RECOMBINANT INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS

Abstract: The present invention provides recombinant infectious bovine rhinotracheitis (IBR) viruses useful in vaccines to protect bovines from diseases particular to them, including infectious bovine rhinotracheitis and bovine respiratory disease complex. The present invention further provides methods for distinguishing an animal vaccinated with a vaccine of the present invention from an animal infected with a naturally-occurring IBR virus. The present invention also provides isolated DNA encoding the gpE glycoprotein, the gpG glycoprotein, and US2 genes of an IBR virus. The present invention further provides homology vectors for producing recombinant IBR viruses.

Description: RECOMBINANT INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS 5 Within this application several publications are -* referenced by arabic numerals within parentheses. Full < citations for these publications may be found at the end * of the specification immediately preceding the claims. 10 The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. 15 Field of the Invention The present invention involves recombinant infectious bovine rhinotracheitis (IBR) viruses useful in vaccines to protect bovines from naturally-occurring infectious 20 bovine rhinotracheitis virus- and other bovine diseases. Background of the Invention The ability to isolate viral DNA and clone this isolated 25 DNA into bacterial plasmids has greatly expanded the approaches available to make viral vaccines. The methods used to make the present invention involve modifying cloned viral DNA sequences by insertions, deletions and single or multiple base changes. The modified DNA is 30 then reinserted into the viral genome to render the virus non-pathogenic. The resulting live virus may then be used in a vaccine to elicit an immune response in a host animal and to protect the animal against a disease. •) 35 One group of animal viruses, the herpesviruses or Herpetoviridae , is an example of a class of viruses amenable to this approach. These viruses contain 100,000 to 200,000 base pairs of DNA as their genetic material. Importantly, several regions of the genome have been identified that are nonessential for the replication of virus in vitro in cell culture. Modifications in these regions of the DNA may lower the pathogenicity of the virus, i.e., attenuate the virus. For example, inactivation of the thymidine kinase gene renders human herpes simplex virus non-pathogenic (28) , and pseudorabieε virus of swine non-pathogenic (29) . Removal of part of the repeat region renders human herpes simplex virus non-pathogenic (30,31). A repeat region has been identified in Marek's disease virus that is associated with viral oncogenicity (32) . A region in herpesvirus saimiri has similarly been correlated with oncogenicity (33) . Removal of part of the repeat region renders pseudorabies virus non-pathogenic (U.S. Patent No. 4,877,737, issued October 31, 1989). A region in pseudorabies virus has been shown to be deleted in naturally-occurring vaccine strains (11,3) and it has been shown that these deletions are at least partly responsible for the lack of pathogenicity of these strains. It is generally agreed that herpesviruseε contain non- essential regions of DNA in various parts of the genome, and that modifications 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 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 is not readily apparent. Infectious bovine rhinotracheitis (IBR) virus, an alpha- herpesvirus with a class D genome, is an important pathogen of cattle. It has been associated with respiratory, ocular, reproductive, central nervous system, enteric, neonatal, and dermal diseases (34) . Cattle are the normal hosts of IBR virus, however it also infects goats, swine, water buffalo, wildebeest, mink, and ferrets. Experimental infections have been established in mule deer, goats, swine, ferrets, and rabbits (35) . Conventional modified live virus vaccines have been widely used to control diseases caused by IBR virus. However, these vaccine viruses may revert to virulence. More recently, killed virus IBR vaccines have been used, but their efficacy appears to be marginal. IBR virus has been analyzed at the molecular level as reviewed in Ludwig (36) . A restriction map of the genome is available in this reference, which will aid in the genetic engineering of IBR according to the methods provided by the present invention. As reported in the current literature, IBR virus has been engineered to contain a thy idine kinase deletion (43,44) and a deletion in the gill gene (45,46). However, no evidence has been presented for the deletions in the US2, repeat, gpG, or gpE regions. In the subject application, we demonstrate the usefulness of such deletions for both the attenuation of IBR virus and for the development of gene deleted marker vaccines. As with other herpesviruses, IBR virus 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 with disease-causing wild type virus. The development of differential vaccines and companion diagnostic tests has proven valuable in the management of pseudorabies disease (47) . A similar differential marker vaccine would be of great value in the management of IBR disease. The construction of differential diagnostics has focused on the deletion of glycoproteins. Theoretically, the glycoprotein chosen to be the diagnostic marker should have the following characteristics: (1) the glycoprotein and its gene should be non-essential for the production of infectious virus in tissue culture; (2) the glycoprotein should elicit a major serological response in the animal; and (3) the glycoprotein should not be one that makes a significant contribution to the protective immunity. Four major IBR virus glycoproteins (gl, gll, gill, and gIV) have been described in the literature (48) . Three of these genes, gl, gill, and gIV, have been sequenced and shown to be homologous to the HSV glycoproteins gB, gC, and gD, respectively. Although it has been suggested that the gll protein is analogous to HSV gE, no sequence evidence has been presented to confirm that suggestion (48) . The gB and gD homologueε are essential genes and would not be appropriate as deletion marker genes. The gC gene of herpesviruses has been shown to make a significant contribution to protective immunity as a target of neutralizing antibody (49) and as a target of cell-mediated immunity (50) . Therefore, the gC gene is not desirable as a deletion marker gene. As indicated above, Kit et al. (45) have described the deletion of the IBR virus gill as a marker gene. It would be expected that such a deletion would compromise the efficacy of an IBR vaccine. For pseudorabies virus (PRV) the criteria for a deletion marker gene are best met by the glycoprotein X (51) . Wirth et al. (52) suggests the existence of a "gX ho ologue of HSV-l" in the IBR virus. It is not clear what is meant by this because although there is a PRV gX gene, there is no reported HSV-l gX gene or gX homologous gene. In any case, no sequence evidence is presented to support this suggestion. We present clear evidence of homologues of PRV gX (HSV-2 gG) and PRV gl (HSV gE) in IBR virus and demonstrate their usefulness as diagnostic markers. The present invention provides a method of producing a fetal-safe, live recombinant IBR virus which comprises treating viral DNA from a naturally-occurring live IBR virus so as to delete from the virus DNA corresponding to the US2 region of the naturally-occurring IBR virus. The present invention also provides viruses in which (1) DNA corresponding to the US2 region of naturally-occurring IBR virus has been deleted, and (2) DNA encoding gpG and/or gpE has been altered or deleted. Such viruses are useful in vaccines which need diagnostic markers and are safe for use 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 immunogens to animals (53). The herpesviruses are attractive candidates for development as vectors because their host range is primarily limited to a single target species (54), and they have the capacity for establishing a latent infection (55) that could provide for stable in vivo expression of a desired cloned polypeptide. Herpesviruses have been engineered to express a variety of foreign gene products, such as bovine growth hormone (56) , human tissue plasminogen activator (57), and E. coli β-galactosidase (58,59). In addition, possible immunogenic polypeptideε have been expressed by herpesviruses. Whealy et al. (60) expressed portions of the human immunodeficiency virus type 1 envelope glycoprotein in pseudorabies virus (PRV) as fusions to the PRV glycoprotein III. The hepatitis B virus surface antigen (61) and a hybrid human malaria antigen from Plasmodium f lciparum have been expressed in herpeε simplex virus type 1 (HSV-l) (62)*. The IBR viruses described above may be used as vectors for the insertion of geneε encoding antigenε from microorganisms causing important cattle diseases. Such recombinant viruses would be multivalent vaccines protecting against IBR as well as other diseases. Kit et al. (63) have described the expresεion of a Foot and Mouth disease antigen in IBR virus. In some of the prior applications from which the subject application claims priority (which precedes the Kit publication by at least three years) , we deεcribed the use of IBR virus to express several foreign genes including the _?. coli β-galactoεidase (lacZ) gene, the TN5 neomycin resistance gene, and antigens from bovine rota virus, and parainfluenza type 3 virus (see U.S. Serial No. 06/933,107, filed November 20, 1986 and U.S. Serial No. 07/078,519, filed July 27, 1987). These applications precede the Kit publication by at least three yearε. The viruεeε described in this application provide a combination of attenuation, differentiation and ultivalency. These properties make such viruses useful as vaccines for the management of cattle diseases. The present invention provides recombinant infectious bovine rhinotracheitis (IBR) viruses useful in vaccines * 5 to protect bovines from infectious bovine rhinotracheitis , and other bovine diseases. The present invention further -_ , provides methods for distinguishing an animal vaccinated with the vaccine of the present invention from an animal infected with a naturally-occurring IBR virus. The 10 present invention also provides isolated DNA encoding the gpE glycoprotein of IBR virus and isolated DNA encoding the gpG glycoprotein of IBR virus. The present invention also provides a method of producing a fetal-safe, live recombinant IBR virus which comprises treating viral DNA 15 from a naturally-occurring live IBR virus so as to delete from the virus DNA corresponding to the US2 region of the naturally-occurring IBR virus. The present invention also provides isolated DNA encoding 20 the US2 gene of an IBR virus. The present invention further provides a homology vector for producing a recombinant IBR virus by inserting foreign DNA into the genomic DNA of an IBR virus which comprises a double- stranded DNA molecule consisting essentially of double- 25 stranded foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant IBR is introduced and at one end of the foreign DNA, double-stranded IBR viral DNA homologous to genomic DNA located at one side of a site on the genomic DNA which is 30 not essential for replication of the IBR virus and at the other end of the foreign DNA, double-stranded IBR viral DNA homologous to genomic DNA located at the other side κ < of the same site on the genomic DNA. 35 The present invention also provides for a homology vector for producing a recombinant IBR virus by deleting DNA which encodes a detectable marker which had been inserted into the genomic DNA of an IBR virus comprising a double- stranded DNA molecule consisting essentially of double- stranded IBR viral DNA homologous to the genomic DNA which flanks on each side the DNA to be deleted. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA from the US2 gene, the gpE glycoprotein gene and the gpG glycoprotein gene has been deleted so that upon replication, the recombinant IBR virus produces no gpE glycoprotein and no gpG glycoprotein. The invention also provides a vaccine which comprises an effective immunizing amount of a recombinant virus protective against bovine respiratory disease complex and a suitable carrier. Brief Deεcription of the Ficrαreε Figure 1 Details of the IBR Cooper Strain. Diagram of IBR genomic DNA showing the unique long, internal repeat, unique short, and Terminal repeat regions. Restriction maps for the enzymes Hindlll, EcoRI, and XJal are indicated (7) . Fragments are lettered in order of decreasing size. The unique short region is also expanded for inclusion of more detail. The location of several genes is also indicated, they are unique short 2 (US2) , immediate early genes (IE) (20) , glycoprotein G (gpG) , glycoprotein IV (gpIV) (17), glycoprotein E (gpE) . Note that due to the inversion of the short region, which includes the unique short, internal, and terminal repeats, four half molar indiII fragments are present (HindiII D, C, F, and H) . Figure 2 Details of S-IBR-002. Diagram of S-IBR-002 genomic DNA showing the unique long, internal repeat, unique short, and Terminal repeat regions. Restriction maps for the enzymes Hindlll , EcoRI , and Xbal are indicated (7) . Fragments are lettered in order of decreasing size. The EcoRI B and F fragments are expanded for inclusion of more detail. The -800 BP repeat deletions are indicated by deltas. Note that due to the inversion of the short region, which includes the unique short, internal, and terminal repeats, four half molar Hindlll fragments are present (Hindlll D, C, F, and H) . Figure 3 DNA sequence of the IBR Unique Short 2 gene. The sequence of the first 1080 base pairs of the Hindlll K fragment, reading from the Hindlll K/Hindl±I O junction, are shown. The unique short 2 (US2) gene is transcribed toward the Hindlll ¥./ Hindlll 0 junction as indicated in Figure 1. The sequence has been reversed and complemented in order to show the translation εtart and termination of US2 gene. Figure 4 Homology between the IBR US2 protein and the US2 proteins of HSV-l, PRV, HSV-2, and MDV. (a) Matrix plot of the amino acid sequence of the IBR US2 protein (309) against the amino acid sequence of the HSV-l US2 protein (291) (8) . (b) Alignment of the conserved region between IBR US2 protein, HSV-l US2 protein, PRV US2 protein (256 amino acids) (21) , HSV-2 US2 protein (291) (9) , and MDV US2 protein (270 amino acids) (1) . Figure 5 Details of the Nasalgen deletion. Diagram of IBR genomic DNA showing the unique long, internal repeat, unique short, and terminal repeat regions. A restriction map for the enzyme Hindlll is indicated. Fragments are lettered in order of decreasing size. The unique short region is expanded for inclusion of more detail. The location of the deletion in the Nasalgen Hindlll K fragment is shown. Three regions of DNA sequence are also shown. The first line shows the first 60 base pairs upstream of the Hindlll 0/ Hindlll D junction in the IBR Cooper strain. The second line shows the first 60 base pairs upstream of the Hindlll K/Hindlll D junction in the Nasalgen strain. The third line shows 60 base pairs flanking the DNA encoding amino acid 59 of the IBR US2 gene in the IBR Cooper strain. Figure 6 Details of S-IBR-027. Diagram of S-IBR-027 genomic DNA showing the unique long, internal repeat, unique short, and terminal repeat regions. Restriction maps for the enzymeε Hindlll, EcoRI, and Xbal are indicated (7) . Fragments are lettered in order of decreasing size. The unique short region is also expanded for inclusion of more detail. The location of several genes is also indicated, they are unique short 2 (US2) , immediate early genes (IE) (20) , glycoprotein G (gpG) , glycoprotein IV (gpIV) (17) , glycoprotein E (gpE) . The unique short region and repeat region deletions are indicated by deltas. The location of the approximately 1200 BP deletion of the US2 gene is shown in the expanded region. Note that due to the inversion of the short region, which includes the unique short, internal, and terminal repeats, four half molar Hindlll fragments are present (Hindlll D, C, F, and H) . Figure 7 Detailed description of the DNA insertion in Homology Vector 129-71.5. Diagram showing the orientation of DNA fragments assembled in plasmid 129-71.5. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. 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 heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, () , refer to amino acids, and restriction sites in brackets, [], indicate the remnants of sites which were destroyed during construction. The following abbreviationε are used: polyadenylation signal (pA) , infectious bovine rhinotracheitis virus (IBR) , Herpes simplex virus type 1 (HSV-l) , thymidine kinase (TK) , neomycin resistance (NE0) , bacterial transposon Tn5 (Tn5) . Figure 8 DNA sequence of the IBR glycoprptein G gene. The sequence of approximately 1400 base pairs of the Hindlll K fragment, starting approximately 2800 base pairs downstream of the Hindlll K Hindlll 0 junction, are shown. The glycoprotein G (gpG) gene is transcribed away from the Hindlll K/ Hindlll 0 junction as indicated in Figure 1. The translational start and termination of the gpG gene are indicated. Figure 9 Homology between the IBR gpG protein, the gpX protein of PRV and the gpG protein of HSV-2. (a) Matrix plot of the amino acid sequence of the IBR gpG protein (441) against the amino acid sequence of the PRV gpX protein (498) (12) . (b) Alignment of the conserved region between IBR gpG protein, PRV gpX protein, and HSV-2 gpG protein (699) (9) . Note that IUPAC- IUB Biochemical Nomenclature Commission conventions are used. Figure 10 Western blot of proteinε released into the medium of IBR and PRV infected cells, showing the absence of gpG in S-PRV-013, S-IBR-035, S- IBR-036, S-IBR-037, and S-IBR-038 but its presence in S-PRV-160 and wild type S-IBR-000. Lanes (A) 0.5 μg purified gpG, (B) blank lane, (C) S-PRV-160, (D) S-PRV-013, (E) pre-stained molecular weight markers, (F) 0.5 μg purified gpG, (G) S-IBR-038, (H) S-IBR-037, (I) S-IBR- 036, (J) S-IBR-035, (K) S-IBR-000, (L) uninfected MDBK cells, (M) pre-stained molecular weight markers. Media samples were prepared as described in the PREPARATION OF HERPESVIRUS CELL LYSATES. The concentrated media from the infection of one 6 cm dish of infected cells was loaded in each sample lane except for samples S-PRV-013 and S-PRV-160 for which the media from two 6 cm dishes were loaded. Figure 11 Detailed description of the DNA insertion in Plasmid 459-12.6. Diagram showing the orientation of DNA fragments assembled in plasmid 459-12.6. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. 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 heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, () , refer to amino acids, and restriction sites in brackets, [], indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique glycoprotein G (gpG) , glycoprotein III (gpIII) , glycoprotein X (gpX) , polyadenylation signal (pA) , infectious bovine rhinotracheitis virus (IBR) , pseudorabies virus (PRV) , and human cytomegalovirus (HCMV) . Figure 12 Detailed description of the DNA insertion in Homology Vector 439-01.31. Diagram showing the orientation of DNA fragments assembled in plasmid 439-01.31. The origin of each fragment iε indicated in the table. The sequences located at each of the junctions between fragments iε alεo shown. 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 heavy bar. The location of several gene coding regions and regulatory elements iε also given. The following two conventions are used: numbers in parentheses, () , refer to amino acids, and restriction sites in brackets, [], indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique short 2 (US2) , glycoprotein G (gpG) , glycoprotein IV (gpIV) , polyadenylation signal (pA) , infectious bovine rhinotracheitis virus (IBR) , pseudorabieε virus (PRV) , and human cytomegalovirus (HCMV) . Figure 13 Detailed description of the DNA insertion in Homology Vector 439-21.69. Diagram showing the orientation of DNA fragments assembled in plasmid 439-21.69. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments iε also shown. The reεtriction εites 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 heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, () , refer to amino acids, and restriction sites in brackets, [], indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique short 2 (US2) , glycoprotein G (gpG) , glycoprotein IV (gpIV) , polyadenylation signal (pA) , infectious bovine ' rhinotracheitis virus (IBR) , pseudorabieε virus (PRV) , and human cytomegalovirus (HCMV) . Figure 14 Detailed description of the DNA insertion in Homology Vector 439-70.4. Diagram showing the orientation of DNA fragments assembled in plasmid 439-70.4. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. 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 heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, () , refer to amino acids, and restriction sites in brackets, [], indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: glycoprotein G (gpG) , glycoprotein IV (gpIV) , and infectious bovine rhinotracheitiε viruε (IBR) . Figure 15 DNA sequence of the IBR glycoprotein E gene. The sequence of 2038 base pairs of the IBR unique short region, starting approximately 1325 base pairs upstream in the Hindlll K/ Hindlll F junction in the Hindlll K fragment, are shown. The glycoprotein E (gpE) gene is transcribed toward the Hindlll K/Hindlll F junction as indicated in Figure 1. The translation start and termination of the gpE gene are indicated. Note that IUPAC-IUB BiochemicalNomenclature Commissionconventions are used. Figure 16 Homology between the IBR gpE protein and the gpE protein of HSV-l, the gpl protein of VZV, and the gl protein of PRV. (a) Matrix plot of the amino acid sequence of the IBR gpE protein (617) against the amino acid sequence of the PRV gl protein (577) (64) . (b) Alignment of the conserved region between IBR gpE protein, PRV gl protein, and VZV gpl protein (37) . Figure 17 Detailed description of a plasmid containing the gpE gene. Diagram showing the orientation of DNA fragments to be assembled in the gpE- containing plasmid. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown. The restriction sites used to generate each fragment are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, () , refer to amino acids, and restriction sites in brackets, [], indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique glycoprotein E (gpE) , glycoprotein IV (gpIV) , and infectious bovine rhinotracheitis virus (IBR) . Figure 18 Detailed description of the DNA insertion in the homology vector 536-03.5. Diagram showing the orientation of DNA fragments to be assembled in the homology vector. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. 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 heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, () , refer to amino acids, and restriction siteε in bracketε, [], indicate the remnants of siteε which were deεtroyed during conεtruction. The following abbreviations are used: glycoprotein E (gpE) , immediate early promoter (IE) , infectious bovine rhinotracheitis virus (IBR) , and pseudorabies virus (PRV) . Figure 19 Construction of Recombinant S-IBR-004 Virus. S-IBR-004 is an IBR recombinant virus carrying an inserted foreign gene (NEO) under the control of the PRV gpX promoter. A new XJal site was created at the short unique region and the original Sad site was deleted. Figure 20 Conεtruction of Recombinant S-IBR-008 Viruε. S-IBR-008 iε a recombinant IBR viruε that haε a bovine rotavirus glycoprotein gene and the plaεmid vector inεerted in the XJbal εite on the unique long region. A site specific deletion was created at the [Sad] site due to the loss of NEO gene in the small unique region. Figure 21 Sequence of the PI-3 (SF-4 Strain) HN Gene. Note that IUPAC-IUB Biochemical Nomenclature Commission conventionε are uεed. Figure 22 Details of S-IBR-018 Construction. A. First line showε the IBR (Cooper Strain) BamHI- C fragment map. Second line showε the conεtruction of the alpha-4 promoter on the PI- 3 HN gene and itε insertion into the Hindlll site in BamHI-C. Also shown are the beta-gal and neomycin (NEO) gene constructions under the control of the gX promoter that were put into the XJbal site and used as selectable markers to purify the recombinant viruε. B. The BamHI-C fragment map of S-IBR-018 after inεertion of the PI-3 HN, beta-gal, and neomycin geneε. C. The S-IBR-018 genome showing the location of the three inserted foreign genes. Legend: B = BamHI; H = Hindlll; X = XJbal; S = StuI; UL = unique long region; US - unique short region; IR = internal repeat region; TR = terminal repeat region. Figure 23 Details of S-IBR-019 Construction. A. First line showε the IBR (Cooper Strain) BamHI- C fragment map. Second line shows the conεtruction of the alpha-4 promoter on the PI- 3 F gene and its insertion into the Hindlll site in BamHI-C. Also shown are the beta-gal and neomycin (NEO) gene constructionε under the control of the gX promoter that were put into the XJbal εite and used as selectable markers to purify the recombinant virus. B. The BamHI-C fragment map of S-IBR-019 after insertion of the PI-3 F, beta gal, and neomycin genes. C. The S-IBR-019 genome showing the location of the three inserted foreign genes. Figure 24 Detailed description of the DNA insertion in Homology Vector 591-21.20. The diagram shows the orientation of DNA fragments assembled in plasmid 591-21.20. The origin of each fragment is described in the MATERIALS AND METHODS section. The sequences located at the junctions between each fragment are shown. 