NZ276234A - An attenuated replicating non pathogenic flavivirus (bovine viral diarrhoea virus - bvdv) vaccine - Google Patents

Description

New Zealand No. 276234 International No.

TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 05.11.1993; Complete Specification Filed: 31.10.1994 Classification:^) C12N7/01; C12N15/40; A61K39/12 Publication date: 26 January 1998 Journal No.: 1424 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Viral vector with bovine viral diarrhea virus (BVDV) antigens Name, address and nationality of applicant(s) as in international application form: PHARMACIA & UPJOHN COMPANY, a company organised under the laws of the United States of America of 301 Henrietta Street, Kalamazoo, Michigan 49001, United States of America New Zealand No. International No. 276234 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Viral vector with bovine viral diarrhea virus (BVDV) antigens Name, address and nationality of applicant(s) as in international application form: THE UPJOHN COMPANY, of 301 Henristta Street, Kalamazoo, Ml 49001, United States of America two 95/12682 PCTAJS94/12198 276234 VIRAL VECTOR WITH BOVINE VIRAL DIARRHEA VIRUS (BVDV) ANTIGB35 BACKGROUND OF THE INVENTION Field of the Invention 5 Thin invention relates to the field of Bovine Viral Diarrhea Virus (BVDV). and vaccines for the treatment thereof.

Information PiBclosure van Zyl, ILetal. Live Attenuated Pseudorabies Virus Expressing Envelope Glycoprotein El of Hog Cholera Virus Protects Swine against both Pseudorabies and 10 Hog Cholera, Jounwl ofVirolog", VoL 65, No. 5, pp. 2761-2765 (1991). U.S. patent 4,703,011, Kit, M., an* 311,3., Thymidine Kinase Deletion Mutants of Bovine Herpesvirus-1, issued 27 October 1987. U.S. patent 4,824,667, Eat, M,, and Kit, S. Thymidine Kinase Deletion Mutants of Bovine Herpesvirus-1, Vaccines Against Infectious Bovine Khinotracheitis Containing Same and Methods for the Production 15 and Use of Same, issued 25 April, 1989. Collett, M.S., et al., Proteins Encoded by Bovine Viral Diarrhea Virus: The Genomic Organization of a Pestivirus, Virology, Vol. 165 pp. 200-208 (1988). Collett, M.S., et al., Molecular Cloning and Nucleotide Sequence of the Pestivirus Bovine Viral Diarrhea Virus, Virology, VoL 165 pp. 191-199 (1988).

TWlrfn-mipd Bovine viral diarrhea virus (BVDV) is a Pestivirus belongii % to the family of the Flaviviridae. It causes a number of different conditions in sheep, goats, and especially cattle. The symptoms depend upon the age, physiological and virological state of the animal. In young susceptible calves and young adults it causes a 25 disease which is characterized by high morbidity and low mortality. The symptoms can include fever, depression, occulo-nasal discharges, diarrhea and occasionally oral ulcerations. Apart from these primary effects the virus also causes immunosuppression. Although primary BVDV infections are normally relatively mild, the virus may potentiate or enhance the pathogenicity of other co-infecting 30 microorganisms.

In older or susceptible animals, BVDV causes similar symptoms to those described above for younger susceptible calves. In addition, in pregnant animals the virus has the ability to cross the placenta and infect the fetus. The outcome of this infection depends upon the age of the fetus and whether it is at a stage where its 35 imTmiTiA eystem is fully competent The possible outcome of infections include fetal reabsorption, abortion, mummification, congenital defects, birth defects, calves bom which are persistently infected with BVDV and completely normal calves. Calves born which are persistently infected with BVDV, represent the most important segment of this BVDV pathogenesis complex. Persistently infected animals shed 5 large amounts of virus into their environment which can infect susceptible animaic Furthermore, even though persistently infected animals are ixnmunotolerant to the virus which infected them in utero, they do develop disease when infected with other closely related BVDV biotypes. These infections are characterized by low morbidity (because relatively speaking there will not be many pregnant animals infected at the 10 right during pregnancy to produce BVDV persistently infected normal calves), but high mortality. This disease syndrome is known as mucosal disease and often manifests itself as a peracute condition with calves dying of a profuse watery diarrhea which contains large amounts of fresh blood.

The importance of this virus and it's widespread presence in the cattle 15 population has led to the development of many vaccines in the attempt to try to prevent BVDV infection. These vaccines have been built on the traditional concepts of inactivation or attenuation but, because of the behavior of BVDV, they have many significant drawbacks.

It is generally accepted that inactivated vaccine preparations are not as 20 effective as attenuated live vaccines. Inactivated antigen from inactivated vaccine preparation undergoes exogenous processing. After injection into the the antigen becomes part of Hie animal's soluble protein mileau. The antigen enters antigen presenting cells through pinocytotic mechanisms and this usually produces antibodies. Unfortunately, because antibodies cannot gain entry into cells, they 25 normally only interrupt viral life cycles when mature virus is released from the cell. On the other hand, antigen from live virus which replicates inside cells, undergoes endogenous processing and this mechanism produces the preferred cell mediated immune responses. Cell mediated immune responses can recognize cells infected with viruses and have the potential of interrupting the virus life cycle at a much SO earlier stage. Cell mediated responses are thus thought to be extremely important in the immunological defense to many viral infections.

Because of the cell mediated response, attenuated live products such as vaccines should induce good cell mediated responses. With BVDV, attenuation of the virus to produce the live vaccine does not always prevent that vaccine virus from 35 causing the immunosuppression normally associated with field isolates. Roth J A.

WO 95/12682 PCT/US94/12198 and Kaeberle M.L., Suppression of Neutrophil and Lymphocyte Function Induced by a Vaccinal Strain of Bovine Viral Diarrhea Virus With or Without the Administration of ACTH, American Journal of Veterinary Research, Vol. 44 pp. 2366-2372 (1983). The failure of the vaccine to stop the immunosuppression response 5 creates a serious drawback to the vaccine. An animal owner may be vaccinating HTiimala to protect against a disease but because of the properties of the vaccine the owner provides an opportunity for other diseases to afflict the animals. This forces the owner to use inactivated BVDV vaccines, which because of the way in which the immune system operates, are not particularly effective.

In summary, inactivated vaccines are safe but not particularly effective while the attenuated live vaccines are more effective but under certain conditions may not be very safe.

This invention combines the effectiveness of the attenuated live vaccines with the safety of the inactivated vaccines. Bovine herpesvirus type 1(BHV-1) is another 15 major pathogen of cattle which produces respiratory disease. Thus, in common with BVDV, BHV-1 also replicates at a mucosal surface We take the gene which codes for gp53, a mqjor glycoprotein of the BVDV virus and against which the host produces substantial immune responses, and express it in bovine herpes virus -1 (BHV-1), this recombinant virus (BHWBVDVgp53) is used as a vaccine against 20 BVDV. Donis, R.O. and Dubovi, E.J., Glycoproteins of Bovine Viral Diarrhoea- Mucosal Disease Virus in Infected Bovine Cells, Journal of General Virology, VoL 68, pp. 1607-1616 (1987) and Magar, R., et al., Bovine Viral Diarrhea Virus Proteins: Heterogeneity of Cytopathogenic and Noncytopathogenic Strains and Evidence of 53K Glycoprotein Neutralization Epitope, Veterinary Microbiology, VoL 16, pp. 303-25 314. Cited references are incorporated herein by reference.

SUMMARY OF THE INVENTION.

A replicating nonpathogenic virus, for preventing disease caused by Bovine Viral Diarrhea Virus (BVDV), where said replicating nonpathogenic virus comprises: a gene or gene combination taken from a BVDV virus, and said replicating 30 nonpathogenic virus functionally expresses said gene or gene combination.

