin 415

GGG

Gly 3 AGG JArg

AAT

Asn

GAT

Asp 240

CTC

Leu

CAT

His

GGG

Cly

TTC

Phe

GAA

Giu 320

GCT

Ala

GTG

Val1

AAT

Asn

AGA

Arg

AAG

Lys 400

CGG

Arg

AAT

Asn 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 175 ATA GCA ATG GAG Ile Ala Met Giu 420 AAT GAG GGA CGG ACA TCA GAC ATG CGA ACG GAA GTT ATA AGG ATG ATG 1344 -367- Asn Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Val Ile Arg Met Met 435 440 445 GAA AGT GCA AAG CCA GAA GAT TTG TCC TTC CAG GGG CGG GGA GTC TTC Glu Ser Ala Lys Pro Glu Asp Leu Ser Phe Gin Gly Arg Gly Val Phe 450 455 460 GAG CTC TCG GAC GAA AAG GCA ACG AAC CCG ATC GTG CCT TCC TTT GAC Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp 465 470 475 480 ATG AGT AAT GAA GGG TCT TAT TTC TTC GGA GAC AAT GCA GAG GAG TAT Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485 490 495 GAC AAT TGA ATT C Asp Asn Ile 500 INFORMATION FOR SEQ ID NO:231: SEQUENCE CHARACTERISTICS: LENGTH: 500 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 1392 1440 1488 1501 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231: Met Asn Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Gly 1 5 10

I

a a Gly Ile Leu Arg 65 Glu Tyr Lys Ala Asp 145 Pro Giu Gly Ser Met His Arg Glu Thr 130 Al a Arg Arg Gly Asp Val1 Pro Arg Glu Al a Thr Met Gin Ile Tyr Leu Ser Val 100 Ile Gly Tyr Cys Asp Gly Glu Ser Al a Asp Arg Leu Gin Ser 165 Ala Thr Arg Phe Gly Arg 55 Ala Phe 70 Gly Lys Gly Lys Arg Val Thr His 135 Arg Thr 150 Leu Met Glu Tyr 40 Leu Asp Asp) Trp Trp, 120 Ile Arg Gin Ile 25 Ile Ile Glu Pro Met 105 Arg Met Ala Gly Arg Gin Gin Arg Lys 90 Arg Gin Ile Leu Ser 170 Al a Met Asn Arg 75 Lys Glu Al a Trp Val 155 Thr Ser Cys Ser 60 Asn Thr Leu Asn His 140 Arg Leu Val Thr Ile Lys Gly Ile Asn 125 Ser Thr Pro Gly Glu Thr Tyr Gly Leu 110 Gly Asn Gly Arg Arg Leu Ile Leu Pro Tyr Glu Leu Met Arg 175 Met Lys Giu Glu Ile Asp Asp Asn Asp 160 Ser Gly Ala Ala Ala Ala Val Lys Gly Val Gly Thr Ile Ala Met Glu 185 190 -368- Leu Ile Arg 195 Met Ile Lys Arg *0* S. 5 S a

S.

Gly Ile 225 Gin Ile Lys His Lys 305 Asn Ala Vai Giu Tyr 385 Ala Asn Asn Giu Giu 465 Met Asp *Glu 210 Leu Val Phe Ser Asp 290 Leu Pro Phe Pro Asn 370 Trp Ser Leu Glu Ser 450 Leu Ser Asn Asn Lys Arg Leu Cys 275 Phe Leu Al a Glu Arg 355 Val1 Al a Al a Pro Gly 435 Ala Ser Asn Gly Gly Glu Al a 260 Leu Giu Gin His Asp 340 Gly Glu Ile Gly Phe 420 Arg Lys Asp Glu Ile 500 Arg Lys Ser 245 Arg Pro Arg Asn Lys 325 Leu Lys Al a Arg Gin 405 Giu Thr Pro Giu Gly 485 Arg Phe 230 Arg Ser Al a Glu Ser 310 Ser Arg Leu Met Thr 390 Ile Arg Ser Glu Lys 470 Ser Thr 215 Gin Asn Ala Cys Gly 295 Gin Gin Ile Ser Asp 375 Arg Ser Ala Asp Asp 455 Ala Tyr Gly 200 Arg Thr Pro Leu Val1 280 Tyr Val Leu Ser Thr 360 Ser Ser Val Thr Met 440 Leu Thr Phe Ile Asn Asp Arg Ile Aila Gly Ile 265 Tyr Ser Phe Val1 Ser 345 Arg Ser Gly Gin Val1 425 Arg Ser Asn Phe Aila Aia Asn 250 Leu Gly Leu Ser Trp 330 Phe Gly Thr Gly Pro 410 Met Thr Phe Pro Gly 490 Tyr Gin 235 Al a Arg Leu Val1 Leu 315 Met Ile Val Leu Asn 395 Thr Ala Giu Gin Ile 4*7S Asp Giu 220 Arg Glu Gly Al a Gi y 300 Ile Al a Arg Gin Lys 380 Thr Phe Alia Val1 Gly 460 Val1 PAsn Asn 205 Arg Al a Ile Ser Val 285 Ile Arg Cys Gly Ile 365 Leu Asn Ser Phe Ile 445 Arg Pro Ala Phe Met Met Glu Val1 270 Ala Asp Pro His Lys 350 Al a Arg Glm Val Ser 430 PArg

