CA2141422C

CA2141422C - Recombinant equine herpesviruses

Info

Publication number: CA2141422C
Application number: CA 2141422
Authority: CA
Inventors: Mark D. Cochran
Original assignee: Schering Plough Ltd
Current assignee: MSD International Holdings GmbH
Priority date: 1992-08-07
Filing date: 1993-08-06
Publication date: 2006-12-05
Anticipated expiration: 2013-08-06
Also published as: CA2141422A1

Abstract

The present invention relates to a non-naturally occurring, recombinant equine herpesvirus. The invention also relates to a recombinant equine herpesvirus capable of replication which comprises viral DNA from a species of equine herpesvirus and foreign DNA, the foreign DNA being inserted into the equine herpesviral DNA at a site which is not essential for replication of the equine herpesvirus. The invention also relates to DNA encoding the US2 protein of an equine herpesvirus. The invention relates to homology vectors for producing recombinant equine herpesviruses which produce recombinant equine herpesviruses by inserting foreign DNA into equine herpesviral DNA. The invention further relates to a method of producing a fetal-safe, live recombinant equine herpesvirus.

Description

Within this application, several publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims.
Background of the Inventi~
The present invention involves recombinant equine herpesviruses useful in the preparation of vaccines to protect horses from various species of naturally-occurring infectious equine herpesvirus. The equine herpesvirus is a member of the family herpesviridae, which are co~only known as the herpesviruses.
Generally, herpesviruses contain 100,000 to 200,000 base pairs of DNA as their genetic material, and several areas of the genomes of various members have been identified that are not essential for the replication of virus in vitro in cell culture. Modifications of these regions of the DNA have been known to lower the pathogenicity of the virus, i.e. to attenuate the virus when it infects an animal species. For example, inactivation of the thymidine kinase gene of either human herpes simplex virus (29) or pseudorabies virus of swine (38) renders these herpesviruses less pathogenic.

Removal of specific regions of the repeat region of a human herpes simplex virus have been shown to render the virus less pathogenic (32, 39). Furthermore, a repeat region has been identified in Marek's disease virus that is associated with viral oncogenicity (13). A region in herpesvizus saimiri has similarly been correlated with oncogenicity (21). Removal'of a specific region of the repeat region renders pseudorabies virus less pathogenic (US Pat. 4,877,737). A region in pseudorabies virus has been shown to be deleted in naturally-occurring vaccine strains (22). These deletions are at least in part responsible for the lack of pathogenicity of these strains.
It is generally agreed that herpesviruses contain non-essential regions of DNA in various parts of the genome, and that modification of these regions can attenuate the virus, leading to a non-pathogenic strain from which a vaccine may be derived. The degree of attenuation of the 2o virus is important to the utility of the virus as a vaccine. Deletions which cause too much attenuation of the virus will result in a vaccine that fails to elicit an adequate immune response. Although several examples of attenuating deletions are known, the appropriate combination of deletions for any herpesvirus is not readily apparent.
Major economic losses to the equine industry result from infection by two species of equine herpesvirus (17).
3o These two equine herpesvirus species, currently identified in the literature as EHV-1 and EHV-4, belong to the herpesvirus sub-family alpha-herpesvirus and are characterized by a class D genome (33). Formerly, both species were identified as EHV-1 and further differentiated as EHV-1 subtype 1 (EHV-1) and EHV-1 subtype 2 (EHV-4) respectively. EHV-1 is the primary cause of abortion in pregnant mazes and EHV-4 is the primary cause of respiratory disease in foals and yearlings. Currently available products are not designed to address both disease syndromes, with the result that these products are marginally effective.
EHV-1 and EHV-4 have been analyzed at the molecular level. Restriction maps of the genomes of EHV-1 and EHV-4 have been reported (42 and 8).
Although several of the herpesviruses have been genetically engineered, no examples of recombinant EHV
have been reported.
EFIV can become latent in healthy animals which makes them potential carriers of the virus. For this reason, it is clearly advantageous to be able to distinguish animals vaccinated with non-virulent virus from animals infected 2o with disease-causing wild-type or naturally-occurring virus. The development of differential vaccines and companion diagnostic tests has proven valuable in the management of pseudorabies disease (Federal Register, Vol. 55, No. 90, pp. 19245-19253). A similar differential marker vaccine would be of great value in the management of EHV caused disease.
The present invention provides a method of producing a fetal-safe, live recombinant EHV virus which comprises 3o treating viral DNA from a naturally-occurring live EHV so as to delete from such viral DNA, DNA corresponding to the US2 gene of the naturally-occurring EHV. The present invention also provides viruses in which (aj DNA
corresponding to the US2 gene has been deleted, and (b) DNA encoding gpG, gpE, and/or TK has been altered or _;~ ~.
deleted. Such viruses are useful for the creation of vaccines which require diagnostic markers and safety in pregnant animals.
The ability to engineer DNA viruses with large genomes, such as vaccinia virus and the herpesviruses, has led to the finding that these recombinant viruses can be used as vectors to deliver vaccine. antigens and therapeutic agents for animals. The herpesviruses are attractive to candidates for development as vectors because their host range is primarily limited to a single target species (16) and they have the capacity for establishing latent infection (7) that could provide for stable in vivo expression of a foreign gene. Although several herpesvirus species have been engineered to express foreign gene products, recombinant equine herpesviruses expressing foreign gene products have not been constructed. The equine herpesviruses described above may be used as vectors for the delivery of vaccine 2o antigens from microorganisms causing important equine diseases. Such multivalent recombinant viruses would protect against EHV as well as other diseases. Similarly the equine herpesviruses may be used as vectors for the delivery of therapeutic agents. 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 equine herpesvirus replication. This limits the therapeutic agent in the first analysis to either DNA, RNA or protein. There are examples of therapeutic agents from each of these classes of compounds in the form of anti-sense DNA, anti-sense RNA (19), ribozymes (41), suppressor tRNAs (3), interferon-inducing double stranded RNA and numerous examples of protein therapeutics, from hormones, e.g., insulin, to lymphokines, e.g., interferons and interleukins, to 21 4 14 2 2 _5'i natural opiates. The discovery of these therapeutic agents and the elucidation of their structure and function does not necessarily allow one to use them in a viral vector delivery system, however, because of the experimentation necessary to determine whether an appropriate insertion site exists.

The invention provides a non-naturally occurring, recombinant equine herpesvirus. The invention provides isolated DNA encoding the US2 protein of an equine herpesvirus.
The invention provides a zecombinant equine herpesvirus capable of replication which comprises viral DNA from a l0 species of a naturally-occurring equine herpesvirus and foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant equine herpesvirus is introduced, the foreign DNA being inserted into the naturally-occurring equine herpesviral DNA at a site which is not essential for replication of the equine herpesvirus.
The invention provides a homology vector for producing a recombinant equine herpesvirus by inserting foreign DNA
into a genome of an equine herpesvirus which comprises a double-stranded DNA molecule consisting essentially of:
a) a double-stranded foreign DNA sequence encoding RNA
which does not naturally occur in an animal into which the recombinant equine herpesvirus is introduced: b)at one end of the foreign DNA sequence, double-stranded equine herpesviral DNA homologous to genomic DNA located at one side of a site on the genome which is not essential for replication of the equine herpesvirus: and c) at the other end of the foreign DNA, double-stranded equine herpesviral DNA homologous to genomic DNA located at the other side of the same site on the genome.
The invention provides a method of producing a fetal-safe, live recombinant equine herpesvirus which comprises treating viral DNA from a naturally-occurring live equine _.. 2141422 herpesvirus so as to delete from the virus DNA
corresponding to the US2 region of the naturally-occurring equine herpesvirus.
FIGURE 1 Details of the EHV1 Dutta Strain. Diagram of EHV1 genomic DNA showing the unique long, internal repeat, unique short, and Terminal repeat regions. A restriction map for the enzyme ~IiI is indicated (42). Fragments are lettered in order of decreasing size. The unique short region and the thymidine kinase to region are expanded showing the locations of fragments III b, SRI d, k and c. The location of several genes is indicated they are thymidine kinase (Tk), unique short 2 (US2), glycoproteins G (gpG), D (gpD), I (gpI), and E
(gpE) (1).
FIGURE 2 Details of the EHV4 Dutta Strain. Diagram of EHV4 genomic DNA showing the unique long, internal repeat, unique short, and Terminal repeat regions. Restriction maps for the enzymes ~,gRI, p~I and p~gI are indicated.
Fragments are lettered in order of decreasing size. The unique short region and the thymidine kinase region are expanded showing the locations of fragments CHI c, d. The locations of two genes are also indicated, they are thymidine kinase (Tk) (27, 28) and unique short 2 (US2).
FIGURE 3 Homology between the equine herpesvirus US2 proteins and the US2 Proteins of HSV-1, PRV, ~ 14'~ 4~ '~
HSV-2, and MDV. (a) Matrix plot of the amino acid sequence of the EHV-4 US2 protein (324 amino acids) (SEQ ID NO: 4) against the amino acid sequence of the HSV-1 US2 protein (291 amino acids) (24). (b) Alignment of the conserved region (SEQ ID NO: 7) between EHV-1 US2 protein (303 amino acids) (SEQ ID NO: 2), EHV-4 US2 protein (S$Q ID NO: 8), HSV-1 US2 protein (SEQ ID NO: 9j, PRV US2 protein (SEQ.
ID NO: 11) (256 amino acids) (49) HSV-2 US2 protein (SEQ ID NO: 10) (291 amino acids) (25), MDV US2 protein (SEQ ID NO: 12) (270 amino acids) (4), and IBR US2 (SEQ ID NO: 13).
FIGURE 4 Detailed description of the DNA insertion in Homology Vector 450-46.84. The diagram shows the orientation of DNA fzagments assembled in plasmfd 450-46.84. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 14), junction B (SEQ ID
NO: 15) , and junction C (SEQ ID NO: 17) . The restriction sites used to generate each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a double bar. The location of several gene coding regions and regulatory elements is also given.
The following two conventions are used:
numbers in parenthesis ( ) refer to amino acids, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations nre used,. equine herpesvirus 1 (EHV1), thymidine kinase (TK), glycoprotein H
(gpA), and poly adenylation signal (pA).
FIGURE 5 Detailed description of the DNA insertion in Homology Vector 467-21.19. The diagram shows the orientation of DNA fragments assembled in plasmid 467-21.19. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 19), junction B (SEQ ID
NO : 2 0 ) and j unction C ( SEQ ID NO : 2 3 ) . The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the US2 gene coding region is also given. The following two conventions are used: numbers in parenthesis () refer to amino acids, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, equine herpesvirus 1 (EHV1) and unique short 2 (US2).
FIGURE 6 Detailed description of the DNA insertion in Homology Vector 536-85.30. The diagram shows the orientation of DNA fragments assembled in plasmid 536-85.30. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 24), junction B (SEQ ID
NO: 25), and junction C (SEQ ID NO: 26). The restriction sites used to generate each fragment are indicated at the appropriate 2141~~2 junction. The location of the gpD, MGP, and US3 gene coding regions ayes also given.
Restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, equine herpesvirus 1 (EHV1), membrane glycoprotein (MGP),, unique short 3 (US3) glycoprotein D (ggD).
to FIGURE 7 Detailed description of the DNA insertion in Homology Vector 495-61.39. The diagram shows the orientation of DNA fragments assembled in plasmid 495-61.39. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 27), junction B (SEQ ID
NO: 28), and junction C (SEQ ID NO: 31). The restriction sites used to generate each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a double bar. The location of the TK and gpH gene coding regions 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, equine herpesvirus 4 (EHV4) and glycoprotein H (gpH).
FIGURE 8 Detailed description of the DNA insertion in Homology Vector 523-38.9. The diagram shows 2 ~ 4 ~ 4 ~ ~ -11-the ori8ntation of DNA fragments assembled in plasmid 523-38.9. The origin of each fragment is described in the Materials and Methods asction. The aequsnces located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 33j, junction B (SEQ ID
NO: 34j, and junction C (SEQ ID NO: 36j. The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the US2 gene coding region is also given. The following two conventions are used: numbers in parenthesis (j refer to amino acids, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, equine herpesvirus 4 (EHV4j and unique short 2 (US2j.
FIGURE 9 Detailed description of the DNA insertion in Homology Vector 580-57.25. The diagram shows the orientation of DNA fragments assembled in plasmid 580-57.25. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment are shown, including junction A (SEQ ID N0: 37j, junction B (SEQ ID
NO: 38j, and junction C (SEQ ID NO: 39j. The restriction sites used to generate each fragment are indicated at the appropriate 3o junction. The location of the US9 gene coding region is also given. The following two conventions are used: numbers in parenthesis (j refer to amino acids, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, equine herpesvirus 4 (EHV4) and unique short 9 (US9).
FIGURE 10 Detailed description of the marker gene insertion in Homology Vector 467-22.A12. The diagram shows the orientation of DNA fragments assembled in the marker gene. The origin of each fragment is described in the Materials and Methods section:~~The sequences located at the l0 junctions between each fragment and at the ends of the marker gene are shown, including junction A (SEQ ID NO: 40), junction B (SEQ ID
NO: 41), junction C (SEQ ID NO: 43) and junction D (SEQ ID NO: 43). The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the lacZ gene coding region is also given. The following two conventions are used:
numbers in parenthesis ( ) refer to amino acids, 2o and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, pseudorabies virus (PRV), lactose operon Z gene (lacZ), Escherichia coli (E.coli), poly adenylation signal (pA), and glycoprotein X (gpX).
FIGURE 11 Detailed description of the marker gene insertion in Homology Vector 523-42.A18. The diagram shows the orientation of DNA fragments assembled in the marker gene. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment and at the ends of the marker gene are shown, including junction A (SEQ ID NO: 46), junction B (SEQ ID
NO: 47), junction C (SEQ ID NO: 49), and junction D (SEQ ID NO: 51) . The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the lacZ gene coding region is also given. The following two comientions 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, peeudorabies virus (PRV), lactose operon Z gene (lacZ), Escherichia cola (E.coli), poly adenylation signal (pA) and glycoprotein X (gpX).
FIGURE 12 Detailed description of the marker gene insertion in Homology Vector 552-45.19. The diagram shows the orientation of DNA fragments 2o assembled in the marker gene. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment and at the ends of the marker gene are shown, including junction A (SEQ ID N0: 52), junction 8 (SEQ ID
NO: 53), junction C (SEQ ID NO: 55) and junction D (SEQ ID NO: 57). The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the uidA gene coding region is also given. The following two conventions are used:
numbers in parenthesis ( ) refer to amino acids, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, pseudorabies virus (PRV), uronidase A gene (uidA), ~,scherichia cola (E.coli) , herpes simplexvirus type 1 (HSV
1), poly adenylation signal (pA), and glycoprotein X (gpX).
FIGURE 13 Detailed description of the marker gene insertion in Homology Vector 593-31.2. The diagram shows the orientation of DNA fragments l0 assembled in the marker gene. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment and at the ends of the marker gene are shown, including junction A (SEQ ID NO: 58), junction B (SEQ ID
NO: 59), junction C (SEQ ID NO: 61), and junction D (SEQ ID NO: 63). The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the lacZ gene coding region is also given. The following two conventions are used:
numbers in parenthesis ( ) refer to amino acids, and restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, pseudorabies virus (PRV), lactose operon Z gene (lacZ), Escherichia coli (E.coli), poly adenylation signal (pA) and glycoprotein X (gpX).

WO 94/03628 PCT/US93/f7424 The present invention provides a non-naturally occurring, recombinant equine herpesvirus. The invention further provides that this recombinant equine herpesvirus is of the species EHV-1 and EHV-4.
For purposes of this invention, the term "equine herpesvirus" includes, but is not limited to, the species l0 EHV-1 and EHV-4. These species were previously referred to in the literature as EHV-l, subtype 1 and EHV-1 subtype 2, respectively.
The invention further provides a recombinant equine herpesvirus wherein a DNA sequence which is not essential for replication of the virus has been deleted from the genomic DNA of the virus.
For purposes of this invention, "a DNA sequence which is not essential for replication of the virus" is a sequence located on the genome where it does not serve a necessary function for viral replication. Examples of necessary sequences include the following: complex protein binding sequences, sequences which code for reverse transcriptase or an essential glycoprotein, DNA sequences necessary for packaging, etc.
One embodiment of the present invention provides a recombinant equine herpesvirus wherein the deleted DNA
sequence is deleted from a gene which encodes a polypeptide of the virus. Preferably, the deleted sequence is deleted from the US2 gene of the virus. The present invention provides an example of such a recombinant equine herpesvirus designated S-lEHV-002.
The S-lEHV-002 virus has been deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms far 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 2358. Preferably, the deleted DNA sequence is deleted from the gene which encodes the gpG glycoprotein.
Preferably, the deleted DNA sequence is deleted from the gene which encodes the gpE glycoprotein. Preferably, the deleted DNA sequence is deleted from the thymidine kinase gene of the virus. The present invention provides an example of such a recombinant equine herpesvirus designated S-lEHV-001. The S-lEHV-001 has been deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC
Accession No. VR 2357. The present invention provides a further example of such a recombinant equine herpesvirus designated S-4EHV-001. The S-4EHV-001 has been deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC
Accession No. VR 2361.
The invention also provides a recombinant equine herpesvirus with a deleted DNA sequence deleted from the thymidine kinase gene of the virus and a second DNA
sequence which is not essential for replication of the virus deleted from the genomic DNA of the virus. An embodiment of this invention is a recombinant equine herpesvirus wherein the second deleted DNA sequence is ...
' WO 94/03628 ~ . PCT/US93/07424 21 4 14 2 2 -1~-deleted from the US2 gene of the virus. The present invention provides an example of such a recombinant equine herpesvirus designated S-lEBV-004. The S-18HV-004 virus has been deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under with ATCC Accession No. VR
2360. The present imrention provides an example of such a recombinant equine herpesvirus designated S-4Exv-002.
The S-4EHV-002 virus has been deposited on March 12; 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No.
VR 2362. The present invention provides a further example of such a recombinant equine herpesviurs designated S-4EHV-023. The S-4EHV-023 has been deposited on August 5, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC
Accession No. VR 2426.
The invention also provides a recombinant equine herpesvirus with a deleted DNA sequence deleted from the thymidine kinase gene of the virus, a second deleted DNA
sequence deleted from the US2 gene of the virus and a third DNA sequence which is not essential for the replication of the virus deleted from the genomic DNA of the virus. An embodiment of this invention is a recombinant equine herpesvirus wherein the deleted third ..~ ~ ~~ 4~~

DNA sequence is deleted from the gpG gene of the virus.
The present, invention provides an example of such a recombinant equine herpesvirus designated S-1EHV-003.
The S-lEHV-003 has been deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the ~atant Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No.
VR 2359. A further embodiment of this invention is a recombinant equine herpesvirus wherein the deleted third DNA sequence is deleted from the gpE gene of the virus.
The present invention provides isolated DNA encoding the US2 protein of an equine herpesvirus.
The present invention provides a recombinant equine herpesvirus capable of replication which comprises viral DNA from a species of a naturally-occurring equine herpesvirus and foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant equine herpesvirus is introduced, the foreign DNA being inserted into the naturally-occurring equine herpesviral DNA at a site which is not essential for replication of the equine herpesvirus.
For purposes of this invention, "a recombinant equine herpesvirus capable of replication" is a live equine herpesvirus which has been generated by the recombinant methods well known to those of skill in the art, e.g. , the methods set forth in HOMOLOGOUS RECOMBINATION
PROCEDURE FOR GENERATING RECOMBINANT EHV in Materials and Methods and has not had genetic material essential for the replication of the recombinant equine herpesvirus deleted.

~~ 4~4~2 ., _ -19-For purposes of this invention, "an insertion site which is not essential for replication of the equine herpesvirus" is a location in the genome where a sequence of DNA is not necessary for viral replication. Examples of DNA sequences which are essential include the following: complex protein binding sequences, sequences which code for reverse transcriptase or an essential glycoprotein, DNA sequences necessary for packaging, etc.
The invention further provides foreign DNA encoding RNA
which encodes a polypeptide. Preferably, the polypeptide is antigenic in an animal into which the recombinant equine herpesvirus is introduced. In one embodiment of the invention, the polypeptide is a detsctable marker.
Preferably, the polypeptide is ,~,,. coli B-galactosidase.
Preferably, the polypeptide is E.E. coli 8-glucuronidase.
The present invention provides an example of such a recombinant equine herpesvirus designated S-4EHV-004.
2o For purposes of this invention, this antigenic polypeptide is a linear polymer of more than 10 amino acids linked by peptide bonds which stimulates the animal to produce antibodies.
In one embodiment of the invention, the polypeptide is a polypeptide normally produced by an equine herpesvirus, a Streptococcus eaui bacterium, an Infectious Anemic Virus, an equine influenza virus or an equine encephalitis virus. Preferably, the naturally occurring equine herpesvirus is EHV-1 and the foreign DNA is derived from EHV-4. Preferably, the naturally-occurring equine herpesvirus is EHV-4 and the foreign DNA is derived from EHV-1. Preferably, the foreign DNA encodes a gp8, gpC, gpD or gpFi glycoprotein.