Embodiments of this invention include the following: A virus where said replicating nonpathogenic virus is attenuated, is selected from attenuated Bovine Herpes Virus type 1 (BHV-" auated adenoviruses, attenuated bovine mammillitis virus, attenuated bovine papillomavirus, or attenuated pseudorabies virus. A virus where 35 said replicating nonpathogenic virus is attenuated and contains and expresses any WO 95/12682 PCT/US94/12198 combination of the following genes: the genes that code for gp48, gp25, pl4 capsid protein, p20 N-terminal protease ard pl26/p80 protein. A virus where the attenuation is created by rnairing the thymidine kinase (tk) gene nonfimctionaL A virus where a signal peptide is inserted proceeding the gene or gene 5 combination that codes for gp53 in said Bovine; Herpes Virus type 1 (BHV-1). A virus where said gene that codes for gp53 is inserted into the inactivated thymidine kinase (tk) gene site. A virus where the functionally expressing gene or gene combination, used to create the virus, comprises a recombined plasmid with intact viral DNA, said plaamid comprising: a) a BHV-1 genomic DNA fragment containing 10 the thymidine kinase (tk) gene and having a deletion to the thymidine kinase (tk) gene, b) a promoter/polyadenylation signal inserted in the thymidine kinase (tk) gene deletion, c) a signal peptide gene sequence preceding a gp53 gene or gene combination all of which is inserted between the promoter and the polyadenylation signal A virus where said plasmid is made from a plasmid having the 15 characteristics of plasmid pHAS4. A virus where said signal peptide gene sequence is taken from any well characterized signal peptide sequences such as any of the thirty-nine examples of well characterized signal peptide sequences found in Perlman, D., et aL, J. Mai. BioL VoL 167 pp. 391-409 (1983), incorporated by reference. A virus where said signal peptide gene sequence is taken from 20 Psuedorabies Virus gin gene (PRV) and/or Bovine Growth Hormone (BGH).

A \iru8 where a plasmid is selected from the following plasmids, a) pBHVtkex-l::BGH/p5S; b) pBHVtkex-l::gIII/p53; c) pBHVtkex-3::BGH/p53; or d) pBHVtkex-3: :gm/u53. A virus that produces the product of a functionally expressing gene or gene combination is selected from one of the following viruses, 25 Tll-3, Tll-6, or Tll-8. A virus where the functionally expressing gene or gene combination, used to create the virus, comprises a recombined plasmid with intact viral DNA, said plasmid comprising: a) a BHV-1 genomic DNA fragment containing the thymidine kinase (tk) gene and having a deletion to the thymidine kinase (tk) gene, b) a promoter/polyadenylation signal inserted in the thymidine kinase (tk) 30 gene deletion, c) a gp53 gene or gene combination inserted between the promoter and the polyadenylation signaL A virus where the plasmid is pBHVtkex-3::p53. A virus selected from one of the following viruses, T2-3#3 or T2-2#5. A vaccine for preventing disease caused by Bovine Viral Diarrhea Virus (BDVD) comprising a pharmaceutically effective amount of the viruses described herein and a carrier. 35 A vaccine as described above for preventing disease caused by Bovine Viral 4748.P CP 27 6 2 3 4 Diarrhea Virus (BDVD) comprising a pharmaceutically effective amount of a virus described above and a carrier, said carrier comprising any physiological buffered medium, i.e. about pH 7.0 to 7.4 containing firom about 2.5 to 15% serum which does not contain antibodies to BHV.

A method of Immunizing a non-human animal against Infectious disease caused, by Bovine Viral Diarrhea Virus (BDVD) comprising administering to an animal a pharmaceutically effective amount of a virus or vaccine described herein.

A process of preparing a virus described herein comprising: a) isolation of a functionally expressing gene or gene combination that causes BVDV, b) inserting said gene or gene combination into a replicating nonpathogenic virus, c) selecting a live-virus that functionally expresses the product of said gepe or gene combination.

A method of preparing a virus described herein where the functionally expressing gene or gene combination, used to create the virus, is produced by a process comprising the recombination of a plasmid with intact viral DNA, said plasmid comprising: a) a BHV-1 genomic DNA fragment containing the thymidine kinase (tk) gene and having a deletion to the thymidine kinase (tk) gene, b) inserting into the thymidine kinase (tk) gene deletion of said plasmid a promoter/polyadenylation signal, c) inserting a gp53 gene or gene combination between the promoter and the polyadenylation signal, d) transfecting cells with said plasmid to produce a recombinate virus containing said functional gene or gene combination inserted into a live virus that does not cause immunosuppression in the usual host and expressing said functional gene or gene combination.

A method of preparing a virus described herein where the functionally expressing gene or gene combination, used to create the virus, is produced by a process comprising the recombination of a plasmid with intact viral DNA, said plasmid comprising: a) a BHV-1 genomic DNA fragment containing the thymidine kinase (tk) gene and having a deletion to the thymidine kinase (tk) gene, b) inserting into the thymidine kinase (tk) gene deletion of said plasmid a promoter/polyadenylation signal, c) inserting a gp53 gene or gene combination preceded by a signal peptide gene sequence between the promoter and the polyadenylation signal, d) transfecting cells with said plasmid to produce a recombinate virus containing said functional gene or gene combination inserted into a live virus that does not cause immunosuppression in the usual host and expressing said functional gene or gene combination.

WO 95/12682 PCT/US94/12198 BRIEF DESCRIPTION OF THE DRAWINGS.

Figure 1. Construction of the shuttle vectors for inserting foreign genes into BHV-1. Figure 2. Strategy for appending signal peptide sequences to the BVDV gp53 gene. Figure 3. Maps of the five shuttle plasmids for inserting gp53 into BHV-1 5 a. EXAMPLE L pBHVtkex-3::p53. b. EXAMPLE 2: pBHVtkex-l::BGH/p53 c. EXAMPLE 3: pBHVtkex-l::gIII/p53 d. EXAMPLE 4: pBHVtkex-3::BGH/p53 e. EXAMPLE 5:pBHVtkex-3::gEI3/p53 Figure 4. Predicted transcript maps of the BHV-l/gp53 recombinant viruses.

Figure 5. Northern blots showing transcription of gp53 messenger RNAs in the BHV-1 recombinants.

Figure 6. Immunoprecipitations showing expression of gp53 protein in the BHV-1 recombinants.

DESCRIPTION OF THE PREFERRED EMBODIMENTS.

All of the terms used below will be readily understood by one skilled in the art In many places the name of the manufacturer of equipment or reagents are provided in parenthesis after the equipment or reagent is named. Commonly used terms, reagents and buffers such as "plasmids," "Klenow Fragments," "religating 20 blunt ends," "Tris," chelating buffers Buch as EDTA and EGTA, and commonly used chromatography columns are referred to without further explanation.

In the descriptions of the construction of the compounds used in this invention, standard molecular biological techniques were used and are briefly named or described here. Detailed explanations of these techniques can be found in 25 standard laboratory manuals such as "Molecular Cloning: a Laboratory Manual" (1989), Sambrook, et. al., Cold Spring Harbor Press, Cold Spring Harbor, New York, or "Current Protocols in Molecular Biology" (1991), Ausubel, F. M., et al., eds., Wiley Intersdence, New York.

This invention combines the effectiveness of the attenuated live vaccines with 30 the safety of the inactivated vaccines. We take the gene which codes for gp53, a major glycoprotein of the BVDV virus and against which the host produces substantial immune responses, and express it in bovine herpesvirus -1 (BHV), this recombinant virus (BHV/BVDVgp53) is used as a vaccine against BVDV.

Bovine herpesvirus (BHV) is another mqjor pathogen of cattle which produces 35 respiratory disease. Thus, in common with BVDV, BHV also replicates at a mucosal WO 95/12682 PCT/US94/12198 surface. With BVDV, replication is mainly at the gut mucosal interface with less replication at the respiratory interface. With BHV it is the respiratory interface which dominates. The common mucosal immune system ensures that immune responses produced at one surface will be effective at other surfaces. Thus the 5 recombinant virus of this invention, BHV/BVDVgp53, will, when administered to cows, sheep or goats, preferably via the intranasal route, replicate in the respiratory mucosae and produce an immune response.