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 gene coding region is also given. The following two conventions are used: numbers in parenthesis () refer to amino acids, and restriction siteε in bracketε [] indicate the remnants of sites which were destroyed during construction. The following abbreviation iε used, infectious bovine rhinotracheitis virus (IBR) . Figure 25 Detailed description of the marker gene insertion in Homology Vector 591-46.12. 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. The restriction siteε 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 conventionε 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, pseudorabieε viruε (PRV) , uronidaεe A gene (uidA) , Escherichia coli (E. coli) , herpes εimplex viruε type 1 (HSV-l) , poly adenylation signal (pA) , and glycoprotein X (gpX) . Legend: B - BamHI; H = Hindlll; X = XJbal; S = StuI; UL = unique long region; US = unique short region; IR = internal repeat region; TR - terminal repeat region. Detailed Deεcription of the Invention The present invention provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR viruε in which DNA encoding gpG glycoprotein haε been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein. The DNA encoding gpG glycoprotein may be deleted or foreign DNA may be inserted into the DNA encoding gpG glycoprotein. The DNA encoding gpG glycoprotein may be deleted and foreign DNA may be inserted in place of the deleted DNA encoding gpG glycoprotein. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein and no gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted or foreign DNA may be inserted into the DNA encoding gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted and foreign DNA may be inserted in place of the deleted DNA encoding gpE glycoprotein. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein, DNA corresponding to the US2 region of the naturally- occurring IBR virus has been deleted, and DNA encoding the gpE glycoprotein has been altered or deleted. The present invention also provides a recombinant IBR viruε compriεing viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus haε been deleted, and (2) DNA encoding gpG glycoprotein haε been altered or deleted. The DNA encoding the gpG glycoprotein may be deleted or foreign DNA may be inεerted in place of the deleted DNA encoding gpG glycoprotein. Foreign DNA may be inserted in place of the deleted DNA corresponding to the US2 region of the naturally-occurring IBR virus. The present invention also provides S-IBR-037, a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally- occurring IBR virus has been deleted, and (2) DNA encoding gpG glycoprotein has been deleted. S-IBR-037 was deposited on April 16, 1991 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 Accesεion No. VR 2320. The present invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR viruε has been deleted and a foreign DNA sequence which encodes Escherichia coli β- galactosidase has been inserted in place of the deleted DNA encoding gpG glycoprotein, and (2) DNA encoding gpG glycoprotein has been altered or deleted. The present invention also provides two examples of such viruses, S- IBR-035 and S-IBR-036. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR viruε produceε no gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted or foreign DNA may be inεerted in the DNA encoding gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted and foreign DNA may be inserted in place of the deleted DNA encoding gpE glycoprotein. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produceε no gpE glycoprotein and DNA correεponding to the US2 region of the naturally- occurring IBR virus has been deleted. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA in the unique short region of the naturally-occurring IBR virus has been deleted. Foreign DNA may be inserted into the DNA of the recombinant IBR virus. The foreign DNA may be inserted into the XJbal site in the long unique region. The foreign DNA may be a sequence which encodes bovine rotavirus glycoprotein 38; this sequence may be inserted into the XJbal site in the long unique region. The present invention provides S-IBR-008, a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted and in which a foreign DNA sequence which encodes bovine rotavirus glycoprotein 38 has been inserted into the Xbal site in the long unique region. S-IBR-008 was deposited on June 18, 1986 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 2141. The present invention further provides a recombinant IBR viruε comprising viral DNA from a naturally-occurring IBR virus from which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted and (2) at least a portion of both repeat sequences has been deleted. The present invention further provides an example of such a recombinant virus, designated S-IBR- 027. S-IBR-027 was deposited on April 17, 1991 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 2322. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which at least a portion of both repeat sequences haε been deleted. The present invention further provides a recombinant IBR virus compriεing viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences haε been deleted and (2) DNA encoding one or more EcoRV restriction siteε has been deleted. The present invention further provides an example of such a recombinant virus, designated S-IBR-002. S-IBR-002 was deposited on June 18, 1986 pursuant to the Budapest Treaty on the International Deposit of Microorganiεms 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 2140. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted and (2) wherein foreign DNA haε been inεerted into the DNA of the recombinant IBR viruε. The foreign DNA may be a sequence which encodes the Tn5 NEO gene. The preεent invention further provideε S-IBR-020, a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted and (2) wherein a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the DNA of the recombinant IBR virus. The present invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted, (2) wherein a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the DNA of the recombinant IBR virus, and (3) wherein at least a portion of the thymidine kinase gene haε been deleted. The present invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted, (2) wherein a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the DNA of the recombinant IBR virus, and (3) wherein at least a portion of the thymidine kinase gene has been deleted. The subject invention provides an example of such a recombinant viruε, designated S-IBR- 028. S-IBR-028 was deposited on May 14, 1991 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 2326. The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the viral DNA. The Tn5 NEO gene may be under the control of an inserted, upstream, pseudorabies virus glycoprotein X promoter. The subject invention further provides an example of a recombinant virus wherein the Tn5 NEO gene is under the control of an inserted, upstream, pseudorabies virus glycoprotein X promoter, designated S-IBR-004. S-IBR-004 was deposited on May 23, 1986 pursuant to the Budapest Treaty on the International Deposit of Microorganismε 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 2134. The subject invention further provides a recombinant IBR viruε comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Escherichia coli β-galactosidaεe and Tn5 NEO genes, and the parainfluenza type 3 virus hemagglutinin gene, HN, has been inserted into the viral DNA. The subject invention provides an example of such a recombinant virus, designated S-IBR-018. The subject invention further provides a recombinant IBR viruε compriεing viral DNA from a naturally-occurring IBR viruε in which a foreign DNA sequence which encodeε the Escherichia coli β-galactosidase and Tn5 NEO genes, and the parainfluenza type 3 viruε fuεion gene, F, haε been inserted into the viral DNA. The subject invention provideε an example of εuch a recombinant virus, designated S-IBR-019. The recombinant viruses of the subject invention were derived from the Cooper Strain. However, other IBR viruseε, such as the LA strain or the 3156 strain, may also be used. The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of any of the recombinant viruses of the present invention. The vaccine may contain either inactivated or live recombinant virus. Suitable carriers for the recombinant virus are well known in the art and include proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as hydrolyzed proteins, lactose, etc. Preferably, the live vaccine is created by taking tissue culture fluids and adding stabilizing agents such as εtabilized, hydrolyzed proteinε. Preferably, the inactivated vaccine uεeε tiεεue culture fluidε directly after inactivation of the virus. The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein. The subject invention provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus compriεing viral DNA from a naturally-occurring IBR viruε in which DNA encoding gpG glycoprotein has been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR viruε produces no gpG glycoprotein and no gpE glycoprotein. The subject invention also provides a vaccine which compriseε a εuitable carrier and an effective immunizing -aaount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein, DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and DNA encoding the gpE glycoprotein has been altered or deleted. The subject invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally- occurring IBR virus haε been deleted, and (2) DNA encoding gpG glycoprotein has been altered or deleted. The subject invention alεo provideε a vaccine which compriεes a suitable carrier and an effective immunizing amount of a recombinant IBR viruε compriεing viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produceε no gpE glycoprotein. The εubject invention provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR viruε in which DNA encoding gpE glycoprotein haε been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein and DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted. The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA corresponding to the US2 region of the naturally- occurring IBR virus has been deleted. The subject invention provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which at least a portion of both repeat sequences has been deleted. The subject invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the viral DNA. The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodeε the Eεcherichia coli β- galactosidase and Tn5 NEO genes, and the parainfluenza type 3 virus hemagglutinin gene, HN, haε been inserted into the viral DNA. The subject invention also provides a vaccine which compriseε a εuitable carrier and an effective immunizing amount of a recombinant IBR viruε comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Escherichia coli β- galactbsidase and Tn5 NEO genes, and the parainfluenza type 3 virus fusion gene, F, has been inserted into the viral DNA. All of the vaccines described hereinabove and hereinbelow may contain either inactivated or live recombinant viruε. The vaccineε may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneouε, intraperitoneal, or intravenouε injection. Alternatively, the vaccine may be administered intranasally or orally. The present invention also provides a method of immunizing an animal against infectious bovine rhinotracheitis viruε which comprises administering to the animal an effective immunizing dose of any of the vaccines of the present invention. The animal may be a bovine. The subject invention also provides a method for distinguishing an animal vaccinated with a vaccine which comprises an effective immunizing amount of a recombinant virus of the present invention from an animal infected with a naturally-occurring IBR virus which compriεeε analyzing a sample of a body fluid from the animal for the presence of gpG glycoprotein of IBR virus and at least one other antigen normally expressed in an animal infected by a naturally-occurring IBR virus, identifying antigens which are present in the body fluid, and determining whether gpG glycoprotein is present in the body fluid. The presence of antigens which are normally expressed in an animal by a naturally-occurring IBR virus and the absence of gpG glycoprotein in the body fluid is indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus. The presence of antigenε and gpG glycoprotein in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigens and gpG glycoprotein. One of the vaccines that is useful in this method is a vaccine which compriseε a suitable carrier and an effective immunizing amount of a recombinant virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein. Another vaccine that is useful in this method is a vaccine which compriseε a suitable carrier and an effective immunizing amount of a recombinant IBR viruε compriεing viral DNA from a naturally-occurring IBR viruε in which DNA encoding gpG glycoprotein haε been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein and no gpE glycoprotein. Yet another vaccine that is useful in this method is a vaccine which compriseε a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR viruε in which DNA encoding gpG glycoprotein haε been altered or deleted εo that upon replication the recombinant IBR virus produces no gpG glycoprotein, DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and DNA encoding the gpE glycoprotein haε been altered or deleted. Still another vaccine that iε useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus haε been deleted, and (2) DNA encoding gpG glycoprotein haε been altered or deleted. The present invention also provides a method for distinguiεhing an animal vaccinated with a vaccine which comprises an effective immunizing amount of a recombinant virus of the present invention from an animal infected with a naturally-occurring IBR virus which compriseε analyzing a εample of a body fluid from the animal for the preεence of gpE glycoprotein of IBR viruε and at least one other antigen normally expressed in an animal infected by a naturally-occurring IBR virus, identifying antigens which are present in the body fluid and determining whether gpE glycoprotein is present in the body fluid. The presence of antigens which are normally expressed in an animal by a naturally-occurring IBR virus and the absence of gpE glycoprotein in the body fluid is indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus. The presence of antigens and gpE glycoprotein in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigens and gpE glycoprotein. One of the vaccines useful in this method iε a vaccine which compriseε a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein and no gpE glycoprotein. Another vaccine that is useful in this method is a vaccine which compriseε a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted εo that upon replication the recombinant IBR viruε produceε no gpG glycoprotein, DNA correεponding to the US2 region of the naturally- occurring IBR viruε haε been deleted, and DNA encoding the gpE glycoprotein haε been altered or deleted. Yet another vaccine that iε useful in this method iε a vaccine which compriseε a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein haε been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein. Still another vaccine that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein and DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted. The present invention also provides isolated DNA encoding the gpG glycoprotein of IBR virus. The subject invention also provideε purified recombinant gpG glycoprotein encoded by the DNA encoding the gpG glycoprotein of IBR virus. The subject invention further provides a recombinant cloning vector which comprises the DNA encoding the gpG glycoprotein of IBR virus. The subject invention also provides a recombinant expression vector which comprises the DNA encoding the gpG glycoprotein of IBR virus. The subject invention provides a host cell which comprises the recombinant expression vector which comprises the DNA encoding the gpG glycoprotein of IBR virus. The subject invention also provides a method of producing a polypeptide which compriseε growing the host cell which compriεeε the recombinant expreεεion vector which comprises the DNA encoding the gpG glycoprotein of IBR viruε under conditions such that the recombinant expression vector expresses gpG glycoprotein and recovering the gpG glycoprotein so expressed. The subject invention also provides an antibody directed to an epitope of the purified gpG glycoprotein of IBR virus encoded by the DNA encoding the gpG glycoprotein of IBR virus. The antibody may be a monoclonal antibody. The εubject invention also provideε a method of detecting the presence or absence of gpG glycoprotein of IBR virus in a sample which comprises contacting the sample with an antibody directed to an epitope of the purified gpG glycoprotein of IBR virus encoded by the DNA encoding the gpG glycoprotein of IBR virus under conditions such that the antibody forms a complex with any gpG glycoprotein present in the seunple and detecting the presence or absence of such complex. The sample may be bovine- derived. The subject invention also provides isolated DNA encoding the gpE glycoprotein of IBR virus. The subject invention also provideε purified recombinant gpE glycoprotein encoded by the DNA encoding the gpE glycoprotein of IBR viruε. The εubject invention further provideε a recombinant cloning vector which comprises the DNA encoding the gpE glycoprotein of IBR virus. The subject invention provides a recombinant expression vector which compriseε the DNA encoding the gpE glycoprotein of IBR viruε. The subject invention also provideε a host cell which comprises the recombinant expression vector which compriseε the DNA encoding the gpE glycoprotein of IBR viruε. The εubject invention also provides a method of producing a polypeptide which comprises growing the host cell which compriεeε the recombinant expression vector which compriseε the DNA encoding the gpE glycoprotein of IBR viruε under conditionε such that the recombinant expression vector expresseε gpE glycoprotein and recovering the gpE glycoprotein so expressed. The subject invention also provides an antibody directed to an epitope of the purified gpE glycoprotein of IBR virus encoded by the DNA encoding the gpE glycoprotein of IBR viruε. The antibody may be a monoclonal antibody. The subject invention also provides a method of detecting the presence or absence of gpE glycoprotein of IBR virus in a sample which compriseε contacting the εample with an antibody directed to an epitope of the purified gpE glycoprotein of IBR virus encoded by the DNA encoding the gpE glycoprotein of IBR virus under conditions such that the antibody forms a complex with any gpE glycoprotein present in the seunple and detecting the presence or absence of such complex. The sample may be bovine- derived. The subject invention also provides a method of producing a fetal-safe, live recombinant IBR virus which compriseε treating viral DNA from a naturally-occurring live IBR virus so as to delete from the viruε DNA correεponding to the US2 region of the naturally-occurring IBR viruε. The subject invention also provides a recombinant pseudorabies virus designated S-PRV-160. The subject invention also provides an antibody which directed to an epitope of the recombinant pseudorabies virus designated S-PRV-160. The subject invention alεo provides isolated DNA encoding the US2 gene of an IBR viruε. The preεent invention further provideε a homology vector for producing a recombinant IBR viruε by inεerting foreign DNA into the genomic DNA of an IBR viruε which compriεes a double- stranded DNA molecule consisting essentially of double- stranded foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant IBR is introduced, with at one end of the foreign DNA, double-stranded IBR viral DNA homologous to genomic DNA located at one side of a site on the genomic DNA which is not eεεential for replication of the IBR virus and at the other end of the foreign DNA, double-stranded IBR viral DNA homologous to genomic DNA located at the other side of the same site on the genomic DNA. The double-stranded foreign DNA may further comprise a promoter. The promoter can be from HSV-l o 4 immediate early gene. Human cytomegalovirus immediate early gene or pεeudorabies virus glycoprotein X gene. The double- stranded foreign DNA may further comprise a polyadenylation signal. The polyadenylation signal may be from HSV-l thymidine kinase gene or pseudorabies virus glycoprotein X gene. The subject invention also provides a homology vector wherein the RNA encodes a polypeptide. The polypeptide may be a detectable marker such as Escherichia coli β-galactosidaεe or bacterial transposon neomycin reεistance protein. The DNA which encodes the polypeptide may be flanked on each side by restriction siteε permitting said DNA to be cut out with a restriction endonuclease which cuts at a limited number of εiteε on the genome. The subject invention further provides for a homology vector wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 860 bp Ncol to BamHI subfragment of the Hindlll A fragment of IBR virus and the downstream double-stranded IBR viral DNA iε homologous to genomic DNA present within the approximately 1741 bp Bglll to StuI subfragment of the Hindlll A fragment of IBR virus. The εubject invention further provideε a homology vector wherein upstream double-stranded foreign DNA which comprises a promoter and downstream double-stranded foreign DNA which comprises a polyadenylation signal flank on each side double-stranded foreign DNA which encodes a detectable marker. The invention further a homology vector wherein the upstream promoter is homologous to genomic DNA present within the approximately 490 bp Pv ~ .II to BamHI subfragment of the BamHI N fragment of HSV-l and the downstream polyadenylation signal is homologous to genomic DNA present within the approximately 784 bp Smal to Smal subfragment of the BamHI Q fragment of HSV-l. The invention further provides a homology vector wherein the DNA which encodes a detectable marker is homologous to the approximately 1541 bp Bglll to BamHI fragment of Tn5. The subject invention also provides a homology vector wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 3593 bp Hindlll to Xhol subfragment of the Hindlll K fragment of IBR virus and the downstreεun double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 785 bp Xhol to Ndel subfragment of the Hindlll K fragment of IBR virus. The invention further provideε a homology vector wherein upstream double-stranded foreign DNA which comprises a promoter and downstream double-stranded foreign DNA which comprises a polyadenylation signal flank on each side double-stranded foreign DNA which encodeε a detectable marker. This upstream promoter is homologous to genomic DNA present within the approximately 1191 bp Avail to PstI subfragment of the XJbal