The present invention also provides a recombinant equine herpesvirus capable of replication which comprises viral DNA from a species of a naturally-occurring equine herpesvirus and foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant equine herpesvirus is introduced, the foreign DNA being inserted into the naturally-occurring equine herpesviral DNA at a site which is not essential for replication of the equine herpesvirus with the DNA sequence which is not essential for replication of the virus deleted from the genomic DNA of the virus. In one embodiment of the present invention, the deleted DNA sequence is deleted from a gene which encodes a polypeptide of the virus.
Preferably, the foreign DNA is inserted into the naturally-occurring herpesviral DNA at a site where a DNA
sequence has been deleted. Preferably, the deleted DNA
sequence is deleted from the US2, Tk, and gpE genes of the virus.
In one embodiment of the present invention, the naturally-occurring equine herpesvirus is EHV-4 and the antigenic polypeptide is or is from the gpD and gpB gene of the EHV-1 species of equine herpesvirus. The present invention provides an example of such a recombinant equine herpesvirus designated S-4EHV-010.
In another embodiment of the present invention, the naturally- occurring equine herpesvirus is EHV-4 and the antigenic polypeptide is or is from the hemagglutinin and neuraminidase genes of a subtype of equine influenza A
virus. Preferably, the subtype of equine influenza A
virus serotype is A1. Preferably, the subtype is further characterized as an isolate of the A1 subtype of equine influenza A virus. Preferably, the isolate is Influenza A/equine/Prague/56. The present invention provides an -21- 214-1 ~ 2 ~
example of such a recombinant equine herpesvirus designated S-4EHV-011.
In another embodiment of the present invention, the subtype of equine influenza A virus is A2. Preferably, the subtype is further characterized as an isolate of the A2 subtype of equine influenza A virus. Preferably, the isolate is Influenza A/equine/Miami/63.
Preferably, the isolate is Influenza to A/equine/Rentucky/81. Preferably, the isolate is Influenza A/equine/Alaska/91. The present invention provides examples of such recombinant equine herpesviruses designated S-4EHV-012, S-4EHV-013 and S-4EHV-014, respectively.
The present invention provides a homology vector for producing a recombinant equine herpesvirus by inserting foreign DNA into a genome of an equine herpesvirus which comprises a double-stranded DNA molecule consisting essentially of: a) a double-stranded foreign DNA sequence encoding RNA which does not naturally occur in an animal into which the recombinant equine herpesvirus is introduced: b) at one end of the foreign DNA sequence, double-stranded equine herpesviral DNA homologous to genomic DNA located at one side of a site on the genome which is not essential for replication of the equine herpesvirus: and c) at the other end of the foreign DNA
sequence, double-stranded equine herpesviral DNA
homologous to genomic DNA located at the other side of the same site on the genome. In one embodiment of the invention, the equine herpesvirus is EHV-1.
In another embodiment of the present invention, the equine herpesvirus is EHV-4. Preferably, the site on the genome which is not essential for replication is present within a DNA sequence included within the US2, TR, gpG or gpE gene. In one embodiment of the present invention, the double-stranded equine herpesviral DNA is homologous to genomic DNA present within the EHV-1 III restriction fragment b. Preferably, the double-stranded equine herpesviral DNA is homologous to a ~3A restriction sub-fragment and a $~EII: ~to p~I restriction sub-fragment.
In another embodiment of the present invention, the double-stranded equine herpesviral DNA is homologous to genomic DNA present within the EHV-1 ~I~iI restriction fragment n. Preferably, the double-stranded equine herpesviral DNA is homologous to genomic DNA present within the ~IiI to ~I restriction sub-fragment and the SRI to p~I restriction sub-fragment. In a further embodiment of the present invention, the double-stranded equine herpesviral DNA is homologous to genomic DNA
present within the EHV-1 SRI restriction fragment k.
Preferably, the double-stranded equine herpesviral DNA is homologous to genomic DNA present within the SRI to g r~II restriction sub-fragment and the p~I to ~FII
restriction sub-fragment. In another embodiment of the present invention, the double-stranded equine herpesviral DNA is homologous to genomic DNA present within the EHV-4 CHI restriction fragment c. Preferably, the double-stranded equine herpesviral DNA is homologous to genomic DNA present within the g~II to ~ggI restriction sub-fragment and the g~II to ~I restriction sub-fragment.
In a further embodiment of the present invention, the double-stranded herpesviral DNA is homologous to genomic DNA present within the EHV-4 ~IiI restriction fragment d. Preferably, the double-stranded herpesviral DNA is homologous to genomic DNA present within the ~I to g,~s I
restriction sub-fragment and the p~I to VIII
restriction sub-fragment. In another embodiment of the present invention, the double-stranded herpesviral DNA is ._ 214-1 ~-2 2 homologous to genomic DNA present within the EHV-4 SRI
restriction fragment j. Preferably, the double-stranded herpesviral DNA is homologous to genomic DNA present within the g,~RI to g~II restriction sub-fragment and the ggy~I to ~gI restriction sub-fragment.
The present invention alsov provides a homology vector wherein the foreign DNA to be inserted corresponds to DNA
encoding the gpH, gpB, gpD or gpC gene of an equine herpesvirus EHV-1 species. The present invention also provides a homology vector wherein the foreign DNA to be inserted corresponds to DNA encoding gpH, gpB, gpD or gpC
glycoprotein of an equine herpesvirus EHV-4 species.
The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant equine herpesvirus of the present invention and a suitable carrier.
Suitable carrier$ for the equine herpesvirus, which would be appropriate for use with the recombinant equine herpesviruses of the present invention, are well known in the art and include proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as stabilized, hydrolyzed proteins, lactose, etc.
For purposes of this invention, an "effective immunizing amount" of the recombinant equine herpesvirus of the 3o present invention is an amount necessary to stimulate the production of antibodies by the equine in which the virus was introduced in numbers sufficient to protect the equine from infection if it was confronted by a wild-type equine herpesvirus or other equine virus which the recombinant equine herpesvirus is directed to.

The present invention also provides a method of immunizing an equine which comprises administering an effective immunizing dose of the vaccine of the present invention.
For purposes of this invention, the vaccine may be administered by any of~,the methods well known to those skilled in the art; for example, by intramuscular, subcutaneous, intraperitoneal or intravenous injection.
Alternatively, the .vaccine may be administered intranasally or orally.
The present invention also provides for a method for testing an equine to determine whether the equine has been vaccinated with the vaccine of the present invention or is infected with a naturally-occurring equine herpesvirus which comprises: (a) obtaining from the equine to be tested a sample of a suitable body fluid:
(b) detecting in the sample the presence of antibodies to 2o equine herpesvirus, the absence of such antibodies indicating that the equine has been neither vaccinated nor infected: and (c) for the equine in which antibodies to equine herpesvirus are present, detecting in the sample the absence of antibodies to equine herpesviral antigens which are normally present in the body fluid of an equine infected by the naturally-occurring equine herpesvirus but which are not present in a vaccinated equine, the absence of such antibodies indicating that the equine was vaccinated and is not infected. In one embodiment of the invention, the equine herpesviral antigen not present in the vaccinated equine is gpE
glycoprotein.
The present invention provides a method of producing a fetal-safe, live recombinant equine herpesvirus which comprises treating viral DNA from a naturally-occurring live equine herpesvirus so as to delete from the virus DNA corresponding to the US2 region of the naturally-occurring equine herpesvirus.
The present invention also provides a host cell infected with the recombinant equine herpsevirus of the present invention. In one embodiment, the host cell is a mammalian cell. Preferably, tha mammalian cell is a vero to 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 Materials and Methods.
Methods for constructing, selecting and purifying recombinant equine herpesviruses are detailed below in Materials and Methods.

2141~~~

PREPARATION OF EHV VIRUS STOCK SAMPLES. S-lEFiV-000 and S-4EHV-000 are fresh isolates of EI;V-1 and EFiV-4 , respectively, and were obtained from Dr. S. R. Dutta (College of Veternary Medicine, University of Maryland, College Park, MD . 20742). EHV virus stock samples were prepared by infecting Vero cells at a multiplicity of infection of 0.01 PFU/cell in Dulbecco's Modified Eagle Medium (DMEM) containing 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components were obtained from Irvine Scientific or equivalent supplier, and hereafter are referred to as complete DME
medium) plus 1% fetal bovine serum. After cytopathic effect was complete, the medium and cells were harvested and the cells were pelleted at 3000 rpm for 5 minutes in a clinical centrifuge. Cells were resuspended in 1/10 the original volume of medium, and an equal volume of skim milk (9% skim milk powder in FI20 weight/volume) was added. The virus samples were frozen at -70'C. The titers were approximately 10a PFU/ml for EHV-1 and approximately 10~ PFU/ml for EHV-4.
PREPARATION OF HERPESVIRUS DNA. For herpesvirus DNA
preparation, a confluent monolayer of Vero cells in a 25 cm2 flask or 60 mm petri dish was infected with 100 ~cl of virus sample. After overnight incubation, or when the cells were showing 100% cytopathic effect, the cells were scraped into the medium. The cells and medium were 3o centrifuged at 3000 rpm for 5 minutes in a clinical centrifuge. The medium was decanted, and the cell pellet was gently resuspended in 0.5 ml solution containing 0.5%
NONIDET P-40~' (octyl phenol ethylene oxide condensate containing an average of 9 moles of ethylene oxide per molecule) (NP-40, purchased from Sigma Chemical Co., St.
Louis, MO.). The sample was incubated at room temperature for 10 minutes. Ten ~1 of a stock solution of RNase A (Sigma) were added (stock was 10 mg/ml, boiled for 10 minutes to inactivate DNAse). The sample was centrifuged to pellet nuclei. The DNA pellet was removed with a pasteur pipette or wooden stick and discarded.
The supernatant fluid was decanted into a 1.5 ml Eppendorf tube containing 25 ~cl of 20~ sodium dodecyl sulfate (Sigma) and 25 ~Cl proteinase-R (10 mg/ml:
Boehringer Mannheim). The sample was mixed and incubated at 37'C for 30-60 minutes. An equal volume of water-saturated phenol was added and the sample was mixed briefly. The sample was centrifuged in an Eppendorf minifuge for 5 minutes at full speed. The upper aqueous phase was removed to a new Eppendorf tube, and two volumes of absolute ethanol were added and the tube put at -20'C for 30 minutes to precipitate nucleic acid. The sample was centrifuged in an Eppendorf minifuge for 5 minutes. The supernatant was decanted, and the pellet was air dried and rehydrated in -16 ~l HZO. For the preparation of larger amounts of DNA, the procedure was scaled up to start with a 850 cm2 roller bottle of Vero cells. The DNA was stored in 0.01 M tris pH 7.5, 1 mM
EDTA at 4'C.
MOLECULAR BIOLOGICAL TECHNIQUES. Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels, ligation, phosphorylation with kinase, treatment with phosphatase, growth of bacterial cultures, transformation of bacteria with DNA, and other molecular biological methods are described by Maniatis et al. (1982) and Sambrook et al. (1989). The polymerise chain reaction (PCR) was used to introduce restriction sites convenient for the manipulation of various DNAs. The procedures used are described by Innis et al (1990). In general, amplified fragments were less than 500 base pairs in size and critical regions of amplified fragments were confirmed by DNA sequencing. Except as noted, these techniques were used with minor variations.
LIGATION. DNA was joined together by the action of the enzyme T4 DNA ligase (HRL). Ligation reactions contained various amounts of DNA (from 0.2 to 20~Cg), 20mM Tris pH
7. 5, lOmM MgCl2, lOmM dithiothreitol (DTT) , 200 ~M ATP and units T4 DNA ligase in 10-20 ~tl final reaction volume.
15 The ligation proceeded for 3-16 hours at 15'C.
DNA SEQUENCING. Sequencing was performed using the USB
Sequenase Rit and 35S-dATP (NEN). Reactions using both the dGTP mixes and the dITP mixes were performed to 20 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. The sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with Superclone and Supersee programs from Coral Software.
SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis et al. DNA
was blotted to nitrocellulose filters and hybridized to appropriate labeled DNA probes. Probes for southern blots were prepared using either the Nonradioactive DNA

._ Labeling and Detection Kit of Boehringer Mannheim or the nick translation kit of Bethesda Research Laboratories (BRL). In both cases the manufacturer's recommended procedures were followed.
DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS. The method is based upon the calcium phosphate procedure of Graham and Van der eb (1973) with the following modifications. Virus and/or Plasmid DNA were diluted to 298 ~1 in 0.01 M Tris pH 7.5, 1mM EDTA. Forty ~1 2M CaCl2 was added followed by an equal volume of 2X HEPES
buffered saline (lOg N-2-hydroxyethyl piperazine N'-2-ethanesulfonic acid (HEPES), 16g NaCl, 0.748 KC1, 0.25g Na2HP0,~~ 2H20, 2g dextrose per liter HZO and buffered with NaOH to pH 7.4). The mixture was then incubated on ice for 10 minutes, and then added dropwise to an 80%
confluent monolayer of Vero cells growing in a 60 mm petri dish under 5 ml of medium (DME plus 1% fetal bovine serum). The cells were incubated 4 hours at 37'C in a humidified incubator containing 5% COZ. The cells were then washed once with 5 ml of 1XPBS (1.158 Na2HP0~, 0.2g KHZPO~, 0. 8g NaCl, 0.2g KC1 per liter H20) , once with 5 ml of 20% glycerol/PBS (v/v), once more with 5 ml 1XPBS, and then fed with 5ml of medium (DME plus 2% fetal bovine serum). The cells were incubated at 37'C as above for 3-7 days until cytopathic effect from the virus was 50-100%. 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 SCREEN FOR RECOMBINANT
HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. This method relies upon the 2~.~~4~~

homologous recombination between herpesvirus DNA and plasmid homology vector DNA which occurs in tissue culture cells co-transfected with these elements. From 0.1-1.0 ~g of plasmid DNA containing foreign DNA flanked by appropriate herpesvirus cloned sequences (the homology vector) were mixed with approximately 0.3~cg of intact herpesvirus DNA. The DNAs were diluted to 298 ~cl in 0.01 M Tris pH 7.5, imM EDTA and transfected into Vero cells according to the DNA TRANSFECTION FOR GENERATING
RECOMBINANT VIRUS (see.above).
DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS. Rather than using homology vectors and relying upon homologous recombination to generate recombinant virus, we have also developed the technique of direct ligation to engineer herpesviruses. In this instance, a cloned foreign gene did not require flanking herpesvirus DNA sequences but only required that it have restriction sites available to cut out the foreign gene fragment from the plasmid vector. A compatible restriction enzyme was used to cut herpesvirus DNA. A
requirement of the technique is that the restriction enzyme used to cut the herpesvirus DNA must cut at a limited number of sites. For EHV-4 the restriction enzymes g~gI or p~I would be appropriate ( see FIGURE 2 ) .
Restriction sites previously introduced into herpesviruses by other methods may also be used. The herpesvirus DNA is mixed with a 30-fold molar excess of plasmid DNA (typically 5~g of virus DNA to 10~g of plasmid DNA), and the mixture is cut with the appropriate restriction enzyme. The DNA mixture is phenol extracted and ethanol precipitated to remove restriction enzymes, and ligated together according to the ligation procedure detailed above. The ligated DNA mixture is then resuspended in 298 girl 0.01 M Tris pH 7.5, 1mM EDTA and transfected into Vero cells according to the DNA
TRANSFECTION FOR GENERATING RECOMBINANT VZRUS (see above) .
PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM
SUBGENOMIC DNA FRAGMENTS. The ability to generate herpesviruses by cotransfection of cloned overlapping subgenomic fragments has been demonstrated for pseudorabies virus (48) and for herpssvirus of turkeys (47). If deletions and/or insertions are engineered directly into the subgenomic fragments prior to the cotransfection, this procedure results in a high frequency of viruses containing the genomic alteration, greatly reducing the amount of screening required to purify the recombinant virus. Ws anticipate utilizing this technique to engineer foreign gene insertions into specific attenuating deletions tUS2, TR, and gpE) in EHV-4. In the first step of this procedure deletions are introduced into separate viruses via homologous recombination with enzymatic marker genes as described below. The homology vector used in this step is constructed such that the enzymatic marker gene is flanked by a restriction enzyme site that does not cut EHV-4 in the region of the DNA to be deleted. In the second step a library of overlapping subgenomic fragments, capable of regenerating wild-type virus, is constructed from randomly sheared 4EHV-000 DNA. In the third step subgenomic fragments are cloned from each of 3o the individual recombinant viruses containing attenuating deletion/marker gene insertions, which were generated in the first step. In each case the subcloned fragment corresponds in size and location to one of the wild-type subgenomic fragments constructed in the second step.
This is accomplished by screening a library of randomly 21~~. 4~~

-3~2=
sheared recombinant virus DNA subclones with probes generated from the ends of the appropriate wild-type subgenomic fragment. The restriction sites which had been engineered to flank the marker genes in the first step are now utilized to replace the marker genes in each subgenomic fragment with various foreign genes (such as lEHV gpB, lEHV gpD, equihe influenza HA, or equine influenza NA). In the fourth step cotransfection of the appropriate overlapping wild type and deletion/insertion to derived subgenomic fragments permits the generation of recombinant EHV-4 viruses incorporating any desired combination of deletions and/or insertions.
SCREEN FOR RECOMBINANT ~iERPESVIRUS EXPRESSING ENZYMATIC
MARI~R GENES. When the E.coli B-galactosidase (lacZ) or 8-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 (300 ~g/ml) into the agarose overlay during the plaque assay. For the lacZ marker gene the substrate BLUOGAL' (halogenated indolyl-B-D-galactosidase, GIBCO-Bethesda Research Iabs) was used.
For the uidA marker gene the substrate X-Glucuro Chx (5-bromo-4-chloro-3-indolyl-B-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 Vero cells and purified by further blue plaque isolation. In recombinant virus strategies in which the enzymatic 3o 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.

SELECTION OF AKA-T RESISTANT VIRUS. Many nucleoside analogs inhibit alpha-herpesvirus replication. One such antiviral drug is arabinosylthymine (Ara-T; Rayo Chemicals, Canada). Resistance of EHV mutants to Ara-T
is due to mutations in the viral TR, so that TR negative (TR-) viruses are selected. The transfection stocks were grown on Vero cells in the presence of 200 ~g/ml Ara-T in complete DME medium plus 1~ fetal bovine serum. The selection was repeated one to two times. The virus stocks generated from Ara-T selection were assayed by thymidine plaque autoradiography (37, 38). Plaques picked from positive stocks were assayed for TK deletion by the SOUTHERN B?.OTTING OF DNA procedure. Note that TK
negative viruses constructed utilizing Ara-T selection (S-1EHV-001 and S-4EHV-001) exhibited changes in restriction fragments not related to the TK locus.
Differences were observed in BamHI fragments c, d, and g in S-4EHV-ool and fragment p in S-1EHV-ool. Since similar changes were not observed in S-4EHV-004 in which the TK deletion was introduced without Ara-T selection, we feel that this procedure is a less desirable procedure for the selection of recombinant viruses.
CONSTRUCTION OF DELETION VIRUSES. The strategy used to construct deletion viruses involved the use of either homologous recombination and/or direct ligation techniques. Initially a virus was constructed via homologous recombination, in which the DNA to be deleted was replaced with a marker gene such as E.coli 8-galactosidase (lacZ) or 8-glucuronidase (uidA). A second virus was then constructed in which the marker gene was deleted either by homologous recombination or via direct ligation. The advantage of this strategy is that both viruses may be purified by the SCREEN FOR RECOMBINANT
HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The first 2~4~4~2 virus is purified by picking blue plaques from a white plaque background, the second virus is purified by picking white plaques from a blue plaque background.
CLONING OF EQUINE INFLUENZA VIRUS HEMAGGLUTININ AND
NEURAMINIDASE GENES. .The equine influenza virus hemagglutinin (HA) and'Neuraminidase (NA) genes may be cloned essentially as 'described by Ratz et al. for the HA
gene of human influenza virus. Viral RNA prepared from virus grown in MDBK cells is first converted to cDNA
utilizing an oligo nucleotide primer specific for the target gene. The cDNA is then used as a template for PCR
cloning (51) of the targeted gene region. The PCR
primers are designed to incorporate restriction sites which permit the cloning of the amplified coding regions into vectors containing the appropriate signals for expression in EHV. One pair of oligo nucleotide primers will be required for each coding region. The HA gene coding regions from the serotype 2 (H3) viruses (Influenza A/equine/Miami/63, Influenza A/equine/Rentucky/81, and Influenza A/equine/Alaska/91) would be cloned utilizing the following primers 5' -GGGTCGACATGACAGACAACCATTATTTTGATAC-3' (SEQ ID NO: 64) for cDNA priming and combined with 5'-GGGTCGACTCAAATGCAAATGTTGCATCTGAT-3' (SEQ ID NO: 65) for PCR. The HA gene coding region from the serotype 1 (H7) virus (Influenza A/equine/Prague/56) would be cloned utilizing the following primers 5'-GGGATCCATGAACACTCAAATTCTAATATTAG-3' (SEQ ID NO: 66) for cDNA priming and combined with 5'-GGGATCCTTATATACAAATAGTGCACCGCA-3' (SEQ ID NO: 67) 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) would be cloned utilizing the following primers 5'-GGGTCGACATGAATCCAAATCAAAAGATAA-3' (SEQ ID NO: 68) for cDNA priming and combined with 5'-GGGTCGACTTACATCTTATCGATGTCAAA-3' (SEQ ID NO: 69) for PCR.
The NA gene coding region from the serotype 1 (N7) virus (Influenza/A/aquine/Prague/56) would be cloned utilizing the following primers 5'-GGGATCCATGAATCCTAATCAAAAACTCTTT-3' (SEQ ID NO: 68) for cDNA priming and combined with 5'-GGGATCCTTACGAAAAGTATTTAATTTGTGC-3' (SEQ ID NO: 71) for PCR. Note that this general strategy may be used to clone the coding regions of HA and NA genes from other strains of equine influenza A virus.
HOMOLOGY VECTOR 450-46.B4. The plasmid 450-46.84 was constructed for the purpose of deleting a portion of the EHV-1 thymidine kinase gene. It may also be used to insert foreign DNA into EHV1. It contains a unique ~I
restriction enzyme site into which foreign DNA may be inserted. It may be constructed utilizing standard recombinant DNA techniques (23 and 34), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in figure 4. The plasmid vector is derived from an approximately 2978 base pair FiI to VIII restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 779 base pair S~au3A restriction sub-fragment of the EHV1 $g~II
restriction fragment b (42). Fragment 2 is an approximately 1504 base pair ~EII to gg~I restriction sub-fragment of EHV1 III restriction fragment b (42).
HOMOLOGY VECTOR 467-21.19. The plasmid 467-21.19 was constructed for the purpose of deleting a portion of the EHV1 unique short 2 gene. It may also be used to insert foreign DNA into EHV1. It contains a unique ~r RI
restriction enzyme site into which foreign DNA may be inserted. It may be constructed utilizing standard ~l~~g~2~