Prior to the expression of the BVDVgp53 gene in BHV, the thymidine kinase gene was deleted from the BHV virus using a process known to attenuate the virus. 10 The BHV, a live attenuated virus, will replicate and produce a cell mediated response. As part of that replicative process, the BVDV gp53 gens will be expressed and, because the virus is inside the cell, the correct processing for a cell mediated response to the BVDV gp53 part of the recombinant virus will also occur. Most importantly, this response will occur without the possible side effects of 15 immunosuppression, as only part of the BVDV virus is present. Thus, the invention combines the efficacy of an attenuated live virus vaccine for BVDV, with the safety of an inactivated preparation.

The examples in the procedures section are provided for illustrative purposes and are in no way intended to limit the scope of the present invention. All media 20 and buffer solutions were made up in glass distilled water unless otherwise indicated.

Compositfong Administrations - A pharmaceutically effective amount of the vaccine of the present invention can be employed along with a pharmaceutically acceptable carrier or diluent as a vaccine against BHV-1 and BVDV in animals, such 25 as bovine, sheep and goats.

Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include any physiological buffered medium, ie., about pH 7.0 to 7.4, containing from about 2.5 to 15% serum which does not contain antibodies to BHV, ie., is seronegative for BHV. Serum which, does not contain gamma globulin 30 is preferred to serum which contains gamma globulin. Examples of serum to be employed in the present invention include fetal calf serum, lamb serum, horse serum, swine serum, and goat serum. Serum protein such as porcine albumin or bovine serum albumin (hereinafter "BSA") in an amount of from about 0.5 to 3.0% can be employed as a substitute for the serum. However, it is desirable to avoid the 35 use of foreign proteins in the carrier or diluent which will induce allergic responses WO 95/12682 PCT/US94/12198 in the animal being vaccinated.

The virus may be diluted in any of the conventional stabilizing solutions containing phosphate buffer, glutamate, casitone, and sucrose or sorbose, or containing phosphate buffer, lactose, dextran and glutamate.

It is preferred that the vaccine viruses of the present invention be stored at a titer of at least 105 to 10® PFU/ml at -70°C to -90°G or in a lyophilized state at 2°C to 7°C. The lyophilized virus may be reconstituted for use with sterile distilled water or ""fag an aqueous diluent containing preservatives such as gentamidn and amphotericin B or penicillin and streptomycin.

The useful dosage to be administered will vary depending upon the age, weight and species of the afiimal vaccinated and the mode of administration. A suitable dosage can be, for example, about 104,5 to 107 PFU/animal, preferably about lO4,5 to lO5*5 PFU.

The vaccines of the present invention can be administered intranasally, 15 intravaginally or intramuscularly. Intranasally is the preferred mode of administration.

Utility of the Invention - This invention is intended to provide the user with an effective vaccine for prevention of BVDV caused disease, where the vaccine can be safely and efficaciously administered intramuscularly, intranasally, or 20 intravaginally. Intranasally may be the preferred route of administration.

The vaccines of this invention are created with the intention of treating disease, preferably through prevention. By prevent or prevention applicant means to keep the host from developing symptoms of the disease or to mitigate the effects of the disease, that is to avert the typical diseased state. Prevention implies decisive 25 action to stop, impede or delay the onset of disease. Prevention can include the following concepts: to hinder, frustrate, to obstruct; to intercept, possibly prohibit, impede or preclude. Preclude would suggest the onset of the disease state either does not occur or the disease pathogen is largely ineffectual in causing the disease state. Prevent or prevention can indicate the disease state is forBtalled, meaning SO that anticipatory action to prevent or hinder the disease has occurred but the conditions creating the disease have not been eliminated.

The usefulness cf this invention will be illustrated by the ability of the vaccine to provide effective protection against the spread of BVDV disease in its various manifestations. Because the vaccine uses gp53, a major glycoprotein of 35 BVDV, and one against which the host produces a substantial immune response, the vaccine will confer substantial benefits upon the treated potential host. Another object of the invention is to provide a BVDV vaccine which can be administered safely to calves and to pregnant cows in all stages of pregnancy.

Measures of Activity - The vaccine uses gp53, a mqjor glycoprotein of BVDV, 5 and one against which the host should produces a substantial immune response. Others have shown that gp53 is highly immunogenic. Donis, R.O. and Dubovi, E.J., Glycoproteins of Bovine Viral Diarrhoea-Mucosal Disease Virus in Infected Bovine Cells, Journal of General Virology, VoL 68, pp. 1607-1616 (1987). It is well known that agents that produce substantial immune responses can make effective vaccines. 10 Magar, R, et al., Bovine Viral Diarrhea Virus Proteins: Heterogeneity of Cytopathogenic and Nonqytopathogenic Strains and Evidence of 53K Glycoprotein Neutralization Epitope, Veterinary Microbiology, VoL 16, pp. 303-314. The vaccines of this invention contain genes that express large quantities of gp53, this is shown in figure 5. Because of the expression of large quantities of gp53 the vaccines of this 15 invention will confer substantial benefits upon the treated potential host Preferred Compounds - Any BHV-1 virus attenuated with a tk deletion and carrying the gp53 gene, the gp53 gene being preceded by a signal peptide, that expresses abundant amounts of gp53, should be a preferred suitable vaccine candidate. It appears the signal peptide sequence may be taken from any suitable 20 source. We chose to examine two different signal peptides to ensure the best localization of the gp53 protein in vivo. We chose two candidates we call "Tll-6", embodied in Example 2, and "Tll-S", embodied in "^xample 3 for vaccine trials. The former virus was deposited to the ATCC under the designation UC VR-58. The latter, Tll-S" plasmid was also deposited. The virus we labeled "Tll-S" might 25 contain truncated forms of the tk transcript and this might suggest, but does not necessarly mean, that it would be less attractive as a vaccine candidate. A large number of existing cell lines are persistently infected with non-cytopathic BVDV from passage in media containing fetal bovine serum taken from infected calves. For this invention, it is imperative that viruses used as live, attenuated vaccines are free 30 of contaminating BVDV.

Preparation of the Compounds Construction of expression shuttle vectors for gene insertion into Bovine herpesvirus type-1 (BHV-1).

We constructed two shuttle vectors to allow insertion of foreign genes into 35 BHV-1. Although this inventioti showB the utility of BHV-1 as a vector for BVDV WO 95/12682 PCT/US94/12198 genes many other viruses could fill the same role. Other examples from cattle, sheep and goats would include cow, goat and sheep pox viruseadenoviruses, bovine mammillitis virus, bovine papillomavirus, and pseudorabies virus. A nonpathogenic virus refers to any virus which has the ability to replicate in one of its 5 host species but does not produce any signs of disease in that species. Such nonpathogenic viruses might arise from pathogenic parent viruses by natural mutation, might be mutagenized by, for instance, chemicals or light to produce a nonpathogenic virus, or could be rendered non-pathogenic through the use of recombinant DNA technologies. See, 1) Mapping Neutralization Domains of Viruses, 10 E.Wimmer, EA Tfonini, and D.C. Diamondand 2) Immunogenidty of Vaccine Products and Neutralizing Antibodies, E Norrby. Both articles are in Edited by Notions and Oldstone Published by Springer-Verlag New York Inc. 1986.

Since we intended to attenuate BHV-1 by inactivating the viral thymidine kinase (tkXM. Kit, et aL, US Patent 4,703,011, (1983 )), we decided to use the BHV-15 1 tk gene for the site of insertion. This approach not only insured the complete inactivation of the viral tk, but als" allowed us to select recombinant, tk-negative virus by established methods. M. F. Shih, et aL, Proc Natl Acad Sd USA, 81:5867-5870 (1984). Other methods to attenuate BHV-1, such as deletion of other non-essential genes would also be applicable to this particular invention. We started 20 with plasmid pHAS4 which contains a 2.7kb Sail subfragment of the BHV-1 HindTII-A fragment cloned into plasmid pUC18. E. Petrovkis, unpublished data. M. Engels, et aL, Virus Res, 6:57-73 (1986); J. E. Mayfield, et aL, J Virol, 47:259-264 (1983); A. L. Meyer, et aL, Biochim Biophye Acta, 1090:267-9 (1991). As shown in Fig. 1, this Sail fragment contained the entire tk gene, as well as a portion of the 25 upstream gene homologous to the HSV-1UL24 gene, and a portion of the glycoprotein H gene. L. J. Bello, et aL, Virology, 189:407-414 (1992); J. G. Jacobson, et aL, J Virol, 63:1839-1843 (1989); M. Kit, et aL, US Patent 4,703,011, (1983); A. L. Meyer, et aL, Biochim Biophys Acta, 1090:267-9 (1991).