B fragment of HCMV and the downstream polyadenylation sequence is homologous to genomic DNA present within the approximately 753 bp Sail to Ndel subfragment of the BamHI #7 fragment of PRV. The DNA which encodes a detectable marker is homologous to the approximately 3347 bp Ball to BamHI fragment of pJF751. The invention further provides a homology vector wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 888 bp Aflul to Smal subfragment of the Hindlll K fragment of IBR virus and the downεtream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 785 bp Xhol to Ndel subfragment of the Hindlll K fragment of IBR virus. The upstream double- stranded foreign DNA may comprise a promoter and double- stranded foreign DNA which comprise a polyadenylation signal flank on each side double-stranded foreign DNA which encodes a detectable marker. The subject invention also provideε a homology vector wherein the upstream promoter is homologous to genomic DNA present within the approximately 1191 bp Avail to PstI subfragment of the XJbal B fragment of HCMV and the downstreεun polyadenylation signal iε homologous to genomic DNA preεent within the approximately 753 bp Sail to Ndel εubfragment of the BamHI ≠l fragment of PRV. The DNA which encodeε a detectable marker iε homologous to the approximately 3347 bp Ball to BamHI fragment of pJF571. The present invention further provides a homology vector wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 1704 bp Smal to Smal subfragment of the Hindlll K fragment of IBR virus and the downstreεun double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 742 bp Nhel to Bgll subfragment of the Smal 2.5KB fragment of IBR virus. The present invention further provides a homology vector wherein upstream double-stranded foreign DNA which comprises a promoter and downstream double-stranded foreign DNA which comprises a polyadenylation signal flank on each side double-stranded foreign DNA which encodes a detectable marker. The upstream promoter iε homologouε to genomic DNA preεent within the approximately 413 bp Sail to BamHI subfragment of the BamHI #10 fragment of PRV and the downstreεun polyadenylation signal is homologous to genomic DNA present within the approximately 754 bp Ndel to Sail subfragment of the BamHI #7 fragment of PRV. The detectable marker is homologous to the approximately 3010 bp BamHI to PvuII ' fragment of pJF751. The present invention provides for a homology vector for producing a recombinant IBR virus by deleting DNA which encodes a detectable marker which had been inserted into the genomic DNA of an IBR virus comprising a double- stranded DNA molecule consisting essentially of double- stranded IBR viral DNA homologous to the genomic DNA which flank on each side the DNA to be deleted. The subject invention further provides a homology vector wherein the upstream double-stranded IBR viral DNA iε homologouε to genomic DNA present within the approximately 888 bp Mlul to Smal subfragment of the Hindlll K fragment of IBR virus and the downstreεun double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 785 bp Xhol to Ndel subfragment of the Hindlll K fragment of IBR virus. The present invention also provides a method of immunizing an animal against infectious bovine rhinotracheitis virus which comprises administering to the animal an effective immunizing dose of any of the vaccines of the present invention. The animal may be a bovine. The subject invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR viruε from which at least a portion of both repeat sequences have been deleted, εpecifically, wherein DNA encoding one or more EσoRV reεtriction sites has been deleted, and wherein foreign DNA has been inserted into the DNA of the recombinant virus. The foreign DNA may be a DNA sequence which encodes bovine viral diarrhea virus glycoprotein gp53. The subject invention provides an example of such a recombinant IBR virus, designated S-IBR-032. The subject invention provides a recombinant IBR virus compriεing viral DNA from a naturally-occurring IBR viruε from which DNA from the US2 gene, the gpE glycoprotein gene εud the gpG glycoprotein gene have been deleted so that upon replication, the recombinant IBR virus produces no gpE glycoprotein and no gpG glycoprotein. A Foreign DNA sequence may be inserted in place of the deleted DNA which encodes gpE glycoprotein. The foreign DNA sequence that may be inserted can be a foreign DNA sequence which encodes Escherichia coli β-galactosidase. The subject invention provides εui example of such a recombinant virus, designated S-IBR-039. The subject invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA from the US2, gpE glycoprotein gene, the gpG glycoprotein gene and the thymidine kinase gene has been deleted so that upon replication, the recombinant IBR virus produces no gpE glycoprotein, no gpG glycoprotein and no thymidine kinase. The subject invention provides an example of such a recombinant virus, designated S-IBR-045. A foreign DNA sequence may be inserted in place of the deleted DNA encoding gpE glycoprotein. The foreign DNA sequence may encode Escherichia coli β-galactosidaεe. The subject invention provides an example of such a recombinant virus, designated S-IBR-044. The foreign DNA sequence may encode bovine viral diarrhea virus gp53 glycoprotein. The subject invention provides an example of such a recombinant virus, designated S-IBR-046. The foreign DNA sequence may encode Parainfluenza virus type 3 fusion protein and Parainfluenza virus type 3 hemagglutinin protein. The subject application provides an example of such a viruε, designated S-IBR-047. The foreign DNA sequence may encode Bovine respiratory syncytial virus fusion protein, Bovine respiratory syncytial virus attachment protein and Bovine respiratory syncytial virus nucleocapsid protein. The subject invention provides an example of such a recombinant virus, designated S-IBR- 049. The foreign DNA sequence may encode Pasteurella haemolvtica leukotoxin and Pasteurella haemolvtica iron regulated outer membrane proteins. The subject invention provides an example of such a recombinant virus, designated S-IBR-051. The subject invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA from the US2 gene, the gpE glycoprotein gene, the gpG glycoprotein gene and the thymidine kinase gene have been deleted so that upon replication, the recombinant IBR virus produces no gpE glycoprotein, no gpG glycoprotein and no thymidine kinase. The subject invention provides for a foreign DNA sequence inserted in place of the DNA which encodes thymidine kinase. The foreign DNA sequence may encode Escherichia coli β-glucuronidase. The present invention further provides a recombinant virus wherein a foreign DNA sequence is inserted in place of the DNA encoding gpE glycoprotein. The foreign DNA sequence may encode Escherichia coli β-galactosidase. The present invention further provides an example of such a recombinant virus, designated S-IBR-043. The subject invention also provides a vaccine which comprises an effective immunizing amount of any of the recombinant viruseε of the preεent invention and a εuitable carrier. The vaccine may contain either inactivated or live recombinant virus. The present invention provideε a vaccine which compriεeε an effective immunizing amount of recombinant viruε protective against bovine respiratory disease complex and a suitable carrier. A recombinant virus may be a recombinant IBR virus and the recombinant virus can consist esεentially of any or all of the recombinant viruses of the present invention. The εubject invention alεo provideε for a vaccine which comprises an effective immunizing amount of a recombinant virus and non-recombinant virus protective against bovine respiratory diseaεe complex and a suitable carrier. The subject invention further provides a vaccine which compriseε an effective immunizing amount of a recombinant IBR virus and non-recombinant virus protective against bovine respiratory diseaεe complex and a suitable carrier. The recombinant IBR viruε can conεist esεentially of any or all of the recombinant viruses of the subject invention. For purposes of this invention, the infectious diseases that contribute to bovine respiratory disease complex include infectious bovine rhinotracheitis, parainfluenza type 3 virus, bovine viral diarrhea virus, bovine respiratory syncytial virus and Pasteurella haemolvtica. For purposeε of the preεent invention, non-recombinant viruεeε can include, but are not limited to, conventionally derived viruεeε which include killed viruε, inactivated bacterinε, and modified live viruseε. The εubject invention further provides for a method of immunizing an animal against infectious bovine rhinotracheitiε which compriseε adminiεtering to the animal an immunizing doεe of any of the vaccineε of the present invention. The subject invention further provides a method of immunizing an animal against Parainfluenza type 3 which comprises administering to the animal an immunizing dose of the vaccine of the present invention that contains the IBR virus encoding antigens for Parainfluenza type 3 viruε. The subject invention further provides a method of immunizing an animal against bovine viral diarrhea which comprises administering to the animal an immunizing dose of the vaccine of the present invention that contains the IBR virus encoding antigenε for bovine viral diarrhea viruε. The εubject invention further provideε a method of immunizing an animal against bovine respiratory syncytial virus disease which comprises administering to the animal an immunizing dose of the vaccine of the present invention that contains the IBR viruε encoding antigenε for bovine respiratory syncytial virus. The subject invention further provides for a method of immunizing an animal against Pneumonic pasteurellosis which compriεeε administering to the animal an immunizing dose of the vaccine of the present invention that contains the IBR virus encoding antigenε for Pasteurella haemolvtica. The invention further provides a method of immunizing an animal against bovine respiratory disease complex which comprises administering to an animal an immunizing dose of the vaccine containing the recombinant IBR viruses of the present invention or the recombinant viruseε of the present invention and non-recombinant viruses. For purposes of this invention, the animal may be a bovine. The invention further provides a method for distinguishing an animal vaccinated with a vaccine which comprises an effective immunizing amount of a recombinant virus of the present invention from an animal infected with a naturally-occurring IBR virus which compriseε analyzing a sample of a body fluid from the animal for the presence of gpE glycoprotein of IBR virus and at least one other antigen normally expressed in an animal infected by a naturally-occurring IBR viruε, identifying antigenε which are preεent in the body fluid and determining whether gpE glycoprotein is present in the body fluid, the presence of antigens which are normally expresεed in an animal by a naturally-occurring IBR viruε and the absence of gpE glycoprotein in the body fluid being indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus. Materialε and Methods PREPARATION OF IBR VIRUS STOCK SAMPLES. IBR virus stock sampleε were prepared by infecting MDBK 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 an equivalent εupplier, and hereafter are referred to aε complete DME medium) pluε 1% fetal bovine serum. After cytopathic effect was complete, the medium and cells were harveεted and the cells were pelleted at 3000 rpm for 5 minutes in a clinical centrifuge. Cells were reεuεpended in 1/10 the original volume of medium, and an equal volume of skim milk (9% skim milk powder in H 2 0 weight/volume) was added. The virus sample was frozen at -70°C. The titers were usually about 10 8 PFU/ l. PREPARATION OF HERPESVIRUS DNA. For herpesvirus DNA preparation, a confluent monolayer of cellε (MDBK for IBR viruε or Vero for PRV) in a 25 cm 2 flaεk or 60 mm petri dish was infected with 100 μl of virus sample. After overnight incubation, or when the cells were showing 100% cytopathic effect, the cells were scraped into the medium. The cellε and medium were centrifuged at 3000 rpm for 5 minuteε in a clinical centrifuge. The medium was decanted, and the cell pellet was gently resuspended in 0.5 ml of solution containing 0.5% NONIDET P-40 1 " (NP- 40, purchased from Sigma Chemical Co., St. Louis, MO). The sample was incubated at room temperature for 10 minutes. Ten μl of a stock solution of RNase A (Sigma) was added (εtock was 10 mg/ml, boiled for 10 minuteε 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 μl of 20% sodium dodecyl sulfate (Sigma) and 25 μl proteinase-K (10 mg/ml; Boehringer Mannheim) . The sεunple was mixed and incubated at 37°C for 30-60 minutes. An equal volume of water-saturated phenol waε added and the sample waε 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 β C for 30 minutes to precipitate nucleic acid. The sample was centrifuged in an Eppendorf minifuge for 5 minuteε. The supernatant was decanted, and the pellet was washed with -300 μl of 80% ethanol, followed by centrifugation in an Eppendorf minifuge for 5 minutes. The supernatant was decanted, and the pellet was air dried and rehydrated in -16 μl H-O. For the preparation of larger amounts of DNA, the procedure was scaled up to start with a 850 cm 2 roller bottle of MDBK cells. The DNA was stored in 0.01 M tris pH 7.5, 1 mM EDTA at 4°C. PREPARATION OF HERPESVIRUS CELL LYSATES. For cell lysate preparation, serum free medium waε used. A confluent monolayer of cells (MDBK for IBR viruε or Vero for PRV) in a 25 cm 2 flaεk or a 60 mm petri dish was infected with 100 μl of virus sample. After cytopathic effect waε complete, the medium and cellε were harvested and the cells were pelleted at 3000 rpm for 5 minutes in a clinical centrifuge. For media sampleε medium waε concentrated approximately 10-fold by filtration with a centricon-10 microconcentrator (Amicon) . For cell samples the cell pellet waε reεuεpended in 250 μl of disruption buffer (2% sodium dodecyl sulfate, 2% β- mercaptoethanol) . The sampleε were sonicated for 30 secondε on ice and εtored at -20 e C. WESTERN BLOTTING PROCEDURE. Samples of lysateε, controls and protein standardε were run on a polyacrylamide gel according to the procedure of Laemmli (2) . After gel electrophoresis the proteins were transferred according to Sambrook (14) . The primary antibody waε a mouεe hyper-immune serum raised against chemically-synthesized gpG peptides (amino acids 232-252 and 267-287) linked to keyhole limpet hemocyanin. The εecondary antibody waε a goat anti-mouse alkaline phosphatase coupled antibody. MOLECULAR BIOLOGICAL TECHNIQUES. Techniques for the manipulation of bacteria and DNA, including such procedures as digeεtion with restriction endonucleaseε, gel electrophoresis, extraction of DNA from gelε, ligation, phosphorylation with kinase, treatment with phosphataεe, growth of bacterial cultureε, transformation of bacteria with DNA, and other molecular biological methods are described by Maniatis (6) . Except as noted, these were used with minor variation. LIGATION. DNA was joined together by the action of the enzyme T4 DNA ligase (BRL) . Ligation reactionε contained variouε amountε of DNA (from 0.2 to 20μg) , 20mM Tris pH 7.5, lOmM MgCl 2 , lOmM dithiothreitol (DTT) , 200 μM ATP and 20 units T4 DNA ligase in 10-20 μl final reaction volume. The ligation proceeded for 3-16 hours at 15°C. DNA SEQUENCING. Sequencing waε performed using the BRL Sequenase Kit and 35 S-dATP (NEN) . Reactions using both the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, compressed areas were resolved on formamide gels. Templates were double-εtranded plaεmid subcloneε or single stranded M13 subcloneε, and primerε were either made to the vector juεt outside the insert to be sequenced, or to previously obtained sequence. Sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with Superclone and Supersee programs from Coral Software. SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis (6) . 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 Laboratorieε (BRL) . In both caεeε the manufacturerε' recommended procedureε were followed. DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS. The method iε baεed upon the calcium phosphate procedure of Graham and Van der Eb (24) with the following modifications. Viruε and/or plaεmid DNA were diluted to 298 μl in 0.01 M Tris pH 7.5, ImM EDTA. Forty μl 2M CaCl- was added followed by an equal volume of 2X HEPES buffered saline (lOg N-2-hydroxyethyl piperazine N'-2- ethaneεulfonic acid (HEPES), 16g NaCl, 0.74g KC1, 0.25g Na 2 HP0 4 * 2H 2 0, 2g dextroεe per liter H 2 0 and buffered with NaOH to pH 7.4). The mixture waε then incubated on ice for 10 minuteε, and then added dropwise to an 80% confluent monolayer of MDBK or rabbit skin (RS) cellε growing in a 60 mm petri diεh under 5 ml of medium (DME pluε 2% fetal bovine serum) . The cells were incubated 4 hours at 37°C in a humidified incubator containing 5% CO.. The cells were then washed with three 5 ml aliquots of 1XPBS (1.15g Na 2 HP0 4 , 0.2g KH 2 P0 4 , 0.8g NaCl, 0.2gKClper liter H 2 0) , and fed with 5 ml of medium (DME plus 2% fetal bovine serum) . The cellε 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 εtock was referred to as a tranεfeetion εtock and waε subsequently screened for recombinant virus by the BLUOGAL" SCREEN FOR RECOMBINANT IBR VIRUS. HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. This method relies upon the homologous recombination between herpesvirus DNA and plasmid homology vector DNA which occurs in tisεue culture cells co-tranεfected with these elements. From 0.1-1.0 μg of plasmid DNA containing foreign DNA flanked by appropriate herpeεviruε cloned sequences (the homology vector) were mixed with approximately 0.3 μg of intact herpesvirus DNA. The DNAs were diluted to 298 μl in 0.01 M Triε pH 7.5, ImM EDTA and transfected into MDBK 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 herpeεviruε DNA εequenceε 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 was that the restriction enzyme used to cut the herpesvirus DNA must cut at a limited number of sites. We have used XJbal, which cuts IBR virus DNA in one place. We have also used EcoRV which cuts IBR virus DNA in two places. For PRV we have used XJbal and Hindlll, both of which cut in two placeε. Restriction sites previously introduced into herpesviruses by other methods may also be used. The herpesvirus DNA was mixed with a 30-fold molar excess of plasmid DNA (typically 5μg of virus DNA to lOμg of plasmid DNA) , and the mixture was cut with the appropriate restriction enzyme. The DNA mixture was phenol extracted and ethanol precipitated to remove restriction enzymes, and ligated together according to the ligation procedure detailed above. The ligated DNA mixture waε then reεuεpended in 298 μl 0.01 M Tris pH 7.5, ImM EDTA and transfected into cells (MDBK or RS for IBR viruε and Vero for PRV) according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS (εee above) . The direct ligation procedure may also be used to delete DNA from herpesviruseε. Non-esεential DNA which is flanked by appropriate restriction enzyme siteε may be deleted by digeεting the virus DNA with such enzymes and religation. The frequency of engineered viruses generated by the direct ligation procedure is high enough that screening can be accomplished by restriction enzyme analysiε of randomly picked plaques from the tranεfection εtock. BLUOGAL" SCREEN FOR RECOMBINANT HERPESVIRUS. When the E. coli β-galactoεidase (lacZ) marker gene was incorporated into a recombinant virus the plaques containing recombinants were visualized by a simple assay. The chemical BLUOGAL * (GIBCO-Betheεda Research Labs) was incorporated (200 μg/ml) into the agarose overlay during the plaque assay, and plaques that expressed active β-galactosidase turned blue. The blue plaques were then picked onto fresh cells (MDBK for IBR virus and Vero for PRV) and purified by further blue plaque isolationε. In recombinant virus strategies in which the E. coli β-galactosidase marker gene iε removed, the aεsay involves plaque purifying white plaques from a background of parental blue plaques. In both caseε viruses were typically purified with three rounds of plaque purification. SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. When the E. coli β-galactosidase (lacZ) or β-glucuronidase (uidA) marker gene was incorporated into a recombinant virus the plaques containing recombinantε were visualized by a simple aεsay. The enzymatic εubεtrate was incorporated (300 μg/ml) into the agarose overlay during the plaque assay. For the lacZ marker gene the substrate BLUOGAL * (halogenated indolyl-β-D- galactoεidaεe, Bethesda Research Labs) was used. For the uidA marker gene the substrate X-Glucuro Chx (5-bromo-4- chloro-3-indolyl-β-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 cells and purified by further blue plaque isolation. In recombinant virus strategies in which the enzymatic marker gene is removed the assay involves plaque purifying white plaques from a background of parental blue plaques. In both cases viruses were typically purified with three rounds of plaque purification. ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS. A third method for screening the recombinant virus stock was to look directly for the expression of the foreign gene with antibodies. Herpesvirus plaques were spotted and picked by inserting a toothpick through the agarose above the plaque and scraping the plaque area on the dish. Viruses were then rinsed from the toothpick by inserting the toothpick into a well of a 96-well micro-titer dish (Falcon Plasticε) containing a confluent monolayer of tisεue culture cellε that had been washed 3 times in DME medium without serum. It was important for the virus to grow without serum at this stage to allow the immunological procedure to work. After cytopathic effect waε complete, the plateε were put at -70°C to freeze and lyse the cells. The medium was thawed, and the freeze/thaw procedure was repeated a second time. Then 50-100 microliters of medium were removed from each well and filtered under vacuum through a nitrocelluloεe membrane (S&S BA85) using a DotBlot- apparatuε (BRL) . The filter blotε were soaked in a blocking solution of 0.01 M Tris pH 7.5, 0.1 M NaCl, 3% bovine εerum albumin at room temperature for two hours with shetking. The filter blotε were then placed in a εealable bag (Sears SEAL-A-MEAL " or equivalent) , and 10 mis of the blocking solution that contained 10 microliters of antibody specific for the foreign protein were added. After overnight incubation at room temperature with shaking, the blot waε waεhed 3 timeε with 100 mlε 0.01 M Triε, pH 7.5, 0.1 M NaCl, 0.05% Tween 20 detergent (Sigma). The blot was put in another sealable bag and 10 mlε blocking εolution containing 10 6 counts per minute of 1 5 I-protein A (New England Nuclear) were added. After allowing the protein A to bind to the antibody for 2 hours at room temperature with shaking, the blot was washed as above, dried, and overlayed with an X-ray film and an intensifying screen (Dupont) and autoradiographed for 1-3 days at -70°C. The film waε developed by εtandard procedureε. Viruε from the positive wells which contained the recombinant virus waε further purified. SELECTION OF G418 RESISTANT IBR VIRUS. The antibiotic G418 (GIBCO) haε a wide range of inhibitory activity on protein synthesiε. However, recombinant viruses expressing the aminoglycoεidaεe 3'-phosphotranεferase, encoded by the NEO gene of the tranεposable element Tn5, are resistant to G418. The transfection stocks of recombinant viruseε were grown on MDBK cellε in the preεence of 500 μg/ml G418 in complete DME medium pluε 1% fetal bovine εerum. After one or two days at 37 β C, plaques from the disheε inoculated with the highest dilution of virus were picked for viruε stocks. The selection was repeated a second or third time. The virus stockε generated from the G418 selection were tested for NEO gene insertion by the SOUTHERN BLOTTING OF DNA hybridization procedure described above. CONSTRUCTION OF DELETION VIRUSES. The strategy used to conεtruct deletion viruεeε involved the uεe of either homologouε recombination and/or direct ligation techniques. Initially a virus waε conεtructed via homologouε recombination, in which the DNA to be deleted waε replaced with a marker gene such as E. coli β- galactosidase (lacZ) or β-glucuronidaεe (uidA) . A εecond viruε waε then conεtructed in which the marker gene waε 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 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. Several homology vectors were constructed for the purpose of deleting the gpG, gpE and Tk gene coding regions. A detailed description of these homology vectors follows. HOMOLOGY VECTOR 129-71.5. The plasmid 129-71.5 was constructed for the purpose of deleting a portion of the TK gene coding region from the IBR virus. It incorporates a selectable marker, the bacterial transposon neomycin resistance gene, flanked by IBR virus DNA. Upstream of the marker gene is an approximately 860 base pair fragment of IBR virus DNA which ends with εequenceε encoding amino acidε 1-62 of the TK primary translation product. Downεtreεun of the marker gene is an approximately 1741 base pair fragment of IBR virus DNA which begins with sequences encoding amino acids 156-367 of the TK primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding for amino acids 63-155 of the TK primary translation product with DNA coding for the marker gene. Note that the marker gene will be under the control of the herpes simplex type 1 alpha-4 immediate early gene promoter (5) . A detailed description of the plasmid is given in Figure 7. It waε conεtructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6) . It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequenceε indicated in Figure 7. The plaεmid vector iε derived from an approximately 2975 baεe pair Smal to Hindlll restriction fragment of pSP65 (Promega) . Fragment 1 is an approximately 860 base pair Ncol to BamHI restriction fragment of the IBR viruε Hindlll restriction fragment A (7) . This fragment iε located on an approximately 5500 baεe pair Clal to Nrul fragment contained in the IBR virus Hindlll A fragment. Fragment 2 is an approximately 490 base pair Pvαill to BamHI restriction sub-fragment of the HSV-l BamHI restriction fragment N (5) . Note that the HSV-l oriS region has been removed from thiε fragment by deletion of the εequenceε between the Smal siteε located 1483 and 128 base pairs away from the PvuII end (10) . Fragment 3 iε an approximately 1541 baεe pair Bglll to BamHI restriction fragment of plasmid pNEO (P.L. Biochemicals, Inc.) . Fragment 4 iε an approximately 784 baεe pair Smal to Smal restriction sub-fragment of the HSV-l BamHI restriction fragment Q (10) . Note that this fragment iε oriented εuch that the polyadenylation sequence (AATAAA) iε located closest to junction D. Fragment 5 is an approximately 1741 base pair Bglll to StuI restriction sub-fragment from the IBR Hindlll restriction fragment A (7). PLASMID 459-12.6. The plasmid 459-12.6 waε generated for the purpoεe of conεtructing a recombinant cloning vector which expreεεeε the IBR viruε glycoprotein G. Thiε waε accomplished by inserting the IBR virus gpG gene into S- PRV-013 (U.S. Serial No. 07/823,102 filed January 27, 1986). Plasmid 459-12.6 contains a chimeric gene under the control of the IBR viruε gpG promoter. The chimeric gene expresses a fusion protein consisting of the first 362 amino acids of IBR virus gpG fused to amino acidε 421-467 of the PRV gpIII (13) followed by amino acidε 480-498 of the PRV gpX (12) . The chimeric gene iε flanked by Hindlll restriction siteε. When this plasmid is uεed with S-PRV-013 and the reεtriction enzyme Hindlll according to the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS the resulting recombinant will express the IBR virus gpG. A detailed description of the plasmid is given in Figure 11. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6) . It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequenceε indicated in Figure 11. The plasmid vector is derived from an approximately 2999 base pair XJbal to XJbal restriction fragment of a hybrid cloning vector derived from pSP64 and pSP65 (Promega) . The hybrid cloning vector was constructed by joining approximately 1369 base pair Pvul to Smal fragment from pSP64 with the approximately 1652 base pair Pvul to Smal fragment from pSP65. Fragment 1 is an approximately 182 base pair PstI to EcoRV reεtriction εub-frag ent of the HCMV XJbal reεtriction fragment B (16) . Fragment 2 iε an approximately 2121 base pair Mlul to Xhol restriction sub-fragment of the IBR virus Hindlll restriction fragment K (7) . Fragment 3 is an approximately 121 base pair Xhol to BamHI restriction sub-fragment of the PRV BamHI restriction fragment #2 (3) . Fragment 4 is an approximately 760 base pair Ndel to Sail restriction sub¬ fragment of the PRV BamHI restriction fragment #7 (3) . HOMOLOGY VECTOR 439-01.31. The plaεmid 439-01.31 waε conεtructed for the purpose of deleting a portion of the gpG gene coding region from the IBR virus. It incorporates an E. coli β-galactosidase marker gene flanked by IBR viruε DNA. Downstreεun of the marker gene is an approximately 3593 base pair fragment of IBR viruε DNA which ends with sequences encoding the first 262 amino acids of the gpG 'primary translation product. Upstream of the marker gene is an approximately 785 base pair fragment of IBR virus DNA which begins with εequenceε encoding the laεt 80 amino acidε of the gpG primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS it will replace the DNA coding for εunino acids 263-361 of the gpG primary translation product with DNA coding for the marker gene. Note that the β-galactosidase (lacZ) marker gene will be under the control of the human cytomegalovirus immediate early gene promoter. A detailed description of the plasmid is given in Figure 12. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6) . It may be constructed by joining restriction fragmentε from the following sources with the synthetic DNA sequences indicated in Figure 12. The plasmid vector is derived from an approximately 2965 base pair Hindlll to Smal restriction fragment of pSP64 (Promega) . Fragment 1 is an approximately 3593 base pair Hindlll to Xhol restriction fragment of the IBR Hindlll restriction fragment K (7) . Fragment 2 iε an approximately 753 baεe pair Sail to Ndel restriction fragment of the PRV BamHI restriction fragment #7 (3) . Note that this fragment waε resected with Exonuclease III/Si nuclease digestion such that approximately 57 base pairs were removed from the Ndel end. Fragment 3 is an approximately 3347 baεe pair Ball to BamHI restriction fragment of plasmid pJF751 (38) . Fragment 4 is an approximately 1191 base pair Aval to PstI restriction fragment from the HCMV XJbal restriction fragment E (16) . Fragment 5 is an approximately 785 base pair Xhol to Ndel restriction fragment from the IBR Hindlll restriction fragment K (7) . Note that the lacZ marker gene is flanked by XJbal sites located at Junction B and Junction E in this plasmid permitting the marker gene to be cut out with XJbal. HOMOLOGY VECTOR 439-21.69. The plasmid 439-21.69 was constructed for the purpose of deleting a portion of the gpG gene coding region from the IBR virus. It incorporates an E.coli β-galactosidase (lacZ) marker gene flanked by IBR virus DNA. Downstreεun of the marker gene is an approximately 888 base pair fragment of IBR virus DNA which begins approximately 1042 base pairs upstream of the initiation codon of the gpG gene and ends approximately 154 base pairs upstream of the initiation codon of the gpG gene. Upstream of the marker gene is an approximately 785 base pair fragment of IBR virus DNA which begins with sequences encoding the last 80 amino acids of the gpG primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS it will replace the DNA coding for amino acids 1-361 of the gpG primary translation product with DNA coding for the marker gene. Note that the β-galactosidaεe (lacZ) marker gene will be under the control of the human cytomegalovirus immediate early gene promoter. A detailed description of the plasmid is given in Figure 13. It waε conεtructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6) . It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figure 13. The plasmid vector is derived from an approximately 2965 base pair Hindlll to Smal restriction fragment of pSP64 (Promega) . Fragment 1 iε an approximately 3593 base pair Hindlll to Xhol restriction fragment of the IBR Hindlll reεtriction fragment K (7) . Fragment 2 iε an approximately 753 base pair Sail to Ndel restriction fragment of the PRV BamHI restriction fragment #7 (3) . Note that this fragment was resected with Exonuclease III/SI nuclease digestion such that approximately 57 base pairs were removed from the Ndel end. Fragment 3 is an approximately 3347 base pair Ball to BamHI restriction fragment of plasmid pJF751 (38) . Fragment 4 is an approximately 1191 base pair Aval to PstI restriction fragment from the HCMV XJbal restriction fragment E (16) . Fragment 5 iε an approximately 785 baεe pair Xhol to Ndel restriction fragment from the IBR Hindlll restriction fragment K (7) . Note that the lacZ marker gene is flanked by XJbal siteε located at Junction B and Junction E in thiε plaεmid permitting the marker gene to be cut out with XJbal. HOMOLOGY VECTOR 439-70.4. The plasmid 439-70.4 was constructed for the purpose of deleting the E.coli β- galactoεidaεe (lacZ) marker gene from S-IBR-035 viruε. It incorporateε two regions of IBR viral DNA which flank the marker gene in S-IBR-035. The first region is an approximately 888 base pair fragment of IBR virus DNA which beginε approximately 1042 base pairs upstream of the initiation codon of the gpG gene and ends approximately 154 base pairs upstream of the initiation codon of the gpG gene. The second region is an approximately 785 base pair fragment of IBR virus DNA which begins with sequences encoding the last 80 amino acids of the gpG primary tranεlation product. When thiε plaεmid iε uεed in conjunction with S-IBR-035 DNA according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS it will delete the DNA coding for the E. coli β-galactoεidaεe (lacZ) marker gene. A detailed deεcription of the plaεmid is given in Figure 14. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6) . It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figure 14. The plasmid vector is derived from an approximately 2965 base pair Hindlll to Smal restriction fragment of pSP64 (Promega) . Fragment 1 is an approximately 3593 base pair Hindlll to Xhol restriction fragment of the IBR Hindlll restriction fragment K (7) . Fragment 2 iε an approximately 785 baεe pair Xhol to Ndel reεtriction fragment from the IBR Hindlll reεtriction fragment K (7) . IBR VIRUS gpE PLASMID. A plaεmid may be generated for the purpose of conεtructing a recombinant cloning vector which expreεεeε the IBR viruε glycoprotein E (gpE) . Thiε plaεmid may be uεed to insert the IBR virus gpE gene into S-PRV-002 (U.S. Patent No. 4,877,737). The plasmid will contain the gpE gene flanked by XJbal reεtriction sites. When this plasmid is used with S-PRV-002 and the restriction enzyme XJbal according to the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS the resulting recombinant will express the IBR virus gpE. A detailed description of the plasmid is given in Figure 17. It may be constructed, utilizing standard recombinant DNA techniques (6) , by joining restriction fragments from the following sources. The plasmid vector is derived from an approximately 2999 base pair XJbal to XJbal restriction fragment of a hybrid cloning vector derived from pSP64 and pSP65 (Promega) . The hybrid cloning vector was constructed by joining an approximately 1369 base pair Pvul to Smal fragment from pSP64 with the approximately 1652 base pair Pvul to Smal fragment from pSP65. Fragment 1 is an approximately 3647 base pair Ndel to Hindlll reεtriction sub-fragment of the IBR virus Hindlll restriction fragment K (7) . Fragment 2 is an approximately 832 base pair Hindlll to SacJ restriction sub-fragment of an IBR virus 2400 base pair Smal restriction fragment. This Smal fragment has been cloned into the Smal site of the plasmid pSP64 (Promega) . This plasmid is designated PSY1645. PSY1645 waε deposited on July 16, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposeε 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. 68650. Note that the lacZ marker gene iε flanked by XJbal εiteε located at Junction B and Junction E in thiε plaεmid permitting the marker gene to be cut out with Xbal . HOMOLOGY VECTOR 536-03.5. The plaεmid 536-03.5 was constructed for the purpose of deleting a portion of the gpE gene coding region from the IBR virus. It incorporates an E.coli β-galactoεidase (lacZ) marker gene flanked by IBR virus DNA. Upstream of the marker gene is an approximately 1704 base pair fragment of IBR virus DNA which ends with sequences encoding amino acids 1-76 of the gpE primary translation product. Downstream of the marker gene is an approximately 742 base pair fragment of IBR virus DNA which begins with εequenceε encoding amino acidε 548-617 of the gpE primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding for amino acids 77-547 of the gpE primary translation product with DNA coding for the marker gene. Note that the β- galactosidase (lacZ) marker gene will be under the control of the PRV gpX. A detailed deεcription of the plasmid is given in Figure 18. It may be constructed utilizing standard recombinant DNA techniques (6) , by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figure 18. The plasmid vector is derived from an approximately 2975 base pair Smal to Hindlll restriction fragment of psP65 (Promega) . Fragment 1 is an approximately 1704 base pair Smal to Smal restriction sub-fragment of the IBR Hindlll restriction fragment K (7) . Fragment 2 is an approximately 413 base pair Sail to BamHI reεtriction fragment #7 (3) . Fragment 5 iε an approximately 742 base pair Nhel to Bςrll sub-fragment of an IBR viruε 2400 base pair Smal fragment. This Smal fragment haε been cloned into the Smal εite of the plaεmid pSP64 (Promega) . Thiε plaεmid iε deεignated PSY1645. PSY1645 waε depoεited on July 16, 1991 with the American Type Culture Collection. Note that the lacZ marker gene is flanked by XJbal sites located at Junction B and Junction E in this plasmid permitting the marker gene to be cut out with XJbal. VACCINATION STUDIES IN CALVES WITH INACTIVATED IBR VIRUS. Calves, seronegative to IBR virus, were housed in facilities secure from IBR virus exposure. Groups of four calves were vaccinated intramuscularly with vaccines containing 10 7*3 or io 8,0 plaque forming units of inactivated IBR virus formulated with an oil adjuvant. A second vaccination was given 21 days later; four calves were maintained as unvaccinated controls. At 21 days after the second vaccination, animals were challenged intranasally with virulent wild-type IBR virus. After vaccination and challenge, animals were observed and the injection site was palpated weekly. Blood samples were taken on days 0, 7, 21, 28, and 42 post vaccination. After challenge, animals were observed daily for clinical signε of IBR. Blood sampleε were taken on days 7 and 13 post challenge. Nasal swabε were collected on dayε 3, 6, 9, and 12 poεt challenge. PURIFICATION OF IBR VIRUS gpG. gpG was purified from the tissue culture medium of infected MDBK cells. Confluent MDBK cells in serum-free medium were infected at a multiplicity of infection equal to 5, with wild-type, Cooper strain of IBR virus. The cells and media were harvested at approximately twenty-two hours after infection, when the cells showed considerable cytopathic effect and the fluids were centrifuged at 5000 rpm for 15 minutes. The supernatant fluid was concentrated approximately 10- fold by ultrafiltration through an Amicon ym-30 membrane, and dialyzed against lOmM NaP0 4 pH 7.2. The dialysate was treated for 20 minutes at 0°C with 70% perchloric acid to a final concentration of 0.2M perchloric* acid, then centrifuged at 12,000 rpm for 20 minutes. The supernatant fluid waε then dialyzed againεt 20mM Tris pH 9.5. The acid-soluble proteins were separated by column chromatography on a DEAE-Sephacel anion exchange column using a liner gradient elution : 0 to 100% A to B where A = 20mM Tris pH 9.5 and B = 20mM Triε pH 9.5/800 mM NaCl. The gpG eluted at approximately 35-40% B. Peak fractionε were aεεayed by Weεtern blot using anti gpG peptide sera. Reactive fractionε were combined and dialyzed against 5 mM Tris pH 7.0. The sample was then concentrated 10-fold by lyophilization and stored at -20-C. ELISA ASSAY. A standard enzyme-linked immunosorbent assay (ELISA) protocol waε uεed to determine the immune εtatus of cattle following vaccination and challenge. A purified gpG antigen solution (100 μl at 1 ng/μl in PBS) was allowed to absorb to the wells of microtiter dishes for 18 hours at 4°C. The coated wellε were rinsed one time with PBS. Wellε were blocked by adding 250 μl of PBS containing 1% BSA (Sigma) and incubating 1 hour at 37°C. The blocked wellε were rinεed one time with PBS containing 0.02% Tween 20. 50 μl of test serum (previously diluted 1:2 in PBS containing 1% BSA) were added to the wellε and incubated 1 hour at 37 β C. The antiεerum waε removed and the wells were washed 3 times with PBS containing 0.02% Tween 20. 50 μl of a solution containing anti-bovine IgG coupled to horseradiεh peroxidaεe (diluted 1:500 in PBS containing 1% BSA, Kirkegaard and Perry Laboratorieε, Inc.) waε added to viεualize the wellε containing antibody againεt the εpecific antigen. The solution was incubated 1 hour at 37°C, then removed and the wells were washed 3 times with PBS containing 0.02% Tween 20. 100 μl of εubεtrate solution (ATBS, Kirkegaard and Perry Laboratories, Inc.) were added to each well and color was allowed to develop for 15 minuteε. The reaction waε terminated by addition of 0.1M oxalic acid. The color waε read at absorbance 410nm on an automatic plate reader. PROCEDURE FOR GENERATING MONOCLONAL ANTIBODIES. To produce monoclonal antibodies, 8 to 10 week old BALB/c female mice were vaccinated intraperitoneally seven times at two to four week intervals with 10 7 PFU of S-PRV-160. Three weeks after the last vaccination, mice were injected intraperitoneally with 40 μg of purified gpG. Spleens were removed from the mice three days after the last antigen dose. Splenocytes were fused with mouse NSl/Ag4 plaεmacytoma cells by the procedure modified from Oi and Herzenberg, (39) . Splenocytes and plasmacytoma cells were pelleted together by centrifugation at 300 x g for 10 minutes. One ml of a 50% solution of polyethylene glycol (m.w. 1300-1600) was added to the cell pellet with εtirring over one minute. Dulbecco's modified Eagles'ε medium (5 ml) waε added to the cellε over three minuteε. Cellε were pelleted by centrifugation at 300 x g for 10 minuteε and resuspended in medium with 10% fetal bovine serum and containing 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine (HAT) . Cells (100 μl) were added to the wells of eight to ten 96-well tissue culture plates containing 100 μl of normal spleen feeder layer cells and incubated at 37°C. Cells were fed with fresh HAT medium every three to four days. Hybridoma culture supernatants were tested by the ELISA ASSAY in 96-well microtiter plateε coated with 100 ng of purified gpG. Supernatants from reactive hybridomaε were further analyzed by black-plaque assay and by Western Blot. Selected hybridomaε were cloned twice by limiting dilution. Ascetic fluid waε produced by intraperitoneal injection of 5 X 10 6 hybridoma cells into pristane-treated BALB/c mice. METHOD FOR cDNA CLONING BOVINE ROTAVIRUS gp38 GENE. The Calf Nebraεka εtrain of bovine rotaviruε (USDA) was propagated on MA-104 cells (Rhesus monkey kidney cells from MA Bioproducts) . Confluent monolayerε were infected at a multiplicity of infection of greater than 10 in DMEM containing 5 micrograms/ml trypsin. Cells were incubated with virus for 48 hours or until a cytopathic effect was obtained. Media and cell debriε were collected and centrifuged at 10,000 x g for 20 minuteε at 4°C. The εupernatant containing the rotaviruε waε then centrifuged at 10,000 x g in a preparative Beckman Ti45 rotor at 4°C. Viruε pellets were resuspended in SM medium (50 mM Tris- HC1 pH 7.5, 100 mM KCl, 10 mM MgCl-) and homogenized lightly in a Dounce-type homogenizer. The resuspended virus was centrifuged at 10,000 x g for 10 minuteε then loaded onto 25-50% CsCl gradientε in SM buffer. Gradientε were centrifuged at 100,000 x g for 4 hourε at 20°C. The two blue-white bandε representing intact virions and cores of rotaviruε were collected, diluted, and the CεCl gradient procedure waε repeated a second time. Virus obtained from the εecond gradient waε dialyzed overnight against SM buffer at 4°C. Dialyzed bovine rotaviruε waε twice extracted with an equal volume of SDS/phenol, then twice more with chloroform: isoεunylalcohol (24:1). The double stranded RNA was precipitated with ethanol in the presence of 0.2 M sodium acetate, centrifuged and reεuεpended in water. The yield waε typically 100 micrograms from 1,000 cm 2 of infected cells. 