G

1 y Ser Glu *Trp, Cys Met Asp 255 Ala Ser Pro Asn Ser 335 Lys Ser Ser Gin Gin 415 Gly Met Val Phe Glu 495 Arg Asn Asp 240 Leu His Gly Phe Glu 320 Ala Val1 Asn Arg Lys 400 Arg Asn Met Phe Asp 480 Tyr

Claims (23)

1. A recombinant swinepox virus comprising a foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within an approximately 2.0 kb HindIII to BglII subfragment of the HindIII M fragment of the swinepox virus genomic DNA.

2. The recombinant swinepox virus of claim 1, wherein the foreign DNA sequence is inserted within a BglII site located within the approximately 2.0 kb HindIII to BglII subfragment of the swinepox virus genomic DNA.

3. The recombinant swinepox virus of claim 1, wherein the foreign DNA is inserted into a NdeI site located within the open reading frame encoding the swinepox virus thymidine kinase.

4. The recombinant swinepox virus of claim 2, wherein the 20 foreign DNA is inserted into open reading frame encoding the swinepox virus 01L gene.

5. A recombinant swinepox virus comprising a foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within a HindIII N fragment of the swinepox virus genomic DNA and is capable of being expressed in a swinepox virus infected host cell. 30 6. The recombinant swinepox virus of claim 5, wherein the foreign DNA sequence is inserted within an approximately kb HindIII to BamHI subfragment of the HindIII N fragment of the swinepox virus genomic DNA. 3 5

7. The recombinant swinepox virus of claim 5, wherein the foreign DNA sequence is inserted within an approximately P:\OPER\F;Is22K2(4-spe.doc-2Mi5/02 370 1.2 kb BamHI to HindIII subfragment of the HindIII N fragment of the swinepox virus genomic DNA.

8. The recombinant swinepox virus of claim 6, wherein the foreign DNA sequence is inserted into an open reading frame within an approximately 2.0 kb HindIII to BamHI subfragment of the HindIII N fragment of the swinepox virus genomic DNA.

9. The recombinant swinepox virus of claim 8, wherein the open reading frame encodes a I7L gene. The recombinant swinepox virus of claim 6, wherein the foreign DNA sequence is inserted within a EcoRV restriction endonuclease site within the approximately 2.0 kb HindIII to BamHI subfragment of the swinepox virus genomic DNA.

11. The recombinant swinepox virus of claim 6, wherein the foreign DNA sequence is inserted within a SnaBI S 20 restriction endonuclease site within the approximately Skb HindIII to BamHI subfragment of the swinepox virus genomic DNA.

12. The recombinant swinepox virus of claim 7, wherein the foreign DNA sequence is inserted into an open reading frame within an approximately 1.2 kb HindIII to BamHI subfragment of the HindIII N fragment of the swinepox virus genomic DNA. 30 13. The recombinant swinepox virus of claim 12, wherein .o the open reading frame encodes a I4L gene.