A 424bp deletion was introduced into the tk gene by digesting pHAS4 with 30 BglH and Xhol, filling in the ends with the Klenow Fragment of DNA polymerase I (Klenow) and religating the resulting blunt ended fragments. This manipulation restored the BglH recognition site, but not the Xhol site (Fig. 1). The resulting plasmid was named pHAS4ABX. This deletion was chosen because it does not impede on the previously identifed transcription initiation sites for the UL24 • 35 homolog which overlaps the 5' end of the tk gene. L. J. Bello, et aL, Virology, WO 95/12682 PCT/US94/12198 189:407-414 (1992); J. G. Jacobson, et aL, J Virol, 63:1839-1843 (1989 ). Numerous other deletions within the BHV-1 tk gene would be possible. To facilitate later dnning manipulations, we eliminated the Hindm site in the pUC18 vector by digesting pHAS4ABX with Hindlll, filling in the cohesive ends with Klenow, and 5 religating the blunt ends.

We obtained a 1775bp cassette containing the Human cytomegalovirus (CMV) major immediate early promoter and the bovine growth hormone polyadenylation sequence. R. J. Brideau, et aL, J Qen Virol, 74:471-477 (1993 ). These gene expression signals are commonly used for high levels of expression of foreign genes 10 in a number of different systems, but other promoter/polyadenylation signal pairs could also be used in this context The cassette, in vector pSCL-DHFR, is bounded by unique EcoRI and BglH sites and contains, between the promoter and the polyadenylation signal, unique TTinflTTT and Sail restriction sites for cloning of foreign genes. The pSCLrDHFR vector was digested with EcoRI, then filled in and 15 ligated to a BamHI linker (New England Biolabs, Beverly, Massachusetts). This manipulation regenerated the EcoRI site. The construct was then digested with BamHI and BglH and the released cassette was ligated into the Bglll site of pHAS4ABX (Fig. 1). The li 'ons were transformed into E. coli strain DHSa. We isolated recombinant plasmids that contained the p3CL insert in both orientations 20 relative to the BHV-1 tk gene by mapping of asymmetric restriction sites. These two constructs, designated pHAS4ABXex-l and pHAS4ABXex-3 (Fig. 1), contained then, a strong promoter and polyadenylation signal bounded by the BHV-1 tic gene and flanking regions to allow homologous recombination into the BHV-1 genome.

Figure 1. Construction of shuttle vectors for inserting foreign genes into 25 BHV-1. FHAS4 is a 2.7kb subfragment from the BHV-1 HindllT-A fragment. The BglH/XhoI subfragment to be deleted is shown. The deletion derivative of pHAS4 is pHAS4ABX. The deleted thymidine kinase (tk) gene is shown as a dark stippled box. The cassette containing the promoter and polyadenylation signal is shown just below pHASiABX. The CMV immediate early promoter is shown as a light stippled 30 box, and the Bovine Growth Hormone (BGH) polyadenylation signal is shown as a striped box. Finally, the inserts of the two expression shuttle plasmids, pHAS4ABXexl and pHAS4ABXex3 are shown.

Addition of Signal Peptide Sequences to BVDV gp53 gene.

A cDNA containing the BVDV gp53 gene from strain 2724, a noncytopathic 35 strain, has been previously described. Kennedy, M et al, abstracts of the American WO 95/12682 PCT/US94/12198 College of Veterinary Microbiologists, 1992 workshop. Since the BVDV RNA genome is normally translated as one long polyprotein and then post-translationally modified into the various viral proteins, the gp53 portion of the BVDV genome does not contain the usual signal peptide required for translocation of the protein to the cell 5 membrane, where the protein is normally expressed. Nonetheless, the cDNA was successfully expressed in both cell-free systems and baculovirus, and the protein appeared to be translocated, glycosylated and anchored in both systems, despite the lack of a conventional signal peptide. We decided, however, to evaluate expression of gp53 in BHV-1 both with and without various signal peptides.

In order to attach nucleotide sequences encoding signal peptides to the gp53 gene, we introduced a BamHI Bite into 5' end of the p53 gene by site directed mutagenesis, as follows: The p53 gene was blunt-end ligated into the filled-in BamHI site of plasmid pSP72 (Promega Corp., Madison, Wisconsin), thus removing all BamHI sites from the resulting plasmid. We introduced a single base change, a 15 C to a G, 11 bases in from the initiation codon used by the cDNA, using a synthetic oligonucleotide and the "Double Take" site directed mutagenesis kit (Stratagene, La Jolla CA) according to the manufacturer's instructions. This base change introduced a unique BamHI site into the gene without altering the gp53 amino acid sequence (Fig. 2 section B). The base change was verified by nucleotide sequencing, and the 20 resulting plasmid was called pP53mut We inserted, into pP53mut sequences, encoding signal peptides from the PRV gUI gene (A K Bobbins, et aL, J Virol, 58:339-347 (1986)) and from Bovine growth hormone. R. P. Woychik, et aL, Nucl Acids Res, 10:7197-7210 (1982). (Figure 2 section A) Complentary oligonucleotides encoding the two signal peptides were synthesized such that annealed oligos had 25 Sail cohesive ends 5* and BamHI cohesive ends 3' (Fig 2 section A). These signal peptide cassettes were ligated into pP53mut digested with BamHI and Sail, and. transformed into DH5o. We confirmed the correct insertion of the signal peptide cassettes by nucleotide sequencing.

Complementary oligonucleotides encoding any well characterized signal 30 peptide can be used in this invention. Thirty-nine examples of well characterized signal peptide sequences found in Perlman, D., et aL, J. Mol. Biol VoL 167 pp. 391-409 (1983). Incorporated by reference. These and any other well characterized signal peptides should be suitable for use as embodiments of this invention.

Figure 2. Strategy for appending signal peptide sequences to the BVDV 35 gp53 gene. Section A: Synthetic oligonucleotides corresponding to the signal WO 95/12682 PCT/DS94/12198 peptide sequences of Bovine Growth Hormone (BGH), and Pseudorabies virus gill (PRV glH). Complementary oligonucleotides were synthesized such that the annealed pairs had Sallsites on th^ 5' ends and BamHI sites on the 3* ends. The deduced amino acid sequences of the signal peptides are also shown. In each case 5 the predicted cleavage sites for the signal peptides are just after the alanine (A), three amino acids from the ends. Codons for two amino add residues (FJ? in BGH; P,S in glH) firom the original native proteins were left on the signal peptide sequences to ensure correct clf&vage.

Section B: Site directed Mutagenesis of the cDNA encoding the BVDV gp53 10 gene. The first 60 nudeotides of the gp53 cDNA and the corresponding amino acid sequence are shown. A single base pair, shown by the arrow, was changed to create a BamHI restriction site in the sequence, shown in the box. This change does not change the amino add sequence. The cDNA was then digested with BamHI as shown, allowing in frame ligation to either of the signal peptide sequences shown in 15 section A.

Other expression gene fragments in addition to gp53.

Expression of other BVDV gene or gene combinations in a live virus vector are also embodiments of this invention. This would include any and all BVDV proteins to which a vaccinated animal could elicit an immune response. Examples 20 indude, but are not limited to, the other two BVDV surface glycoproteins, gp48 and gp25 (Collett, M.S., et al., Virology 165:200-208 (1988)), the pl4 capsid protein (Thiel, H.J., et aL, J. Virol. 65:4705-4712 (1991)), and the p20 N-terminal protease. Wiskerchen, M., et al., J. ViroL 65:4508-4514 (1991). This group of proteins, along with the gp53 gene, can be expressed together from a single cDNA molecule, the 25 expressed polyprotein will process itself correctly into the separate proteins.

Another BVDV protein candidate to express in a vaccine is the nonstructural pl25/p80 protein (Deregt, D., et &L, Can. J. Microbiol. 37:815-122 (1991)), which elidts a significant antibody response in infected cows.