160 microgramε of double-stranded bovine rotaviruε RNA obtained from the above procedure waε mixed with one microgrεun each of two εynthetic oligonucleotide primerε in a volume of 160 microliter (εequenceε of primerε were: 5 # -GGGAATTCTGClAGGTClAC TClATACAATTCTAATCTAAG-3' * and 5'-GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCCGTTTGGCTA-3') derived from the published sequence of bovine rotavirus (40) . The RNA-pri er mixture was boiled for 3 minutes in a water bath then chilled on ice. Additions of 25 microliterε of 1 M Triε-HCl pH 8.3, 35 microliterε of 1 M KCl, 10 microliterε of 0.25 M MgCl 2 , 7 microliters of 0.7 M 2-mercaptoethanol, 7 microliters of 20 mM dNTP's, and 6 microliters of reverεe tranεcriptaεe (100 unitε) were made εequentially. The reaction waε incubated at 42°C for 1.5 hourε then 10 microliters of 0.5 M EDTA pH 8.0 was added and the solution was extracted once with chloroform:phenol (1:1). The aqueous layer was removed and to it 250 microliters of 4 M ammonium acetate and 1.0 ml of 95% ethanol was added, the mixture was frozen in dry ice and centrifuged in the cold. The resulting pellet was resuspended in 100 microliters of 10 mM Triε- HCl pH 7.5 and the ammonium acetate precipitation procedure waε repeated. The pellet waε resuspended in 100 microliterε of 0.3 M KOH and incubated at room temperature overnight, then at 37°C for 2 hourε. The solution was brought to neutral pH by addition of 10 microliters of 3.0 M HCl and 25 microliters of 1.0 M Tris-HCl pH 7.5 The resulting single-εtranded cDNA waε then precipitated two timeε by the above-described ammonium acetate-ethanol procedure. The pellet obtained was resuspended in 50 microliters of 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, boiled in a water bath for 2 minutes, then incubated at 59°C for 16 hours. The solution waε lyophilized to a volume of 15 microliterε and the reεulting double-εtranded cDNA waε run on a 1.0% agaroεe gel (Sigma agaroεe Type II) . The ethidium bromide-εtained DNA migrating at 1,000-1,100 baεe pair length was excised from the gel and electroeluted in a CBS electroeluter device. The solution was lyophilized, and the cDNA waε resuspended in 25 microliters of water. To this εolution waε added 2 microliterε of 1.0 M Triε- HCl pH 7.5, 2 microliterε of 1 M KCl, 1 microliter of 0.25 M MgCl 2 , 1 microliter of 20 mM dNTP'ε and 5 units of E. coli DNA polymerase I. The reaction waε incubated at room temperature for 15 minutes, then chloroform/phenol extracted and ammonium acetate-ethanol precipitated as described above. The resulting cDNA waε tailed with dCTP uεing terminal deoxynucleotide tranεferaεe (BRL buffer and enzyme uεed) . The reaction was stopped with 2 microliters of 0.5 M EDTA, chloroform/phenol extracted and precipitated with sodium acetate in the preεence of 10 microgramε of carrier tRNA. The resuspended cDNA waε mixed with 200 ng of dGMP-tailed Pst I cut pBR322 (BRL catalog #5355SA) in 200 microliters of 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, . heated to 65 β C for 5 minutes, then 57 β C for 2 hours. The annealed cDNA-vector pBR322 was tranεformed onto E. coli DH-1 cellε prepared for high efficiency tranεformation. Colonieε that showed senεitivity to ampicillin and tetracycline resistance were grown and DNA was prepared and cut with Pst I to determine the size of the cDNA insert. Several clones having Pst I inserts of 1,050-1,100 base pairs were analyzed and found to have identical reεtriction enzyme digest patterns. For one of these clones, the 1,100 base pair PstI insert was εubcloned into a M13 phage sequencing vector. Part of the DNA sequence of thiε clone was determined and was found to be identical to the published sequence (40) . cDNA CLONING. cDNA cloning refers to the methodε uεed to convert RNA moleculeε into DNA moleculeε following state of the art procedures. Applicants' methodε are described in Gubler and Hoffman (23) . Bethesda Research Laboratories (Gaithersburg, MD) have designed a cDNA Cloning Kit that is very similar to the procedures uεed by applicantε and containε the beεt set of reagents and protocols to duplicate our -eεultε. For cloning viruε RNA εpecies, a host cell line senεitive to infection by the viruε waε infected at 5-10 plague forming unitε per cell. When cytopathic effect was evident, but before total destruction, the medium was removed and the cells were lysed in 10 mis lysis buffer (4 M guanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0, 0.5% N-lauroyl sarcosine, 0.1 M beta- mercaptoethanol) . The cell lysate was poured into a sterilized Dounce homogenizer and homogenized on ice 8-10 times until the solution was homogenouε. For RNA purification, 8 mlε of cell lysate were gently layered over 3.5 mlε of CεCl solution (5.7 M CsCl, 25 mM sodium citrate pH 7.0) in a Beck an SW41 centrifuge tube. The εamples were centrifuged for 18 hours at 20°C at 36,000 rpm in a Beckman SW41 rotor. The tubes were put on ice and the supernatantε from the tubes were carefully removed by aεpiration to leave the RNA pellet undisturbed. The pellet waε reεuεpended in 400 microliters glass distilled water, and 2.6 mis of guanidine solution (7.5 M guanidine-HCl, 25 mM sodium citrate pH 7.0, 5 mM dithiothreitol) were added. Then 0.37 volumes of 1 M acetic acid were added, followed by 0.75 volumes of cold ethanol and the sample was put at - 20°C for 18 hours to precipitate RNA. The precipitate was collected by centrifugation in a Sorvall centrifuge for 10 min at 4°C at 10,000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, recentrifuged at 13,000 rpm, and the supernatant saved. RNA was reextracted from the pellet 2 more times as above with 0.5 ml distilled water, and the supernatants were pooled. A 0.1 volume of 2 M potasεium acetate εolution waε added to the εample followed by 2 volumes of cold ethanol and the sample was put at -20°C for 18 hours. The precipitated RNA was collected by centrifugation in the SS34 rotor at 4°C for 10 minutes at 10,000 rpm. The pellet waε dissolved in 1 ml distilled water and the concentration taken by absorption at A260/280. The RNA was stored at -70 β C. mRNA containing polyadenylate tails (poly-A) was selected using oligo-dT cellulose (Pharmacia #27 5543-0) . Three milligrams of total RNA waε boiled and chilled and applied to a 100 mg oligo-dT cellulose column in binding buffer (0.1 M Tris pH 7.5, 0.5 M LiCl, 5 mM EDTA pH 8.0, 0.1% lithium dodecyl sulfate) . The retained poly-A + RNA was eluted from the column with elution buffer (5 mM Triε pH 7.5, 1 mM EDTA pH 8.0, 0.1% sodium dodecyl sulfate) . This mRNA was reapplied to an oligo-dT column in binding buffer and eluted again in elution buffer. The sample was precipitated with 200 mM sodium acetate and 2 volumes cold ethanol at -20°C for 18 hours. The RNA was resuspended in 50 microliters distilled water. Ten micrograms poly-A + RNA waε denatured in 20 mM methyl mercury hydroxide for 6 minuteε at 22 β C. Beta- mercaptoethanol was added to 75 mM and the sample waε incubated for 5 min at 22 β C. The reaction mixture for firεt strand cDNA syntheεiε in 0.25 ml contained 1 microgrεun oligo-dT primer (P-L Biochemicals) or 1 microgram synthetic primer, 28 units placental ribonuclease inhibitor (Betheεda Research Labs #5518SA) , 100 mM Tris pH 8.3, 140 mM KCl, 10 mM MgCl-, 0.8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia) , 100 microcuries 32 P- labelled dCTP (New England Nuclear #NEG-013H) , and 180 units AMV reverse transcriptaεe (Molecular Geneticε Resources #MG 101) . The reaction waε incubated at 42°C for 90 minuteε, and then was terminated with 20 mM EDTA pH 8.0. The sample was extracted with an equal volume of phenol/chloroform (1:1) and precipitated with 2 M ammonium acetate and 2 volumes of cold ethanol -20°C for 3 hours. After precipitation and centrifugation, the pellet was dissolved in 100 microliters distilled water. The sample was loaded onto a 15 ml G-100 Sephadex column (Pharmacia) in buffer (100 mM Triε pH 7.5, l mM EDTA pH 8.90, 100 mM NaCl). The leading edge of the eluted DNA fractionε were pooled, and DNA waε concentrated by lyophilization until the volume was about 100 microliters, then the DNA was precipitated with ammonium acetate plus ethanol as above. The entire first strand sample was used for second strand reaction which follow the Gubler and Hoffman (23) method except that 50 microgramε/ml dNTP'ε, 5.4 units DNA polymeraεe I (Boehringer Mannheim #642-711) , and 100 unitε/ml E. coli DNA ligase (New England Biolabβ #205) in a total volume of 50 microliterε were used. After εecond strand synthesiε, the cDNA was phenol/chloroform extracted and precipitated. The DNA was resuspended in 10 microliters distilled water, treated with 1 icrogrεun RNase A for 10 minutes at 22°C, and electrophoresed through a 1% agarose gel (Sigma Type II agarose) in 40 mM Tris-acetate buffer pH 6.85. The gel was stained with ethidium bromide, and DNA in the expected size range was excised from the gel and electroeluted in 8 mM Tris- acetate pH 6.85. Electroeluted DNA was lyophilized to about 100 microliters, and precipitated with ammonium acetate and ethanol as above. The DNA was resuspended in 20 microliters water. Oligo-dC tails were added to the DNA to facilitate cloning. The reaction contained the DNA, 100 mM potassium cacodylate pH 7.2, 0.2 mM dithiothreitol, 2 mM CaCl 2 , 80 micromoles dCTP, and 25 units terminal deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 microliters. After 30 minutes at 37°C, the reaction was terminated with 10 mM EDTA, and the sample waε phenol/chloroform extracted and precipitated as above. The dC-tailed DNA sample waε annealed to 200 ng plasmid vector pBR322 that contained oligo-dG tails (Bethesda Research Labs #5355 SA/SB) in 200 microliterε of 0.01 M Triε pH 7.5, 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65°C for 2 minuteε and then 57 β C for 2 hours. Fresh competent E. coli DH-1 cells were prepared and transformed as described by Hanahan (41) using half the annealed cDNA sample in twenty 200 microliter aliquots of cells. Transformed cells were plated on L-broth agar plates plus 10 micrograms/ml tetracycline. Colonieε were εcreened for the preεence of inserts into the ampicillin gene using Ampscreen (Bethesda Research Labs #5537 UA) , and the positive colonies were picked for analysis. POLYMERASE FILL-IN REACTION. DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KCl, 5 mM MgCl 2 , and 400 micromolar each of the four deoxynucleotides. Ten units of Klenow DNA polymerase (BRL) were added and the reaction was allowed to proceed for 15 minutes at room temperature. The DNA was then phenol extracted and ethanol precipitated aε above. HOMOLOGY VECTOR 523-78.72. The plasmid 523-78.72 was constructed for the purpose of deleting a portion of the gpE gene coding region from the IBR virus. It may also be used to insert foreign DNA into IBR. Plasmid 523- 78.72 may be constructed by digestion of the plasmid 536- 03.5 with the enzyme XJbal followed by religation to remove the lacZ marker gene. HOMOLOGY VECTOR 591-21.20. The plasmid 591-21.20 was conεtructed for the purpoεe of deleting a portion of the IBR thymidine kinase gene. It may also be used to insert foreign DNA into IBR. It contains a unique Bglll restriction enzyme εite into which foreign DNA may be inεerted. It may be constructed utilizing standard recombinant DNA techniques (6, 14) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in figure 24. The plasmid vector is derived from an approximately 2999 base pair Sail to Sail restriction fragment of pSP64 (Promega) . Fragment 1 is an approximately 1400 base pair Sail to Narl restriction subfragment contained on the approximately 2700 base pair Sall-Sall restriction subfragment of the IBR Hindlll restriction fragment A (72) . Fragment 2 iε an approximately 1215 base pair Bgllll to Sail reεtriction εubfragment contained on the approximately 2700 baεe pair Sall-Sall reεtriction εubfragment of the IBR Hindlll restriction fragment A (72). HOMOLOGY VECTOR 552-46.12. The plasmid 591-46.12 was constructed for the purpose of deleting a portion o the Tk gene coding region from the IBR virus. It incorporates an E. coli β-glucuronidase (uidA) marker gene flanked by IBR virus DNA. The uidA marker gene waε inεerted into the homology vector 591-21.20 at the unique Bglll site. The marker gene is oriented in the same direction as the Tk gene in the homology vector. A detailed description of the marker gene is given in figure 25. It may be constructed utilizing standard recombinant DNA techniques (6, 14) by joining restriction fragments from the following sources with the εynthetic DNA εequenceε indicated in figure 25. Fragment 1 iε an approximately 404 base pair Sail to EcoRI restriction subfragment of the PRV BamHI reεtriction fragment #10 (3). Note that the EcoRI εite waε introduced at the location indicated in figure 12 by PCR cloning. Fragment 2 iε an approximately 1823 base pair EcoRI to Smal fragment of the plasmid pRAJ260 (Clonetech) . Note that the EcoRI and Smal siteε were introduced at the locationε indicated in figure 25 by PCR cloning. Fragment 3 is an approximately 784 base pair Smal to Smal restriction subfragment of the HSV-l BamHI restriction fragment Q (10) . Note that this fragment is oriented such that the polyadenylation sequence (AATAAA) is located closest to junction C. CLONING OF BOVINE VIRAL DIARRHEA VIRUS gp53 GENE. The bovine viral diarrhea (BVDV) gp53 gene waε cloned essentially aε described earlier (see cDNA CLONING) using the random priming method (6) . Viral RNA prepared from BVDV Singer strain grown in MADIN-DARBY bovine kidney (MDBK) cellε waε converted to cDNA uεing the random priming method. The cDNA waε used for second strand reaction (23) and the resulting double stranded DNA was uεed cloned aε described in the cDNA CLONING procedure. From this procedure a series of clones were obtained that comprised parts of the genome of BVDV. The location of the gene for gp53 gene has been published (66) and this sequence information was used to locate and isolate the gp53 encoding region from the 449 kilodalton primary translation product open reading frame contained in the complete cDNA clone. The gp53 encoding gene of BVDV waε also cloned essentially as described by Katz et al. for the HA gene of human influenza virus. Viral RNA prepared from the Singer strain of BVDV virus grown in MDBK cellε waε first converted to cDNA utilizing an oligo nucleotide primer specific for the target gene. The cDNA waε then uεed aε a template or polymerase chain reaction (PCR) cloning (67) of the gp53 gene. The PCR primers were designed to incorporate restriction endonuclease enzyme sites that permit the cloning of the εunplified coding region into vectors that contain the appropriate signals for gene expression in IBR. The gp53 gene of the Singer strain of BVDV was cloned using the following oligo nucleotide primerε: 5'-CATAGATCTTGTGGTGCTGTCCGACTTCGCA-3' for cDNA priming and waε combined with 5'- CGTGGATCCTCAATTACAAGAGGTATCGTCTAC-3' for PCR amplification. Note that thiε general εtrategy may be uεed to clone the gp53 coding region from BVDV εtrainε other than Singer. CLONING OF BOVINE RESPIRATORY SYNCYTIAL VIRUS FUSION PROTEIN AND NUCLEOCAPSID PROTEIN GENES. The bovine respiratory virus (BRSV) fusion (F) , attachment (G) , an nucleocapsid protein (N) genes may be cloned essentially as described by Katz et al. for the HA gene of human influenza. Viral RNA prepared from virus grown in bovine naεal turbinate (BT) cellε is first converted to cDNA utilizing an oligo nucleotide primer specific for the target gene. The cDNA is then used as a template for polymerase chain reaction (PCR) cloning (67) of the targeted region. The PCR primers are designed to incorporate restriction siteε which permit the cloning of the εunplified coding regionε into vectorε containing the appropriate signals for expression in IBR. One pair of oligo nucleotides will be required for each coding region. The N gene coding region from the BRSV εtrain A51908 (or 391-2??) would be cloned utilizing the f o l l o w i n g p r i m e r ε : 5 ' - AAAAGAT<^AGC^GGT(_AAACTAAATGA(^CITTCAAC-3 ' for cDNA p r i m i n g a n d c omb i n e d w i t h 5 ' - CGTGGATCC-_ACTC_ACAATTCCΛClATC-_TTATCrrTTGGGATT-3' for PCR. The G gene coding region from the BRSV εtrain A51908 (or 391-2??) would be cloned utilizing the following primers: 5'-TATAGATCraCΛTACCCATCΛTσiT_-AATTCAAGACATTA-3' for cDNA p r i m i n g a n d c omb i n e d w i t h 5 ' - CGTGGATCCATAATTAGATGTTATATGGAGGTGTGTTG-3' for PCR. The F gene from εtrain A51908 (or 391-2??) of BRSV would be cloned utilizing the following primerε: 5'- TATAGATCTAAC_AGCCΑTGAG , GATGATCATCAGCATTATC-3 ' for CDNA p r i m i n g a n d c om b i n e d w i t h 5 ' - CGTGGATCCTTCTGAG'GTTTAGATTGTAAACATTATGCA-3' for PCR. Note that this general strategy may be used to clone the coding region of F and N genes from other εtrainε of BRSV. CLONING OF PASTWI LLA HAEMOLYTICA LEUKOTOXIN AND IRON REGULATED OUTER MEMBRANE PROTEIN(S) . The Pasteurella haemolytica εtrain Al leukotoxin gene may be cloned from a genomic DNA sample. Genomic DNA is prepared from P. haemolytica Al cells grown in culture (68) by the methods deεcribed in Maniatiε et al. (1982) . The purified P. haemolytica DNA iε then uεed as a template for polymerase chain reaction (PCR) cloning (67) of the targeted leukotoxin gene. The PCR primers are designed so that restriction endonuclease sites are incorporated that allow the cloning of the 102 kilodalton toxin portion of the gene into vectors containing the appropriate signals for expresεion in IBR. The P. haemolytica Al (ATTC 43279 biotype A, εerotype 1) leukotoxin gene would be cloned utilizing the following primerε: - 5'- TATAGATCTTAGACTTACAACCCTAAAAAAC-3' and 5'- CGTGGATCCIAACTCTATAATGTGTGAAACAATATAG-3' for PCR. Note that thiε general εtrategy may be uεed to clone the coding regionε for the leukotoxin gene of all P. haemolytica serotypes. The P. haemolytica Al iron regulated outer membrane proteinε (IRP) of 3 major polypeptideε with molecular weightε of 35, 70 and 100 kilodaltonε. The DNA coding for the array of P. haemolytica genes can be cloned in Escherichia coli uεing plaεmid vectorε eεεentially as deεcribed in Maniatiε et al. (1982) . The clone library iε conεtructed by partial digeεtion of the genomic DNA. The IRP genes can be isolated from this library of P. haemolytica clones by screening for the production of iron regulated outer membrane antigenε by a colony enzyme-linked immunoεorbent assay blot method with antiεerum that is specific to the IRPs. This antiserum may be obtained by eluting antibodies derived from polyclonal antiserum raised against whole P. haemolytica or membrane enriched fractionε but selectively bound to the IRPs on Western blots (69) . The specificity of the antibodies can be verified by immunoblot screening of P. haemolytica polypeptides from iron restricted and iron induced cultures. EXAMPLES Example 1 S-IBR-002 S-IBR-002 iε an IBR viruε that haε a deletion of approximately 800 bp in the repeat region of the genome. Thiε deletion removeε the only two EcoRV restriction εiteε on the viruε genome and an adjacent Bglll site (Figure 2) . To construct this viruε, the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS was performed. Purified IBR virus DNA (Cooper strain) digested with EcoRV restriction enzyme was mixed with Dral-restriction enzyme-digested plasmid DNA containing the E.coli β- galactosidase (lacZ) gene under the control of the HSV-l TK promoter. After ligation the mixture was used to tranεfect animal cellε and the tranεfection stock was screened for recombinant IBR virus by the SOUTHERN BLOTTING OF DNA procedure. The final result of the purification waε the recombinant IBR viruε designated S- IBR-002. It was shown by Southern hybridization that this virus doeε not carry any foreign geneε. Restriction enzyme analysis also showed that the insertion sites (EcoRV) in both repeats were deleted. Figure 2 shows the restriction map of the EcoRI B fragment which contains the EcoRV reεtriction εiteε and the map of S-IBR-002 which lackε the EcoRV sites. S-IBR-002 was deposited on June 18, 1986 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposeε of Patent Procedure with the Patent Culture Depoεitory of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2140. A study was conducted to determine the safety and serological reεponse of young calves following intramuscular administration of S-IBR-002. These results are presented in Table 1. Three calves were inoculated intramuεcularly with 10 7 PFU of S-IBR-002. Clinical signs of IBR and febrile response were absent in these calves, as well as in the contact control calf. All three calves developed significant neutralizing antibody to IBR virus but the contact control remained seronegative. These results suggest that s-IBR-002 is useful aε a vaccine against IBR diseaεe. Table 1. Serologic and Clinical Response of Young Calves Following Vaccination with S- IBR-002 Virus Calf Qinical Virus Antibody Titer Construct # and Isolation" Days Post Inoculation Febrile response 0 14 21 28 28 NONE (-) <2 <4 S-IBR-002 30 NONE (-) <2 <4 <2 94 NONE Θ <2 <4 8 Control 32 NONE (-) <4 <4 <4 <2 <4 -From nasal swabs and peripheral blood leukocytes. Examole 2 Unique Short 2 αene The unique short region of IBR viruε containε a gene homologouε to the US2 gene of several other herpesviruεeε. In the studies described below we show that deletion of the IBR unique short 2 gene (US2) may render the virus safe for use in pregnant cows, aε determined by direct fetal inoculation. Obεerving that the Nasalgen IBR vaccine strain will not cause abortion when used in IBR-susceptible pregnant cowε at variouε stages of gestation (18,65), we attempted to determine the genomic lesion responsible for this property. We characterized the genome of this virus by restriction mapping and DNA sequence analysis. It was determined that a major portion of the IBR virus US2 gene was deleted from the Nasalgen virus. Restriction mapping of the Nasalgen virus indicated that the Hindlll K fragment contained an approximately 800 base pair deletion. The deletion waε localized to the end of the Hindlll K Fragment located next to the Hindlll O fragment (see Figure 1) . Therefore, the Hindlll K fragment from the Cooper strain waε εubcloned and this region waε sequenced. The first 1080 base pairs of the fragment were found to contain an open reading frame (ORF) coding for 309 εunino acids (see Figure 3) . The ORF is 68% G+C and encodes a protein with a predicted molecular weight of 46,094. Comparison of the sequence of the predicted protein with sequences of gene products of HSV-l, PRV, HSV-2, and MDV in the unique short region indicated that this ORF is homologouε to the herpeεviruε US2 gene (see Figure 4) . Although the function of the herpesvirus US2 gene is not known, the gene has been shown to be nonesεential for growth of HSV in cell culture (4,19). The US2 gene haε also been shown to be deleted in the PRV vaccine εtrainε Norden and Bartha (11) . The Hindlll K fragment from the Naεalgen virus was subcloned and the deletion region waε sequenced. When the sequence obtained from the Nasalgen strain was compared to the sequence obtained from the Cooper strain (εee Figure 5) , it waε poεsible to determine that amino acids 59 to 309 of the US2 gene had been deleted. It was also determined that most of the Hindlll 0 fragment had also been deleted. Cattle studieε have shown that the Naεalgen viruε will not cauεe abortion when used in IBR-susceptible pregnant cows at various stages of gestation (18) . Since the only major difference between the wild-type IBR strain and the Nasalgen strain resides in the deletion of the US2 gene, this gene may be involved in the fetal virulence observed for the wild type virus. Exa ple 3 S-IBR-027 s-IBR-027 is an IBR virus that has a deletion of approximately 800 bp in the repeat regions and approximately 1200 bp in the short unique region of the genome. The deletion in the short unique region removes the US2 gene (Figure 6) . The repeat deletion was derived from the parental virus S-IBR-002 and is described in Example 2. To construct this virus, the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS was performed. A homology vector containing the bacterial tranεpoεon Tn5 NEO (aminoglycosidase 3'- phosphotranεferase) gene under the control of the HSV α4 promoter flanked by sequences from the IBR virus TK gene was constructed. The IBR virus homology regions were derived from the TK gene. The upεtream homology included the first εunino acid of the TK gene (15) and extended approximately 800 base pairs upstream of the TK coding region. The downstream homology included εunino acids 156 to 357 and extended downstream of the TK coding region approximately 60 base pairs. S-IBR-002 DNA was mixed with the homology vector and transfected into rabbit skin cells as indicated in the methods. The transfection stock was selected according to the SELECTION OF G418 RESISTANT IBR VIRUS. Individual clones were picked after one round of selection and analyzed by the SOUTHERN BLOTTING OF DNA procedure. When a probe derived from the NEO gene was used in this analysis, one clone was found which did not hybridize to the NEO probe but had a Hindlll restriction digestion pattern clearly distinct from the parental S-IBR-002. Further analysis indicated that the NEO had not been inserted into the TK region. however an approximately 1200 base pair deletion had occurred in the Hindlll K fragment. In order to characterize the Hindlll K deletion, that fragment waε εubcloned and subjected to restriction mapping. Utilizing a serieε of oligonucleotide probes derived from the wild type εequence it waε determined that approximately 1200 baεe pairs were deleted from the end of the Hindlll K fragment adjacent to the Hindlll Kf Hindlll 0 junction (see Figure 6) . This deletion removes the entire coding region of the US2 gene. S-IBR- 027 was deposited on April 17, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposeε of Patent Procedure with the Patent Culture Depoεitory of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2322. Direct fetal inoculation iε the most sensitive test for determining the safety of live, IBR vaccines as regards their use in pregnant cows or in calves nursing pregnant cows. Three viruε conεtructε were tested for fetal safety by inoculating directly into the bovine fetuε, following laparotomy to expoεe the uterus. Abortion occurring within seven days after inoculation was considered to be surgically-induced. If fetuses aborted after this time, tissue sampleε were removed and cultured for the presence of the IBR construct. Caesarean εectionε were performed on cowε with fetuses surviving for greater than 30 dayε poεt-inoculation. Fetal tiεεue waε removed for viruε culturing and blood samples were taken for evaluation of serum antibody to IBR viruε. The S-IBR-027 construct described above was tested, aε well aε two other conεtructε, S-IBR-020 and S-IBR-028. The S-IBR-020 conεtruct waε derived from the Cooper strain of IBR virus by making deletions in the repeat regions of the DNA and by inεerting the Tn5 NEO gene. The S-IBR-028 construct was derived from the Cooper strain of IBR virus by making deletions in the repeat region of the DNA and in the TK gene. The Tn5 NEO gene waε alεo inεerted into the TK deletion. The following results were obtained from studies with the three virus constructs. In the studies with S-IBR-020, two fetuses were inoculated, one at approximately 130-140 days geεtation and the other at approximately 170-180 dayε geεtation. The younger fetuε aborted twenty dayε after inoculation, but viruε could not be recovered from tissue sampleε of thiε fetuε (Table 2) . The other fetuε waε live and appeared normal when it was surgically removed 60 days post-inoculation. In studies with S-IBR- 027, four fetuses, ranging in age from 125 days to >250 days, were