recombinant DNA techniques (23, 34) by joining restriction fragments from the following sources as indicated in FIGURE 5. The plasmid vector is derived from an approximately 2983 base pair CHI to p~I
restriction fragment of pSP65 (Promega). Note that the SRI site has been removed from the plasmid vector by nuclease S1 digestion:v Fragment 1 is an approximately 767 base pair I~to ~I restriction sub-fragment of the EHV1 I restriction fragment n (42). Fragment 2 is an approximately .1283 base pair SRI to g~I
restriction sub-fragment of EHV1 I restriction fragment n (42).
IiOMOLOGY VECTOR 536-85.30. The plasmid 536-85.30 was constructed for the purpose of deleting the EIiV1 glycoprotein G gene. It was used to insert foreign DNA
into EHVi. It contains a pair of ~I restriction enzyme sites into which foreign DNA may be inserted. It may be constructed utilizing standard recombinant DNA techniques (23 and 34) by joining restriction fragments from the following sources as indicated in FIGURE 6. The plasmid vector is derived from an approximately 2643 base pair E~RI to p~I restriction fragment of pNE8193 (New England Biolabs). Fragment 1 is an approximately 2292 base pair SRI to III restriction sub-fragment of the EI~V1 SRI restriction fragment k (42). Fragment 2 is an approximately 1077 base pair ~I to CHI restriction sub-fragment of EHV1 E~RI restriction fragment k (42).
HOMOLOGY VECTOR 495-61.39. The plasmid 495-61.39 was constructed for the purpose of deleting a portion of the EHV-4 thymidine kinase gene. It may also be used to insert foreign DNA into EHV-4. It contains a unique ~I
restriction enzyme site into which foreign DNA may be inserted. It may be constructed utilizing standard r WO 94/03628 ~~ ~ PCT/US93/07424 recombinant DNA techniques (23, 34) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGURE 7. The plasmid vector is derived from an approximately 2988 base pair ,~,~I to III restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 830 base pair III to gg~I restriction sub-fragment of the EHV-4 ~gHI
restriction fragment c (8). Fragment 2 is an approximately 1220 base pair g~II to ;~I restriction sub-fragment of EHV-4 ~FiI restriction fragment c (8).
HOMOLOGY VECTOR 523-38.9. The plasmid 523-38.9 was constructed for the purpose of deleting a portion of the EHV4 unique short 2 gene. It may also be used to insert foreign DNA into EHV4. It contains a unique p~I
restriction enzyme site into which foreign DNA may be inserted. It may be constructed utilizing standard recombinant DNA techniques (23, 34) by joining restriction fragments from the following sources as indicated in FIGURE 8. The plasmid vector is derived from an approximately 2984 base pair ~,I to VIII
restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 1098 base pair ~I to p~I restriction sub-fragment of the EHV4 SRI restriction fragment g (8). Fragment 2 is an approximately 2799 base pair gg~I
to VIII restriction sub-fragment of EHV4 ,CHI
restriction fragment d (8).
HOMOLOGY VECTOR 580-57.25. The plasmid 580-57.25 was constructed for the purpose of deleting the EHV4 gpE
gene. It may also be used to insert foreign DNA into EHV4. It contains a unique CHI restriction enzyme site into which foreign DNA may be inserted. It may be constructed utilizing standard recombinant DNA techniques (23, 34), by joining restriction fragments from the 2~g~~.42~~

following sources as indicated in figure 9. The plasmid vector is derived from an approximately 2973 base pair SRI to III restriction fragment of pSP65 (Promega).
Fragment 1 is an approximately 2046 base pair ~qRI to g~II restriction sub-fragment of the EHV4 SRI
restriction fragment j .(8). Fragment 2 is an approximately 1976 base-vpair ~I to ~pI restriction sub-fragment of EHV4 SRI restriction fragment j (8).
HOMOLOGY VECTOR 467-22.A12. The plasmid 467-22.A12 was constructed for the purpose of deleting a portion of the US2 gene coding region from the EHV-1 virus. It incorporates an E.coli B-galactosidase (lacZ) marker gene flanked by EHV-1 virus DNA. The lacZ marker gene was inserted into the homology vector 467-21.19 at the unique SRI site. The marker gene is oriented opposite to the US2 gene in the homology vector. A detailed description of the marker gene is given in FIGURE 10. It may be constructed utilizing standard recombinant DNA
techniques (23, 34) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGURE 10. Fragment 1 is an approximately 413 base pair ~I to ~FiI restriction sub-fragment of the PRV ~FiI restriction fragment 10 (22). Fragment 2 is an approximately 3010 base pair CHI to g~p~II
restriction fragment of plasmid pJF751 (11). Fragment 3 is an approximately 754 base pair l~gI to S,~,I
restriction sub-fragment of the PRV CHI restriction fragment # 7 ( 2 2 ) .
HOMOLOGY VECTOR 588-81.13. The plasmid 588-81.13 was constructed for the purpose of deleting a portion of the US2 gene coding region from the EHV-4 virus. It incorporates an E.coli ~-galactosidase (lacZ) marker gene flanked by EHV-4 virus DNA. A lacZ marker gene was h inserted as a p~I restriction fragment into the homology vector 523-38.9 at the unique p~I site. The marker gene is oriented in the opposite direction to the US2 gene in the homology vector. A detailed description of the marker gene is given in FIGURE il. It was constructed utilizing standard recombinant DNA techniques (23, 34) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGURE 11.
Fragment 1 is an approximately 413 base pair ,~I to $~HI restriction sub-fragment of the PRV $~FiI
restriction fragment 10 (22). Fragment 2 is an approximately 3010 base pair ~FII to III restriction fragment of plasmid pJF751 (li). Fragment 3 is an approximately 754 base pair ~gI to ;~,,I restriction sub-fragment of the PRV $~IiI restriction fragment #7 ( 22 ) .
HOMOLOGY VECTOR 552-45.19. The plasmid 552-45.19 was constructed for the purpose of deleting a portion of the TR gene coding region from the EHV-4 virus. It incorporates an E.coli B-glucuronidase (uidA) marker gene flanked by EHV-4 virus DNA. The uidA marker gene was inserted into the homology vector 495-61.39 at the unique ~I site. The marker gene is oriented opposite to the TR gene in the homology vector. A detailed description of the marker gene is given in FIGURE 12. It may be constructed utilizing standard recombinant DNA
techniques (23, 34) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figure 12. Fragment 1 is an approximately 404 base pair ,~I to ~gRI restriction sub-fragment of the PRV g~Iil restriction fragment X10 (22). Note that the SRI site was introduced at the loction indicated in FIGURE 12 by PCR cloning. Fragment 2 is an approximately 1823 base pair ~r RI to S.maI fragment of the plasmid pRAJ260 (Clonetech). Note that the SRI and S.maI sites were introduced at the locations indicated in figure 12 by PCR cloning. Fragment 3 is an approximately 784 base pair ~I to ~I restriction sub-fragment of the HSV-1 CHI restriction fragment Q (24). Note that this fragment is oriented such that the polyadenylation sequence (AATAAA) is located closest to junction C.
HOMOLOGY VECTOR 593-31.2. The plasmid 593-31.2 was constructed for the purpose of deleting the gpE gene l0 coding region from the EHV-4 virus. It incorporates an ~,.coli B-galactosidase (lacZ) marker gene flanked by EHV-4 virus DNA. The lacZ marker gene was inserted into the homology vector 580-57.25 at the unique ~aHI site. The marker gene is oriented the same as the deleted gpE gene in the homology vector. A detailed description of the marker gene is given in FIGURE 13. It may be constructed utilizing standard recombinant DNA techniques (23, 34) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in figure 10.
Fragment 1 is an approximately 413 base pair S~.I to CHI restriction sub-fragment of the PRV BamFII
restriction fragment 10 (22). Fragment 2 is an approximately 3010 base pair ~nFiI to g~II restriction fragment of plasmid pJF751 (il). Fragment 3 is an approximately 754 base pair ~I to ,~g~,I restriction sub-fragment of the PRV FII restriction fragment #7 (22).
HOMOLOGY VECTOR 616-40. The plasmid 616-40 was constructed for the purpose of deleting a portion of the EHV-4 thymidine kinase gene. It is also used to insert foreign DNA into EHV-4. It contains a unique 1o I site into which foreign DNA is inserted. The homology vector 616-40 is derived from a cosmid library made of sheared DNA from virus 4EHV-004. A library of subclones containing overlapping EHV subgenomic fragments was generated as follows. 4EHV-004 DNA was sheared and then size selected on a glycerol gradient as described (48) with 40-50 kb fragments chosen as the insert population.
The pooled fractions were diluted twofold with TE, one-s tenth volume of 3M NaAc and 2.5 volumes of ethanol were added, and the DNA was precipitated at 30,000 rpm in a Beckman SW41 rotor for 1 hr. The sheared fragments were polished to give blunt ends by initial treatment with T4 DNA polymerise, using low dNTP concentrations to promote l0 3' overhang removal, followed by treatment with Klenow polymeraae to fill in rocsssad 3' ends. These insert fragments were than ligated to 384-94. Cosmid vector 384-94 is a derivative of pHC79 from Gibco BRL, Inc. from which the tetracycline resistance gene was deleted by 15 restriction endonuclease digestion with VIII and gy~I, and a DNA linker containing the ~I-~IiI-~I
restriction sited was insertod. Cosmid vector 384-94 was digested with I, made blunt by treatment with Klenow polymerise and treated with calf intestinal phosphatase.
20 The ligation mixture containing cosmid vector 384-94 and 4EHV-004 genomic DNA fragments was then packaged using Gigapack XL packaging extracts (Stratagene). Ligation and packaging were as recommended by the manufacturer.
Colonies were grown in overnight cultures and cosmid DNA
25 was extracted (23,34). Cosmid DNA was analyzed by restriction endonuclease digestion with ~I. The cosmid DNA clones were screened for the presence of a 3.0 kb NotI fragment indicating the presence of the PRV gX
promoter-uidA foreign gene insert into a lNotI site within 30 the TR gene deletion. One cosmid, 607-21.16, containing the TR gene deletion with an insertion of the uidA gene was isolated. The cosmid, 607-21.16, was digested with NotI to remove the gX promoter/uidA gene and religated to obtain the homology vector, 616-40. The homology vector, digested with ~I to remove the gX promoter/uidA gene and religated to obtain the homology vector, 616-40. The homology vector, 616-40, contains DNA sequences surrounding the TR gene of approximately 22,600 base pairs which includes approximately 4000 base pairs of SRI a fragment, approximately 600 base pairs of the entire SRI q fragment;~and approximately 18,000 base pairs of the g~RI ~ w fragment. The vector is derived from an approximately 4,430 base pair I restriction fragment from cosmid vector, 384-94 (derived from pHC79 Gibco-BRL) . Homology vector 616-40 contains the 653 base pair deletion in the TR gene with a unique t~I site and no additional marker gene inserted.
HOMOLOGY VECTOR 593-20.5. The plasmid 593-20.5 was constructed for the purpose of deleting the EHV4 gpE gene and inserting the B-glucuronidase (uidAj marker gene under the control of the PRV gX promoter. It is also used to insert other foreign DNA including the equine influenza HA and NA genes into EHV4. It was constructed using standard recombinant DNA techniques (23, 34), by joining restriction fragments from the following sources.
The plasmid is derived from an approximately 2973 base pair SRI to HincII restriction fragment of pSP65 (Promega) . Fragment 1 is an approximately 2046 base pair c-oRI to III restriction subfragment of the EHV4 SRI
restriction fragment j (8). Fragment 2 is an approximately 3011 base pair ~amHI fragment containing the PRV gX promoter, uidA gene, and the HSV-1 polyadenylation site. Fragment 3 is an approximately 1976 base pair ~spI to ~pI restriction sub-fragment of EHV4 ~c RI restriction fragment j.

~...

The deletion of the US2 gene in an Equine herpesvirus renders a recombinant equine herpesvirus safe for use in pregnant equines, that is, it renders the virus incapable of causing abortion of the fetus.
We have characterized the unique short regions of EHV-1 and EHV-4 by DNA sequence analysis. SEQ ID NO: 1 shows the sequence of the first 1322 bases of the iI
fragment n (see FIGURE 1) reading away from the ~IiI n -~pHI d junction. This sequence contains a 303 amino acid ORF which exhibits homology to several other herpesvirus US2 genes (see FIGURE 3). SEQ ID NO: 3 shows the 1252 bases of sequence which starts 198 bases upstream of the VIII site located approximately in the middle of the EI~V-4 SRI g fragment (see FIGURE 2) . The sequence reads back toward the SRI g - ~gRI b junction and contains a 324 amino acid ORF. After we sequenced the unique short region, we found that it contained a US2 gene with homology to several other herpesvirus US2 genes (see FIGURE 5). Since we determined the location and sequence of the US2 gene in the equine herpes virus, we can delete the US2 gene of EHV-1 and EHV-4 and attenuate as well as render the virus safe for use in pregnant horses.

The homology vector 450-46.B4 is a plasmid used for attenuating EHV-1 via inactivation of the TR gene.
Inactivation of the TR gene is accomplished by a deletion of DNA which encodes Tk from EHV-1. Plasmid 450-46. B4 carries a copy of the TR gene (31) into which an approximately 202 by deletion between amino acids 115 and 182 has been introduced. The plasmid, used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS and the SELECTION OF ARA-T
RESISTANT VIRUS, generates an EHV-1 containing a deleted TK gene.
Plasmid 450-46.84 is also useful for inserting foreign DNA into EHV-1. The plasmid contains a unique ~I
restriction site located at the site of the deletion.
Foreign DNA cloned into this site results in a plasmid which should be used according to the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS to generate an EHV-1 containing the foreign DNA. Note that if an appropriate marker gene (e. g.
E.coli lac2) is inserted into the homology vector, then a recombinant virus is generated without the SELECTION OF
ARA-T RESISTANT VIRUS.
For the procedures described above to be successful, it is important that the deletion/insertion site be in a region non-essential to the replication of the EHV-1 and that the site be flanked with equine herpesvirus DNA
appropriate for mediating homologous recombination between virus and plasmid DNAs. Note that the deletion was designed so that it is limited to a specific portion of the TK coding region. This region contains amino acids important foz TK enzymatic activity. The deletion does not remove sequences that are involved with flanking genes which are important for efficient viral growth (12). We have demonstrated that the insertion/deletion site in homology vector 450-46.B4 inserts foreign DNA
into EHV-1 as represented by the two recombinant EHV-1 viruses in EXAMPLES 7 and 9.

The homology vector 467-21.19 is a plasmid used for attenuating EHV-1 via inactivation of the US2 gene.
Inactivation of the US2.gene is accomplished by deletion of US2 encoding DNA from EHV-1. Plasmid 467-21.19 carries a copy of the US2 gene into which an approximately 93 by deletion between amino acids 174 and 205 has been introduced. The plasmid should be used according to the CONSTRUCTION OF DELETION VIRUSES to generate an EHV-1 containing a deleted US2 gene.
Plasmid 467-21.19 is also useful for the insertion of foreign DNA into EFIV-1. The plasmid contains a unique SRI restriction site located at the site of the deletion. Foreign DNA cloned into this site results in a plasmid which should be used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS to generate an EHV-1 containing foreign DNA.
For the procedures described above to be successful, it is important that the deletion/insertion site be in a region non-essential to the replication of the EHV-1 and that the site be flanked with equine herpesvirus DNA
appropriate for mediating homologous recombination between virus and plasmid DNAs. Note that the deletion was designed so that it is limited to the unique short region and does not remove sequences from the internal or terminal repeats. We have demonstrated that the insertion/deletion site in homology vector 467-21.19 inserts foreign DNA into EHV-1 as represented by the two recombinant EHV-1 viruses in EXAMPLES 7 and 9.

The homology vector 536-85.30 is a plasmid used for attenuating EHV-1 by removing the glycoprotein G (gpG) gene and a portion of the unique short region large membrane glycoprotein (MGPj gene. Plasmid 536-85.30 carries a portion of the unique short region into which a deletion of approximately 2384 base pairs which removes the entire gpG coding region and the N-terminal 307 amino acids of the MGP has been engineered. The plasmid may be used according to the CONSTRUCTION OF DELETION VIRUSES to generate a gpG/MGP deleted EHV-1.
Plasmid 536-85.30 is also useful for the insertion of foreign DNA into EHV-1. The plasmid contains a pair of ;~I restriction sites located at the site of the deletion. Foreign DNA cloned into these sites results in a plasmid which should be used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS to generate an EHV-1 containing foreign DNA.

WO 94/03628 ~ 1 ~.~, ~ ~ PCT/US93/07424 The homology vector 495=61.39 is a plasmid used for attenuating EHV-4 via inactivation of the TR gene.
Inactivation of the TR gene ie accomplished by deletion of DNA which encodes Tk from EHV-4. Plasmid 495-61.39 carries a copy of the TK gene (27) into which an approximately 653 by deletion between amino acids 98 and 317 has been engineered. The plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS and the SELECTION OF ARA-T
RESISTANT VIRUS to generate an EHV-4 with a deletion of the gene which encodes Tk.
Plasmid 495-61.39 is also useful for the insertion of foreign DNA into EHV-4. The plasmid contains a unique I restriction site located at the site of the deletion. Foreign DNA cloned into this site results in a plasmid which should be used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS to generate an EHV-4 virus containing foreign DNA. Note that if an appropriate marker gene (e.g. E.coli lacZ) is inserted into the homology vector, then a recombinant virus is generated without the SELECTION OF ARA-T RESISTANT VIRUS.
For the procedures described above to be successful, it is important that the deletion/insertion site be in a region non-essential to the replication of the EHV-4 and that the site be flanked with equine herpesvirus DNA
appropriate for mediating homologous recombination between virus and plasmid DNAs. Note that the deletion was designed so that it is limited to a specific portion of the TK coding region. This region contains amino acids important for TK enzymatic activity. The deletion does WO 94/03628 PCf/US93/07424 not remove sequences that are involved with flanking genes which are important for efficient viral growth (18, 12) .

The homology vector 523-38.9 is a plasmid used for attenuating EHV-4 via inactivation of the US2 gene.
Inactivation of the US2 gene is accomplished by deletion DNA which encodes US2 from EHV-4. Plasmid 523-38.9 carries a copy of the US2 gene into which an approximately 711 by deletion between amino acids 131 and 324 has been engineered. The plasmid should be used according to the CONSTRUCTION OF DELETION VIRUSES to generate an EHV-4 with a deletion of the gene which encodes US2.
Plasmid 523-38.9 is also useful for the insertion of foreign DNA into EHV-4. The plasmid contains a unique g~I restriction site located at the site of the deletion. Foreign DNA cloned into this site results in a plasmid which should be used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS to generate an EHV-4 containing foreign DNA.
For the procedures described above to be successful, it is important that the deletion/insertion site be in a region non-essential to the replication of the EHV-4 and that the site be flanked with equine herpesvirus DNA
appropriate for mediating homologous recombination between virus and plasmid DNAs. Note that the deletion was designed so that it is limited to the unique short region and does not remove sequences from the internal or terminal repeats. We have demonstrated that the insertion/deletion site in homology vector 523-38.9 inserts foreign DNA into EHV-4 as represented by the two recombinant EHV-4 viruses in EXAMPLES 13 and 14.

We have determined that the deletion of the glycoprotein E gene from the equine herpesvirus is useful in attenuating the virus for use in a vaccine for horses and for providing a negative serological marker.
l0 The homology vector 580-57.25 is a plasmid used to attenuate EHV-4 by removing the glycoprotein E (gpE) gene (8 and SEQ ID NOS: 5 ~ 6). Plasmid 580-57.25 carries a portion of the unique short region into which a deletion of approximately 1694 base pairs, which removes the entire gpE coding region, has been engineered. The plasmid may be used according to the CONSTRUCTION OF
DELETION VIRUSES to generate an EHV-4 virus with a deletion of the gene which encodes gpE.
Plasmid 580-57.25 is also useful for the insertion of foreign DNA into EHV-4. The plasmid contains a unique I restriction site located at the site of the deletion. Foreign DNA cloned into this site results in a plasmid which should be used according to the IiOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS to generate an EHV-4 containing foreign DNA.