Insertion of the BVDV gp53 gene into the BHV-1 expression vectors. 30 The p53 gene, either with or without added Bignal peptide sequences, was ligated into the HindTII insertion sites of pHAS4ABXex-l and pHAS4ABXex-3 by filling in all the respective cohesive ends of vectors and inserts followed by blunt end ligation. The ligations were transformed in E. coli strain DH5a. We wanted to eventually evaluate the expression of gp53 in BHV-1 in various orientations and •35 with at least two different signal peptides to ensure that we achieved the most efficient expression. The transformed colonies were screened by colony hybridization using as a probe the p53 insert labelled with Digaxygenin-dUTP. The "Genius" DNA hybridization system (Boeringer Mannheim Biochemicals (BMB), Indianapolis, IN) was used for this and all other DNA hybridizations described in the characterization 5 of this invention. Positive recombinants were then screened by restriction analysis for those carrying the gp53 gene in the proper orientation relative to the CMV promoter and BgH polyadenylation signal. Five plasmids were isolated, which are schematically depicted in Figs. 3A-E. Their descriptions are as follows.

EXAMPLE 1. pBHVtkex-3::p5S: contains the BVDV gp53 gene inserted 10 between the CMV promoter and the BGH polyadenylation signal of pHAS4ABXex-3 with no added signal peptide. In this construct the original gp53 gene, PRIOR to site directed mutagenesis, was inserted. See Fig. 3A This plasmid was then used to construct the virus T2-3#.

EXAMPLE 2. pBHVtkex-l::BGH/p53: contains the mutagenized gp53 gene 15 preceded by the BGH signal peptide sequence inserted into pHAS4ABXex- 1. See Fig. SB. This plasmid was used to create the virus Tll-6. This virus was deposited.

EXAMPLE 3. pBHVtkex-l::gIH/p53: contains the mutagenized gp53 gene preceded by the PRV gin signal peptide sequence inserted into 20 pHAS4ABXex-l. See Fig. SC. This plasmid was used the create the virus Tll-3. This plasmid was deposited.

EXAMPLE 4. pBHVtkex-3::BGH/p53: contains the mutagenized gp53 gene preceded by the BGH signal peptide sequence inserted into pHAS4ABXex-3. See Fig. SD.

EXAMPLE 5. pBHVtkex-3::glIFp53: contains the mutagenized gp53 gene preceded by the FRV glH signal peptide sequence inserted into pHAS4ABXex-3. See Fig. 3E. This plasmid was used to create the virus Tll-8. This plasmid was deposited.

Figures 3A-E. Complete maps of the five shuttle plamids for inserting gp53 30 into BHV-1. The gp53 gene is shown as a solid band, th* BHV-1 sequences are shown as dark stippled bands, the CMV promoter region is shown as a light stippled band, and the BGH polyadenylation signal region is shown as a striped band. The plasmid vector, pUC18, is shown as a thin line. In each case the direction of transcription of gp53 relative to the original direction of transcription of BHV-1 tk is 35 shown. The various signal peptide sequences are indicated.

WO 95/12682 PCT/US94/12198 a. EXAMPLE 1. pBHVtkex-3::p53. b. EXAMPLE 2. pBHVtkex-l::BGH/p53 c. EXAMPLE 3. pBHVtkex-l::gIII/p53 d. EXAMPLE 4. pBHVtkex-3::BGH/p53 5 e. EXAMPLE 5. pBHVtkex-3::gIII/p53 These, and all other possible insertions of the BVDV gp53 gene into the BHV-1 tk gene are embodiments of this invention. These plasmids and any plasmids created in this manner are known as "Principal Plasmid Vectors" and are the plasmid vectors used to create the virus vaccines of this invention. 10 Introduction of the gpffS gene into BHV-1 Iowa".

The five expression shuttle plasmids carrying gp53 were linearized by Xbal and cotransfected into Bovine Turbinate (BT) cells with unit length DNA from BHV-1 strain Iowa (tk positive) by the standard CaP04 method (R. L. Graham, et al., Virology, 52:456-467 (1973 )) as modified by Cai (W. Cai, et aL, J Virol, 61:714-721 15 (1987 )). The cells were obtained from ATCC. The transfections were then subjected to two rounds of selection either on 143tk" cells (S. K. Mittal, et aL, J Gen Virol, 70:(1989)), or on Rab (BU) cells (S. Kit, et aL, Virology, 130:381-389 (1983 )) in the presence of lOOug/ml 5-Bromo-2'-Deoxyuridine (BDUR, Sigma Chemical Company, St Louis, Missouri) to isolate virus no longer expressing tk. This is a standard 20 procedure described previously. M. Kit, et aL, US Patent 4,703,011, (1983). Other tk' cell lines permissive for growth of BHV-1 can also be used. After the two rounds of BDUR passage, transfections that still showed cytopathic effect were infected onto BT cells under complete media with 1% low melting agarose to obtain single plaques. Multiple single plaques were picked from each transfection and the viral 25 DNAs were screened for the p53 gene by dot-blot DNA hybridization. Although not all transfections survived the BDUR passages (particularly those on the 143 tk' cells, as these cells are only marginally permissive for BHV-1 viral growth), those that did survive yielded 100% recombinant virus. Four different recombinant viruses were isolated and further characterized: SO EXAMPLE 1. T2-3#3 and T2-2#5 (two identical, but independently isolated viral clones): BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-8::p53 recombined Contains the BVDV gp53 gene with no added signal peptide sequence situated between the CMV promoter and the BGH polyadenlyation signal, with transcriptional orientation in the 35 same direction as the BHV-ltk gene.

WO 95/12682 PCT/US94/12198 EXAMPLE 2. Ti.1-6 (This virus was submitted to ATCC tinder the designation UC VR-58): BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-l::BGH/p53 recombined. Contains the BVDV gp53 gene with the BGH signal peptide sequence situated between the 5 CMV promoter and the BGH polyadenlyation signal, with transcriptional orientation in the opposite direction relative to the BHV-1 tk gene.

EXAMPLE 3. Tll-3: BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-l::gm/p53 recombined. Contains the BVDV gp53 gene with the PRV gin signal peptide sequence Bituated between the 10 CMV promoter and the BGH polyadenlyation signal, with transcriptional orientation in the opposite direction relative to the BHV-1 tk gene.

EXAMPLE 5. Tll-8: BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-3::gin/p53 recombined. Contains the BVDV gp53 gene with the PRV gin signal peptide sequence situated between the 15 CMV promoter and the BGH polyadenlyation signal, with transcriptional orientation in the same direction as the BHV-1 tk gene.

A virus was not isolated firom cotransfections with "Iowa" DNA and plasmid pBHVtkex3:-3GH/p53, EXAMPLE 4, but this prophetic virus, could be easily created, it and any other BHV-1 viruses containing the BVDVgp53 gene inserted 20 into thymidine kinase gene are embodiments of this invention. We purified DNA from each of these viruses and checked for the proper insertions in the proper orientations by Southern Hybridization using both the gp58 gene and the CMV promoter/BgH polyadenylation cassette as probes (data not shown). All four of the viruses cairied the complete promoter/gene/polyadenylation cassettes in the BHV-1 25 tk gene, deleted as predicted, based on restriction fragment sizes. As a control, with these transfections, we also transfected the pHAS4ABX plasmid with BHV-1 "Iowa" unit length DNA and isolated a tk-negative progeny carrying the 424bp deletion in tk (also verified by Southern Hybridization). This virus is named IowaABX. All of these viruses were plaque purified twice by limiting dilution on BT cells. 30 A large number of existing cell lines are persistently infected with non- cytopathic BVDV from passage in media containing fetal bovine serum taken from infected calves. For this invention, it is imperative that viruses used ao live, attenuated vaccines are free of contaminating BVDV. In order to ensure that the BHV-1 viruses carrying the BVDV sequences were not contaminated with non-35 cytopathic BVD virus, we prepared DNA from each of the viruses (including the WO 95/12682 PCT/US94/12198 parent strain Iowa and lowaABX) and subjected the DNA preps to extensive RNAse treatment using a cloned RNAse (RNAse ONE, Fromega Corporation, Madison, Wisconsin). Since BVDV has only RNA as its genetic material, this manipulation should eliminate any possible contaminating BVDV sequences firom the viral DNA 5 preps. We then transfected these RNAsed viral DNAs into certified BVD-free MDBK ftftlln (ATCC) and picked virus plaques from the transfections to use in further manipulations.