inoculated (Table 2). All fetuses survived and appeared normal. In studies with S-IBR-028, three fetuseε, ranging in age from 140 days to >250 days, were inoculated. The youngest and eldest fetuseε survived and appeared normal, however the fetus inoculated at 160-170 days gestation aborted nine days after inoculation. Direct fetal inoculation is the most sensitive test for measuring the safety of live, IBR viruses used in pregnant cows. To date, the gene(s) involved in fetal virulence has not been reported. We have engineered IBR viruses with deletions in three different regions of IBR virus DNA and then determined the effect of the gene deletion. All three virus constructs tested have a deletion in the repeat region of the DNA and two constructs do not have TK activity. One fetus inoculated with each of the TK- constructs has aborted. In contrast, the construct with deletions in the repeat regions and the US2 gene (S-IBR-027) haε been inoculated into four fetuseε with no adverse reactions. Table 2. Safety of IBR Viruses for Bovine Fetuses "Approximate age at time of virus inoculation We have shown that S-IBR-027 is safe for fetal inoculation in contrast to S-IBR-020 and S-IBR-028 which are not. Although all three viruεeε were engineered by similar approacheε, the distinguishing difference of S- IBR-027 is the deletion of the US2 gene. We have also shown that the Nasalgen virus, which was generated by independent methods and is also safe for use in IBR- susceptible pregnant cows, has been deleted in the US2 gene. Although the S-IBR-027 and Nasalgen have the similar property of fetal safety, S-IBR-027 offers additional advantages. The major portion of the US2 gene (251 out of 309 amino acids) has been deleted in the Nasalgen virus. This deletion would clearly inactivate the gene, however the remaining portion of the gene may make it more likely to revert to virulence via recombination with other viruses. The complete coding region of the US2 has been deleted from S-IBR-027 making it less likely that this gene could be restored and revert the virus to virulence. The S-IBR-027 construct also carries an important deletion in the repeat region, which is not present in the Nasalgen virus. A deletion in the analogous region of the pseudorabies virus (PRV) has been shown to be valuable in attenuating PRV for swine (see U.S. Patent No. 4,877,737). This deletion has also been shown to attenuate IBR for cattle as seen in the testing of S-IBR-002 (see Example 1) . * g ^ ~ ip]e 4 S-IBR-028 S-IBR-028 is an IBR viruε that haε a deletion of approximately 800 bp in the repeat regionε and approximately 250 bp in the TK region of the genome. The deletion in the TK region inactivateε the TK gene. The repeat deletion waε derived from the parental viruε S- IBR-002 and iε described in Example 2. To construct this virus, the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS was performed. A homology vector containing the bacterial transposon Tn5 NEO (aminoglycoεidaεe 3'- phosphotrε_nsferase) gene under the control of the HSV-l α4 gene promoter flanked by sequences from the IBR virus TK gene was constructed. The IBR viruε homology regions were derived from the TK gene. The upstreεun homology included εunino acidε 1 to 62 of the TK gene (15) and extended approximately 674 base pairs upstream of the TK coding region. The downstream homology included εunino acidε 156 to 357 and extended downstreεun of the TK coding region approximately 1138 base pairs. S-IBR-002 DNA was mixed with the homology vector 129-71.5 and transfected into rabbit skin cells as indicated in the methods. The transfection stock was selected according to the SELECTION OF G418 RESISTANT IBR VIRUS. Individual cloneε were picked after two rounds of selection and analyzed by the SOUTHERN BLOTTING OF DNA procedure. Several cloneε were assayed for TK activity by a 1 C-thymidine incorporation assay (29) . One clone which was negative for TK activity was chosen and characterized by digestion with Hindlll and XJbal. The restriction endonuclease analysis confirmed that the NEO gene had been inserted into the TK gene. This clone, designated S-IBR-028, was deposited on May 14, 1991 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 2326. ExaffiPle 5 Glycoprotein G gene Deletion of the PRV gpX gene has been shown to be valuable both aε an attenuating leεion and aε a negative εerological marker (see U.S. Serial No. 192,866, filed May 11, 1988 now U.S. Patent No. 5,047,237 issued September 10, 1991). In the studies described below we show that the unique short region of IBR virus contains a gene homologous to the gpX gene of PRV. The sequence of an approximately 1400 base pair region of the IBR Hindlll K fragment (see Figure 8) , located approximately 2800 base pairs downstream of the Hindlll Kf Hindlll 0 junction was determined. This region was found to contain an ORF coding for 441 εunino acids translated in the direction away from the Hindlll K/HindIII O junction (see Figure 1) . The ORF is 69% G+C and encodes a protein with a predicted molecular weight of 58,683. Comparison of the sequence of the predicted protein with sequences of gene products of HSV-2 and PRV in the unique short region indicated that this ORF is homologous to the herpesvirus gpG gene (see Figure 9) . The complete gpG gene resides on an approximately 2800 base pair Jflul to Ndel sub-fragment of the IBR virus Hindlll K fragment. Thiε subfragment has been cloned as a blunt ended fragment into the plasmid pSP64. This plasmid is designated PSY1643. PSY1643 was deposited on July 16, 1991 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. 68652. This plasmid may be used to confirm the sequence of the gpG gene. The sequence of the gpG gene may also be confirmed by comparing the appropriate DNA sequence of the wild type virus S-IBR-000 (Cooper strain with the sequence of the gpG deleted virus S-IBR- 037 (ATCC Accession No. 2320) . To confirm the expression of the IBR virus gpG gene product, cells were infected with IBR virus and εampleε of media from infected cultures were subjected to SDS- polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. The anti-serum used was a mouεe hyper-immune serum raised against chemically-synthesized gpG peptides (amino acids 242-254 and 269-289) linked to keyhole limpet hemocyanin. Aε εhown in Figure 10, gpG iε prominent in the media of cells infected with wild type virus (S-IBR-000) , but is not detected in media of mock infected cells. Example 6 S-PRV-160 S-PRV-160 is a pseudorabies virus that has a deletion in the TK gene in the long unique region, a deletion in the repeat region, and an approximately 1414 base pair deletion in the gpX coding region. The gene for E.coli β-galactosidase (lacZ gene) was inserted in the place of the gpX gene and iε under the control of the gpX promoter. A chimeric gene coding for an IBR viruε gpG, PRV gpIII and PRV gpX fusion protein was inserted at the Hindlll sites located in each repeat. S-PRV-160 was constructed utilizing plasmid 459-12.6, pseudorabieε viruε S-PRV-013 (εee U.S. Serial No. 823,102, filed January 27, 1986 now U.S. Patent No. 5,068,192 issued November 26, 1991 and U.S. Serial No. 07/192,866, filed May 11, 1988 now U.S. Patent No. 5,047,237 issued September 10, 1991) and the restriction enzyme Hindlll in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Several clones were screened by digeεtion with Hindlll for the presence of the Hindlll band containing the chimeric gene insert from plasmid 459-12.6. One clone exhibiting the correct Hindlll insert band was choεen and designated S-PRV-160. S-PRV-160 was conεtructed εo that it would express precisely the gpG specific εunino acidε that were deleted in S-IBR-037. Thiε allows the gpG fuεion protein expressed in S-PRV-160 to be used as an antigen to identify antibodies directed against the wild type virus as opposed to antibodies directed againεt S-IBR-037. Note that gpX, the PRV homologue of IBR viruε gpG, haε been deleted from S-PRV-160 to prevent any confuεion reεulting from croεε reactivity that might exiεt between the two proteinε. To confirm that S-PRV-160 doeε express IBR virus gpG, a Western blot analysiε waε performed. Aε can be seen in Figure 10, gpG specific antibody does react with an appropriately sized media protein from S- PRV-160. S-PRV-160 may also be utilized as an antigen for the production of gpG εpecific monoclonal antibodieε. Such antibodieε are useful in the development of diagnostic tests specific for the gpG protein. Monoclonal antibodieε were generated in mice utilizing S-PRV-160 according to the PROCEDURE FOR GENERATING MONOCLONAL ANTIBODIES. One of these antibodies' clone 3-1G7 was shown to react εpecifically with purified gpG in the gpG ELISA aεsay. S-IBR-035 S-IBR-035 is an IBR virus that has two deletions in the εhort unique region of the genome. The firεt deletion iε approximately 2500 baεe pairε and begins in the Hindlll K fragment approximately 1750 base pairs downstream of the Hindlll Of Hindlll K junction and extends back through that junction. This deletion removes the US2 gene. The εecond deletion iε approximately 294 baεe pairε and begins in the Hindlll K fragment approximately 3900 base pairs downstreεun of the Hindlll K/Hindlll O junction and extends back toward that junction. This deletion removes amino acidε 263 to 361 of the gpG gene. The gene for E. coli β-galactosidase (lacZ gene) waε inserted into the deletion in the gpG gene and is under the control of the HCMV immediate early promoter. s-IBR-035 was derived from S-IBR-000 (Cooper strain) . This waε accompliεhed utilizing the homology vector 439- 01.31 (see Materials and Methods) and virus S-IBR-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was εcreened by the BLUOGAL" SCREEN FOR RECOMBINANT HERPESVIRUS. The final result of blue plaque purification was the recombinant virus designated S-IBR- 035. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the β-galactosidase (lacZ) marker gene and the deletion of approximately 294 base pairs of the gpG gene. It was also confirmed that an approximately 2500 base pair deletion had occurred in the region of the US2 gene. Example 8 S-IBR-P?*? S-IBR-036 is an IBR viruε that haε two deletions in the short unique region of the genome. The first deletion is approximately 2500 base pairs and is similar to the deletion in S-IBR-035 (see Exεunple 7) which removes the US2 gene. The second deletion is approximately 1230 base pairs and beginε in the Hindlll K fragment approximately 3900 base pairs downstreεun of the Hindlll 0/ Hindlll K junction and extends back toward that junction. This deletion removes amino acids 1 to 361 of the gpG gene. The gene for E.coli β-galactoεidaεe (lacZ gene) was inserted into the deletion in the gpG gene and is under the control of the HCMV immediate early promoter. S-IBR-036 was derived from S-IBR-000 (Cooper strain) . This was accomplished utilizing the homology vector 439- 21.69 (see Materials and Methods) and virus S-IBR-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. The final result of blue plaque purification was the recombinant virus designated S-IBR- 036. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the β-galactosidase (lacZ) marker gene and the deletion of approximately 1230 base pairs of the gpG gene. It was also confirmed that an approximately 2500 base pair deletion had occurred in the region of the US2 gene (see above) . 1- ^-ιη ' e 9 g-IPfi-Q37 S-IBR-037 is an IBR virus that has two deletions in the short unique region of the genome. The first deletion iε approximately 2500 baεe pairε and begins in the Hindlll K fragment approximately 1750 base pairs downstreεun of the Hindlll Of Hindlll K junction and extends back through that junction. This deletion removes the US2 gene. The second deletion is approximately 1230 base pairs and begins in the Hindlll K fragment approximately 3900 base pairs downstream of the Hindlll Of Hindlll K junction and extends back toward that junction. Thiε deletion removeε amino acidε 1 to 361 of the gpG gene. S-IBR-037 waε derived from S-IBR-035. Thiε waε accomplished utilizing the homology vector 439-70.4 (see Materials and Methods) and virus S-IBR-035 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the BLUOGAL * SCREEN FOR RECOMBINANT HERPESVIRUS. The result of white plaque purification was the recombinant virus designated S-IBR-037. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the deletion of the β-galactosidaεe (lacZ) marker gene and the deletion of approximately 1230 baεe pairs of the gpG gene. It waε also confirmed that an approximately 2500 base pair deletion had occurred in the region of the US2 gene (see above) . S-IBR-037 was deposited on April 16, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposeε 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 Accesεion No. VR 2320. To test the efficacy of S-IBR-037 aε an inactivated IBR virus vaccine in protecting εusceptible calves against virulent IBR virus challenge, a study was performed according to the VACCINATION STUDIES IN CALVES WITH INACTIVATED IBR VIRUS. The following results were observed. Virus neutralization antibody titers were elicited in animals after the first vaccination (see Table 3) . Antibody titers were not significantly different between animals that received a vaccine dose of 10 7,3 virus and animals vaccinated with 10 8,0 virus. After the second vaccination, mean antibody titers increased to 1:19 and 1:32, respectively, for the 10 7,3 and lθ 8,0 vaccine groups. Control animals remained seronegative to IBR virus throughout the vaccination period. Antibody titers in both vaccinate groups showed an increase typical of an anamnestic response after challenge with virulent IBR virus. By 13 days post challenge, mean antibody titers were 1:152 and 1:215 for the 10 7*3 and lθ 8,0 vaccinate groups respectively. In contrast, mean antibody titers in challenged control animals were 1:4 at 7 dayε and 1:8 at 13 dayε poεt challenge. Nasal swabs were collected from challenged animals to determine whether vaccination decreaεed the time of viruε εhedding (Table 4) . The moεt dramatic difference between vaccinates and control animals waε obεerved at 12 dayε poεt challenge. At thiε time, seventy-five percent of control animalε continue to shed, whereas, only twenty- five percent of both vaccinate groups shed virus. Virus was not iεolated from control or vaccinated groups at 15 days post challenge. Table 3. Generation of virus neutralizing antibody in animals vaccinated with inactivated S-IBR-037 vaccine. * Statistically greater than controls (p 0.05) "Expressed as reciprocal of dilution. Table 4. Isolation of IBR virus from vaccinated and unvaccinated control animals after challenge with virulent IBR virus. Table 5. Vaccinated animals demonstrate reduced clinical signs of IBR. Number of ulcers. c Days with serous discharge. d Days with mucopurulent discharge. e Days with > 2°F above baseline temperature. f Anϊm-1 exhibited mucopurulent discharge on the day of challenge and for 13 days post challenge. Statistically greater than controls (p 0.05) Animalε were observed daily for 13 days post challenge for clinical signs of IBR infection. Clinical disease was evaluated with respect to attitude, the number of ulcers, extent of serious and mucopurulent discharge and the number of days with elevated temperature. The reεultε presented in Table 5 show that vaccinated animals exhibited less severe disease than did unvaccinated control animals. Control animals showed clinical depression ("Attitude" in Table 5) for 4.5 days compared with 1 to 1.5 dayε for vaccinated animalε. The amount and extent of serous discharge was substantially reduced in both vaccinate groups compared with controls. The extent of mucopurulent discharge was also reduced in vaccinated animals, although to a lesser degree. However, vaccinate animal #36 did have mucopurulent discharge on the day of challenge and is not consistent with the results for other vaccinates. None of the vaccinates exhibited temperatures of >2°F above baseline. In contrast, all control animals exhibited elevated temperatures of ≥2°F over baseline and 2 of 4 control animals had temperatures of 104°F and above. Vaccination of calves with inactivated S-IBR-037 vaccine protected the animals against virulent wild-type IBR viruε challenge. Viruε neutralization titerε were statistically greater in vaccinated than in control animals. An anamnestic response in antibody titer was observed 7 days post challenge, indicating the development of humoral memory response. Except for 7 days post challenge, neutralization titers between the 10 7 * 3 and 10 8 '° vaccinate groups were not εtatiεtically different. Fewer vaccinated animalε shed virulent challenge virus than control animals. These resultε suggest that virulent IBR virus is cleared more rapidly in vaccinated than in unvaccinated animals. Clinical εymptomε of IBR viruε infection were alεo reduced in vaccinated animals. After challenge, both vaccinate groups exhibited fewer days of depressed attitude, reduced serous discharge, and no elevated temperature compared with controls. In order to εhow that gpG antibody is produced in vaccinated calves following exposure to wild-type virus, serum sεunpleε tεϋen pre- and poεt-expoεure to wild-type viruεeε were subjected to the ELISA assay. Samples taken at the day of challenge and at 13 days post-challenge were analyzed. As seen in Table 6, the post-challenge absorbance readings for gpG increase for each animal (ratio of > 1.0), indicating that within 13 days of infection a detectable immune responεe to gpG iε preεent. Table 6. Detection of antibody to gpG in serum of animals vaccinated with S-IBR-037 and challenged with wild type. .Ammals were challenged with 10 76 PFU of wild type IBR virus. Pre-challenge serum from day of challenge, post-challenge serum from 13 days post challenge. Data reflects the average of the ratio of absorbance readings for three independent ELISA determinations. Examole 10 S-IBR-038 S-IBR-038 is an IBR virus that has two deletions in the εhort unique region of the genome. The firεt deletion iε approximately 2500 base pairε and beginε in the Hindlll K fragment approximately 1750 base pairs downstreeun of the Hindlll 0/HindIII K junction and extends back through that junction. This deletion removes the US2 gene. The εecond deletion iε approximately 294 base pairs and begins in the Hindlll K fragment approximately 3900 base pairs downstream of the Hindlll K/Hindlll 0 junction and extends back toward that junction. This deletion removes amino acids 261 to 359 of the gpG gene. S-IBR-038 resulted from the removal of the marker gene from S-IBR-035 (see above) . This was accomplished by digestion of S-IBR-035 with Xbal as described in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The structure of S-IBR-035 was confirmed by restriction enzyme analysiε with Hindlll, BamHI and XJbal. Example 11 Glycoprotein E gene Deletion of the PRV gl gene haε been εhown to be valuable both aε an attenuating leεion and a negative εerological marker (3,42). In the studies described below we show that the unique short region of IBV virus contains a gene homologous to the gl gene of PRV. The εequence of 2038 base pairs of the IBR unique short region, starting approximately 1325 baεe pairε upεtream of the Hindlll K/Hindlll F junction in the Hindlll K fragment waε determined. Thiε region waε found to contain an ORF coding for 617 amino acidε translated in the direction away from the Hindlll K/ Hindlll 0 junction (see Figure 1). The ORF is 70.5% G+C and encodes a protein with a predicted molecular weight of approximately 88,980. Comparison of the sequence of the predicted protein with εequenceε of gene productε of HSV- 1, VZV, and PRV in the unique short region indicated that this ORF iε homologouε to the herpesvirus gpE gene (see Figure 16) . The DNA encoding the gpE gene has been cloned in two plasmidε, PSY1644 and PSY1645. The amino-terminal half of the gene (encoding amino acids 1-276) was cloned as an approximately 2300 base pair fragment resulting from a partial Smal digest of wild type S-IBR-000 (Cooper Strain) DNA. This fragment was inserted into the plasmid pSP64 to yield PSY1644. This plasmid, designated PSY1644, waε depoεited on July 16, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposeε 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 Accesεion No. 68651. The carboxyl-terminal half of the gene (encoding amino acids 277-617) was cloned aε an approximately 2400 baεe pair Smal fragment. The fragment was inserted into the plasmid pSP64 to yield PSY1645. This plaεmid, deεignated PSY1645, waε depoεited on July 16, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganismε for the Purposeε 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. 68650. These plasmids may be used to confirm the sequence of the gpE gene. Example 12 Pseudorabies virus expressing IBR viruε gpE A pseudorabies viruε analogouε to S-PRV-160 may be conεtructed for the purpose of expresεing the IBR viruε gpE. Thiε may be accomplished by inserting the gene coding for IBR virus gpE into S-PRV-002 (U.S. Patent No. 4,877,737) . Such an expression vector may be constructed utilizing the IBR virus gpE plasmid described in the methods section, pseudorabies virus S-PRV-002 and the restriction enzyme XJbal in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Viruses resulting from this procedure may be screened by digestion with XJbal for the presence of the XJbal band containing the IBR viruε gpE gene. The gpE protein expreεεed from this vector may be used aε an antigen to identify antibodieε directed againεt the wild type viruε aε opposed to antibodieε directed against gpE deleted viruses. This virus may also be utilized as an antigen for the production of gpE specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the gpE protein. Monoclonal antibodies may be generated in mice utilizing thiε virus according to the PROCEDURE FOR GENERATING MONOCLONAL ANTIBODIES. Exampl 1 Glycoprotein E deleted IBR viruses The HOMOLOGY VECTOR 536-03.5 was used to generate various gpE-deleted IBR viruses. Utilizing the general strategy described in CONSTRUCTION OF DELETION VIRUSES, a gpE deletion of approximately 1410 base pairs (amino acids 77-547) waε introduced into two different IBR viruε backbones, S-IBR-000 (Cooper Strain) and S-IBR-037. The virus resulting from the S-IBR-000 parent contains the gpE deletion alone. The virus resulting from the S-IBR- 037 pεirent contains the gpE deletion in conjunction with the US2 and gpG deletions. The lacZ marker gene may be removed from these viruses utilizing the procedures outlined in the methods section. These gpE-deleted viruses are of great value as IBR vaccineε. Their combination of different deletions will provide the varying degrees of attenuation which are required for a superior vaccine. These viruses will also provide a negative serological marker which may be used to distinguish vaccinated from infected animals. The virus containing both gpG and gpE deletions should be of even greater value by having two negative markers. The availability of two negative markers permits one marker to be used as a confirmatory test, greatly increasing the reliability of such a diagnostic determination. Example 14 S-IBR-004 S-IBR-004 is an IBR recombinant virus carrying an inserted foreign gene, Tn5 NEO (aminoglycoside 3'- phosphotransferase) gene, under the control of the pseudorabies virus (PRV) glycoprotein X promoter. To conεtruct thiε viruε, the Hindlll K DNA fragment from wild type IBR viruε waε cloned into the plasmid pSP64 at the Hindlll site. This plaεmid waε designated pSY524. A map of the Hindlll K fragment is shown in Figure 19. The DNA from the Xhol site to the Hindlll site and containing the Ndel site from pSY524 was cloned into plasmid pSP65 and called pSY846. The Ndel to EcoRI fragment was removed from pSY846 by digestion with Ndel and EcoRI restriction enzymes, followed by POLYMERASE FILL-IN REACTION and LIGATION. The resulting plasmid was called pSY862. The plaεmid pNEO (P.L. Biochemicals, Inc.) contains the aminoglycoεide 3'-phosphotranεferase (NEO) gene and confers reεiεtance to ampicillin and neomycin on E. coli hoεtε. The coding region of this gene (Bglll-BamHI fragment) was isolated and cloned between the PRV gpX promoter and the HSV-TK poly A sequence in a plasmid called pSY845. The NEO gene construct in pSY845 waε excised with Hindlll, made blunt ended by the POLYMERASE FILL-IN REACTION, and