14. The recombinant swinepox virus of claim 7, wherein the foreign DNA sequence is inserted within a BglII restriction RA endonuclease site within the approximately 1.2 kb HindIII to BamHI subfragment of the swinepox virus genomic DNA. P:\OPER\F;s\22 6264-spedoc-2/A)5/U2 371 A recombinant swinepox virus comprising a foreign DNA sequence inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within a HindIII K fragment of the swinepox virus genomic DNA and is capable of being expressed in a swinepox virus infected host cell.

16. The recombinant swinepox virus of claim 15, wherein the foreign DNA sequence is inserted into an approximately 3.2 kb subfragment of the HindIII K fragment of the swinepox virus genomic DNA.

17. The recombinant swinepox virus of claim 16, wherein the foreign DNA is inserted into an open reading frame within an approximately 3.2 kb subfragment of the HindIII K fragment of the swinepox virus genomic DNA.

18. The recombinant swinepox virus of claim 17, wherein 20 the open reading frame encodes a B18R gene.

19. The recombinant swinepox virus of claim 17, wherein Sthe open reading frame encodes a B4R gene. 9

20. The recombinant swinepox virus of any of claims 1, or 15, wherein the foreign DNA encodes a polypeptide.

21. The recombinant swinepox virus of claim 20, wherein 4.. ~the polypeptide is antigenic. S. 22. The recombinant swinepox virus of any of claims 1, or 15, further comprising a foreign DNA sequence which encodes a detectable marker. RAL, 23. The recombinant swinepox virus of claim 22, wherein the detectable marker is E. coli beta-galactosidase. P.\OPERas\22 24 -spe doc-2Xm5/)2 372

24. The recombinant swinepox virus of claim 22, wherein the detectable marker is E. coli beta-glucuronidase.

25. The recombinant swinepox virus of any of claims 1, or 15, wherein the foreign DNA encodes a cytokine.

26. The recombinant swinepox virus of claim 25, wherein the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN).

27. The recombinant swinepox virus of claim 25, wherein the cytokine is selected from the group consisting of interleukin-2, interleukin-6, interleukin-12, interferons, granulocyte-macrophage colony stimulating factors and interleukin receptors.

28. The recombinant swinepox virus of claim 21 wherein the antigenic polypeptide is derived from the group consisting 20 of human herpesvirus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, human herpesvirus-6, human 9 herpesvirus-7, human influenza, human immunodeficiency virus, rabies virus, measles virus, hepatitis B virus, and 25 hepatitis C virus. g* *30 9