Transcriptional analysis of the gp58 recombinants* We prepared RNA firom each of the recombinant viruses and the parent BHV-10 1 strain Iowa and evaluated transcription of gp53 by Northern hybridization. A diagram of the possible message species and the probes used is shown in Fig. 4.

Figure 4. Predicted transcripts of the BHV-l/gp53 recombinant viruses later shown in Figure. 6. The two probes are 1) the gp53 cDNA and 2) the Sall/BglTI portion of pHAS4 (Bhown above the maps). The first map shows the predicted 15 transcripts from viruses Tll-3 and Tll-6, and the second map Bhows the predicted transcripts from Tll-8. The sites of transcript initiation for tk and UL24 are shown for reference.

All of the gp53 recombinant viruses made a 1.6kb message that hybridized with a 32P-labelled gp53 probe, the size predicted for transcription initiation at the 20 CMV promoter and termination at the BgH polyadenylation site, fig. 6, probe 1. The T2-3#3 and T2-2#f< virus are not shown. As additional major bands, Tll-S and Tll-6 made an 8.5kb transcript and Tll-8 and T2-3#S made a 5.6kb transcript. These transcripts were unique to the recombinant viruses, and were consistent with messages initiating at the CMV promoter, reading through the BgH poly adenylation 25 signal and terminating at the UL24 or tk/gH polyadenylation signals, respectively.

Hybridization with the upstream and downstream probes confirmed the identity of these longer messages. The p53 probe did not hybridize to Iowa, IaABX or mock infected RNAs. As a quantitation control we used probe pHAS6, an 867bp sail fragment that maps downstream of the tk open reading frame and is internal to SO the gH gene. A. L. Meyer, et aL, Biochim Biophys Acta, 1090:267-9 (1991). All of the viruses made equivalent amounts of the S.lkb gH message (data not shown). This probe also hybridized to the longer p53 messages in Tll-8 and T2-3-3, and to the 4.3kb tk message in Iowa, which is 3' coterminal with the gH transcript. L. J. Bello, et aL, Virology, 189:407-414 (1992).

To examine the transcription patterns upstream of the gp53 insertions, we used a probe that consisted of the pHAS4 fragment from the upstream Sail site to the Bglll site in the tk gene, the beginning of the deletion in the recombinant viruses (probe 2). All of the viruses made a message of approximately 4.4kb which we deduced to be UL24 (Fig. 5, probe 2). This message, however, was smaller than 5 the 5.2kb UL24 message in BHV-1 strain Cooper described by Bello, et al (L. J. Bello, et al., Virology, 189:407-414 (1992 )) and comigrated with the tk message in the wild-type strain Iowa. Although we did not evaluate these comigrating messages further by single stranded probes, we detected a tk transcript of 4.2 kb only in the Iowa DNA with probe pHAS6 and we detected similarly sized 10 transcripts in all the viral RNAs with the upstream probe, even though these other viruses cannot be making a wild-type sized tk transcript In Tll-8 and Tll-6, the upstream probe did not detect any truncated forms of tk message and hybridzed to only the UL24 message and the the 8.5kb p53 message. In Tll-8, on the other hand, the probe hybridized to four additional (minor) bands of approximately 5.0, 15 3.7,1.8, and l.Okb.

Figure 5. Northern blots showing transcription of gp53 messenger RNAs in the BHV-1 recombinant viruses. The first panel shows transcripts hybridizing to probe 1, the pg5S cDNA, and the second panel shows transcripts hybridizing to probe 2, the Sall/Bgll subfragment of pHAS4. KEY: M«Mock infected cells, I»BHV-20 1 "Iowa" infected cells, 3,6,8*Tll-3, Tll-6 and Tll-8 infected cells. RNA size standards, in kilobases (kB) are given to the left of each panel Expression of BVDV gp53 protein In BHV-1.

We evaluated expression of gp53 protein in the BHV-1 recombinants by immunoprecipitation (IP). Detailed procedures for IPs can be found in standard 25 references such as "Current Protocols in Molecular Biology", Ausubel, F. M., et aL, eds., Wiley Interscience, New York. BT cells infected with the BHV-1 recombinants were metabolically labelled with ^S-methionine (Amersham, Arlington Heights, Illinois). The viral infected cells were lysed and soluble proteins were reacted with hyperimmune serum from bovine or goat against BVDV. VMRD, Pullman, 30 Washington. Antigen/antibody complexes were precipitated staph A (Immunopredpitin, Gibco/BRL, Gaithersburg, Maryland,) or protein A sepharose 4B (Pharmacia, Uppsala, Sweden). Immunoreactive proteins were resolved by SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography.

Figure 6 shows that all three of the recombinant viruses carrying the gp53 35 gene preceded by a signal peptide sequence made significant amounts of the protein.

WO 95/12682 PCT/US94/12198 We did not detect any expression of gp53 from T2-3#3, or T2-2#5 the viruses carrying the gp53 gene, but lacking a signal peptide, even though this virus synthesized considerable amounts of gp53 messenger RNA. The: clones t2-3#3 and T2-2#5 are independently isolated clones, which rules out the possibility that one 5 particular virus had a defect that precluded gp53 expression (data not shown). The possiblity remains that gp53 is being synthesized from T2-3, but is rapidly degraded, or that our antibody does not detect unprocessed forms of the protein.

Figure 6. Immunopredpitated proteins showing expression of gp53 in the BHV-1 recombinants. Labelled proteins were precipitated with polyclonal bovine-10 anti-BVDV serum, this serum also had minor reactivity with BHV-1 antigens. KEY: 3,6,8=Tll-3, Tll-6, and Tll-8 infected cell proteins, IA=BHV-1 "Iowa" infected cell proteins, M«Mock infected cell proteins. MW«approximate protein molecular weight standards, in Kilodaltons.

The gp53 protein bands in Tll-3, Tll-6 and Tll-8 were broad, suggesting 15 that the proteins were processed, and they appeared to be equivalent but not identical in size to the gp53 protein in NADL (data not shown). Removal of the N-linked sugars from the BVDV-NADL and BHV-1 expressed gp53 proteins by digestion with N-glycansase (Genzyme, Cambridge, Massachusetts) did not resolve the size difference in the proteins, but the proportional reduction in size of the 20 proteins suggested that the native and recombinant forms of gp53 were processed similarly. The slight size difference between the recombinant and native proteins could be due to the fact that the gp53 gene in the BHV-1 viruses came from a different BVD strain which could have a gp53 of a slightly different size, or the dDNA gp53 clone might not contain the exact amino acids processed from the BVDV 25 polyprotein into native gp53.

The present invention is not to be limited in scope by the cell lines deposited or the embodiments disclosed herein which are intended as single illustrations of one aspect of the invention and any which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention, in addition to 30 those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

It is also to be understood that all base pair and amino acid residue numbers and sizes given for nucleotides and peptides are approximate and used for the 35 purposes of description.

WO 95/12682 PCT/US94/12198 All documents cited herein are incorporated by reference.

Deposit of Genetic One skilled in the art should be able to reconstruct all the various embodiments of this invention by utilizing only the written description. However, 5 for the sake of completeness, to ensure enablement, and to provide every opportunity for others to malcA and use this invention, certain genetic constructs of this invention have been deposited at recognized depositories in accordance with the Budapest Treaty.

A virus was deposited with the American Type Culture Collection, 12301 10 Parklawn Drive, Rockville, Maryland, zip code 20852, USA. That deposit was designated UC VR-58 by the Upjohn Company and given the following number by the depository, ATCC No. VR2436, it corresponds to the virus described herein as "Tll-6," also known as "Example 2." This deposit was received by the American Type Culture Collection depository on 28 October 1993.