cloned into the Sad εite of plaεmid pSY862. The final product was called pSY868. Wild type IBR DNA was mixed with pSY868 DNA and the mixture was transfected into rabbit skin cells to generate recombinant IBR. The recombinant IBR virus carrying a functional NEO gene was then isolated and purified according to the SELECTION OF G 18 RESISTANT IBR VIRUS method. S-IBR-004 recombinant IBR waε shown to expresε the NEO gene by the fact that cellε infected with thiε viruε were reεiεtεmt to the toxicity of G418. A detailed map of the plaεmid conεtruction iε εhown in Figure 19. The structure of S-IBR-004 is also shown in Figure 19. S- IBR-004 waε deposited on May 23, 1986 pursuant to the Budapest Treaty on the International Deposit of Microorganismε for the Purpoεes of Patent Procedure with the Patent Culture Depoεitory of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Acceεεion No. VR 2134. Example 15 S-.BR-QQP S-IBR-008 is an IBR virus that has a deletion in the short unique region, and an insertion of the bovine rotavirus glycoprotein 38 (gp38) gene in the Xbal site in the long unique region. The bovine rotavirus gp38 gene was cloned utilizing the METHOD FOR cDNA CLONING BOVINE ROTAVIRUS gp38 GENE. The bovine rotavirus gp38 gene was then engineered to contain herpesvirus regulatory signalε as shown in Figure 20. This was accomplished by cloning the gp38 gene BamHI fragment contained in pSY1053 between the BamHI and Bglll sites in pSY1052. The resulting plasmid, pSY1023, contained the PRV gpX promoter in front of the gp38 gene, and the HSV-l TK polyadenylation signal behind the gp38 gene. The entire construct was flanked by Xbal sites to allow for the insertion of the XJbal fragment into IBR by direct ligation. S-IBR-004 was the starting virus for the generation of S- IBR-008. S-IBR-004 DNA and pSY1023 DNA were mixed together, cut with XJbal, and transfected into rabbit skin cells according to the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened for recombinant virus by the ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS procedure using antibodies prepared against the rotavirus gp38 protein. One of the viruses purified by this screen was S-IBR-008, which has the following characteristicε. It containε the rotaviruε gp38 gene pluε the plasmid DNA inserted into the XJbal site in the long unique region of the virus genome, but no longer contains the NEO gene of parent S- IBR-004 in the unique short region. In fact, a small deletion was created in the unique short region at the location of the NEO gene, aε evidenced by the abεence of an XJbal εite at thiε location in S-IBR-008. s-IBR-008 waε shown to be expressing the rotavirus gp38 gene by εinalyεis of RNA transcription in infected cells, and by the ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS procedure using antibodieε specific for the gp38 gene. S-IBR-008 was depoεited on June 18, 1986 purεuant to the Budapest Treaty on the International Deposit of Microorganismε for the Purpoεeε 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 2141. The structure of S-IBR-008 is shown in Figure 20. Sxample 16 g-IBR-Q . S S-IBR-018 is an IBR virus that has three foreign genes inserted: the E.coli beta-galactosidaεe gene and the neomycin resistance gene in the XJbal site in the unique long region, and the parainfluenza type 3 (PI-3) virus hemagglutinin gene (HN) in the Hindlll site in the unique long region immediately adjacent to the Xbal site. For cloning the PI-3 HN gene, the SF-4 strain of PI-3 was grown in MADIN-DARBY bovine kidney (MDBK) cells in culture and RNA was extracted from infected cellε. The RNA waε used in a reverse transcription protocol as outlined in the cDNA CLONING procedure using poly-dT as primer for reverse tranεcriptase. From this procedure, a εeries of clones was obtained that comprised parts of the genome of the PI-3 virus. The location of the gene for the human PI-3 HN gene has been published (25,26) and this information was used to locate the gene in applicants' bovine PI-3 clones. The entire open reading frame of the bovine PI-3 HN gene was sequenced by applicants and is given in Figure 21. The HSV ICP4 promoter was used to express the PI-3 HN gene and the HSV TK poly-A signal was used to terminate transcription. The engineering of this construct was done aε εhown in Figure 22 A and B. The construct contained (5' to 3') the HSV ICP4 promoter, the ICP4 TATA box, the ICP4 cap site, a fusion within the ICP4 5' untranslated region to the PI-3 HN gene at the Hhal εite, the HN gene εtart codon, the HN εtructural gene, the HN stop codon, a fusion within the HN 3' untranslated region to the HSV TK untranslated 3' region, and the HSV TK poly-A signal sequence. Thiε plaεmid alεo contained the beta-galactosidase (lacZ) gene under the control of the PRV gpX promoter with the gpX poly-A termination εignal, aε well as the neomycin resistance gene under the control of the gpX promoter with the TK poly-A termination signal. These latter two genes were cloned in tandem at the XJbal site in BamHI-C fragment (Figure 22 A and B) . This BamHI-C fragment contained the homology regions for use in the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure. After the trans ' fection step in the procedure, the resulting recombinant virus from the transfection stock was selected for by the SELECTION OF G418 RESISTANT IBR VIRUS procedure, followed by the BLUOGAL * SCREEN FOR RECOMBINANT HERPESVIRUS procedure, and subsequently analyzed for the insertion of the PI-3 HN gene by the SOUTHERN BLOTTING OF DNA procedure. The viruε that resulted from this screening was designated S-IBR-018. S-IBR-018 was depoεited on July 21, 1987 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposeε of Patent Procedure with the Patent Culture Depoεitory of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Acceεsion No. VR 2180. The structure of S-IBR-018 is shown in Figure 22 C. g-IPfi-Q19 S-IBR-019 is an IBR virus that has three foreign genes inserted: the E.coli beta-galactosidase (lacZ) gene and the neomycin resistance gene in the XJbal site in the unique long region, and the parainfluenza type 3 (PI-3) virus fuεion gene (F) in the Hindlll site in the long unique region adjacent to the XJbal site. For cloning the PI-3 F gene, the SF-4 strain of PI-3 was grown in MDBK cellε in culture and RNA was extracted from infected cells. The RNA waε used in a reverse tranεcription protocol as outlined in the cDNA CLONING procedure using poly-dT as primer for reverse transcriptase. From this procedure, a series of clones was obtained that comprised parts of the genome of the PI-3 virus. The location of the gene for the Sendai viruε F gene haε been published (27) and this comparative sequence information was uεed to locate the homologouε gene in applicants' bovine PI-3 clones. The HSV alpha-4 promoter waε uεed to express the PI-3 F gene and the HSV TK poly-A signal was used to terminate transcription. The conεtruct contained (5' to 3') the HSV alpha-4 promoter, the alpha-4 TATA box, the alpha-4 cap εite, a fuεion in the alpha-4 5' untranεlated region to the PI-3 F gene, the F εtart codon, the F structural gene, the F εtop codon, a fuεion in the F 3' untranεlated region to the HSV TK 3' untranεlated region, and the TK poly-A εignal εequence. Thiε plasmid also contained the beta-galactosidase (lacZ) gene under the control of the PRV gpX promoter with the gpX poly-A termination signal, as well as the neomycin resistance gene under the control of the gpX promoter with the TK poly-A termination εignal. Theεe latter two geneε were cloned in tandem at the XJbal site in BamHI-C fragment (Figure 23 A and B) . This BamHI-C fragment contained the homology regions for use in the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure. After the transfection step in the procedure, the resulting recombinant viruε from the tranεfection stock waε εelected for by the SELECTION OF G418 RESISTANT HERPESVIRUS procedure, followed by the BLUOGAL * SCREEN FOR RECOMBINANT HERPESVIRUS procedure, and subεequently analyzed for the insertion of the PI-3 F gene by SOUTHERN BLOTTING OF DNA procedure. The virus that resulted from this screening was designated S-IBR-019. The structure of S-IBR-019 is shown in Figure 23 C. Examole 18 S-IBR-032 S-IBR-032 is an IBR virus that haε two foreign geneε inεerted: the Escherichia coli beta-galactosidase (lacZ) gene with the bovine viral diarrhea virus (BVDV) gp53 gene fused to the lacZ C-terminus and inserted in the long unique region at the XJbal restriction endonuclease site. For cloning the BVDV gp53 gene, the Singer strain of BVDV was grown in MADIN-DARBY bovine kidney (MDBK) cells in culture and the RNA was extracted from infected cells. The RNA was used in a reverse transcriptase procedure as outlined in the cDNA CLONING procedure using random primers for reverse transcriptase. From this procedure, a series of clones was obtained that comprised parts of the genome of BVDV. The location of the gene for BVDV gp53 has been published (66) and this comparative sequence information was used to locate the homologous gene in the applicant's BVDV clones. The PRV gpX promoter was used to express lacZ with a region of BVDV gp53 fused to the C-terminus, and the PRV poly-A signal was used to terminate transcription. A plasmid construct was engineered that contained (5' to 3') the PRV gpX promoter and then the coding region consisting of amino acid codons 1-7 of the PRV gpX gene, 10-1024 of the Escherichia coli lacZ gene, and 684-758 of the BVDV major open reading frame, and the PRV poly-A sequence. Thiε lacZ fuεion gene cassette was then excised from the plaεmid vector at the flanking Xbal εiteε and cloned into the unique XJbal εite in IBR-002 using the in vitro ligation method described in CONSTRUCTION OF DELETION VIRUSES. After the transfection Step in DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure, the reεulting recombinant viruε waε screened and iεolated from the tranεfection εtock using the BLUOGAL * SCREEN FOR RECOMBINANT HERPESVIRUS procedure, and subεequently analyzed for the inεertion of the BVDV gp53 region by SOUTHERN BLOTTING OF DNA procedure. The viruε that reεulted from this screening was designated S- IBR-032. Example 19 S-IBR-039 s-IBR-039 is an IBR virus that has three deletions in the short unique region of the genome. The first deletion is approximately 2500 base pairs and begins in the Hindlll K fragment approximately 1750 base pairs downstream of the Hindlll 0/ Hindlll K junction and extends back through that junction. This deletion removes the US2 gene. The second deletion is approximately 1230 base pairs and begins in the Hindlll K fragment approximately 3900 base pairs downstream of the Hindlll O/Hind K junction and extends back toward that junction. This deletion removes εunino acids l to 361 of the gpG gene. The third deletion is approximately 1410 base pairs and removes amino acids 77-547 of the gpE gene. S-IBR-039 was derived from S-IBR-037. This was accomplished utilizing the homology vector 536-03.5 (see MATERIALS AND METHODS) and virus S-IBR-037 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 reεult of blue plaque purification waε the recombinant viruε designated S-IBR-039. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA procedure. This analysiε confirmed the inεertion of the β-galactoεidase (lacZ) marker gene and the deletion of approximately 1230 base pairε of the gpG gene. It waε also confirmed that an approximately 1410 base pair deletion haε occurred in the region of the gpE gene (εee above) . Thiε viruε will be useful as a vaccine to protect cattle from infection with IBR viruε. The deletions of the glycoproteins G and E genes from this viruε alεo provideε two negative εerological markerε for differentiating it from wild type IBR. ' g-xample go g-.PR-Q45 S-IBR-045, a recombinant IBR virus with deletions in the Tk, US2, gpG and gpE genes may be constructed in the following manner. S-IBR-045 would be derived from S-IBR- 039 (see example 19) through the construction of two intermediate viruses. The first intermediate virus, S- IBR-043, would be constructed utilizing the homology vector 591-46.12 (see MATERIALS AND METHODS) and virus S- IBR-039 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock would be screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (uidA substrate) . The resulting virus would have deletions of the Tk, US2, gpG and gpE genes and insertion of lacZ gene in the gE gene deletion. Finally, S-IBR-045 would be constructed, utilizing the homology vector 523-78.72 (see MATERIALS AND METHODS) and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock would be screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (lacZ substrate) . This virus will be useful as a vaccine to protect cattle from infection with IBR. The combination of deletions will provide the appropriate attenuation which is required for a superior vaccine. This virus will alεo provideε two negative εerological markerε which may be uεed to distinguish vaccinated from infected animals. The availability of two negative markers permits one marker to be used as a confirmatory test, greatly increasing the reliability of such a diagnostic determination. Examole 21 S-.BR--Q46 S-IBR-046, a recombinant IBR virus with deletions in the Tk, US2, gpG and gpE genes and the bovine viral diarrhea virus gp53 gene inεerted in place of the gpE gene, may be constructed in the following manner. S-IBR-046 would be derived from S-IBR-044 (see example 20) . It would be constructed utilizing the homology vector 523-78.72, into which the bovine viral diarrhea virus gp53 gene has been inserted, and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Note that the bovine diarrhea virus gene would be cloned using techniques described in the methods section. The gp53 gene would be placed under the control of the HCMV immediate early promoter. The transfection εtock would be screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (lacZ substrate) . This virus will be useful as a vaccine to protect cattle from infection with IBR virus and bovine viral diarrhea virus. Examole 22 S-IBR-Q47 S-IBR-047, a recombinant IBR virus with deletions in the Tk, US2, gpG and gpE genes and the parainfluenza type 3 genes for hemagglutinin and fusion protein inserted in place of the gpE gene may be constructed in the following manner. S-IBR-047 would be derived from S-IBR-044 (see example 20) . It would be constructed utilizing the homology vector 523-78.72, into which the parainfluenza type 3 virus hemagglutinin and fuεion geneε haε been inεerted, and viruε S-IBR-044 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Note that the parainfluenza type 3 viruε geneε would be cloned uεing techniques described in the methods section. The transfection stock would be screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (lacZ subεtrate) . This virus will be useful as a vaccine to protect cattle from infection with IBR virus and parainfluenza type 3 virus. Examolβ 23 S-IBR-049 S-IBR-049, a recombinant IBR virus with deletions in the Tk, US2, gpG and gpE genes and the bovine reεpiratory εyncytial viruε geneε for the attachment, nucleocapsid and fuεion proteins inserted in place of the gpE gene may be constructed in the following manner. S-IBR-049 would be derived from S-IBR-044 (see example 20) . It would be constructed utilizing the homology vector 523-78.72, into which the bovine respiratory syncytial virus attachment nucleocapsid and fusion genes had been inserted and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Note that the bovine respiratory syncytial virus genes would be cloned using techniques described in the methods section. The attachment protein gene would be placed under the control of the HCMV immediate early promoter and the fusion and nucleocapsid protein genes would be placed under the PRV gpX promoter. The transfection stock would be screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (lacZ substrate) . This virus will be useful as a vaccine to protect cattle from infection with IBR virus and bovine respiratory syncytial. Example 24 S-IBR-051 S-IBR-051, a recombinant IBR virus with deletions in the Tk, US2, gpG and gpE genes and the Pasteurella haemolytica genes for the leukotoxin and iron regulated outer membrane proteins inserted in place of the gpE gene, may be constructed in the following manner. S-IBR- 051 would be derived from S-IBR-044 (see exεunple 20) . It would be constructed utilizing the homology vector 523- 78.72, into which the Pasteurella haemolytica leukotoxin and iron regulated outer membrane protein genes had been inserted, and virus S-IBR-044 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Note that the Pasteurella haemolytica genes would be cloned using the techniques described in the methods section. The leukotoxin gene would be placed under the control of the HCMV immediate early promoter and the iron regulated outer membrane protein genes would be placed under the PRV gpX promoter. The transfection stock would be screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES for a white plaque recombinant virus (lacZ substrate) . This virus will be useful as a vaccine to protect cattle from infection with IBR virus and Pasteurella haemolytica. E aff l? 25 Shipping Fever Vaccine Shipping fever or bovine respiratory disease (BRD) complex iε manifested aε a reεult of a combination of infectiouε diseases of cattle and additional stress related factors (70) . Respiratory virus infections, augmented by pathophysiological effectε of stress, alter the susceptibility of cattle to Pasteurella organisms that are normally present in the upper respiratory tract by a number of mechanisms. Control of the viral infections that initiate BRD as well as control of the terminal bacterial pneumonia is essential to preventing the disease syndrome (71) . The major infectious diseases that contribute to BRD are: infectious bovine rhinotracheitis virus, parainfluenza type 3 virus, bovine viral diarrhea virus, bovine respiratory syncytial virus, and Pasteurella haemolytica (71) . The applicants' examples 1 through 24 describe vaccine inventions that individually immunize against the various components of BRD. An extension of the applicants' approach is to combine vaccines in a manner so aε to control the array of disease pathogens with a single immunization. To this end, at least two approaches can be taken: first, mixing of the various IBR vectored antigenε (BRSV, PI-3, BVDV and P. Haemolytica) in a single vaccine dose, and secondly, the individual antigens (BRSV, BVDV, PI-3 and P. haemolytica) can be simultaneously cloned into the same IBR backbone virus. Note that a combination of antigens could be included in one or more IBR backbone viruses so as to limit the number of IBR viruses required for BRD protection. Also, . conventionally derived vaccines (killed virus, inactivated bacterins and modified live viruses) could be included as part of the BRD vaccine formulation εhould εuch vaccine components prove to be more effective. Referenceε 1. J.L. Cantello et al.. Journal of Virology £5., 1584- 1588 (1991) . 2. U.K. Laemnli, Nature 227. 680-685 (1970). 3. B. Lomniczi et al., Journal of Virology 4J_, 970-979 (1984) . 4. R. Longnecker and B. Roizman, Science 236. 573-576 (1987) . 5. S. Mackem and B. Roizman, Proc. Natl. Acad. Sci. USA 79., 4917-4921 (1982) . 6. T. Maniatiε et al. , Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Press (1982) . 7. J.E. Mayfield et al., Journal of Virology _|2, 259- 264 (1983) . 8. D.J. McGeoch et al.. Journal of Molecular Biology 181. 1-13 (1985) . 9. D.J. McGeoch et al., Journal of General Virology 68. 19-38 (1987) . 10. D.J. McGeoch et al., Journal of General Virology 69. 1531-1574 (1988) . 11. E.A. Petrovskiε et al. , Journal of Virology 0, 116- 169 (1986). 12. T.J. Rea et al. , Journal of Virology 5_£, 21-29 (1985) . 13. A.K. Robbins et al.. Journal of Virology £8., 339-347 (1986) . 14. J. Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Press (1989) . 15. G.A. Smith et al., Journal of General Virology 71, 2417-2424 (1990) . 16. D.R. Thomsen et al.. Gene 16, 207-217, (1981) 17. S.K. Tikoo et al.. Journal of Virology 64., 5132-5142 (1990) . 18. C.E. Aronεon, ed. , Veterinary Pharmaceuticalε and Biologicalε. Veterinary Medicine Publ. Co., Lenexa, KS, pp. 138-139 (1982-1983). 19. P.C. Weber et al.. Science 236. 576-579 (1987). 20. U.S. Wirth et al., Journal of Virology 6≥, 195-205 (1991) . 21. M. Zijil et al., Journal of Virology 71, 1747-1755 (1990) . 22. L. Villarreal and P. Berg, Science 196. 183-185 (1977) . 23. U. Gubler and B.J. Hoffman, Gene 25. 263-269 (1983). 24. F.L. Grεϋaam and A. Van der Eb, Virology ___., 556-567 (1973) . 25. N. Elango et al.. Journal of Virology ≤Z, 481-489 (1986). 26. M.K. Spriggs and P.L. Collinε, Journal- of Virology ■ 59. 646-654 (1986) . 27. B.M. Blumberg et al. , Journal of General Virology 66. 317-331 (1985) . 28. R. W. Price and A. Kahn, Infection and Immunity 34. 571-580 (1981) . 29. P. B. Tenεer et al. , J. of General Virology 64. 1369-1373 (1983). 30. B. Roizman et al. , Cold Spring Harbor Conference on New Approacheε to Viral Vaccines (September, 1983). 31. R. L. Thompson et al.. Virology 131. 180-192 (1983). 32. K. Fukuchi et al., Proc. Natl. Acad. Sci. U.S.A. 82. 751-754, 1985. 33. J. M. Koomey et al., J. of Virology ≤p_, 662-665, 1984. 34. S.B. Mohanty and S.K. Dutta, Veterinary Virology. Lea and Febiger, Philadelphia (1981) . 35. R. Crandell in Current Veterinary Therapy, pages 543-546, W.B. Saunders, Philadelphia (1981). 36. H. Ludwig in The Herpesviruseε. Vol. 2, B. Roizman, ed. , Plenum Press (1983). 37. A.J. Davison, EMBO Journal, 2 , 2203-2209 (1983). 38. F.A. Ferrari et al., J. of Bacteriology 161, 556-562 1985. 39. V.T. Oi and L.A. Herzenberg, Selected Methods in Cellular Immunology. Freeman Publ. Co., San Francisco (1980). pp. 351-372. 40. S. Ihara et al., Virology 122., 268-278 (1982). 41. D. Hanahan, Molecular Biology 166. 557-580 (1983) . 42. M.W. Mellencεunp et al., J. of Clinical Microbiology 22, 2208-2213 (1989). 43. Kit et al., U.S. Patent No. 4,824,667, issued April 25, 1989. 44. Kit et al., U.S. Patent 4,703,011, issued October 27, 1987. 45. Kit et al., The Veterinary Record 127. 363-364 (1990) . 46. European Patent Publication EP 0 326 127 A2, published August 2, 1989. 47. Federal Register, Vol. 55, No. 90, pp. 19245-19253 (May 9, 1990) . 48. Fitzpatrick et al., J. of Virol. 6_2, 4239-4288 (1988). 49. T. Ben-Porat et al., Virol. 151, 325-334 (1986). 50. F. Zuckerman et al., in Vaccination and Control of Auieszkv'ε Disease. Ed. J. van Oirεchot, Kluwer, London (1989). pp. 107-117. 51. L.E. Poεt et al., J. Reprod. Fert. Suppl. 4 , 97-104 (1990) . 52. Wirth et al., J. Of Virol. 6_i, 4882-4889 (1989). 53. B. Moss, Science 2i2, 1662-1667 (1991). 54. R.W. Honesε, J. of General Virology 6J5, 2077-2107 (1984) . 55. Cook & Stevenε, J. of General Virology .31, 75-80 (1976) . 56. Deεrosiers et al., Molecular and Cellular Biology 5_, 2796-2803 (1985) . 57. Thomεen et al. , Gene 57, 261-265 (1987). 58. Weir and Narayanan, Nucleic Acidε Research 16. 10267-10282 (1988) . 59. Spaete and Mocarski, Proceedingε of the National Academy of Sciences U.S.A. ___L, 7213-7217 (1987). 60. Whealy et al. , Journal of Virology §2., 4185-4194 (1988) . 61. Shih et al., Proceedingε of the National Academy of Scienceε U.S.A. fil, 5867-5870 (1984). 62. Edwards et al., in Technolpqjcal A -anς.-.s in Vagc-ine Development. pp. 223-234, Alan Riss Inc. (1988). 63. Proceeding of the 94th Annual Meeting of the United States Animal Health Association, pp. 66-75 (1990) . 64. E.A. Petrovskiε et al. , Journal of Virology £0_, 185- 193 (1986) . 65. Todd et al., U.S. Patent No. 4,132,775, issued January 2, 1979. 66. M.S. Collett et al., Journal of Virology ££, 200- 208, (1988). 67. M.A. Innis et al., PCR Protocols: A Guide to Methods and Applications, 84-91, Academic Press, Inc. San Diego (1990) . 68. R.D. Walker, et al., Am. J. Vet Res. j5!>, 1230-1234 (1984) 69. E. Harlow, and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York (1988) . 70. CA. Hjerpe, The Bovine Respiratory Diseaεe Complex. In: Current Veterinary Therapy 2: Food Animal Practice. Ed. by J.L. Howard, Philadelphia, W.B. Saunderε Co., 1986, pp 670-680. 71. F. Fenner, et al., "Mechanisms of Disease Production: Acute Infections", Veterinary Virology. Academic Presε, Inc., Orlando, Florida, 1987, pp 183-202. 72. T. Inque, et al., Journal of General Virology 7£, 919-934 (1989).