3029. The recombinant swinepox virus of claim 21, wherein 9.. the antigenic polypeptide is hepatequine influenza virus core protein Sor hepatitis B virus surface protein.hemagglutinin. S. 30. The recombinant swinepox virus of claim 21, wherein the antigenic polypeptide is equine influenza virus neuraminidase or hemagglutinin. 31. The recombinant swinepox virus of claim 21, wherein I- the antigenic polypeptide is selected from the group P.\OPER\Fas226264-sp, doc-2 A/ 052 373 consisting of equine influenza type A/Alaska 91 neuraminidase, equine influenza virus type A/Kentucky 92 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, equine influenza type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D. 32. The recombinant swinepox virus of claim 21, wherein the antigenic polypeptide is selected from the group consisting of hog cholera virus glycoprotein El, hog cholera virus glycoprotein E2, swine influenza virus hemagglutinin, swine influenza virus neuraminidase, swine influenza virus matrix, swine influenza virus nucleoprotein, pseudorabies virus glycoprotein B, pseudorabies virus glycoprotein C, pseudorabies virus glycoprotein D, and PRRS virus ORF7. 33. The recombinant swinepox virus of claim 21, wherein 20 the antigenic polypeptide is selected from the group i consisting of infectious bovine rhinotracheitis virus glycoprotein E, bovine respiratory syncytial virus attachment protein (BRSV bovine respiratory syncytial virus fusion protein (BRSV bovine respiratory syncytial 25 virus nucleocapsid protein (BRSV bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase. 34. The recombinant swinepox virus of claim 21, wherein 30 the antigenic polypeptide is bovine viral diarrhea virus S(BVDV) glycoprotein 48 or glycoprotein 53. The recombinant swinepox virus of claim 21, wherein the foreign DNA sequence encodes an antigenic polypeptide which is derived or derivable from a group consisting of feline immunodeficiency virus gag, feline immunodeficiency P:\OPER\Fas.22862(4-spe.doc-28A5/)2 374 virus env, infectious larynotracheitis virus glycoprotein B, infectious larynotracheitis virus glycoprotein I, infectious larynotracheitis virus glycoprotein D, infectious bovine rhinotracheitis virus glycoprotein G, infectious bovine rhinotracheitis virus glycoprotein E, pseudorabies virus glycoprotein 50, pseudorabies virus II glycoprotein B, pseudorabies virus III glycoprotein C, pseudorabies virus glycoprotein E, pseudorabies virus glycoprotein H, Marek's disease virus glycoprotein B, Marek's disease virus glycoprotein D, Newcastle disease virus hemagglutinin or neuraminidase, Newcastle disease virus fusion, infectious bronchitis virus spike, infectious bronchitis matrix, and chick anemia virus. 36. The recombinant swinepox virus any of claims 1, 5 or wherein the foreign DNA sequence is under the control of a promoter. 37. The recombinant swinepox virus of claim 36, wherein S 20 the foreign DNA sequence is under control of an endogenous upstream poxvirus promoter. 38. The recombinant swinepox virus of claim 36, wherein the foreign DNA sequence is under the control of a 25 heterologous upstream promoter. 39. The recombinant swinepox virus of claim 37, wherein the promoter is selected from a group consisting of :g synthetic pox viral promoter, pox synthetic late promoter 30 1, pox synthetic late promoter 2 early promoter 2, pox 01L promoter, pox I4L promoter, pox I3L promoter, pox I2L promoter, pox IlL promoter, and pox ElOR promoter. A recombinant swinepox virus, designated S-SPV-042. 41. A recombinant swinepox virus, designated S-SPV-043. P:\OPER\F;s\2286264-sp.doc-2H/ 15/02 375 42. 43. 44. 46. 47. 48. 49. 20 51. 52. 53. 54. A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant A recombinant swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, swinepox virus, designated designated designated designated designated designated designated designated designated designated designated designated designated designated S-SPV-041. S-SPV-045. S-SPV-046. S-SPV-047. S-SPV-048. S-SPV-049. S-SPV-050. S-SPV-052. S-SPV-053. S-SPV-054. S-SPV-055. S-SPV-060. S-SPV-061. S-SPV-062. g f ft f ft of «f ftf ft t f ft ft ft ft f 56. A homology vector for producing a recombinant swinepox virus by inserting foreign DNA into the viral genome of a swinepox virus which comprises a double-stranded DNA molecule consisting essentially of: a) double stranded foreign DNA not usually present Swithin the swinepox virus viral genome; P:\OPER\F:sl226264-spe.doc-28/(5I12 376 b) at one end the foreign DNA, double-stranded swinepox virus DNA homologous to the virus genome located at one side of the HindIII N fragment of the coding region of the swinepox virus genome; and c) at the other end of the foreign DNA, double- stranded swinepox virus DNA homologous to the viral genome located at the other side of the HindIII N fragment of the coding region of the swinepox virus viral genome. 57. The homology vector of claim 56, wherein the foreign DNA sequence encodes a cytokine. 58. The homology vector of claim 57, wherein the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN) 59. The homology vector of claim 56, wherein the foreign DNA sequence encodes a polypeptide. 60. A homology vector of claim 59, wherein the polypeptide is antigenic. 61. The homology vector of claim 56, wherein the foreign 25 DNA sequence is under control of a promoter. S 62. A vaccine useful for immunizing an animal against an animal pathogen which comprises an effective immunizing amount of the recombinant swinepox virus any of claims 1, 30 or 15 and a suitable carrier. 63. A method of immunizing an animal against an animal pathogen which comprises administering to the animal an effective immunizing dose of the vaccine of claim 62. A recombinant swinepox virus according to any one of P:\OPER\Fs\226264-spe doc-24/06i/02 377 claims 1 to 39 substantially as hereinbefore described with reference to the drawings and/or examples. A homology vector according to any one of claims 56 to 61 substantially as hereinbefore described with reference to the drawings and/or examples. 66. A vaccine according to claim 62 substantially as hereinbefore described with reference to the drawings and/or examples. DATED this 2 4 th day of June 2002 Syntro Corporation by DAVIES COLLISON CAVE Patent Attorneys for the Applicants **o .e