PRFPA1~ATTyT~ OF RECOMBiPIANI ~OCJiNr.
HERPESVIRUS DESIGNATED.S-lEHV-001 ,..
S-lEHV-001 is an equine herpesvirus type 1 (EHV-1) virus that has an approximately 202 base pair deletion in the TR gene. The S-lEHV-001 equine herpesvirus was deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC
Accession No. VR 2357.
S-1EHV-001 was derived from S-1EHV-000 (Dutta strain).
This was accomplished utilizing the homology vector 450-46.B4 (see Materials and Methods) and virus S-lEHV-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. The transfection stock was selected according to the SELECTION OF ARA-T RESISTANT
VIRUS. Individual clones were picked after two rounds of selection and assayed by thymidine plaque autoradiography (37, 38). Plaques picked from TR negative stocks were assayed for TR deletion by the SODTHERN BLOTTING OF DNA
procedure. A plaque which was TR minus by both the thymidine incorporation assay and the southern analysis was chosen and designated S-lEHV-001.
The construction of this virus establishes the EHV-1 thymidine kinase gene as a non-essential gene and a viable site for the insertion of foreign DNA. This virus is useful because the inactivation of the TK gene attenuates the virus.

..

s-1EHV-002 is an squine'herpssvirus type 1 (EHV-1) virus that has two deletions in the short unique region of the genome. The first deletion is approximately 93 base pairs and removes amino acids 174 to 205 of the US2 gene (SEQ ID NO: 1). The second deletion is approximately 2283 base pairs and removes portions of the gpG and MGP
genes from the unique short region. The gene for E.coli B-galactosidase (lacZ gene) was inserted into the deletion in the US2 gene and is under the control of the PRV gpX promoter. The S-1EHV-002 equine herpesvirus was deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit o! Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of tha American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2358.
S-lEHV-002 was derived from S-lEHV-000 (Dutta strain).
This was accomplished utilizing the homology vector 467-22.A12 (see Materials and Methods) and virus S-1EHV-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS
EXPRESSING ENZYMATIC MARRER GENES. The final result of blue plaque purification was the recombinant virus designated S-lEHV-002. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA
procedure. This analysis confirmed the insertion of the B-galactosidase (lacZ) marker gene and the deletion of approximately 93 base pairs of the US2 gene. To characterize the second unique short region deletion, the deleted EcoRI k fragment from S-lEHV-002 was subcloned WO 94/03628 ~ ~ ~'~, PCT/US93/07424 and subjected to DNA sequence analysis. This analysis confirmed a deletion which begins with amino acid 14 of the gpG gene and continues through amino acid 303 of the MGP gene. The deletion occurred such that the remaining 13 amino acids of the gpG gene are in frame with the remaining 494 amino acids of the MGP gene.
The construction of this virus establishes the EHV-1 US2 and gpG genes as non-essential genes and are viable sites for the insertion of foreign DNA. This virus is useful because inactivation of the US2 gene attenuates the virus and the deletion of the glycoprotein G gene from this virus provides a negative serological marker for differentiating it from wild type EHV-1.

r 214..1 ~. ~ 2 S-1EHV-o03 is an equine herpesvirus type 1 (EHV-1) virus that has two deletions in the short unique region and one deletion in the unique long region of the genome. The first deletion is an approximately 202 base pair deletion . .
in the TR gene. The second deletion is approximately 93 base pairs and removes nucleic acids 174 to 205 of the US2 gene (SEQ ID NO: 1). The third deletion is approximately 2283 base pairs and removes portions of the gpG and MGP genes from the unique short region. The gene for E.coli B-galactosidase (lacZ gene) was inserted into the deletion in the US2 gene and is under the control of the PRV gpX promoter. The S-1EHV-003 equine herpesvirus was deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2359.
S-lEHV-003 was derived from S-1EHV-002 (see EXAMPLE 9).
This was accomplished utilizing the homology vector 450-46.B4 (see Materials and Methods) and virus S-lEHV-002 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. The transfection stock was selected according to the SELECTION OF ARA-T RESISTANT
IBR VIRUS. Individual clones were picked after two rounds of selection and assayed by thymidine plaque autoradiography (37, 38). Plaques picked from TK
negative stocks were assayed for TK deletion by the SOUTHERN BLOTTING OF DNA procedure. A plaque which was TK
minus by both the thymidine incorporation assay and the southern analysis was chosen and designated S-lEHV-003.

The construction of this virus establishes that multiple deletions inactivating the TR and US2 genes and removing the gpG genes can be made in a single EHV-1 virus. This virus is useful because the inactivation of the TR and US2 genes attenuates the virus and the deletion of the region which enCOdes glycoprotein G from this virus provides a negative serological marker for differentiating it from wild type EHV-1.
to S-lEHV-004 is an equine herpesvirus type 1 (EHV-1) virus that has one deletion in the long unique region and one deletion in the short unique region of the genome. The first deletion is an approximately 202 base pair deletion to in the TR gene. The second deletion is approximately 93 base pairs and removes DNA encoding nucleic acids 174 to 205 of the US2 gene (SEQ ID NO: 1). The gene for E.coli B-galactosidase (lacZ gene) was inserted into the deletion in the US2 gene and is under the control of'the PRV gpX promoter. The S-lEHV-004 equine herpesvirus was deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2360.
S-lEHV-004 was derived from S-1EHV-001 (see EXAMPLE 8).
This was accomplished utilizing the homology vector 467-22.A12 (see Materials and Methods) and virus S-1EHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS
EXPRESSING ENZYMATIC MARI~R GENES. The final result of 3o blue plaque purification was the recombinant virus designated S-lEHV-004. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA
procedure. This analysis confirmed the insertion of the B-galactosidase (lacZ) marker gene, the deletion of approximately 93 base pairs of the US2 gene, and the approximately 202 base pair deletion of the TK gene.

The construction of this virus establishes that the EHV-1 US2 and TR genes are non-essential and are viable sites for the insertion of foreign DNA. This virus is useful because the inactivation of the TK and US2 genes attenuates the virus.

S-4EHV-0o1 is an equine herpesvirus typ~ 4 (EHV-4) virus that has an approximately 202 bass pair deletion in the TK gene. The S-4EHV-001 equine herpesvirus was deposited on March 12, 1992 pursuant to the Budapest Treaty on the l0 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 2361.
S-4EHV-001 was derived from S-4EHV-000 (Dutta strain).
This was accomplished utilizing the homology vector 450-46.H4 (see Materials and Methods) and virus S-4EHV-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. The transfection stock was selected according to the SELECTION OF ARA-T RESISTANT
IHR VIRUS. Individual clones were picked after two rounds of selection and analyzed by the SOUTHERN BLOTTING
OF DNA procedure. A plaque which was TK minus by the southern analysis was chosen and designated S-4EHV-001.
The construction of this virus establishes the EHV-4 thymidine kinase gene as a non-essential gene and a viable site for the insertion of foreign DNA. This virus is useful because the inactivation of the TR gene attenuates the virus. The construction of this virus also demonstrates that a homology vector derived from EHV-1 can engineer EHV-4 in an analogous manner.

WO 94/03628~~ ~~~, PCT/US93/07424 S-4EHV-002 is an equine herpesvirus type 4 (EHV-4) virus that has one deletion in the long unique region and one deletion in the short unique region of the genome. The first deletion is an approximately 202 base pair deletion in the TR gene. The second deletion is approximately 705 base pairs and removes amino acids 131 to 324 of the US2 gene (SEQ ID NO: 3). The gene for E.coli B-galactosidase (lacZ gene) was inserted into the deletion in the US2 gene and is under the control of the PRV gpX promoter.
The S-4EHV-002 equine herpesvirus was deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC
Accession No. VR 2362.
S-4EHV-002 was derived from S-4EHV-001 (see EXAMPLE 12).
This was accomplished utilizing the homology vector 523-42.A18 (see Materials and Methods) and virus S-4EHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS
EXPRESSING ENZYMATIC MARRER GENES. The final result of blue plaque purification was the recombinant virus designated S-4EHV-002. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA
procedure. This analysis confirmed the insertion of the B-galactosidase (lacZ) marker gene, the deletion of approximately 705 base pairs of the US2 gene, and the approximately 202 base pair deletion of the TK gene.

WO 94/03628 ~ ~ PCT/US93/07424 The construction of this virus establishes the EHV-4 US2 and TR genes as non-essential genes and as viable sites for the insertion of foreign DNA. This virus is useful because the inactivation of the TR and US2 genes attenuates the virus.

WO 94/03628 ~~ PCT/US93/07424 S-4EHV-0o3 is an equine herpesvirus type 4 (EHV-4) virus that has one deletion in the short unique region of the genome. The deletion is approximately 705 base pairs and removes amino acids 131 to 324 of the US2 gene (SEQ ID
No: 3). The gene for E.ccli B-galactosidase (lacZ gene) was inserted into the deletion in the US2 gene and is under the control of the PRV gpX promoter. The S-4EHV-003 equine herpesvirus was deposited on March 12, 1992 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2363.
S-4EHV-003 was derived from S-4EHV-000 (Dutta strain).
This was accomplished utilizing the homology vector 523-42.A18 (see Materials and Methods) and virus S-4EHV-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS
EXPRESSING ENZYMATIC MARI~R GENES. The final result of blue plaque purification was the recombinant virus designated S-4EHV-003. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA
procedure. This analysis confirmed the insertion of the B-galactosidase (lacZ) marker gene and the deletion of approximately 705 base pairs of the US2 gene.
The construction of this virus establishes the EHV-4 US2 gene as non-essential and as a viable site for the .. 2141422 insertion of foreign DNA. This virus is useful because the inactivation of the US2 gene attenuates the virus.

S-4EHV-004 is an equine herpesvirus type 4 (EHV-4) virus that has a deletion of approximately 653 base pairs between amino acids 98 and 317 of the thymidine kinase gene (28). The gene for E.coli B-glucuronidase (uidA
gene) was inserted into.the deletion in the TR gene and is under the control of the PRV gpX promoter.
S-4EHV-004 was derived from S-4EHV-000 (Dutta strain).
This was accomplished utilizing the homology vector 552-45.19 (see Materials and Methods) and virus S-4EHV-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. 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-4EHV-004. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA
procedure. This analysis confirmed the insertion of the B-glucuronidase (uidA) marker gene and the deletion of approximately 653 base pairs of the TK gene.
The construction of this virus establishes that the EHV-4 TK gene is non-essential and is a viable site for the insertion of foreign DNA. This virus is useful because the inactivation of the TK gene attenuates the virus.

_ Recombinant EHV-4 viruses expressing glycoproteins from EHV-1 are utilized in vaccines to protect against infection by both EHV-1 and EHV-4. Similarly, recombinant EHV-1 viruses expressing EHV-4 glycoproteins are utilized in vaccines to protect against infection by both EHV-1 and EHV-4.
S-4EHV-010, a recombinant EHV-4 with deletions in the TR, US2, and gpE genes and with insertions of the genes for EHV-1 gpD and gpB in place of the TR and gpE genes, respectively, is constructed in the following manner. S-4EHV-010 is derived from S-4EHV-004 (see EXAMPLE 15) through the construction of four intsrmadiats viruses.
The first intermediate virus, S-4EHV-005, was constructed similarly to S-4EHV-003, utilizing the homology vector 588-81.13 (see Materials and Methods) and virus S-4EHV-004 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR
GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT
HERPESVIRUS EXPRESSING ENZYMATIC MARI~R GENES for a blue plaque recombinant virus (lacZ substrate). The resulting virus has deletions of the TK and US2 genes and insertions of uidA and lacZ in the TK and US2 gene deletions, respectively. The second intermediate virus 3o S-4EHV-006, was constructed, utilizing the homology vector 523-38.9 (see Materials and Methods) and virus S-4EHV-005 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR
GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT
HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (lacZ substrate). The resulting virus has deletions of the TK and US2 genes and an insertion of uidA gene in the TK gene deletion. The third intermediate virus, S-4EHV-007, is constructed, utilizing the homology vector 593-31.2 (see Materials and Methods) and virus ~-4EHV-006 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS. The transfection stock is screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC
MARI~R GENES for a blue plaque recombinant virus (lacZ
substrate). The resulting virus has deletions of the TK, US2, and gpE genes and insertions of the uidA and lacZ
genes in the TR and gpE gene deletions, respectively.
The fourth intermediate virus S-4EHV-009, is constructed, utilizing the homology vector 580-57.25, into which the EHV-1 gpB gene had been inserted, and virus S-4EHV-007 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT HERPESVIRUS. Note that the EHV-1 gpB gene is cloned as an approximately 3665 by g~gI to ~I sub-fragment of an approximately 5100 by pg~I fragment of EHV-1 (43). The transfection stock is screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC
MARI~R GENES for a white plaque recombinant virus (lacZ
substrate). The resulting virus has deletions of the TR, US2, and gpE genes and insertion of the uidA and EHV-1 gpB genes in the TK and gpE gene deletions, respectively.
Finally, S-4EHV-010 is constructed, utilizing the homology vector 495-61.39, into which the EHV-1 gpD gene is inserted, and virus S-4EHV-009 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS. Note that the EHV-1 gpD gene is cloned as an approximately 1929 by S.S. maI to ~o,RV sub-fragment of the approximately 10,500 by ~FiI D fragment of EHV-1 (1) . The transfection stock is screened by the SCREEN FOR
RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES
for a white plaque recombinant virus (uidA substrate).
This virus is utilized in a vaccine to protect horses from infection with EHV-1 and EHV-4. The deletion of the glycoprotein E gene from this virus provides a negative WO 94/03628 ~ ~ PCT/US93/07424 serological marker for differentiating it from wild type EHV-1 and EHV-4.

WO 94/03628 ~~ ~ PCT/US93/07424 Recombinant poxviruses encoding the hemagglutinin (HA) and the neuraminidase genes (NA) from influenza viruses have been reported to mediate protective immunity against infection with the homologous influenza virus (5, 44).
Delivery of the HA and NA antigens from several subtypes of equine influenza virus via recombinant EHV viruses is utilized to provide protective immunity against equine influenza virus in addition to equine herpesvirus.
S-4EHV-Oll, a recombinant EHV-4 with deletions in the TR, US2, and gpE genes and with the genes for Influenza A/equine/Prague/56 hemagglutinin and neuraminidase of the isolate of equine influenza inserted in place of the gpE
gene is constructed in the following manner. S-4EHV-011 is derived from S-4EHV-023 through the construction of an intermediate virus. S-4EHV-023 was constructed utilizing homology vector 616-40 (see Materials and Methods) and virus S-4EHV-006 in the HOMOLOGOUS RECOMBINATION
PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR
RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES
for a white plaque recombinant virus (uidA substrate).
The intermediate virus, S-4EHV-008, was constructed utilizing the homology vector 593-20.5 (see Materials and Methods) and virus S-4EHV-023 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC
MARRER GENES for a blue plaque recombinant virus (uidA
substrate) . The resulting virus has deletions in the TK, US2, and gpE genes and an insertion of uidA in the gpE
gene deletion. Finally S-4EHV-011 is constructed, utilizing the homology voctor 580-57.25, into which the hemagglutinin and neuraminidase genes of the Influenza A/oquine/Prague/56 isolate of equine influenza were inserted, and virus S-4EHV-008 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS. Note that the influenza virus genes were cloned using the techniques described in the Materials and Methods section. The hsmagglutinin gene was placed under the control of the HCliV immediate early promoter and the neuraminidase gene was placed under the control of the PRV gpX promoter. The transfection stock is screened by the SCREEN FOR RECOMBINANT IiERPESVIRUS
EXPRESSING ENZYMATIC MARRER GENES for a white plaque recombinant virus (uidA substrate). This virus is utilized in vaccines to protect horses from infection with EHV-4 and equine influenza virus. An effective vaccine requires antigens from several different influenza strains. This is accomplished by construction of multiple recombinant viruses expressing IiA and NA from several different influenza strains (see EXAMPLES 18-20).
A more efficacious vaccine is formulated by mixing this recombinant virus with those described in EXAMPLES 18-20.

WO 94/03628 ~~~ PCT/US93/07424 -7p-S-4EHV-012, a recombinant EHV-4 with deletions in the TR, US2, and gpE genes and the genes for hemagglutinin and neuraminidase of the isolate of Influenza A/equine/Miami/63 equine influenza inserted in place of the gpE gene is constructed in the following manner. S-4EHV-012 is derived from S-4EHV-023 (see EXAMPLE 16) through the construction of an intermediate virus. The intermediate virus, S-4EHV-008, was constructed as described in EXAMPLE 17. S-4EHV-012 is constructed, utilizing the homology vector 580-57.25, into which the hemagglutinin and neuraminidase genes of the Influenza A/equine/Miami/63 isolate of equine influenza are inserted, and virus S-4EHV-008 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS. Note that the influenza virus genes were cloned using the techniques described in the Materials and Methods section. The hemagglutinin gene was placed under the control of the HCMV immediate early promoter and the neuraminidase gene was placed under the control of the PRV gpX promoter. The transfection stock is screened by the SCREEN FOR RECOMBINANT HERPESVIRUS
EXPRESSING ENZYMATIC MAR~R GENES for a white plaque recombinant virus (uidA substrate). This virus is utilized in a vaccine to protect horses from infection by EHV-4 and equine influenza virus. A more efficacious vaccine is formulated by mixing this recombinant virus with those described here and in EXAMPLES 17, 19 and 20.

S-4EHV-013, a recombinant EHV-4 with deletions in the TR, US2, and gpE genes and the genes for hemagglutinin and neuraminidase of the Influenza A/equine/Rentucky/81 isolate of equine influenza inserted in place of the gpE
gene is constructed in the following manner. S-4EHV-013 is derived from S-4EHV-023 (see EXAMPLE 16) through the construction of an intermediate virus. The intermediate virus, S-4EHV-008, was constructed as described in EXAMPLE 17. S-4EHV-013 is constructed, utilizing the homology vector 580-57.25, into which the hemagglutinin and neuraminidase genes of the Influenza A/equine/Rentucky/81 isolate of equine influenza is inserted, and virus S-4EHV-008 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS. Note that the influenza virus genes were cloned using the techniques described in the Materials and Methods section. The hemagglutinin gene was.placed under the control of the HCMV immediate early promoter and the neuraminidase gene was placed under the control of the PRV gpX promoter. The transfection stock is screened by the SCREEN FOR RECOMBINANT HERPESVIRUS
EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (uidA substrate). This virus is utilized in a vaccine to protect horses from infection by EHV-4 and equine influenza virus. A more efficacious vaccine is formulated by mixing this recombinant virus with those described here and in EXAMPLES 17, 18 and 20.

rxAwrpr.E 2 0 p Fpp~RATTQN OF RECO~INANT EQUINE
S-4EHV-014, a recombinant EHV-4 with deletions in the TK, US2, and gpE genes and the genes for hemagglutinin and neuraminidase of the Influenza A/equine/Alaska/91 isolate of equine influenza inserted in place of the gpE gene is constructed in the following manner. S-4EHV-014 is derived from S-4EHV-023 (see EXAMPLE 17) through the construction of an intermediate virus. The intermediate virus, S-4EHV-008, was constructed as described in EXAMPLE 17. S-4EHV-014 is constructed, utilizing the homology vector 580-57.25, into which the hemagglutinin and neuraminidase genes of the Influenza A/equine/Alaska/91 isolate of equine influenza were inserted, and virus S-4EHV-008 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS . Note that the inf luenza virus genes were cloned using the techniqusa described in the Materials and Methods section. The hemagglutinin gene was placed under the control of the HCMV immediate early promoter and the neuraminidase gene was placed under the control of PRV gpX promoter. The transfection stock is screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING
ENZYMATIC MARKER GENES for a white plaque recombinant virus (uidA substrate). This virus is useful as a vaccine to protect horses from infection by EHV-4 and equine influenza virus. A more efficacious vaccine is formulated by mixing this recombinant virus with those described here and in EXAMPLES 17, 18 and 19.

STREPTOCOCCUS EQUI
The H protein (14) has been shown to play an important role in the immune response to Streptococcus e~, the causative agent of the severe respiratory disease Strangles. Delivery of this antigen via a recombinant EHV virus would result in strong protective immunity without the poet-vaccinal eequelae that often accompany whole culture and protein extracted Streptococcus eqyi bacterins. It is contemplated that the procedures that have been used to express the marker genes (lacZ and uidA) in S-1EHV-002, S-lEHV-003, S-lEHV-004, S-4EHV-002, S-4EHV-003, and S-4EHV-004 and which are disclosed herein are applicable to the expression of this and other 2o potential Stres~tococcus sa~ui antigens.
Antigens from the following microorganisms are utilized to develop equine vaccines: equine infectious anemia virus, equine encephalitis virus, equine rhinovirus, equine rotavirus, equine viral arteritis, rabies, equine adenovirus pneumonia, African horse sickness, equine coital exanthema, equine papillomatosis, equine cytomegalovirus, leptospirosis, tetanus, anthrax, colibacillosis, salmonellosis, pasteurellosis, ~, brucella-associated disease, actinomycosis, Taylorella er,~iqenitolia, and mycoplasma-associated disease.