Claims: What is claimed is: 1. A recombinant IBR virus comprising viral DNA from a naturally-occurring IBR viruε in which DNA encoding gpG glycoprotein haε been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein. 2. A recombinant IBR viruε of claim 1, wherein DNA encoding gpG glycoprotein has been deleted. 3. A recombinant IBR virus of claim 2, wherein foreign DNA has been inserted in place of the deleted DNA encoding gpG glycoprotein. 4. A recombinant IBR virus of claim 1, wherein foreign DNA has been inεerted into the DNA encoding gpG glycoprotein. 5. A recombinant IBR viruε of claim 1, further characterized in that DNA encoding the gpE glycoprotein haε been altered or deleted so that upon replication the recombinant IBR virus produceε no gpE glycoprotein. 6. A recombinant IBR virus of claims 2, 3, or 4, further characterized in that DNA encoding the gpE glycoprotein haε been altered or deleted εo that upon replication the recombinant IBR viruε produces no gpE glycoprotein. 7. A recombinant IBR virus of claim 5, wherein DNA encoding gpE glycoprotein has been deleted. 8. A recombinant IBR virus of claim 7, wherein foreign DNA iε inεerted in place of the deleted DNA encoding gpE glycoprotein. 9. A recombinant IBR viruε of claim 5, wherein foreign DNA haε been inεerted into the DNA encoding gpE glycoprotein. 10. A recombinant IBR viruε of claim l, further characterized in that (1) DNA correεponding to the US2 region of the naturally-occurring IBR viruε haε been deleted, and (2) DNA encoding the gpE glycoprotein has been altered or deleted. 11. A recombinant IBR virus of claims 2, 3, or 4, further characterized in that (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and (2) DNA encoding the gpE glycoprotein has been altered or deleted. 12. A recombinant IBR viruε comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally- occurring IBR virus has been deleted, and (2) DNA encoding gpG glycoprotein has been altered or deleted. 13. A recombinant IBR virus of claim 12, wherein DNA encoding the gpG glycoprotein has been deleted. 14. A recombinant IBR virus of claim 13 designated S- IBR-037. 15. A recombinant IBR virus of claim 12, wherein foreign DNA is inserted in place of the deleted DNA encoding gpG glycoprotein. 16. A recombinant IBR virus of claim 12, wherein foreign DNA iε inεerted in place of the deleted DNA corresponding to the US2 region of the naturally- occurring IBR virus. 17. A recombinant IBR virus of claim 15, -wherein the foreign DNA is a sequence which encodes Escherichia coli β-galactosidase. 18. A recombinant IBR viruε of claim 17 deεignated s- IBR-035. 19. A recombinant IBR virus of claim 17 designated S- IBR-036. 20. A recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR viruε produceε no gpE glycoprotein. 21. A recombinant IBR virus of claim 20, wherein DNA encoding gpE glycoprotein has been deleted. 22. A recombinant IBR virus of claim 21, wherein foreign DNA is inserted in place of the deleted DNA encoding gpE glycoprotein. 23. A recombinant IBR virus of claim 20, wherein foreign DNA haε been inεerted in DNA encoding gpE glycoprotein. 24. A recombinant IBR viruε of claim 20, further characterized in that DNA correεponding to the US2 region of the naturally-occurring IBR virus has been deleted. 25. A recombinaαt IBR virus of claims 21, 22, or 23, further characterized in that DNA corresponding to the US2 region of the naturally-occurring IBR virus haε been deleted. 26. A recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA in the unique short region of the naturally-occurring IBR virus has been deleted. 27. A recombinant IBR viruε of claim 26, wherein foreign DNA haε been inserted into the DNA of the recombinant IBR virus. 28. A recombinant IBR virus of claim 27, wherein foreign DNA has been inserted into the XJbal site in the long unique region. 29. A recombinant IBR virus of claim 27, wherein the foreign DNA iε a sequence which encodeε bovine rotaviruε glycoprotein 38. 30. A recombinant IBR viruε of claim 29, wherein the sequence which encodes bovine rotavirus glycoprotein 38 has been inserted into the XJbal site in the long unique region. 31. A recombinant IBR virus of claim 30 designated S- IBR-008. 32. A recombinant IBR virus of claim 26, wherein at least a portion of both repeat εequences haε been deleted. 33. A recombinant IBR viruε of claim 32 designated S- IBR-027. 34. A recombinant IBR virus compriεing viral DNA from a naturally-occurring IBR viruε from which at least a portion of both repeat sequences has been deleted. 35. A recombinant IBR viruε of claim 34, wherein DNA encoding one or more EcoRV restriction sites haε been deleted. 36. A recombinant IBR virus of claim 35 designated S- IBR-002. 37. A recombinant IBR virus of claim 34, wherein foreign DNA has been inserted into the DNA of the recombinant IBR virus. 38. A recombinant IBR virus of claim 37, wherein the foreign DNA is a sequence which encodes the Tn5 NEO gene. 39. A recombinant IBR virus of claim 38 designated S- IBR-020. 40. A recombinant IBR virus of claim 38, wherein at least a portion of the thymidine kinase gene has been deleted. 41. A recombinant IBR virus of claim 40, wherein the Tn5 NEO gene is under the control of an inserted, upstream, HSV-l alpha-4 promoter. 42. A recombinant IBR virus of claim 41 designated S- IBR-028. 43. A recombinant IBR virus comprising viral DNA from a naturally-occurring IBR viruε in which a foreign DNA sequence which encodeε the Tn5 NEO gene has been inεerted into the viral DNA. 44. A recombinant IBR viruε of claim 43, wherein the Tn5 NEO gene iε under the control of an inεerted, upεtream, pεeudorabieεviruεglycoproteinXpromoter. 45. A recombinant IBR viruε of claim 44 deεignated S- IBR-004. 46. A recombinant IBR viruε compriεing viral DNA from a naturally-occurring IBR viruε in which a foreign DNA sequence which encodes the Eεcherichia coli β- galactosidase and Tn5 NEO geneε, and the parainfluenza type 3 viruε hemagglutinin gene, HN, haε been inεerted into the viral DNA. 47. A recombinant IBR viruε of claim 46 designated S- IBR-018. 48. A recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Escherichia coli β- galactoεidaεe and Tn5 NEO geneε, and the parainfluenza type 3 viruε fusion gene, F, has been inserted into the viral DNA. 49. A recombinant IBR virus of claim 48 designated S- IBR-019. 50. A vaccine which comprises a suitable carrier and an effective immunizing amount of the recombinant virus of claim 26, 34, 43, 46, or 48. 51. A method of immunizing an animal against infectious bovine rhinotracheitis virus which compriseε administering to the animal an effective immunizing dose of the vaccine of claim 50. 52. A method of claim 51, wherein the animal is a bovine. 53. A method for distinguishing an animal vaccinated with a vaccine which compriseε an effective immunizing amount of a recombinant virus of claims 1, 5, 10, or 12 from an animal infected with a naturally-occurring IBR viruε which comprises analyzing a sample of a body fluid from the animal for the presence of gpG glycoprotein of IBR virus and at leaεt one other antigen normally expressed in an animal infected by a naturally-occurring IBR virus, identifying antigens which are present in the body fluid and determining whether gpG glycoprotein iε present in the body fluid, the presence of antigens which are normally expressed in an animal by a naturally-occurring IBR virus and the absence of gpG glycoprotein in the body fluid being indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus. 54. The method of claim 53, wherein the presence of -αtigens and gpG glycoprotein in the body fluid iε determined by detecting in the body fluid antibodies specific for the antigens and gpG glycoprotein. 55. A method for distinguiεhing an animal vaccinated with a vaccine which compriseε an effective immunizing amount of a recombinant viruε of claimε 5, 10, 20, or 24 from an animal infected with a naturally-occurring IBR viruε which compriseε analyzing a εample of a body fluid from the animal for the preεence of gpE glycoprotein of IBR viruε and at least one other antigen normally expressed in an animal infected by a naturally-occurring IBR virus, identifying antigens which are present in the body fluid and determining whether gpE glycoprotein is preεent in the body fluid, the preεence of antigenε which are normally expressed in an animal by a naturally-occurring IBR viruε and the abεence of gpE glycoprotein in the body fluid being indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus. 56. The method of claim 55, wherein the presence of antigens and gpE glycoprotein in the body fluid is determined by detecting in the body fluid antibodies specific for the antigens and gpE glycoprotein. 57. Isolated DNA encoding the gpG glycoprotein of IBR virus. 58. Purified recombinant gpG glycoprotein encoded by the DNA of claim 57. 59. A recombinant cloning vector which compriseε the DNA of claim 57. 60. A recombinant expreεεion vector which comprises the DNA of claim 57. 61. A host cell which comprises the recombinant expression vector of claim 60. 62. A method of producing a polypeptide which comprises growing the host cell of claim 61 under conditions such that the recombinant expresεion vector expresses gpG glycoprotein and recovering the gpG glycoprotein so expressed. 63. An antibody directed to an epitope of the purified gpG glycoprotein of IBR virus of claim 58. 64. A monoclonal antibody of claim 63. 65. A method of detecting the presence or absence of gpG glycoprotein of IBR virus in a sample which comprises contacting the εample with the antibody of claim 63 under conditions such that the antibody forms a complex with any gpG glycoprotein present in the εample and detecting the presence or absence of such complex. 66. A method of claim 65, wherein the sεunple iε bovine- derived. 67. Iεolated DNA encoding the gpE glycoprotein of IBR viruε. 68. Purified recombinant gpE glycoprotein encoded by the DNA of claim 67. 70. A recombinant cloning vector which compriεes the DNA of claim 67. 71. A recombinant expression vector which comprises the DNA of claim 70. 72. A method of producing a polypeptide which comprises growing the host cell of claim 70 under conditions such that the recombinant expression vector expresseε gpE glycoprotein and recovering the gpE glycoprotein εo expreεεed. 73. An antibody directed to an epitope of the purified gpE glycoprotein of IBR viruε of claim 68. 74. A monoclonal antibody of claim 73. 75. A me'thod of detecting the preεence or absence of gpE glycoprotein of IBR virus in a sample which compriseε contacting the sample with the antibody of claim 73 under conditions such that the antibody forms a complex with any gpE glycoprotein present in the seunple and detecting the presence or absence of such complex. 76. A method of claim 75, wherein the sample is bovine- derived. 77. A method of producing a fetal-safe, live recombinant IBR virus which comprises treating viral DNA from a naturally-occurring live IBR virus so as to delete from the virus DNA corresponding to the US2 region of the naturally-occurring IBR virus. 78. A recombinant pseudorabieε virus designated S-PRV- 160. 79. An antibody which recognizes the recombinant pseudorabieε virus of claim 78. 80. Isolated DNA encoding the US2 gene of an IBR virus. 81. A homology vector for producing a recombinant IBR virus by inserting foreign DNA into the genomic DNA of an IBR virus which comprises a double-stranded DNA molecule consisting essentially of: (a) double-stranded foreign DNA encoding RNA which does not naturally occur in an animal into which the recombinant IBR is introduced; (b) at one end of the foreign DNA, double-stranded IBR viral DNA homologous to genomic DNA located at one side of a site on the genomic DNA which is not essential for replication of the IBR virus; and (c) at the other end of the foreign DNA, double- stranded IBR viral DNA homologouε to genomic DNA located at the other εide of the same site on the genomic DNA. 82. A homology vector of claim 81, wherein double- stranded foreign DNA further compriseε a promoter. 83. A homology vector of claim 82, wherein the promoter is from HSV-l α 4 immediate early gene, Human cytomegalovirus immediate early gene or pseudorabies virus glycoprotein X gene. 84. A homology vector of claim 81, wherein double- stranded foreign DNA further comprises a polyadenylation signal. 85. A homology vector of claim 84, wherein the polyadenylation signal is from HSV-l thymidine kinase gene or pεeudorabieε viruε glycoprotein X gene. 86. A homology vector of claim 81, wherein the RNA encodeε a polypeptide. 87. A homology vector of claim 86, wherein the polypeptide iε a detectable marker. 88. A homology vector of claim 87, wherein the polypeptide iε Eεcherichia coli β-galactosidase or bacterial transpoεon neomycin resistance protein. 89. A homology vector of claim 88, wherein DNA which encodes the polypeptide is flanked on each side by restriction siteε permitting εaid DNA to be cut out with a reεtriction endonuclease which cuts at a limited number of sites on the genome. 90. A homology vector of claim 81, wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 860 bp Nσol to BamHI subfragment of the Hindlll A fragment of IBR virus and the downstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 1741 bp Bglll to StuI subfragment of the Hindlll A fragment of IBR virus. 91. A homology vector of claim 90, wherein upstream double-stranded foreign DNA which compriseε a promoter and downstream double-stranded foreign DNA which comprises a polyadenylation signal flank on each side double-stranded foreign DNA which encodes a detectable marker. 92. A homology vector of claim 91, wherein the upstream promoter iε homologouε to genomic DNA preεent within the approximately 490 bp PvulI to BamHI εubfragment of the BamHI N fragment of HSV-l and the downεtreεun polyadenylation signal is homologous to genomic DNA present within the approximately 784 bp Smal to Smal subfragment of the BamHI Q fragment of HSV-l. 93. A homology vector of claim 91, wherein the DNA which encodes a detectable marker is homologous to the approximately 1541 bp Bglll to BamHI fragment of Tn5. 94. A homology vector of claim 81, wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 3593 bp Hindlll to Xhol subfragment of the Hindlll K fragment of IBR virus and the downstream double- stranded IBR viral DNA is homologous to genomic DNA present within the approximately 785 bp Xhol to Ndel subfragment of the Hindlll K fragment of IBR virus. 95. A homology vector of claim 94, wherein upstream double-stranded foreign DNA which comprises a promoter and downstream double-stranded foreign DNA which compriseε a polyadenylation signal flank on each side double-stranded foreign DNA which encodeε a detectable marker. 96. A homology vector of claim 95, wherein the upεtream promoter iε homologouε to genomic DNA present within the approximately 1191 bp Avail to PstI subfragment of the XJbal B fragment of HCMV and the downεtreεun polyadenylation sequence is homologous to genomic DNA present within the approximately 753 bp Sail to Ndel εubfragment of the BamHI #7 fragment of PRV. 97. A homology vector of claim 95, wherein the DNA which encodeε a detectable marker iε homologouε to the approximately 3347 bp Ball to BamHI fragment of pJF751. 98. A homology vector of claim 81, wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 888 bp Af ul to Smal subfragment of the Hindlll K fragment of IBR virus and the downstreεun double-stranded IBR viral DNA is homologous to genomic DNA preεent within the approximately 785 bp Xhol to Ndel subfragment of the Hindlll K fragment of IBR virus. 99. A homology vector of claim 98, wherein upstream double-stranded foreign DNA which compriseε a promoter and double-εtranded foreign DNA which comprises a polyadenylation signal flank on each side double-stranded foreign DNA which encodes a detectable marker. 100. A homology vector of claim 99, wherein the upεtream promoter is homologous to genomic DNA present within the approximately 1191 bp Avail to PstI subfragment of the XJbal B fragment of HCMV and the downstream polyadenylation εignal iε homologouε to genomic DNA present within the approximately 753 bp Sa l to Ndel subfragment of the BamHI #7 fragment of PRV. 101. A homology vector of claim 99, wherein the DNA which encodes a detectable marker is homologous to the approximately 3347 bp Ball to BamHI fragment of pJF571. 102. A homology vector of claim 81, wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 1704 bp Smal to Smal subfragment of the Hindlll K fragment of IBR virus and the downstream double-stranded IBR viral DNA is homologous to genomic DNA present within the approximately 742 bp Nhel to Bg l subfragment of the Smal 2.5KB fragment of IBR virus. 103. A homology vector of claim 102, wherein upstream double-stranded foreign DNA which comprises a promoter and downstream double-stranded foreign DNA which comprises a polyadenylation signal flank on each side double-stranded foreign DNA which encodeε a detectable marker. 104. A homology vector of claim 103, wherein the upεtream promoter iε homologouε to genomic DNA present within the approximately 413 bp Sail to BamHI subfragment of the BamHI #10 fragment of PRV and the downstream polyadenylation signal is homologous to genomic DNA present within the approximately 754 bp Ndel to Sail subfragment of the BamHI #7 fragment of PRV. 105. A homology vector of claim 103, wherein the detectable marker is homologouε to the approximately 3010 bp BamHI to PvuII fragment of pJF751. 106. A homology vector for producing a recombinant IBR viruε by deleting DNA which encodeε a detectable marker which had been inserted into the genomic DNA of an IBR virus comprising a double-stranded DNA molecule consisting essentially of double-stranded IBR viral DNA homologous to the genomic DNA which flank on each side the DNA to be deleted. 107. A homology vector of claim 106, wherein the upstream double-stranded IBR viral DNA is homologous to genomic DNA preεent within the approximately 888 bp Λflul to Smal subfragment of the Hindlll K fragment of IBR virus and the downstreεun double-stranded IBR viral DNA is homologouε to genomic DNA present within the approximately 785 bp Xhol to Ndel εubfragment of the Hindlll K fragment of IBR virus. 108. A vaccine which comprises a suitable carrier and an effective immunizing εunount of the recombinant virus of claim 1, 5, 10, 12, 20 or 24. 109. A method of immunizing an animal against infectious bovine rhinotracheitis viruε which compriseε administering to the animal an effective immunizing dose of the vaccine of claim 108. 110. A method of claim 109, wherein the animal iε a bovine. 111. A recombinant IBR viruε of claim 37, wherein the foreign DNA is a sequence which encodes Bovine viral diarrhea virus glycoprotein gp53. 112. A recombinant IBR virus of claim 111, designated S- IBR-032. 113. A recombinant IBR viruε comprising viral DNA from a naturally-occurring IBR virus from which DNA from the US2 gene, the gpE glycoprotein gene and the gpG glycoprotein gene has been deleted so that upon replication, the recombinant IBR virus produces no gpE glycoprotein and no gpG glycoprotein. 114. A recombinant IBR virus of claim 113, wherein a foreign DNA sequence is inserted in place of the DNA which encodes gpE glycoprotein. 115. A recombinant IBR virus of claim 114, wherein the foreign DNA sequence encodes Escherichia coli β- galactoεidase. 116. A recombinant IBR virus of claim 115, designated S- IBR-039. 117. A recombinant IBR virus of claim 113, wherein DNA from the thymidine kinase gene has been deleted so that upon replication, the recombinant IBR virus produces no thymidine kinase. 118. A recombinant IBR virus of claim 117, designated S- IBR-045. 119. A recombinant IBR virus of claim 117, wherein a foreign DNA sequence is inserted in place of the DNA encoding gpE glycoprotein. 120. A recombinant IBR virus of claim 119, wherein the foreign DNA sequence encodes Escherichia coli 6- galactosidase. 121. A recombinant IBR viruε of claim 120 designated S- IBR-044. 122. A recombinant IBR viruε of claim 119, wherein the foreign DNA εequence encodeε Bovine viral diarrhea virus gp53 glycoprotein. 123. A recombinant IBR virus of claim 122 designated S- IBR-046. 124. A recombinant IBR virus of claim 119, wherein a foreign DNA sequence is inserted in place of the DNA encoding thymidine kinase. 125. A recombinant IBR virus of claim 124, wherein the foreign DNA sequence inserted in place of the DNA encoding thymidine kinase encodes Escherichia coli β-glucuronidaεe. 126. A recombinant IBR viruε of claim 125, wherein the foreign DNA sequence inserted in place of the DNA encoding gpE glycoprotein encodes Escherichia coli β-galactosidase. 127. A recombinant IBR virus of claim 126, designated S- IBR-043. 128. A recombinant IBR virus of claim 119, wherein the foreign DNA sequence encodes Parainfluenza viruε type 3 fuεion protein and Parainfluenza viruε type 3 hemagglutinin protein. 129. A recombinant IBR viruε of claim 128, designated S- IBR-047. 130. A recombinant IBR virus of claim 119, wherein the foreign DNA sequence encodeε Bovine reεpiratory syncytial virus fusion protein. Bovine respiratory syncytial virus attachment protein and Bovine respiratory syncytial virus nucleocapsid protein. 131. A recombinant IBR virus of claim 130, designated S- IBR-049. 132. A recombinant IBR virus of claim 119, wherein the foreign DNA sequence encodes Pasteurella haemolvtica leukotoxin ' and Pasteurella haemolvtica iron regulated outer membrane proteins. 133. A recombinant IBR virus of claim 132, designated S- IBR-051. 134. A vaccine which comprises an effective immunizing amount of the recombinant IBR virus of claim 118 and a suitable carrier. 135. A vaccine which comprises an effective immunizing εunount of the recombinant IBR viruε of claim 123 and a suitable carrier. 136. A vaccine which comprises an effective immunizing amount of the recombinant IBR virus of claim 129 and a suitable carrier. 137. A vaccine which comprises an effective immunizing amount of the recombinant IBR virus of claim 131 and a suitable carrier. 138. A vaccine which comprises an effective immunizing amount of the recombinant IBR virus of claim 133 and a suitable carrier. 139. A vaccine which compriεes an effective immunizing εunount of a recombinant viruε protective againεt Bovine respiratory diseaεe complex and a εuitable carrier. 140. A vaccine of claim 139, wherein the recombinant viruε iε a recombinant IBR viruε. 141. A vaccine of claim 140, wherein the recombinant viruε consists essentially of any or all of the recombinant IBR viruseε of claims 118, 123, 129, 131 and 133. 142. A vaccine of claim 139 further comprising non- recombinant virus. 143. A vaccine of claim 140 further comprising non- recombinant viruε. 144. A vaccine of claim 143, wherein the recombinant IBR viruε consistε essentially of any or all of the recombinant viruseε of claims 117, 122, 134, 136 and 138. 145. A method of immunizing an animal against infectious bovine rhinotracheitiswhich compriseε administering to the animal an immunizing dose of the vaccine of claim 134, 135, 136, 137 or 138. 146. A method of immunizing an animal against bovine viral diarrhea which compriseε administering to the animal an immunizing dose of the vaccine of claim 135. 147. A method of immunizing an animal against Parainfluenza type 3 which comprises administering to the animal an immunizing dose of the vaccine of claim 136. 148. A method of immunizing an animal against Bovine respiratory syncytial virus disease which comprises administering to the animal an immunizing dose of the vaccine of claim 137. 149. A method of immunizing an animal against Pneumonic pasteurelloεis which comprises administering to the animal an immunizing dose of the vaccine of claim 138. 150. A method of immunizing an animal against bovine respiratory disease complex which comprises administering to an animal an immunizing dose of the vaccine of claim 141. 151. A method of immunizing an animal against bovine respiratory disease complex which comprises administering to an animal an immunizing dose of the vaccine of claim 144. 152. The method of claim 145, wherein the animal is a bovine. 153. The method of claims 146-151, wherein the animal is a bovine. 154. A me-thod for distinguishing an animal vaccinated with a vaccine which comprises an effective immunizing amount of a recombinant virus of claim 118 from an animal infected with a naturally- occurring IBR virus which comprises analyzing a sample of a body fluid from the animal for the presence of gpE glycoprotein of IBR virus and at least one other antigen normally expressed in an animal infected by a naturally-occurring IBR virus, identifying antigens which are present in the body fluid and determining whether gpE glycoprotein is present in the body fluid, the presence of antigens which are normally expressed in an animal by a naturally-occurring IBR virus and the absence of gpE glycoprotein in the body fluid being indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus.

Inventors: COCHRAN MARK D, MACDONALD RICHARD D, COCHRAN, MARK, D., MACDONALD, RICHARD, D.
Applicants: SYNTRO CORP, SYNTRO CORPORATION
Application year: 1992
Main classification: C12N7
IPC codes: A61K39, C07K14, C12N7, C12N15