Several plasmids were deposited with the Agricultural Research Service Culture Collection (NRRL), of the U.S. Department of Agriculture, at 1815 North University Street, Peoria, Blinds, zip code 61604, USA One plasmid was given the Upjohn designation, pUC 1564, E. coli culture UC 15085, referring to pBHVtkex-l::gin\p53, it corresponds to the plasmid used to create the virus described herein 20 as *111-3," also known as "Example 3." This plasmid was given the following number by the depository, NRRL B-21350. Another deposit was given the Upjohn designation, pUC 1565, E. coli culture UC 15086, referring to pBHVtkex-8::gIII\p53, it corresponds to the plasmid used to create the virus described herein as, "T-ll-8," also known as "Example 5." This plasmid was given the following number by the 25 depository, NRRL B-21351. Both of the plasmids were received by the Agricultural Research Service Culture Collection depository on 26 October 1994. 45 55 65 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: The Upjohn Company INVENTORS (For U.S. purposes only): Wardley, Richard C. and Haane8, Elizabeth J. (ii) TITLE OF INVENTION: A Replicating Nonpathogenic Virus Expressing Envelope Glycoproteins from Bovine Viral Diarrhea Virus (BVDV) (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Thomas A Woottoo (1920-32-1), The Upjohn Company (B) STREET: 7000 Portage Road (C) CITY: Kalamazoo 20 (D) STATE: Michigan (E) COUNTRY: U.S.A (F) ZIP: 49001-0199 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patently Release *1.0, Version *1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (Viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Wootton, Thomas A.