WO 94/03628 ~~ PCT/US93/07424 The protocol was used to generate a recombinant equine herpesvirus by combining EHV genomic fragments cloned into cosmids and genomic fragments of wild type helper virus containing less than one plaque forming unit. The presence of wild type EHV genomic DNA in the transfection mixture increases the efficiency of obtaining a recombinant equine herpesvirus. Overlapping subgenomic fragments were cloned from 4EHV-000 (wild type) and 4EHV-004 viral DNA. DNA from cosmid subclones of 4EFiV-000 and 4EHV-004 was digested with the appropriate restriction endonucleases to release the inserts from the cosmid vector. Transfection with an appropriate mixture of these five fragments covering the entire EHV genome and very low concentrations of wild type viral DNA (less than one plaque-forming unit) resulted in 4EHV-004 virus production. One hundred percent of the viruses in the cotransfection stock were recombinant viruses carrying the uidA gene.

1. J. Audonnet, et al., Journal of General Viroloav 71, 2969-2978 (1990).
2. T. Ben-Porat et al., Virolocv i51, 325-334 (1986).
3. R. A. Bhat, et al.. Nucleic Acids Research 17, 1159-1176 (1989) 4. J. L. Cantello, et al., Journal of Viroloav 65, 1584-1588 (1991).

5. T. M. Chambers, et al., yiroloay 169, 414-421 (1988) .

6. C. F. Colle III, et al., Viroloav 188, 545-557 ( 1992 ) .

7. M. L. Cook & J. G. Stevens, Journal of General Viroloav 31, 75-80 (1976).

8. A. A. Cullinane, et al., Journal of General Virolow 69, 1575-1590 (1988).

9. R. C. Desrosiers et al., Molecular and Cellular ~,oloay 5, 2796-2803 (1985).

10. S. J. Edwards, et al., Plasmodium falc~~,parum antigens in recombinant IiSV-1, Technological Advances in Vaccine DevelcZpment, pp. 223-234, Alan Riss, Inc. (1988).

11. F. A. Ferrari, et al., Journal of Bacterioloav 161, 556-562 (1985).

WO 94/03628 ~~ ~~~ PCT/US93/07424 12. A. Forrester, get al~, Journal of Viroloay 66, 314-348 (1992).

13. R. Fukuchi gt al., Proc. Natl. Acad. Sci. U.S.A. 82, 751-754 (1985).

14. J. E. Galan and J.F. Timoney, Infection and Immunity 55, 3181-3187 (1987).

15. F. L. Grahm and A. Van der Eb., Virolav 52, 556-567 (1973).

16. R. W. Honess, ~,ournal of General Virology 65, 2077-2107 (1984).

17. D. R. Hustead, Larcre Animal Veterinarian 46 (2), March/April, 23-24, (1991).

18. J. G. Jacobson, et al., Journal of Virolocrv 63, 1839-1843, (1089).

19. S. Joshi, et al., Journal of Virology 65, 5524-5530 (1991).

20. Rit et al., Proceedings of the 94th Annual Meeting of the United States Animal Health Association, pp.
66-75 (1990).

21. J. M. Koomey gt al., Journal of Virology 50, 662-665 (1984).

22. B. Lomniczi et al., Journal of Virology 49, 970-979 (1984).

23. T. Maniatis, et a ., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982).

24. D. J. McGeoch, et al., Journal of Molecular Bioloav 181, 1-13 (1985).

25. D. J. McGeoch, et al., Journal o~ General Viroloav 68, 19-38 (1987).

26. D. J. McGeoch, et al., Journal of General Viroloav 69, 1531-1574 (1988).

27. L. Nicolson, et al., Journal of General Viroloav 71, 1801-1805 (1990).

28. L. Nicolson, et al., Viroloav 179, 378-387 (1990).

29. R. W. Price and A. Kahn, Infection and Immunity, 31, 571-580 (1981).

30. M. P. Riggio, et al., Journal of Viroloav 63, 1123-1133 (1989).

31. G. R. Robertson and J.M. Whalley, Nucleic Acids Research i6, 11303-11317 (1988).

32. B. Roizman, et al. , Cold Spring Harbor Conference on New Approaches to Viral Vaccines (September 1983).

33. B. Roizman, et al., Archived of Virology 123, 425-449 (1992).

34. J. Sambrook, et al., Molecular Cloning: A
Laboratonr Manual Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

35. M. Shih, et al., Proceedincrs of the National Academy of Sciences U.S.A. 81, 5867-5870 (1984).

1 ~~. 4~~
-~8-36. R. R. Spaete and E.S. Mocarski, Proceedings of the NatinnW Academy of Sciences U.S.A. 84, 7213-7217 (1987).

37. R. B. Tenser, ,et alal. , Tnlmo of General Virology 64, 1369-1373 (1983).

38. R. B. Tenser, et al., Journal of Clinical ~icrobioloav 19, 122-127 (1983).

39. R. L. Thompson ~t al., Virology 131, 180-192 (1983).

40. D. R. Thomsen, et al., Gene 57, 261-265 (1987).

41. M. Wachsman, et al., Journal of General Virology 70, 2513-2520 (1989).

42. J. M. Whalley, et al., Journal of General Virology 57, 307-323 (1981).

43. J. M. Whalley, et al., Journal of General Virolow 70, 383-394 (1989).

44. R. G. Webster, et al., Virology 164, 230-237 (1988).

45. J. P. Weir and P.R. Narayanan, Nucleic Acids Research 16, 10267-10282 (1988).

46. M. E. Whealy, et al., ~Tournal of Virology 62, 4185-4194 (1988).

47. M. A. Wild, et al., 15th International Herpesvirus Workshop, Abstract No. 122, Washington, D.C. (1990).

48. M. Zijil, et ., Journal of Virology 62, 2191-2195 (1988).

49. M. Zijil, et al., Journal of Virolow 71, 1747-1755 (1990) .

50. F. Zuckerman et al., y~~-~-~~~t;or and Control of ~~j~eskv's Disease, pp. 107-117 Ed. J. van Oirschot, Kluwer, London (1989).

51. M. A. Innis, et al., PCR Protocols: A Guide To Methods And A~~lications, pp. 84-91, Academic Press, Inc., San Diego, CA (1990).

52. Katz et al., Journal of Virolow, 64, 1808-1811 (1990).

tEOUENCE LISTING
(1) INFORMATION:
GENERAL

(i) APPIIGNT: Coehran Ph.D., Mark D

(ii) TITLE OF INYENTI011: Recambiennt Equine Nerpesviruc 1 ( i NIBIBER OF tEOtJENCFS: 71 O i i ) (iv) CORREiP01<flENCE ADDRESS:

(A) ADDRESSEE: John P. lR~ite . .

(B) STREET: 30 Rockefeller Plasa 1 (C) CITT: Nar York (D) STATE: New fork (E) COUNTRT: USA

(F) ZIP: 10112 2 (v) COMPUTER READABLE FORM:

(A> IIEDIIIlI TYPE: Floppy disk (B) ~PUTER: 1111 PC eaepatible (C) OPERATING STSTE11: PCDOS/MS-DOS

(D) SOFTIIARE: Patsntln Release l1.0, Persian 1.25 (vi) CURRENT APPLIGTIdI DATA:

(A) APPLIGTION NUMBER:

(B) FILING GATE:

(C) CLASStfIGTION:

(viii) ATTORNET/AGENT INFORMATION:

(A) NAME: Sfiite, John P

(ix) TELECOMMUNIGTI011 INFORMATION:

3 (A) TELEPHONE: (212)977-9550 (B) TELEFAX: (212)661-0525 (C) TELEX: 622523 4 (2) FOR

ID
N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1322 base pairs (B) TYPE: nucleic acid 4 (C) STRANDEDNESS: double (D) TOPOLOGT: linear (ii) MOLECULE TTPE: DNA (penamic) 50 (iii) HTPOTHETIGL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

5 (A) ORGANISM: Equine herpesvirus 1 ~~ 21 4 X42 2 (8) StRAIN: 0utte (C) INDIVIDUAL ISOLATE: S-tEVV-000 (vi i ) IIIIEDIATE
SOURCE:

lB) CLOVE: A3254.N17 (viii) POSITION
IN GENDlEE:

(B) IIAP POSITION: -a3 (C) UNItS: ><G

(ix) FEATUtE:

(A) NAVE/ID:Y: (~S

(B) LOGTI011: 249..1157 (D) OTVER INFORhATl011: /eodo~
start Ii9 /produet 'KJS2 wne prodvet /9~ "US2~
(xi)tEOUENCE DESCRIPTION: SE0 ID
N0:1:

GGATCGCCG AGGGTGtGGG AGGTGGTAGC GGAG~CTGG TGTCGTCIiA

TCGCTCGTAG TGGAAAACG GTCGGTTAGG TGGTCGCATT GTTTATTTTC

2 CCGTCGCGGT GTGCCTATM AGCTAtAtxG CTTG~CAC GGGCAGTCTT
TTtGCMG lis0 GAGTGTGTAt CTAWGCAGC TCTGCTGAM tTTAtGGACT TCGTTCAACC
GCCGTTTG 2'0 tTMtMC ATG CTGCTC TtAATTAG GTtGTTAG GTTGTCGAC AGA 290 GGT

3 Ilet ValVat LauIlethr Wl VelThr VelVelAsp Ar9 0 Gly GC AAAGCATTG CG AAC AGTTCCATC GACGTCWT CG11GT CTG TGG 33a NisLysAleLeu ProAsn ferSerIle AspYelAsp GlyNisLeu Trp 3 15 to 25 30 GAGTTTTTGAGC CW CM TGTtTCGTA TTGGG TCT GM CCGCTT GGA 386 GluPheLeuSer Ar9Gln CysPheril leuAleSer GluProLeu Gly 35 i;0 '5 IleProIleVel VslArp SerAleAsp Leu1yrArp PheSerSer Ser 4 TTATTGACCCTA CG MG CCGtGTAGG CG ATAGTC ACAACCAGG GGG 182 LeuLeuThrLeu ProLys AleCysArp ProIleVal ArpThrArp Gly 5 AlaThrAleIle AleLeu AspArpAsn GlyVelYel TyrNisGlu Asp SO d5 90 ArpIletGlyVsl SerIle GluTrpLeu SerYelleu SerGlyTyr Asn 55 ~ too toy tto MisLau AsnSerter lautleIle AsnGlnPro TyrMisLeu TrpVal LeuGlY AlaAlaAsp LauCysLys ProYalPhe AspLeuIle ProGly 1 ProLys ArpIletYal TyrAlaGlu IleAlaAsp GluPheMis LysSer TGGGG CC1CCCTTC GTGTGtGGA AM CTGTTT GAGAG ATA CG TGG 770 TrpGln ProProPhe YalCysGly LysLauPhe GluThrIle ProTrp 160 165~ t7o ACCACC GTTGAGGT MT GT CCG CTCAAATtA AGAGCGCCG GGTGGA a1a ThrThr ValGluM1s AsnBisPro LeuLysLeu ArOAlaAla GlyGly GM GAC ACCGtAGTG GGtGAGTGT GCCTTTTCC AM GT AGC TCGM1 866 GluAsp ThrYalVsl GlyGluCys GlyPheSer LysNisSer SerAsn SerLeu VatArpPro ProThrVal LysArpYal IleTyrAla ValVsl 3 AspPro AlaArpLeu ArpGluIle ProAlaPro GlyArpPro LeuPro ArpArp ArpProSer GluGlyGly IletArpAls ProArpArS ArOSer CGCGCT CCCGCGGCC GCTCGGTCC ACGGCCGCC GCCGCGACG CCGCCC 105a ArpAla ProAlaAla AlaAroSer ThrAlaAla AlaAlaThr ProPro ArpPro GlyAspPro ArpAlaPro AlaAlaArO ArpAlaGly AspVal ThrTrp NetGluArp lauLeuTrp GlyValPhe GlyArpThr SerThr 5 Art GGTGGTTTC TTGCCTtGG AAAGCCTCGC CTTTAGCCC ACGCCGCC1 tMG 1322 ~

12)INi0R11At10N fORSEC I0 110:2:

ti)tEOIR:NCE CM~RACTERISTICS:

tA) LENGTN: 303 wino acids ti) TrPE: acid wino tD> TOPOLOGT: linear t i IIOLEL1JLE TTPE:protein i ) t xi)fEOUEtiCE DESCRIPTION: tE0ID 110:2:

IletGlyValVal LeuIle ThrWl ValThrVal ValAspAroNis Lys t 5 10 is AlaLauProAsn SerSer IleAsp ValAspGly NisLsuTrpGlu Phe LsuSerArOGln CysPhe ValLeu AlaSerGlu ProLauGlyIle Pro 2 0 35 co as IleValYalArp SsrAla AspLau TyrArfPhe !erterferleu Leu ThrLauProLys AlaCys ArOIro IleYalAro ThrAraGlyAla Thr 2 5 bs 7D 75 ao AlaIleAlaLau AspArp AtnGly ValVat1yr NisGluAspArp Ilet a5 90 95 3 0 GlyValSerIle GluTrp LeuSer ValLauSer GlyTyrAsnNis Leu ' 100 105 110 AsnSerSerLeu Ile11e AsnGln ProTyrNis LauTrpVatLeu Gly AlaAlaAspLau CysLys ProVsl PheAspLeu IleProGlyPro Lys 130 135 1~0 ArpIletVat1yr AlaGlu IleAla AspGluPhe NisLysSer1rp Gln 4 0 145 t5o 155 160 ProProPheVal CysGly LysLeu PheGluThr lleProTrpThr Thr 4 5 ValGluNisAsn NisPro LeuLys LeuArpAla AlaGlyGlyGlu Asp ThrValWl Gly GluCys GlyPhe SerLysNis SerSerAsnSer Leu YalAr0ProPro ThrYal lysArp YalIleTyr AlaValYalAsp Pro AlaAr9LeuArp Glu11e ProAls ProGlyArp ProLeuProArp Arp 5 5 z25 z3o 235 tao Arp ier Glu Gly Gly IletAla Pro ArpArp SerArp Ala Pro Arp Arp Pro Ala Ala Ar9 Ser Thr Ala Ala ThrPro ProArp Pro Ala Ala Ala 260 265 zm Gly Pro Arp Ala Pro Ala Arp Arp GlyAsp valThr Trp Asp Ala Ala 275 2~

1 Ilet Arp Lau lau Trp Gly Pha Gly Thrfar ThrArp 0 Glu Val ArD

p0 p5 300 (2) INFORMATION
fOR

ID
110:3:

1 (i) SEOIR:NCE CHARACTERISTICS:

(A) LENGTH: 1252 pairs base (e) TrPE: nrclaic acid (C) STRAIIDEDNESS:
double (D) TOPOLOGT: linear (ii) MOLECULE TTPE: DNA
(penoeie) (iii) HTPOTHETIGL: NO

2 (iv) ANTI-SENSE: NO

(vi) ORIGIIIAL SOURCE:

(A) ORGAlIISH: Equineharpasvirus (B) STRAIN: OutL

3 (C) IIIDIVIDUAL ISOLATE:S-~EIiV000 (vi I11(EOIATE SOURCE:
i ) (B) CLONE: 197-52.33and x-18.9 (viii)POSITION IN GENOME:

35 (B) IIAP POSITION:

(C) UIIITS: xG

(ix) FEATURE:

(A) NAME/IO:T: CDS

4 (B) LOGTI011: 153..1124 (D) OTHER INFORMATION:/eodon 153 start.

/Product= "U52 perx produet 4 (xi) SEQUENCE DESCRIPTION:SEC ID
5 N0:3:

TMGGGGAGTCTTTTTC ACMGMGT GTGTAGCTAGACGGCTCTG CTGAAliTTTA 120 GGT

Ilet Gly ValVsl Leu Thr Ile WO 94/03628 a ~ ø~ ~ PCT/US93/07424 -.8 ValVslIletValValAsp ArpNisLys AlaLeuPro AspterSer Ile 10 15 m AspValAsp GlyLysLw 1rpGluthe Lw GlyArp Lw CysPhe Vat TTAGCCTG GM CCTCTA GG11ATACG ATAGTGGTG CGTTCTGCt GAC 317 Lw AlaSer GluProLau GlyIlePro IleValVal ArS8erAla Asp 10i0 LS 50 55 CTGTACAM TTTTCtTCG AGTCTCTTA GCCCTGCG AM CG TGC ACG 365 Lw TyrLys PheSerSer SerLauLw AlaLauPro LysAlaCys Aro Pro1leYal ArpThrAro GlyAlaThr AlaIleAla Lw GluArp Asn 75 80 d5 2 GGCGTGATT TATCAAGAG GATAGAATT GCCATTAGT ATAGAGTCG C1T i61 O

GlyValIle TyrGlnGlu AspAr0Ile GlyIleSer ileGluTrp leu TCTGTACTA TCCGCCTAC AACTACCtC AACTCCAGC ATTATCATC MT S09 2 ServalLw SerGtytyr AsnTyrLau AsnSerSer IleIleile Asn tos tto tts ArOProTyr NisLw Trp ValLw Gly AlaAlaAsp Lw CysArO Pro GTGTTCMC CTCATACCG GGCCCCMG CGAATtGTG TATGTGGAG ATC 605 VatPheAsn Lw IlePro GlyProLys AraIleYal TyrValGlu Ile 1G0 1~5 150 GM CATGAG TTtMT AAI1TCTTGGGG CCCAGCTTC GTGTGCGG11MA 653 GluAspGlu PheAsnLys SerTrpGln ProSerPhe ValCysGly Lys 40Lw PheGlu Thr1lePro Lw ThrThr ValAsp1yr LysNisLw Lw 170 t75 t80 LysGlnLys ValLauPro GlyGlnAsp NisProGlu SerAlaArO Ser 45 ta5 t9o tvs TTATTAGA GT AM TG TCtTTtGTA TCTCCCCCG CG MT TTT MG 797 Lw LeuGln NisLysSer SerPheVat SerProPro ProAsnPhe Lys CGGTTAATT TATGCGGTT GTAGACCCT ATGCGtTTA CAAGAGMT TTA a45 ArpLw Ile TyrAlsVat ValAspPro IletArplw GlnGluAsn Lw 22o zzs z3o 55TGTCG CM ATAACTMC AGAAG AM AC1AM AGA CGTTCTAM AM a93 WO
94/03628 P~T/US93/07424 CysPro GlnIle ThrAsnArp ThrLys Thrlys ArpAro SerLriLYs ~5 Z4p Z45 ACTTAT MtGGC CTGTTTTGC GA WG TCTAG CCCAGC CTAMC GAT

ThrTyr AsnGly LeuPheCys GlnGlu !erThr AleSer LeuAsnAsp MG ATG TGTTTT ACTCG ,GG CG TG AMGGC AAAAAC TTGGG CGC 9a9 LysMet CysPhe ThrProGln ProSer LysEly LysAsn LauGlnArp GTTACC ACGTCG ATGGA GCC AACTCt AGATA CG CCT AGCACCCTA 1037 ValSer Thrfer MetGlnAle Asnfer ThrIle ProPro SerThrLeu ~ pp Z95 TCTCCT CGTGG CCTGCLCGG AM CCC AGGM ATGACG TGGMA TG t0:s5 SerPro ArpAla AlaAlaArp LysPro ThrGlu MetThr TrpLysSer 2 CGCCTA CTAGCG CGTCTGTTT Wt ACA AGCCC ACACGT TAAAAGCTTG 1136 O

ArpLeu LeuGly GlyValPhe AspArO ThrAle ArpArf GGGAAGCTCT TtGCTACTG CTGC(~TTTG CGACTCTGG TTTCC1CTW

TACAAACTtC ACGTCTA1CT TTAWGTGA GCTCCGACAT GCTTAGC.CC

(2)INFORMATION FORSE0 ID IIO:A:

(i) SEOUEHCE CHARACTERISTICS:

(A) LEHGTN: 32L
asino acids (B) T>rPE:
amino acid (D) TOPOLOGY: linear (ti) MOLECULE tIPE:protein (xi) SEOUEHCE OESCRIPtIOM:SE0ID IIOa:

4 MetGly Yel Leulle Thr ValMetVal ValAspArpNis Lys 0 Val Yal AlaLeu Asp SerSer Ile ValAspGly LysLeuTrpGlu Phe Pro Asp LeuGly leu CysPhe Vel AlaSerGlu ProLeuGlyIle Pro Arp Leu 35 '0 '5 IleVal Arp SerAla Asp TyrLysPhe SerSerSerLeu leu Yal leu AlaLeu Lys AlaCys Arp IleVslArO ThrAr9GlyAla Thr Pro Pro 5 AlaIle Leu GluArp Asn ValIletyr GlnGluAspArp Ile 5 Ala Gly 2~4~422 Gly !le ier lle Glu Trp ~w fer Yal Lw Eer Gly Tyr Asn Tyr ~w Asn ter ier Ile Ile ila Atn ArO Iro Tyr Nis Lw Ttp Yal lw Gly Ala Ala Asp Lw Cys Aro Pro Val Phe A:n lw t le Pro Gly Pro Lys Arp !le Val Tyr Val Glu Ile Glu Asp Glu PM Asn Lys fer Trp Gln 1 5 Pro Ser Phe Vat Cys Gly lys Lw Phe Glu Thr Ile Pro lw Thr thr Val Asp TYr Lys Nis lw Lw Lys Gln lys Val Lw 1ro Gly Gln Asp His Pro Glu Ser Ala Arp ier Lw Lw Gln Nis Lys Ser ter Phe Val Ser Pro Pro Pro Asn Phe Lys ArO Lw Ile Tyr Ala Val Val Asp Iro Ilet ArO Lw Gln Glu Asn lw Cys Pro Gln Ile Thr Aan ArO Thr lys 3 0 Thr lys Arp Arp Ssr lys Lys Thr Tyr Asn Gly Lw Phe Cys Gln Glu Ser Thr Ala Ser Lw Asn Asp Lys Ilet Cys Phe Thr Pro Gln Pro Ser Lys Gly lys Asn lw Gln ArO Yal Ser Thr Ser Ilet Gln Ale Asn Ser Thr 1le Pro Pro Ser Thr Lw Ser Pro Arp Ala Ala Ala Arp Lys fro Thr Glu Ilet Thr Trp lys Ser ArE Lw lw Gly Gly Val Phe Asp Arp 4 5 Thr Ala ArO ArO
(2) INFORMATION FOR tE0 iD 110:5:
50 (i) SEOIIENCE CHARACTERISTICS:
(A) LENGTH: 1149 base pairs (8) TTPE: nucleic acid (C) STRAIIDEDNESS: double (0) TOPOLOGT: linear 5 5 (ii) IiOLECUIE TTPE: DNA (penomic) -.88.-i i NYPOTNETIGL: 110 i ) (iv) AIITI-fElISE: 110 (vi) oRIGIrA~ souRCE:

(A) ORGAIIIS;11: herpesvirus Equine , t (B) fTRAIII: Dutta (C) IIIDIVIDUAL S-6EIIV-000 . ISOLATE:

(vi IIItEDIAtE fOIJRCE:
i ) (p) CL011E: 667-62.A12 (vi POSITI011 111 GE110iE:
i i ) (B) IIAP POSIT1011:

(c) ulltts: xri (ix) FEATURE:

(A) wAlIE/KET: CDs (8) LOCATI011: 271..1169 2 (D) OTHER INFORIIATI01l:/partial /eodon start. 2T1 /function. ~ambrane plycoprotein"

/product. Glycaprotein E li-ter'irsn /per' ~ppE~

(xi tE0UE11CE OESCRIPT1011:tEp ID 110:5:
) TCTAGMGGTTGMCCCTA MCT~CC GTAAMGAG WAGACTMTMTGGCGGGT 60 TTTTAAAGTTTATGTATTAT TGT1TCTATATATtAMAAT TGTTGAAATATAAATATCTT 120 ATGTMTGTTTAGTTATTC GTWtTGGCACCGTCTTAGG GGAGCTGGTGG1ACTAGGGT 1150 CTG GAC CGC

Ilet Glu leu ser Arp Leu Asp Arp GCT TTC TtT TTT GTA AG GTA CTC WT TGG GTT 342 TTT CTA ATA GCG GGA

Ala Phe Phe Phe Val Thr Val Asp Trp Vat Phe leu Ile Leu Ala Gly Gln Val Glu Leu Thr Ala Trp Met Asp Arp Arp Glu Gly Ala Ile Gly GTT ACG AGG AG

5 Asp Leu Thr Pro Thr Thr Thr ril ~ys Trp 0 Yal Asn Thr Arp Thr Ala TTT GGA CGT ATA GTC

Thr Leu Glu Thr Pro Cys Ala Asp Thr lys Phe Pro Gly Gly Ile Vat 5 eso 65 To _$9_ Thr Vat Cys Val Gln Ala itr Lsu M Glu Asp Asn Ile Ile Ile Gly s0 s!S
MT GC TGTAAC CTACTAACC GGGGAGGt GGCATTGCG CTTGG GAG Sa2 AsnNisCyaAsn lsuLsuThr GlyGluNia GlyIleAla lauAlaGlu TTTMC CTACTT AACGW TCG C'TACM AGG ACCAM WT GTGTACTTT 630 1 PheAsnVslVal AsnGlyfer LsuGlnArp ThrlysAsp ValTyrPhe GTTMT CGAAG GTTTTtCCT ATTCTGGG GM ACCCGC AGCGTGTTA 67S

ValAsnGlyThr ValPM Iro ItsLw Ala GluThrArO ierVallw GlnIleGlnArp AlaThrPro SeerItsAla GlyYalTyr ThrlsuNis ValSerIleAsn GlyNisIts lysNis>ierValWl lau LsuThrVat 2 MG AM CG CG AG CGCGTA GT CTCAAG ACCCCTCG CCCATACTA i322 lysLysProIro ThrAr0Yal NisYallys ThrProPro ProIlelau GTTCCCGG GTT AG CG GAG CG GT AG CATTTCATA GTGCGCGGA a70 3 Wl ProGlnVal thrProGlu AlaNisThr AspPheIle YalArpGly 1a5 190 195 200 TACGC TCGCCC CTATAtCCT CTGGGTGAG TCCTTTGAC CTGTCTGTG 91<!

TyrNisSerArp ValtyrAla ValGlyGlu 8srPheAsp LsuterYal Nis leu Glu ter Nis lle Gln Glu ter ter Phe Asn Ala Glu Ile Gln 220 tZS 230 TGG TAT TAT AtG MT ACG TG TCG TG TG TGC GAT TTG TTT CGA GTT 1014 Trp Tyr Tyr Nst Asn Thr !er ter fer itr Cys Asp Leu Phe Arp Ysl 4 5 TTt GM AG TGC ATT TTT GC CG ACC GLT ATG GCC TGC CTG GC CCC 1062 Phe Glu Thr Cys Its Phe Nis Pro Thr Ala Net Ala Cys Lsu Nis Pro 5 0 Glu Gln Nis Ala Cys Cys Phe Thr ter Pro Val Arp Ale Thr Lye Ile 265 Z70 Z75 2s0 leu Nis Arp Val Tyr Gly Asn Cys &er Asn Arp Gly ter 5 5 Zss 290 (2)INFOwunoN aEO ID
foR wo:6:

(i) SEQUENCECHARACTERISTICS:

(A) 293wino LENCTN: acids (8) acid TtPE:
asino (D) .linear TOPOLOGT:

i i IIOLEWLETYPE:protsin ) (xi) SEQUENCEDESCRIPTION: SE0ID 110:6:

IletGlu Lsu Ssr ArCArliAlathe PhsPhaPhs YslLauIle lau Asp ThrYal Asp Trp GlyValGlnArp ValGluLeu ThrGluGly Lsu Ala 2p 25 30 AlaTrp Ilet Asp GlyArpAspVal LsuThrPro ThrAsnThr Ala 1le 2 35 ~ ~5 ThrThr Val Lys AlaTrpThrPhe LsuGluthr ProProGly Arp Thr 2 CysAla Asp Thr VatLysThrVal CysValGln AlaSerLau 5 Gly Ile CysGlu Asn Ile IleGlyAsnNis CysAsnLau LeuThrGly Asp Ile 9p 95 GluNis ils Lau AlaGluPhsAsn ValValAsn GlyterLsu Gly Ala GlnArp Lys Val TyrPheValAsn GlyThrVal PheProIts Thr Asp LsuAla Thr ter ValLsuGlnIle GlnArpAla ThrProSsr Glu ArO

130 135 1~.0 4 ileAla Val Thr LsuNisValSer 1leAsnGly NitIleLys 0 Gly Tyr NisSer Vel Lsu ThrValLyslye ProProThr ArpYalNis Yal Lau YalLye Pro Pro 1leLeuValPro GlnValThr ProGluAla Thr Pro NisThr Phe Val ArpGtyTyrNis SerArOYsl TyrAleVal Asp Ile 5 195 too toe GlyGlu Phe Leu SerYalNisLeu GluSerNis IleGlnGlu Ser Asp 5 SerSer Asn Glu IleGln1rpTyr TyrIletAsn ThrSerSer 5 Phe Ala -~ 2141422 aD ~ too SerSer M Asp Leu Phe Arp Vel Phe M lle Phe Nis Pro Glu Thr ThrAla llet Ala M ~w Nis fro Glu Ala M M Phe Thr 0ln Nis 26p ?b5 270 SerPro Val ArO Ala Thr ~ys Ile Leu Vsl Tyr Gly Asn M
Ills Aro 275 2b0 2b5 SerAsn Arp Gly fer ..

1 (2)INFORMATION

ID
110:7:

(i) tEOUEYCE CIIARACTERItTiCt:

tA) IEtIGTN: 1b anira acids (B) TTPE: s~iro acid 2 tc) stRAmEDrESS: double (D) TOPOIOGT: linear (it)MOLECULE TYPE: DIIA (oenoote) 2 (tit)NrPOTNETICAL: 110 (iv)ANTI-SEIISE: 110 (vi ORIGIIIAL iDlbtCE:
) 3 tA) ORGAlIISM: Equine herpesvirus1 (b) (TRAIN: Dutta (C) IIID1VIDUAL ItOLATE: S-1ENV-000 (vi POSITION IN GEIIQIE:
i i ) 35 te) IIAP PostTtaN: -b3 (C) UN1TS: xG

(ix)FEATURE:

(A) NAME/KET: Region 4 te) ~acATION: 1..1b (D) OTHER iNFORI1ATI0N: /labelNV1-US2 E

/note. Conservsd rpion of USZ qane stsrtinp at wino acid 123.

(xi ) SE0lIE11CE DESCRIPTION: tE0 ID 110:7:
Nis leu Trp Vel Leu Gly Als Ala Asp lsu M Lys Pro Vel Phe Asp ~eu Ite (2) INFORl111TION FOR SE0 ID NO:b:

_92_ , (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 aairo aeidc (8) TYPE: eino acid (C) STRANDEDNESS: dable (D) TOPOLOGY: linear (ii) IaLECUIE TYPE: DNA ~(panoatC) (iii) NTPOTHETICAL: NO

(iv) AIITIfENSE: 110 (vi) ORIGINAL SDUtCE:

(A) ORCAIIISM: Equine Mrpaavirvt 1 (B) STRAIN: Dutta (C) IIIDIVIDWL ISOLATE: s-~EHV-000 (vi POSIT1011 Iil CENOIE:
i i ) (B) IIAP POSITION: -83 (c) uilTS: xc (ix) FEATURE:

(A) NAIE/~Y: Rpion (g) LOGT1011: 1.18 2 (D) OTHER INFORMATION: /label. ENVY-US2 /note. tonsarvad rpion of tJS2 vane starting at awino acid 123.

(xi) SEQUENCE DETCR1PTION: iE0 ID N0:8:

His Lau Trp Val Lau Gly Ala Ala Asp Lau Cys Arg Pro Val Phe Asn lau Ile (2) INFORMATION
FOR
SEG
ID
110:9:

(i) SEOIIENCE CHARACTERISTICS:

4 (A) LENGTH: 18 aeino acids (8) TYPE: aoino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear 4 (ii) MOLECULE TYPE: DNA (ganauic) (iii) HIP01NETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Herpes sioplex virus 1 (B) STRAIN: 17 55 (viii) POSITION IN GENOf(E:

(B) IUIP POSITION:
(C) UNITS: xC
(ix) FEATIRtE:
(A) NRIIE/1~T: Rpion (B) LOCATION: l..if3 (D) OTHER INfORlIATIdI: /labal~ NSV1~1lS2 /note. ~Coeatrvad ration of US2 gene starting at wino acid 124.~
(xi) tEOUENCE DESCRIPTI011: !EO ID N0:9:
Nis Lau Trp Val Val Oly Ala Ala Asp Lau Cys Val Pro Phe Lau Glu 1yr Als (2) INFORMATION fOR SE0 ID 110:10:
(i) tEOIR:NCE CHARACTERISTICS:

(A) LENGTH: tE awino aeide (B) TYPE: awiro acid 2 (c) sTRANDEDNESS: doubts ID) TOPOLOGT: linear ( i IIOLECLLE T1PE: DIIA (genovie) i ) (iii) NrPOTNETIGL: 110 (iv) ANTItENSE: 110 (vi ORIGINAL SOIRtCE:
) (A) ORGAIIISII: Herpes sisplex virus 2 (B) STRAIN: NG52 (viii)POSITION IN GENOME:

(B) IIAP POSITION: -d8 (C) UNITS: xG

(ix) fEATtRtE:

(A) IIAIIE/I~t: Region (R) LOCATION: t..is (D) OTHER INFORNATION: /label. NSV2US2 /note. Co~erved rpion of U52 gone starting at asiro acid 123.

(xi) lEOtJENCE DESCRIPTION: sE0 ID N0:10:
Nis Leu Trp Val Val Gly Ala Ala Asp Lau Cys Wl Pro Phe Phe Glu Tyr Als _94-(2) INFORItAtI011 FOR

ID
N0:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amiro acids (B) TYPE: wino acid (C) STRAIIDEDNESS: double (D) TOPOLOGY: lir~sr (ii) MOLECULE TYPE: DNA (tic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: 110 1 (vi) oRICINAL sauRCE:

(A) ORGAIIIffI: Paeudorabies virus (B) STRAIN: NIA-3 (viii) POSITION IN GEN01E:

(B) IIAP POSITtON: -'90 (C) UNITS: ><G

(ix) FEATURE:

(A) IIAME/KEY: Region 2 (s) LocATIaN: 1..1e (D) OTHER INFORMATION: /labelPRV-US2 /rote Coroervad region of US2 acne starting at amino acid 148.

(xi) SEQUENCE DESCRIPTION: SE0 ID N0:11:

His Lau Trp Ile Leu Gly Ala Ala Leu M AsP Gln Val Leu Leu Asp Ala Ala (2) INFORMATION
FOR
SEG
ID
N0:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) StRAIIDEDNESS: double 4 (o) tovoLOCY: linear (ii) MOLECULE TYPE: DNA (genomiC) (iii) NTPOTHEtICAL: NO

(iv) ANT!-SENSE: NO

(vi) ORIGINAL SC~tCE:

(A) ORGAIIISM: Msrek's diseasegaaaosherpesvirus 5 (B) STRAIN: 8818 WO 94/03628 ~ PCf/US93/07424 (viii) PosmoN IN

ts) NIAP POSITION: -da (C) UNITS:

tix) FEATI~E:

(A) IIAlIE/KET: Rpian (B) LOCATION: 1..19 tD) OTNER INFORNATION: /labtl MDVUS2 /rote. Constrvad rpion of US2 Bene starting at 1 s~ino acid 132.

lxt tEQUEI1CE OEtQIPTION: fE0 I0 110:12:
>

1 Nis atr Ltu Trp Ilt Val fly Ala Alt Cys Arp Ilt Ala leu 5 Asp lle Olu Cys Ilt (2) INFORMATION
fOR
iE0 ID
110x13:

(t) sEauENCE cNARACTERtsrta:

(A) LENGTN: 1a s~iro acids 25 ti) TYPE: aisino acid (c) sTRANDEDNEn: dovdlt (0) TOP~OOT: linear (ii) MOLECULE TYPE: 0NA (fit) (iii) NYPOTMETICAI: NO

(iv) ANTI-SENSE: 110 3 (vi oRICINAL :oliRCE:
5 ) (A> ORGANI511: Bovine herpesvirus i (B) STRAIN: Cooper (C) IIIDIVIDUAI ISOLATE: S-IIR000 40(viii) POSIT10N IN CENOIIE:

(6) MAP POSITION: -a5 (C) UIIITS: >rG

(ix) FEATURE:

45 tA) BAIE/KEY: Rpion ta) LocATION: 1..1a tD) OTNER iNfORMAT10N: /label.

/rote. Canstrvsd rpion of US2 Qtne starting at aaino acid 115.

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

Nis Met Trp Val Phe Oly Ala Ala Asp Ale Pro Ile Phe Ala Leu Tyr Nis 1la (2) fOR
iEG
ID
110:11:

(i) tE0UE11CE CNARACTERISTICS:

(A) LENGTN: 66 base pli n (B) TYPE: rx~cleic acid (C) STRAlIDEDNESS: dabla 1 (D) TOPOLOGY: Circular O

(ii) MOLECULE TrPE: 011A (Olno~ic) (iii) NTPOTNEtICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGAIIISM: Plaasid (vi IMIIED1ATE SOURCE:
i ) (B) CLOVE: 150-46.14 (Figure 4 Junction A) (xi) SEQUENCE DETCRIPT1011: iE0 ID N0:li:

AGGtCAGCTATAGMTACA CGGAATTCGA GCTCGL'GGG GCATCTCACC GCTTCCCGGG

(2) INFORMATION
fOR
!E0 ID
N0:15:

(1) SEANCE CHARACTERISTICS:

3 (A) LENGTN: 66 base plus (B) TTPE: exulaic acid (C) STRAIIDEDNESS: double (D) TOPOIOCY: circular 4 (ii) MOLECULE TYPE: DNA (genosic) (iii) NTPOTNEtICAI: NO

(iv) ANTI-SENSE: NO

(vi) ORICINAI SQ1RCE:

(A) ORGANIS11: Plasoid (vii) IMMEDIATE SOURCE:

5 (B) CLONE: 450-16.84 (Figure 4 ,lunctian B) (ix) FEATURE:

(A) NAtlE/KET: CDS

(8) LOGTION: 1..66 5 (D) OTHER INFORMATION: /product "Region of deleted thyoidine tierse ~"
(xi ) SEOUEltCE DESCRIPTiOH: tE0 ID 110:15:

ACCACG CCC TAC CTT ATC CTA CAC WT CCT CTA GAG CTC MT iB
TGC ACC

ThrThr Pro Tyr Lw Ile Lw Nis Asp Pro Lw Glu Lw Asn fer Thr CTGGAC GAG CAC GTG CW M

ValAsp Glu Nis Val Arg (2)IIIFORlIATION FOR SE0 I0 110:16:

(i) SEGUEHCE CHARACTERISTICS:

(A) LENGTH: 22 giro acids (B) TrPE: ewino acid 2 0 (D) T0POL0GT: linear (ii) MOLECULE TrPE: protein (xi) SEQUENCE DESCRIPT10H: tE0 ID H0:16:

ThrThr Pro Tyr Lw I le Lw Nit Asp Pro Lw Glu lw Aan Ber . Thr ValAsp Glu Nis Val Ate (2)INFOWIIlTION fOR SEQ ID 110:17:

(i) SEQUENCE CHARACTERISTICS:

3 5 (A) LENGTH: 66 bse pears (B) TtPE: rxuleic acid (C) STRAIIDEDNESS: double (D) tOPOLOGT: circular 4 O (ii) NOIECULE TrPE: 0NA (Beno~sie) (iii) HYPOTHETICAL: HO

(iv) ANTI-SENSE: HO

(vi) ORIGINAL SOIJItCE:

(A) ORGANISlI: Plaswtid (vi i ) IN!(EDIATE SOURCE:

5 0 (B) CLONE: i50-1.6.14 (Figure ' Junction C) (ix) FEATURE:

(A) IIAI~/KET: CDS

(B) LOCATION: 1..30 5 5 (0) OTHER INFORMATI011: /partial _98-/eodon_start~ 1 /product' .Region of ENV1 glycoprotein N gene"
(xi) fEOUENCE DESCRIPTION: SE0 ID 110:17:

TATCTC CCC GT GGG TTT ATG CGC CTG GG CCGAGCTTG CCGTAATGT SO

TyrLau Gly Nis Gly Phe list Gly . L~u Gln GGTGTAGCT
GTTTCC

(2)INFORIIAtION FOR iE0 i0 w0:le:

( i ) SEOIJENCE CHARACTER I ST I CS

(A) LENGTH: 10 aioino acids (B) TTPE: nsino acid (D) TOPOIOGr: linear 2 ( i i ) MOLEQJLE TYPE: protein (xi) SEQUENCE DESCRIPTION: tE0 ID N0:le:

TyrLeu Gly Nis Gly Phe Met Gly Lw Gln (2)INFORMATION fOR SE0 ID N0:19:

(i) SEQUENCE CHARACTERISTICS:

3 (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRAIIDEDNESS: double (D) TOPOLOGY: eirwlar 35 (ii) lIDIEQJLE TYPE: DNA (per~amic) (iii) i(YPOTNETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Plaseid (vii) IMMEDIATE SOURCE:

4 (e) CLONE: 167-21.19 (Figure S Junction A) (xi) SEQUENCE DESCRIPTION: SEG ID N0:19:

GCACGC
5 5 (2) INFORMAT1011 FOR SEC ID N0:20:

~.~ 2 ~ ~ ~ ~.~ z _99-(t) SEOUEIICE CHARACTERISTICS:

(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRAIIDEDNESS: double (D) TOPOLOGY: tireular (ii) MOLECULE TYPE: DNA (ganamic) ( i NYPOTNETIGL: I10 i i ) (iv) ANt1SENSE: I10 (v1 ORIGINAL iDIRtCE:
) (A) ORGANIStI: Plasmid (vi IIIIEDIATE tOIIRCE:
i ) (B) CLONE: X67-21.19 (Figure 5 ~unetion B) ( i FEATtAtE
x ) (A) NA~EncEr: cas (B) LOCJ1T10N: 1..30 (D> OTHER INFORMATION: /partial /codon_start= t /ptnduct ~Itpion of EMV1 US2 gar:e"

(ix) FEATURE:

(A) IIAIIE/I~f: CDS

(B) LOGTION: 33..65 (D) OTHER IIIf0R11AT1011: /partial 3 /eodo~ start. 33 /product. Rpion of ENVt US2 Bane"

3 (xi) DESCRIPTI011:

110:20:

CGA

ValCys Lys Lau Phe Glu thr Ile Pro Asn Ser Leu Vsl Arg Gly 1 5 10 t 5 AG

ProPro Val LYs Arg Thr (z)INFOR~ATIOw FoR seo to Noat:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 smiro acids 5 (B) TYPE: amino acid (0) tOPOLOGT: linear (ii) IIDLECULE TYPE: protein 5 (xi) SEQUENCE DESCRIPTION: SE0 ID N0:2t:

Ysl Cys Gly lys Lau Pha Glu Thr Ile Pro i 5 10 (2) INFORMATION
FOR
tE0 ID
110:22:

(1) tEOUENCE CHARACTERISTICS:

(A) LENGTH: 1i aoino scads (B) tTPE: aoiro acid 1 (D) TOPOLOCT: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: fE0 ID 110:22:

Asn Ser Leu Val Arg Pro Pro Thr Val Lys Arg (2) INFORMATION
FOR
tE0 ID
IIO:Z3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs (B) TTPE: nucleic acid (C) STRAIIDEDNESS: double 2 (D) TOPOLOGT: circular (ii) MOLECULE TrPE: DIIA (genomie) ( i NTPOTNETIGL: NO
i i ) 3 (iv) ANTIsENSE: No (vi) ORIGINAL SOURCE:

(A) ORGANISlI: Plaswid 3 (vii) iM!lEDIATE SOURCE:

(B) CLONE: 467-21.19 (Figure 5 Junction C) 4 O (xi) SEQUENCE DESCRIPTION: SEO ID N0:23:

GTGTA
(2) 1NFORMATIOH FOR SE0 ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LERGTN: 66 base pairs 5 0 (B) TTPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGT: circulsr (ii) MOLECULE TTPE: 0NA (gendeiC) WO 94/03628 ~ ~ PCT/US93/07424 (iii) NrPOTNETIGI: No (iv) ANTI-SENSE: 110 (vi) ORIGINAL fDUtCE:
(A) 0ltGANIiN: Placid (vii) INlIEDIATE SOURCE:
(B) CLOwE: 53b~a5.30 (iiBure b Junction A) (xi) sEallENCE DESCRPTIaII: slEO Io woa~:

GGtCACWCGTTGTAAMC GACGtXCAGT GMTTCACG AGAAACCGAC GTGTMMAC60 TTCTCC M

(2) IIIfORMATION
fOR

ID
110:25:

(i) sEOUENCE cNARACTERISTICS:

(A) LENGTN: 132 base pairs (8) TYPE: nucleic acid (C) fTRAIIDEDIIEit: double 2 (0) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (tic) (iii) NYPOTNETCAL: 110 (iv) AIITI-SEIItE: 110 (vi) ORIGIIIAL iDURCE:

(A) ORWNISN: Plaswid (vi IIIIEDIATE SOURCE:
i ) (8) CLONE: 536-d5.30 (Fiwre 6 Junction i) ao (xi) tE0tJF11CE DESCRIPTION: SE0 ID N0:25:

ACTCTGCTWTGTTGCAGG GWTCCTTM TTMGTCTAG AGTCWCTGT TTAMCCGGT b0 (2) INfOR11AT10N
fOR
SEG
ID
N0:26:

(i) SEOUEIICE CHARACTERISTICS:

(A) LENGtM: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: dale 5 (0) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (panesie) (iii) NTPOTNETICAL: NO
(iv) ANTI-SENSE: 110 (vi) ORIGINAL S0IRtCE:
(A) ORGANISM: Plamid (vi i ) IMMEDIATE SOURCE:
(e) CLOVE: 536-x5.30 (Figure 6 Junction C) lxt) SEOUEIItE DESCRIPT1011: SEO ID 110:26:

GTGGT

(2) INFORMATION
fOR
SEQ
ID
110:27:

(i) tEOUENCE CHARACTERISTICS:

(A) LEIIGTN: 66 bue pairs 2 (B) TTPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGt: circular (ii) MOLECULE TYPE: DNA (peno~ie) ( i NTPOTIIET I CAL : 110 i i ) (iv) AIITi-SENSE: 110 3 tvi) oRIGINAI sauRCE:

(A) ORGANISM: Pluwid (vii) IMMEDIATE SOURCE:

(B) CLONE: i95-61.39 (Figure T Junction A) (xi) SEQUENCE DESCRIPTION: tE0 ID N0:2T:

TGGrcA

(2) INFORMATION
FOR

ID
N0:21S:

5 (i) SEQUENCE CHARACTERISTICS:
O

(A) LENGTH: 66 base pairs (B) TTPE: rxuleic acid (C) STRANDEDNESS: double (D) TOPOLOGT: circular M WO ~ ~ ~ PCT/US93/07424 94/03628 ~

(ti) IIDLECULE TPE: DNA tplno~ic) l i MYPOTIIETIGL: NO
f t ) tiv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:

tA) ORGANISM: Plas~id 1 0 (vii) IMMEDIATE fDURCE:

tB) CLOVE: 495-61.39 tFioure 7 durction B>

(ix) FEATiIItE:

tA) NIIlIE/10:T: C9S

(a) LoGTION: t..t~

(D) OTHER INFORIIATt011: /partial /codort start. 1 /product. "Iteeiorr of dalatad kinast ENV' thymidine ~"

(ix) FEATURE:

(A) NAME/ICEr: CDS

(B) LOGTION: X6..66 (D) OTHER INFORMATION: /partial 2 5 /oodon_start /product= Reeion of dtlatad kinast ENVA thyaidine oene"

3 0 (xi) SEQUENCE DESCRIPTION: SE0 10 N0:28:

GTT WC GCG GG TTA ATA ACT GCGGGG11TCC GTA St GAt TCTAGAGTCC T GTT

Ysl Asp Alt Ala Leu Ile Thr Vo Val Asp tCT

Glu leu Leu Pro Ser (2) INFORMAtI011 FOR SE0 10 N0:29:
(i) SEOI~NCE CHARACTERISTICS:
tA) LENGTH: 8 siaino acids 4 5 (B) TYPE: awino acid (0) TOPOIOGT: (inetr i i ) MOIEQJLE TYPE : proto n (xi) SEQUENCE DESCRIPTION: SE0 ID N0:29:
Vtl Asp Asp Alt Ala Leu Ile Thr t S
t2) INFORIIATiON FOR SEO ID N0:30:

(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 wino acids (B) TYPE: smiro acid (D) TOPOLOGT: linear c i i ) IIOLECtILE TYPE: protein (xi) SEaJENCE OESCRIPTI011: >iE0 ID 110:30:
Val Yal Glu Ser Leu Leu Pro (2) INFORMATION fOR tE0 I0 110:31:
1 5 (i) tEGUENCE CHARACTERISTICS:
(A) LENGTH: 66 bass pairs (B) TTPE: nucleic acid (C) iTRAIR7EDNESt: double (0) TOPOIOGT: circular (ii) NOIECULE TYPE: DNA (penooic) (iii) NTPOTMETICAL: 110 (iv) ANTI-SENSE: 110 tvi ) ORtctNAL iDiJItCE:
tA) oRCANISN: Planid 3 0 <vi i ) INlIEDIATE SOURCE:
(B) CLOVE: A95-61.39 (figure 7 Junction C) tix) FEATURE:
(A) NAhE/KEY: C9S
(B) LOGTION: t..33 (D) OTHER INFORNATION: /partial /COdOn_ftlrt~ 1 /product= ~Repion of ENVi plycoprotein N pene~
(xi) SEaJENCE DESCRIPTION: SE0 ID 110:31:

4 5 Arp Leu Pro Pro Arp Arp Arp Leu Glu Pro Pro CGTMTGTG GTC
(2) INFORlIATION FOR SE0 ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 asino acids 5 5 (B) TrPE: nsino acid WO 94/03628 ~ ~ PGT/US93/07424 (D) TOP0L0GY: (inssr t i i lIOIEtxJLE TYPE: protein ) txi) tEGtJ>:NCE DESCRIPTION: iE0 ID H0:32:

Arp Lsu Pro Pro Arp Arp Arp Lsu Glu Pro Pro (2) IHfORltATIOH
f0R
fEa ID 110:33:

(i) EHCE CHARACTERISTICS:

tA> LEHGTN: 66 bne pairs ti) TYPE: nsclstic acid 1 5 tc) sTRAHDEDNESS: double (D) TOPOIOGT: eirwlar i i ) IIDLECtJLE TYPE: 0HA (Osno~ic) 2 0 tits) HYPOTHETICAL: Ho (iv) AIITIEEHSE: Ho (vi ) ORiGIHAI SOtJItCE:

25 tA) 0RGA111SM: Plu~id tvi t IIIIEDIATE :DUtCE:
) (B) ttOHE: 52338.9 (Fioure 8 ,function A) (xi) SEOtJEHCE DESCRIPTION: fE0 I0 110:33:

ATAGGTACWTTTAGGTG AGCTATAGA ATAGCGGM TTCWGCTCG t~GGGWTC 60 CTCTAG

(2) lNfORMAT1011 fOR

ID
110:34:

(i) SE01JEHCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs (B) TYPE: rx~eltie acid tC) StRAlIDEOHESS: doubt (D) TOPOLOGY: circular (ii) IIOLEWLE TYPE: DHA (9sno~ic) (iii) NYPOTNETIGL: HO

5 (iv) ANTI-SENSE: HO

tvi) ORIGIIIAI TO<JRCE:

(A) ORGANISM: Plas~lid 5 tvii> IMMEDIATE SOURCE:

WO 94/03628 ~ PCT/US93/07424 (8) CLONE: 523'38.9 (Figure 8 Junction B) (ix) FEATURE:
(A) NAlIE/10=Y: GDS
(a) LocAtION: 1..33 (D) OTHER INFORMATION: /pertiel /eodon_start~ 1 /produCt~_ ' ~Repion of deleted EHV4 US2 gene~
1 O (xi) SEQUENCE DESCRIPTION: SEG ID N0:34:

Arg Pro Tyr Nis Leu Trp Yal Leu Gly Als Ala TAACTCTTCC TCG
(2) INFORMATION FOR SEO ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 asino acids (B) TYPE: amino eeid (D) TOPOLOGY: linear (ii) MOLECULE TtPE: protein (xi) SEQUENCE DESCRIPTION: SE0 ID N0:35:
3 0 Arp Pro Tyr Nis Leu Trp Vat Leu Gly Ala Als (2) INFORMATION FOR SE0 ID N0:36:
3 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 beae peira (B) TYPE: nucleic acid (C) STRANDEDNESS: double (0) TOPOLOGT: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) NTPOTNETIGL: NO
4 5 (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Plasmid 5 O (vii) IMMEDIATE SOURCE:
(8) CLONE: 523-38.9 (Figure 8 Junction C>
5 5 (xi) SEQUENCE DESCRIPTION: SEO ID N0:36:

CCCGTGCMCAACAGTCGTC TTtCTCGtCC GAAAAGCTTG GCGTMTGT GGTGTAGCT

GTTTCC

5 (2) INFORMATION
FOR

ID 110:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 best pairs (g) TYPE: nreleic eeid 10 (C) STRAHDEDNESS: dable (D) TOPOIOGT: cireuler ( i i 110lECULE TYPE: DNA (plnollie) ) 15(iii) HYPOTHETICAL: 110 (iv) A11T1-SENSE: 110 (vi) ORIGINAL iWRCE:

(A) ORGAIIISII: Plesid (vii) IIIIEDIATE SQJRCE:

(g) CLONE: SZlO-57.25 (figure 9 .I~nction A) (xi) fEQI>ENCE DEtCRIPTI011: tE0 ID 110:37:

ATTMtAGTMCCTTATGT ATGTAGG TACWTTTAG CTGAGCTAT AGAATAGCG 60 (2) INFORMATION
FOR
SEQ
ID
N0:3if:

(i) SEQIIfNCE CHARACTERISTICS:

3 5 (A) LEIIGTH: bb base pei n (g) TYPES ra~eleic eeid (c) sTRANDEDNEa: dote (D) TOPOLOGt: eireuler 4 0 (ii) IIOIEME TYPE: DNA (penowie) ( i NYPOTHET I GL: 110 i i ) (iv) ANTI-SENSE: 110 (vi) oRIGIIIAt SollRCE:

(A) ORGAIIIfII: Ples~id (vii) IIIiEDIATE SOURCE:

5 0 (g) CLONE: 5i30-57.25 (figure 9 Junction g) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3E:

GAGTCGCAGG

TATGCT M

(2) INFORh11TI0N fOR SE0 1D N0:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRAIIDEDNESS: dable (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (tic) Z5 (iii) HYPOTHETICAL: 110 (iv) ANTI-SENSE: 110 (vi ) ORIGINAL SOtJItCE:

2 (A) ORGANISM: Pl~s~id (vii) INNWIATE SOURCE:

(B) CLONE: 580-57.25 (figure 9 J:nction C) (xi) ~OUENCF DESCRIPTION: fEG ID 110:39:

GATCCCGAGT CTCGCTTCW AAMCCGTGC GACCTf~C CGTMTGTG 60 CCMGCTTGG

(2) INFORNAT1011 f0lt SESa 10 NOaO:

(i) SEQUENCE CNARACTER1STICS:

3 (A) LENGTH: 66 base pairs (R) TYPE: nucleic acid (C) STRANDEDNESS: dable (D) TOPOLOCT: circular 4 (ii) NDLEQJIE TYPE: DNA (gano~ic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGAl11S11: Plasmid (vii) IlIiEDIATE SOURCE:

5 (8) CLONE: 467-Z2.A12 (Figure 10 Jurction A) (xi) SEOLIENCE DESCRIPTION: SE0 ID N0:40:

.._ 2141422 GMTTCGAGC TCGCCCGGCG .~~CCTCTAGA GTCG11CGTCTCCTGGTGCTC 60 GCCGCGCGGG

TTCGAG

(2) INFORMATION FOR SEQ I0 N0:41:

(i) iEOUENCE CHARACTERIS11CS:

tA) LENGTH: 66 base pairs (B) TTPE: nucleic acid 1 (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii)~ MOLECULE TYPE: DrA tOano~ic) 1 (iii) HTPOTHETICAL: NO

(iv) ANT1-SENSE: NO

(vi) ORIGINAL SOURCE:

20 (A) ORGAIII811: Plaswid (vii) IMMEDIATE :DiRtCE:

(B) CLONE: i67-22.A12 (Fipura 10 Junction t) 2 (ix) FEATURE:

tA) NAI~/tc~r: cDs (1) LOCATI011: 16..66 (D) OTHER IrFOR11At1011: /partial /eodon_start 16 3 /products r-tarninal peptide of hybrid protein"

(xt) sEQUErcE DESCRIPtIOr: sE0 ID wostt:

GAT CCC

3 Mat Lys Trp Ala Thr Trp Ila Asp Pro Vat Val Lau CAA CGT CGT GAC TGC

Gln Aro ArS Asp Trp 40 is (z) INFORIIAnar Fa :EO ID roar:

4 (i) SEOtJErCE CHARACTERISTICS:

(A) LENGTH: 1T aaiiro acids (B) TrPE: amino acid (D) TOPOLOGT: linear 5 tii) MOLECULE ttPE: protein txi) SEQUENCE DESCRIPTION: SEC ID r0a2:

Met Lys Trp Ala Thr Trp ile Asp Pro Val Yal ArO Arp Asp Lau Gln 5 t 5 tG is Trp (2) INFORMATION
FOR

ID
N0:43:

(i) SEQUENCE CHARACTERIS1,ICS:

(A) LENGTH: 132 base pears (B) TTPE: nucleic acid (C) STRANDEDNESS: double 1 (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (pena~ic) (iii)HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGAIIISM: Plafwid (vi IMIiEDIATE SOURCE:
i ) (B) CLOVE: 467-22.A12 (figure 10 Junction C) (ix) FEATURE:

(A) NAME/KEY: cps (g) LOCATION: 1..93 (D) OTHER INfORMAtiON: /partial /codon_atart 1 /funetio~ Tranlational finish of hybrid 3 protein' /product' C-ter~i~l peptide"

/standard name= 'Translation of synthetic DNA

(xi)&EOUENCE DESCRIPTi011: SEa ID
No:43:

GAC CAC TCCtGG AGCCCG GTATCG GCG ATC GG CTGAGC GCC
TG GM

Asp Asp SerTrp SerPro Wl Ser Ala Ile GlnLauSer Ala 8er Glu GGT CGC TACGT TACGG tTG GTCTGG TGT AAA GATCTAGM
CM

Gly Arg TyrNia TyrGln ValTrp Cya Lys AspLeuGlu Lau Gln TMGCtAGAG GATCGATCCC CTATGGCWT GTGGGGC 132 (2) iNFOR111lTI0N fOR SE0 iD N0:4G:
(i) SEOI~NCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) tIPE: amino acid (D) TOPOLOGY: linear z 1 ~-~ øz 2 tii) MOLECULE TYPE: protein (xi) SEQUENCE DESCR1PTJON: SEQ ID VOa4:

AspAsp Leu Ser Ala Ser Trp Ser Pro Ser Val Ser Ala Glu Ile Gln GlyArg Lau Glu Tyr Nis Tyr Gln Lau Val Trp Cya Gln Lys Asp Zp S 30 (2)IVFORIIATIOV
fOR

ID
110:15:

(1) SEQUENCE CNARACTERISTICt:

(A> LEVGTN: 66 bast pairs 1 (B) TYPE: nucleic acid tC) STRAVDEDNESS: double t0) TOPOItIGY: cirwlar (ii) MOLECULE TYPE: 0VA t9~oaie) (iii)NYPOTNETICAL: 110 (tv) ANTI-SEVSE: 110 (vt) oRIC111AL souRCE:

tA) ORGAVI111: Ptas~id (vii)IVNE01ATE SOURCE:

(B) CLOVE: i67-Z2.A12 (figure 10 0) ,I:netion (xi) SEOtJEVCE DESCRIPTIOV: iE0 10 VOsiS:

3 AACGACGGCC AGTACCGG<G txTGGTGTCC GtCGACTCTA GGGCGAGCTC 60 GAATTC

(2)IVFORIIATI011 fOlt ID
V0:1,6:

ti) EQtJEVCE CHARACTERISTICS:

(A) LEVG1V: 66 base pairs (1) TYPE: nucleic acid (C) STRAVDEDVESS: double 4 (D) TOPOLOGY: circular (ii) VOLEWLE TYPE: DVA (penoaic) (iii)NYPOTNETICAI: VO

(iv) ANTI-SEVSE: NO

(vi) ORIGINAL SOURCE:

lA) ORGANISM: Plaasid 2~4~422 (vii) IIfhEDIATE SOURCE:

(B) CLONE: 523-42.A1d (Figure 11 J:nction A) (xi) SEQUENCE DESCRIPTION: SEO' Ifl N0:~6:

lO CTCTTC

(2) fOR
SEC

IIOa7:

(i) SEQUENCE CHARACTERISTICS:

1 (A) LENGTH: 66 base pai n (g) TYPE: nucleic acid (C) STRAIIDEDNESS: double (D) tOPOLOGr: cirwlar 2 ( i IIOLEQJIE TYPE: DNA (peno~ie) 0 i ) ( i NIrPOTNETIGL: 110 i i ) (1v) A11T1-tENSE: NO

(vi) ORIGINAL ~JRCE:

(A) ORCAlIISM: Pletlllid (vi III~DIATE SSDUtCE:
i ) 3 (B) CLONE: 523G2.Alis (figure 11 Junction i) (ix) FEATURE:

(A) NAIIE/KET: CD8 (B) LOGTION: 16..66 3 (D) OTHER INFORNATI011: /p~rti~l /eodon_stsrt 16 /produet N-ter~in~l peptide of hybrid protein 4 0 (xi) tEGiIfNCE DESCRIPTION: SE0 ID NO:f7:

Ilet Lys Trp Ale Thr Trp Ile Asp Pro Vsl Val Lau CM CGT CGT WC TGG
Gln Arp Arg Asp Trp (2) INFORlIATION FOR SEG 1D N0:4il:
(i) SEOtJENCE CHARACTERISTICS:
(A) LENGTH: 17 sniino acids 5 5 (B) TTPE: aoino acid (0) TOPOLOGT: linear (fi) MOLECULE TTPE: protein (xi) SEQUENCE DESCRIPTIO11: SE0 ID NOas:
Met Lys Trp Ale Thr Trp Ile Asp Pro Yal Vet Leu Gln Arp Arp Asp T rp (2)INFORMATION
FOR

ID
110:9:

1 (i)SEQUENCE CHARACTERISTICS:

(A) LENGTH: 132 base pairs (B) TYPE: rx~eleic acid (C) STRAIIDEDNESS: double (D) TOPOLOGT: circular (ii)MOLECULE rrPE: oNA (oenonie) ( 111'POTNETICAL: 110 i i i ) (iv)ANTI-SENSE: 110 (vi)ORIGINAL lDiJRCE:

(A) ORGAIIISM: Plasisid 3 (vii)IMMEDIATE SOURCE:

(B) CLONE: 523-i2.Als (fiotxe JunctionC) (ix)FEATURE:

(A) NAI~/~T: CDS

3 (e) LocAtION: t..q3 (D) OTHER INiORMAT1011: /partial /cadon_start= 1 /functiarr =Translational fininshof hybrid protei n"

4 /product= =C-terwinat pepti de=

/standard nsar "Translation of synthetic DNA

e"

4 (xi)fEOUENCE DESCRIPTION: SE0 I0 5 NOa9:

CACGACTCC TGG AGC CCG TG GTA TCG ATC CTG AGC GCC
GCG GM CAG

AspAsp!er Trp Ser Pro Ser Vel Ser Ile Leu Ser Als Ala Glu Gln CAA

GlyArpTyr Nis Tyr Gln Leu Val Trp Lye Lsu Glu Cys Gln Asp 55 tAAGCTAGAG WTCGATCCC CTATGGCGAT GTGCGGC 132 2~4~422 (2)INFORMATION FOR SE0 ID N0:50:

(i) SEOLIENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids (B) TTPE: amino acid (o) ToPOLOCr: Linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE OESCRIPTI011: N0:50:

AspAsp Ser Trp Ser Pro Ser Yal Glu Ile Gln Leu itr Ala Ser Ala GlyArg Tyr Nis Tyr Gln Leu Val Gln Lys Asp Leu Trp Cys Glu (2)INFORMATI011 fOR SE0 ID N0:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pain (B) TYPE: nucleic acid (C) STRAIIDEDNESS: double (0) TOPOLOGT: cirwter ( t i ) MOLECULE TYPE: DNA (gae:omic) (tti) HTPOTHET1CAL: NO

(iv) ANT!-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Plasmid (vi i ) IMMEDIATE SOURCE:

(B) CLOVE: 523-~.2.Ala (figure 11 ~unetion 0) (xi) fEOUENCE DESCRIPTIO11: SE0 ID 110:51:

(2) INfOR11AT1011 FOR SEg ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
5 0 (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGT: circular 5 5 (ii) MOLECULE TTPE: DNA (genanic) (iii) N1POTNET1 GL: NO
(iv) AHTI-SEHSE: HO
(vi) oatcIHAL souRCE:
(A) ORGAHIS11: Plasoid (vii) III~EDIATE SOURCE:
(B) cL011E: 552-X5.19 (figure 12 Junction A) (xi) . SEOUEHCE DESCRIPTION: SE0 1D 110:52:

TCTAGAGTGGCTTGGCCTC WGGGCCGCG GCCGCCTGG GGTCGAGIITC CCCTCCIICGT

CTGGGG

(2) INFORMATION
fOR

IO
110:53:

2 (i) SEOIIEHCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs lB) TYPE: nucleic acid (C) STRAHOEDHESS: double (D) TOPOLOGY: circular (ii) IIDLECULE T1PE: 0HA (genosic) (iii) HYPOTHETICAL: 110 30(iv) AlItI-SENSE: HO

(vi ORIG1HAL S0IJItCE:
) (A) ORCAHIS11: Plaa~id 3 (vi IHI(EDIATE SOURCE:
5 i ) (B) CLONE: 552-45.19 (figure 12 ,lurrtion B) (ix) fEATtJRE:

(A) IWE/KET: CDS

40 (B) IOGTl011: 31..66 (D) OTHER IHfORhAtIOH: /partial /eodon start= 31 /product= H-tensinal peptide of hybrid protein"

(xi) SEQUENCE DESCRiPTIOH: SEG ID H0:53:
GGCCTTTG CGGTCTCG GGCTCMG ATG MT TCC ATG tTA CGT CCT GTA 5i Met Asn Ser Ilet Leu Arg Pro Vel GM ACC CG ACC
Glu Thr Pro thr -116-.
(2)INFORMATION fOR SE0 ID N0:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12. amiro acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 1 (xi) SEOtJENCE DESCRIPT10N: NO:Si:

MetAsn Ser Met lau Arg Pro Val Pro Thr Glu Thr 1 (2)INFORMATION FOR SE0 ID N0:55:

(i) SECUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs (B) TTPE: rxuleic acid 20 (C) STRAIIDEDNESS: double (D) TOPOLOGY: circular (ti) MOLECULE TYPE: DNA (panamic) 25 (iii) NYPOTNETIGL: NO

(iv) ANTI-SENSE: 110 (vi) ORIGINAL SOURCE:

30 (A) ORCAIIISM: Plasmid (vii) IMMEDIATE SOURCE:

(g) CLONE: 552-f5.19 (Figure 12 ~:nctian C) 3 (ix) FEATURE:

(A) NAIE/KEY: GDS

(g) LOGTI011: 1..15 (D) OTHER INFORMAT1011: /partial /codon_atart 1 4 /product. "C-terwinal peptide of hybrid protein (xi) fEOtJENCE DESCRIPTION:
SEg ID N0:55:

GlnGly Gly Lya Gln AAAGCGCAA
G

(2)INFORMATION FOR SE0 tD N0:56:

(i) SEQUENCE CHARACTERISTICS:

5 (A) LENGTH: 5 amino acids WO 94/03628 ~ ~ PCT/US93/07424 tB) TTPE: amino acid tD) TOPOLOGT: linear (ii) NOLECULE TTPE: protein (xi) SEQUENCE DESCRIPTION: SE0 ID 110:56:

Gln Gly ly Lys Gln G

(2) INFORMATION
FOR

ID N0:5T:

(i) SEOIJEHCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs 1 (B) TYPE: nucleic acid (C) STRAltDEOHESS: double tD) TOPOLOGY: Circular (ii) MOLECULE TTPE: DNA tganowie) (iii) NTPOTNETIGI: HO

(iv) ANTI-SENSE: HO

25(vi) oRIGIHAL soliRCE:

(A) ORG1WI:11: Ilwid (vii) III~EDIATE SOUtCE:

tg) CLONE: 552-65.19 (figure 12 ,iu:etian D) (xi) SEQUENCE DESCRIPTIO11: EE0 ID H0:5T:

3 TGCTGCCTTCCt~ATCT CGACCTGGG GCCCGCCGCG GCCCTCGAGG CCMGCTGAC

TCTAGA

(2) IIIfORMATl011 FOIL
SEQ
ID 110:5x:

(i) SEa<JEIICE CHARACTERISTICS:

(A) LENGTH: 66 bse pairs () TYPE: n:cleic acid (C) STRAIIDED11ESS: double 4 (D) TOPOLOGT: circular (ii) MOLECULE TTPE: DNA (genomic) t i i NTP(ltHEt 1 GL: No i ) (iv) ANTI-SENSE: HO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Plasmid (vii) I111EDIATE SOURCE:

(B) CLONE: 593-31.2 (Figure 13 J~s~ction A) (xi) SEQUENCE DESCRIPTION: ' SE0 ID N0:58:

GTCGACTCTAGACTTMTTA ~AGWTCCGGC GCCCCCCCTC GACGTCtGGG GCGCCCGGGT

Z GGTGCT
O

(2) fOR

ID
N0:59:

(i) tEOIJENCE CHARACTERISTICS:

1 (A) LENGTH: 66 base pairs (B) TTPE: rx~cleic acid (C) STRANDEDNESS: double (D> TOPOLOGY: circular 2 ( i MOLECULE 11'PE: DNA (genaisic) 0 i ) (iii) NTPOtHETIGL: NO

(iv) ANTI-SENSE: 110 (vi) ORIGINAL SOURCE:

(A> ORGA111511: Plaaaid (vii) IMMEDIATE SOURCE:

3 (B) CLONE: 593-31.2 (Figure 13 Junction t) (ix) FEATURE:

(A) NAlIE/KET: CDS

(B) LOGTI011: 16..66 3 (D) OTHER INFORMATION: /partial /product= "Nteroinal peptide of hybrid protein"

/gaM" "ib"

4 0 (xi) SEaVENCE DESCRIPTION: SE0 ID N0:59:
CTCGGGCT CMG ATG MG TCG GG ACG tGG ATC G11T CCC GTC GTT TTA 51 Ilat Lys Trp Ala Thr Trp 1le Asp Pro Val Val Lau CAA CGT CGT GAC TGG
Gln Arg Arg Asp Trp (2) INFORMATION FOR SE0 ID N0:60:
(i) SECUENCE CNARACTERIST1CS:
(A) LENGTH: 17 nsino acids 5 5 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SE0 ID NO:60:
Met Lys Trp Ale Thr Trp Ile Asp Pro Val Vel Leu Gln Arp Arp Asp Trp (2) INFORMATION
fOR

ID
Ib:6l:

1 (i) SEQUENCE CNARACTERISTICS:.

(A) LENGTN: 132 base pairs (B) TTPE: nucleic eeid tC) STRANDEDNESS: double (D) TOPOL0G1: eireuler (ti) MOLECULE TTPE: DNA (~rloAic) (iii) NIPOTNETICAL: NO

2 (iv) ANTI-SENSE: N0 (vi) ORIGINAL SO1JRCE:

tA) ORGAl1IE11: Plsswid 3 (vii) IIIIE01ATE SOURCE:

(B) CLONE: 593-31.2 (FiDure 13 ,lunation C) (ix) FEATURE:

(A) NAIiE/KET: CDS

35 te) LouTtaN: 1..93 <D) OTNER INfORlIATION: /partial /praduet C-terninel peptide of hybrid protein /!~

cxt)sEOUENCE oESCRIPTION: sEOIo Noah WC GACTCCTGG AGCCCG GTATCGGCG GM ATCGG CTGAGC GCC t~E
TG

Asp AspSerTrp ierPro VelSerAle GluIleGln LeuSer Ale Ser s to 15 t Gly ArDTyrNis TyrGln rilTrpCys GlnLysAsp LauGlu Leu (2) INFORNATI011 FOR SE0 ID N0:62:

(1) SEQUENCE CNARACTERIST1CS:

(A) LENGTH: 31 amino acids (A) TYPE: amino acid (D) TOPOLOGT: linear (ii) MOLECULE TxPE: protein (xi) SEQUENCE DESCRIPTION: SE0 ID N0:62:

1 Asp Asp Ser Trp Ser Pro Ser Yal Ser Ala Cln Lau Ser 0 Glu Ile Ala Gly ArO Tyr His Tyr Gln Lsu val Trp Cys Asp Lau Glu Gln Lys (2) INFORMATION FOR SE0 ID N0:63:

(i) SEQUENCE CIIARACTERiSTICS:

(A) LENGTH: 33 base pairs (B) TTPE: txuleic acid (C) STRANDEDNESS: sinpla (D) TOPOLOGT: linear ( i i ) NOLECULE TTPE: DNA (oanoieic) (iii) HyPOTNETICAI: NO

(iv) ANTI-SENSE: NO

3 (vi) ORIGINAL iQJrtC:

(A) ORGAIItSII: Synthetic oliporx~claotideprimer 3 (xi) SEaJENCE DESCRIPTIOII: SE0 ID N0:63:

GGGTCGAGTGMGACAACC ATTATTTTGA TAC

(2) INFORMATION
fOR

ID
N0:64:

(i) SEaIENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs (P) TYPE: nielaic acid (C) STRAIIDEDNESS: double 4 (0) TOPOLOGt: circular (ii) MOLECULE TrPE: DNA (Renomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGAIIISM: Plasmid WO 94/03628 ~ ~~ ~ PCT/US93/07424 (vi i ) 111~EDIATE SOURCE:
(e) CLONE: 593-31.2 (Figure 13 Junction D) (xi) SEQUENCE DESCRIPTION: SE0 ID IIO:bi:

(2) 1NFOR11ATION FOR tE0 i0 N0:65:
(i) SEQUENCE ClIARACTERISTICt:
1 5 (A) LENGTH: 32 base pai n (8) TYPE: rx~eltic acid (C) STRANDEDNESS: single (D) TOPOLOGT: linear 2 O (ii) IIOIECUIE TYPE: 0NA (genaoic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi ) ORIGINAL f0lNtCE:
(A) ORCANISIt: Synthetic oligors:claotida prier (xi) SEQUENCE DESCRIPTION: EE0 ID 110:65:

35(2) INFORIIATIDfi f0lt ID N0:66:

(i) SEOUEHCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid 4 (C) StRANDEDNESS: single (D) TOPOLOGY: linear ( i i IIOLEC1lLE TYPE: DNA ( genaei c ) ) 4 (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

50 (A) ORGAIIIS11: Synthetic oligorruclaotide priner (xi) SEQUENCE DESCRIPTION: SEG IO NO: bb:

(2) INFORMATION FOR SEO ID N0:6T:
<i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs-.
(B) TTPE: rx:eleic acid "
(C) STRANDEDNESS: s9nple (D) TOPOLOGY: linear (ii) MOLECULE TrPE: DNA (peno~ic) (iii) NTPOTNETIGL: NO
1 5 (iv) ANTI-SENSE: NO
(vi ) ORIGINAL SOlIitCE:
(A) ORGANISM: Synthetic olipaxxleotide pris~er (xi) SEQUENCE DESCRIPTION: SE0 ID 110:67:

(2) FOR

NO:bi3:

(i) tEOUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TrPE: rx:cleic acid (C) STRANDEDNESS: single (D) TOPOLOG1: linear (ii) MOLECULE TTPE: DNA (penowic) (iii) HYPOTHETIGL: No (iv) ANTI-SENSE: NO

4 (vi) ORIGINAL SOURCE:

(A) ORGAIIISM: Synthetic oliparx~cleotide pr ier (xi) SEDIIfNCE DESCRIPTION: SE0 ID NO:bii:

(2) INFORMATION FOR SE0 ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 bsse pairs (B) TtPE: nucleic acid (C) STRANDEDNESS: single 5 5 (D) TOPOLOGT: linear .. 21~~4~
(ii> MOLECULE TYPE: DNA (penawie) (iii) NYPOTNETIGL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Synthetic oliporx~cltotide prisxr (xi) SEQUENCE DESCRIPTION: SE0 ID 110:69:

GGGTCWCTTAGtCTTATC GATGTCAM

(2) INFORlIATI0Il FOR
SEC
ID
N0:70:

(i) SEQUENCE CNARACTERISTiCS:

(A) LENGtN: 31 bua pairs (B) TYPE: nueltic acid 2 (C) STRANDEDNESS: sinpla (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Dsrfowie) 2 (iii) NYPOTNETICAL: N0 (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

3 (A) ORGAIIIiM: Synthetic olioonuclsotids priwsr (xi) SEQUENCE DESCRIPTION: SE0 ID N0:70:

GGGATCGTGMTCCTMTC AAAMCTCTT T

(2) INfORIIATION
fOR

ID
N0:71:

4 ( t SEClJENCF CHARACTER I ST I CS:
0 ) (A) LENGTH: 31 bast pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sinpls (0) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (panowic) (iii) NYPOTNETIGL: NO

5 (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Synthetic olipanueleotide priwer 21414..~~~

(xi) SEOUEIICE DESCRIpTI011: SE0 ID 110:71:
GGGATCCtTA CGAAAAGTAT TTAATTTGTC C

Claims

What is claimed is:

1. A live recombinant equine herpesvirus which comprises the genomic DNA of equine herpesvirus 4 (EHV-4) from which a DNA sequence has been deleted from the US2 gene.

2. A live recombinant equine herpesvirus 4 (EHV-4) comprising a foreign DNA
sequence inserted into an equine herpesvirus 4 viral genome, wherein the foreign DNA sequence is inserted within a non-essential site of the unique short region of the equine herpesvirus 4 viral genome.

3. The herpesvirus of claim 1 further comprising a deletion of one or more non-essential regions of the DNA sequences outside of the US2 gene.

4. The herpesvirus of claim 3, wherein the DNA sequence is deleted from one or more genes selected from the group consisting of the gene encoding the thymidine kinase (TK), the gene encoding glycoprotein E (gpE), the gene encoding glycoprotein G (gpG) , and the gene encoding the large membrane glycoprotein (MGP).

5. The herpesvirus of claim 1, 3, or 4 that further comprises a foreign DNA
sequence inserted in place of the deleted DNA sequence.

6. The herpesvirus of claim 2 or 5 wherein the foreign DNA sequence encodes a detectable marker.

7. The herpesvirus of claim 6 wherein the detectable marker is .beta.-galactosidase.

8. The herpesvirus of claim 2 or 5 wherein the foreign DNA is from an equine influenza virus.

9. The herpesvirus of claim 2 or 5, wherein the foreign DNA is from a gene encoding equine herpesvirus 1 (EHV-1) glycoprotein B (gpB) or EHV-1 glycoprotein D(gpD).

10. The herpesvirus of claim 8 wherein the foreign DNA is from the hemagglutinin and neuraminidase genes of the Influenza A/equine/Prague/56 isolate of equine influenza virus.

11. The herpesvirus of claim 8, wherein the foreign DNA is from the hemagglutinin and neuraminidase genes of the Influenza A/equine/Miami/63 isolate of equine/influenza virus.

12. The live recombinant herpesvirus of claim 8 wherein the foreign DNA is from the hemagglutinin and neuraminidase genes of the Influenza A/equine/Kentucky/81 isolate of equine influenza virus.

13. The equine herpesvirus of claim 8, wherein the foreign DNA is from the hemagglutinin and neuraminidase genes of the Influenza Alequine/Alaska/91 isolate of equine influenza virus.

14. The herpesvirus of claim 1 having a DNA sequence encoding E. coli .beta.-galactosidase inserted in place of the US2 gene deletion.

15. The herpesvirus of claim 1, 2, or 14 further comprising a deletion in the gene encoding TK.

16. The herpesvirus of claim 14 wherein the gpG gene and a portion of the MGP
gene are also deleted.

17. The herpesvirus of claim 15 wherein the gpG gene and a portion of the MGP
gene are also deleted.

18. The herpesvirus of claim 4 wherein the genes from which the DNA
sequences were deleted are the gene encoding TK, and the gene encoding gpE.

19. The herpesvirus of claim 18 having a DNA sequence encoding the EHV1 gpB
inserted in place of the gpE deletion and EHV1 gpD inserted in place of the TK
deletion.

20. The herpesvirus of claim 18 having a DNA sequence of the hemagglutinin and neuraminidase gene of an equine influenza virus inserted in place of the gpE
deletion.

21. The herpesvirus of claim 20 wherein the hemagglutinin neuraminidase genes are from the influenza A/equine/Prague/56 isolate of equine influenza virus.

22. The herpesvirus of claim 20 wherein the hemagglutinin neuraminidase genes are from the Influenza A/equine/Miami/63 isolate of equine/influenza virus.

23. The herpes-virus of claim 20 wherein the hemagglutinin neuraminidase genes are from the Influenza A/equine/Kentucky/81 isolate of equine influenza virus.

24. The herpesvirus of claim 20 wherein the hemagglutinin neuraminidase genes are from the Influenza A/equine/Alaska/91 isolate of equine influenza virus.

25. The herpesvirus of claim 14 designated by the ATCC Accession No. VR 2363 (S- 4EHV-003).

26. The herpesvirus of claim 15 designated by the ATCC Accession No. VR 2362 (S-4EHV-002).

27. The herpesvirus of claim 15 designated by the ATCC Accession No. VR 2360 (S-1 EHV-004).

28. The herpesvirus of claim 16 designated by the ATCC Accession No. VR 2358 (S-1 EHV-002).

29. The herpesvirus of claim 17 designated by the ATCC Accession No. VR 2359 (S-1 EHV-003).

30. The herpesvirus of claim 19 designated S-4EHV-010 constructed by:

i) inserting a EHV-1 gpD gene into homology vector 495-61.39;

ii) homologous recombination of the vector of step (i) with virus S-4EHV-009; and iii) screening for herpesvirus S-4EHV-010.

31. The herpesvirus of claim 21 designated S-4EHV-011 constructed by:

i) inserting the hemagglutinin and neuraminidase genes of the influenza Alequine/Prague/56 isolate of equine influenza into homology vector 580-57.25;
ii) homologous recombination of the vector of step (i) with virus 5-4EHV-008; and iii) screening for herpesvirus S-4EHV-011.

32. The herpesvirus of claim 22 designated S-4EHV-012 constructed by:

i) inserting the hemagglutinin and neuraminidase genes of the Influenza A/ equine/Miami/63 isolate of equine influenza into an homology vector 580-57.25;
ii) homologous recombination of the vector of step (i) with virus S-4EHV-008; and iii) screening for herpesvirus S-4EHV-012.

33. The herpesvirus of claim 23 designated S-4EHV-013 constructed by:

i) inserting the hemagglutinin and neuraminidase genes of the Influenza A/equine/Kentucky/81 isolate of equine influenza into an homology vector 580-57.25;

ii) homologous recombination of the vector of claim (1) and virus S-4EV-008; and iii) screening for herpesvirus S-4EHV-013.

34. The herpesvirus of claim 24 designated S-4EHV-014 constructed by:

i) inserting the hemagglutinin and neuraminidase genes of the Influenza Alequine/Alaska/91 isolate of equine influenza into an homology vector 580-57.25;
ii) homologous recombination of the vector of claim (1) and virus S-4EV-008; and iii) screening far herpesvirus S-4EHV-014.

35. The herpesvirus of claim 1 wherein said US2 gene has the nucleotide sequence shown in SEQ ID NO: 3.