(B) REGISTRATION NUMBER: 35,004 (C) REFERENCE/DOCKET NUMBER: 4748 40 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 616 385-7914 (B) TELEFAX: 616 385-6897 (C) TELEX: 224 401 UPJOHN (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8083 base pairs 50 (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 60 (vi) ORIGINAL SOURCE: (A) ORGANISM: Bovine viral diarrhea virus (B) STRAIN: 2724 (C) INDIVIDUAL ISOLATE: pBHVtkex-3::p53 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: 40 50 60 65 GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA uO CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180 TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240 TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300 GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360 GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480 GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540 GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720 GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTXG CGTTTGCCCC 780 GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840 CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900 TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960 CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080 ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140 GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200 CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320 GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380 CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440 GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500 TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560 TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620 CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680 GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740 CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800 GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860 CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920 ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980 AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040 gatatcgcca tttttccaaa atatcgttta cgggggatgg 5 caaatatcgc agtttcgata gacatcaagc tggcacatgg ttagccatat tattcattgg acgttgtatc catatcataa tgttgacatt gattattgac 15 agcccatata tggagttccg cccaacgacc cccgcccatt gggactttcc attgacgtca catcaagtgt atcatatgcc gcctggcatt atgcccagta 25 gtattagtca tcgctattac tagcggtttg actcacgggg ttttggcacc aaaatcaacg caaatgggcg gtaggcgtgt cgtcagatcg cctggagacg 35 cgatccagcc tccgcggcaa gctactactg ataacagggg catagccagg aatgatagaa 40 ggattactca catgaaatga taagtttaca tacctctcaa 45 aagagccttg cagaccagtg aacatttgaa atggctgacg agtaagaggg aagttcaata 50 tataggatgg acaggaactg agtagtgcgt gtgtataaga 55 aagaactctg ggggaggatc tggggaccag ctacaataca attccaaaaa agtgaggggt 60 gactggctac agatttgtag acaaggattg gtaaagtgta 65 caaacttggg cctatgcctt . aaagacggca- tgcaccttca agttgatttt tgggcatacg cg/ltagacgc ctttggtgac taggtgacag acgatatgag ccaatgcata tcgatctata ttatatagca taaatcaata tatgtacatt tatattggct tagttattaa tagtaatcaa cgttacataa cttacggtaa gacgtcaata atgacgtatg atgggtggag tatttacggt aagtacgccc cctattgacg catgacctta tgggactttc catggtgatg cggttttggc atttccaagt ctccacccca ggactttcca aaatgtcgta acggtgggag gtctatataa ccatccacgc tgttttgacc gctgatccgt caggggccag tacaagggga cattgactgc ttggcccatt aggagctgaa agctggaaga cacaatggtc gotgcacaag agaaactaga tggtattcaa aaaacttttc actttgaatt tggactctgc caacactgct aaacggaccg tgagctgtat gttagctaat ggtccaaacc attcccttat tctataactg tgatcttgga caggaggccc tgtcgaatct tgccacacta ccccatcggc acggcaccac ttgcaacaga agataggaga cacaatcgta gcaagccata tgagatcata actacacgag gacattaaaa cgatatctgg cgatacgctt 2100 ttgggcgatt ctgtgtgtcg 2160 gctatatcgc cgatagaggc 2220 cattgaatca atattggcca 2280 ttggctattg gccattgcat 2340 catgtccaac attaccgcca 2400 ttacggggtc attagttcat 2460 atggcccgcc tggctgaccg 2520 ttcccatagt aacgccaata 2580 aaactgccca cttggcagta 2640 tcaatgacgg taaatggccc 2700 ctacttggca gtacatctac 2760 agtacatcaa tgggcgtgga 2820 ttgacgtcaa tgggagtttg 2880 acaactccgc cccattgacg 2940 gcagagctcg tttagtgaac 3000 tccatagaag acaccgggac 3060 atggtacagg gcatcctatg 3120 aaacctgaac actcatacgc 3180 ggcctcacca ctgtttggaa 3240 atagcttggt gcaaagacgg 3300 tatcttgcaa ttctgcattc 3360 gaggggcaaa ggcaagggga 3420 ccatgcgatg ccaatcccgt 3480 gccttccaga tggtatgccc 3540 agggacaccc tagacacagc 3600 agacaaggtt gtatcaccca 3660 gggaattgga cttgtgtgac 3720 tgcaagtggt gtggttataa 3780 aagtgtaggt tgaagaatga 3840 gagggtgtag ccatagtacc 3900 caggtcatag ctcttgacac 3960 ccaagtgagg ggcctgtaga 4020 aataaatatt ttgagcccag 4080 WO 9S/12682 40 45 50 55 60 65 agacagttac ttccagcaat acatgctaaa aggagattat caatactggt tcgacctgga 41«u ggtcactgac catcatcggg attacttcgc cgagtccata ttggtggtgg tggtagcttt 4200 actgggtgga agatacgtgc tctggttact ggtaacatac atggtcctat cagaacaaaa 4260 ggccttgggg acccaatatg gggcagggga agtggtgatg atgggtaact tgctaacaca 4320 tgacagtatt gaagtggtga catatttctt gttgttatac ctactgctaa gagaggaggc 4380 tgtaaagaag tgggtcttac tcttatacca ccttgattga ttgaggatca gcttatccag 4440 ggtcgacctc aggcatgcaa gctcagatcc gctgtgcctt ctagttgcca gccatctgtt 4500 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 4560 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 4620 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat 4680 gcggtgggct ctatgggtac ccaggtgctg aagaattgac ccggttcctc ctgggccaga 4740 aagaagcagg cacatcccct tctctgtgac acaccctgtc cacgcccctg gttcttagtt 4800 ccagccccac tcataggaca ctcatagctc aggagggctc cgcttcaatc ccacccgcta 4860 aagtacttgg agcggtctct ccctccctca tcagcccacc aaaccaaacc tagcctccaa 4920 gagtgggaag aaattaaagc aagataggct attaagtgca gagggagaga aaatgcctcc 4980 aacatgtgag gaagtaatga tagaaatcat agaattgaga tctcgaggtg ttcgtgctgg 5040 acgtgtccgc ggcgccagac gcgtgcgcgg ccgccgtact ggacatgcgg cccgccatgc 5100 aggccgcttg cgcggacggg gcggcgggcg cgacgctggc gaccctggcg cgtcagttcg 5160 cgctagagat ggcgggggag gccacggcgg gccctagggg actataaagc tgcccctgcg 5220 ctcgctcgct cgctgcattt gcgccccgat cgccttacgg ggactcggcg ctcggcggat 5280 cccctcccgg ccccgccgcg aagcaggccg ccagacaaaa aaatgcggcg cccgctctgc 5340 gcggcgctat tggcagcggc tgtcctcgcg ctcgccgcgg gcgcccccgc cgccgcccgc 5400 ggcgggggcg ccgaagccag ggcagcacag agacgcccga tacgaaatcg aagagtggga 5460 aatggtggtc ggagccgggc cggccgtgca cacgttcacc atccgctgcc tcgggccgcg 5520 gggcattgag cgcgtggccc acattgcaaa cctcagccgg ctgctggacg ggtacatagc 5580 ggtccacgtt gacgttgcgc gcacctctgg cctgcgggac gccatgtttt tcctgccgcg 5640 cgcggccgtc gactctagag gatccccggg taccgagctc gaattcactg gccgtcgttt 5700 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 5760 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 5820 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg 5880 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 5940 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 6000 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 6060 cgtcatcacc- gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 6120 40 45 50 55 60 65 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 6180 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 6240 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 6300 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 6360 cgctggtgaa agtaaaagat gotgaagatc agttgggtgc acgagtgggt tacatcgaac 6420 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 6480 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 6540 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 6600 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 6660 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 6720 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 6780 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 6840 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 6900 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 6960 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 7020 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 7080 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 7140 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 7200 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 7260 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 7320 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 7380 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 7440 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 7500 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 7560 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 7620 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 76b0 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 7740 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 7800 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 7860 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 7920 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 7980 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 8040 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aga 8083 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8149 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS. double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: Bovine viral diarrhea virus (B) STRAIN: 2724 (C) INDIVIDUAL ISOLATE: pBHVtkex-1::gBGH/p53 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60 CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180 TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240 TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300 GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360 GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480 GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540 40 GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660 45 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720 GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780 GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840 50 CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900 TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960 55 CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080 ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140 60 GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200 CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260 65 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320 GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380 40 45 50 55 60 65 ccgagcccgc gcgcgctctc cgcgtcgtgc GTATCTACCT ggacggcgcg cacgggcasg 1440 GAAAGACAAC aacgggccgc gcgctcgcgg CCGCTTCCAC cgctggggag GGCGTGCTCT 1500 TTTTCCCGGA gccgatggcg tactggcgca CGATGTTTGG tacggacgcc ttaagtggga 1560 tcctcgcggc gtctgcgcga tgcgccgcag cctcgcacgg gagcgcacgc gcgcggcggg 1620 ccggcgcacc gcgcagacgc ggacgcggcg GGCCTGGTTG cgtactacca ggccaggttc 1680 GCGGCCCCGT acttaatttt gcacgcgcgt GTCCGCGCTG ctgcgccgcc TGGGCCGGCG 1740 ccgggcggcg agctggtgga ccctcgtgtt cgaccgccac cccgtggcgc gcgtgcctct 1800 gctacccctt cgcccgctac tgcctccgcg agatcaacgc ggaagatctc aattctatga 1860 tttctatcat tacttcctca catgttggag gcattttctc tccctctgca cttaatagcc 1920 tatcttgctt taatttcttc ccactcttgg aggctaggtt tggtttggtg ggctgatgag 1980 ggagggagag accgctccaa gtactttagc gggtgggatt gaagcggagc cctcctgagc 2040 tatgagtgtc ctatgagtgg ggctggaact aagaaccagg ggcgtggaca gggtgtgtca 2100 cagagaaggg gatgtgcctg cttctttctg gcccaggagg aaccgggtca attcttcagc 2160 acctgggtac ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc 2220 tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa 2280 tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc 2340 aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg cacagcggat 2400 CTGAGCTTGC atgcctgagg tcgaccctgg ataagctgat cctcaatcaa tcaaggtggt 2460 ATAAGAGTAA gacccacttc tttacaocct cctctcttag cagtaggtat aacaacaaga 2520 aatatgtcac cacttcaata ctgtcatgtg ttagcaagtt acccatcatc accacttccc 2580 ctgccccata ttgggtcccc aaggcctttt gttctgatag gaccatgtat gttaccagta 2640 accagagcac gtatcttcca cccagtaaag ctaccaccac caccaatatg gactcggcga 2700 agtaatcccg atgatggtca gtgacctcca ggtcgaacca gtattgataa tctcctttta 2760 gcatgtattg ctggaagtaa ctgtctctgg gctcaaaata tttatttttt aatgtcctcg 2820 tgtagttgaa ggtgcatgcc gtcttttcta caggcccctc acttggtatg atctcatatg 2880 gcttgcaagg cataggccca agtttggtgt caagagctat gacctgtacg attgtgtctc 2940 ctatcttaca ctttaccaat ccttgtggta ctatggctac accctctctg ttgcaagtgg 3000 tgccgtctac aaatctgtag ccagtctcat tcttcaacct acacttgccg atggggtagt 3060 gtggcaaccc ctcacttttt tggaatttat aaccacacca cttgcaagat tcgacagggc 3120 ctcctgtgta ttgtagctgg tccccagtca cacaagtcca attccctcca agatcacagt 3180 tatagagatc ctcccccaga gttctttggg tgatacaacc ttgtctataa gggaatggtt 3240 tggacctctt atacacacgc actactgctg tgtctagggt gtccctatta gctaacatac 3300 agctcacagt tcctgtccat cctatagggc ataccatctg gaaggccggt ccgtttagca 3360 gtgttgtatt gaacttccct cttactacgg gattggcatc gcatgggcag agtccaaatt 3420 40 45 50 55 60 65 caaagtcgtc agccatttca aatgtttccc cttgcctttg cccctcgaaa agttttttga 34t>o ataccacact ggtctgcaag gctcttgaat gcagaattgc aagatatcta gtttctcttg 3540 tgcaccttga gaggtatgta aacttaccgt ctttgcacca agctatgacc attgtgtctt 3600 ccagcttcat ttcatgtgag taatccttcc aaacagtggt gaggccttca gctcctaatg 3660 ggccaattct atcattcctg gctatggcgt atgagtgttc aggtttgcag tcaatgtccc 3720 cttgtacccc tgttatcagt agtagccata ggatccctgg gaaggcgccc accacctgag 3780 tccagggcag gcagagcagg gcgaaagcca ggagcaggga ggtccggggg cctgcagcca 3840 tcatgtcgaa gcttgccgcg gaggctggat cggtcccggt gtcttctatg gaggtcaaaa 3900 cagcgtggat ggcgtctcca ggcgatctga cggttcacta aacgagctct gcttatatag 3960 acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat 4020 tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg 4080 agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac 4140 cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc 4200 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata 4260 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa 4320 tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc 4380 attattgacg tcaatgggcg gggjtcgttg ggcggtcagc caggcgggcc atttaccgta 4440 agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac 4500 tattaataac tagtcaataa tcaatgtcaa catggcggta atgttggaca tgagccaata 4560 taaatgtaca tattatgata tggatacaac gtatgcaatg gccaatagcc aatattgatt 4620 tatgctatat aaccaatgaa taat-itggct aatggccaat attgattcaa tgtatagatc 4680 gatatgcatt ggccatgtgc cagcttgatg tcgcctctat cggcgatata gcctcatatc 4740 gtctgtcacc tatatcgaaa ctgcgatatt tgcgacacac agaatcgccc aagtcaccaa 4800 aggcgtctat cgccatcccc cgtaaacgat ataagcgtat cgccagatat cgcgtatgcc 4860 caaaaatcaa cttttggaaa aatggcgata tcagttacac agaaactcac atcggcgaca 4920 ttttcaatat gccatatttt caaatatcga tttttccaat atcgccatct ctatcggcga 4980 taaacaccac tatcgcgcga catgaattta gtcgggacag aaatctcaaa cgcgtatttc 5040 ggacaaacac acatttzatt attcactgca ggtcgaggaa ttcggatctc gaggtgttcg 5100 tgctggacgt gtccgcggcg ccagacgcgt gcgcggccgc cgtactggac atgcggcccg 5160 ccatgcaggc cgcttgcgcg gacggggcgg cgggcgcgac gctggcgacc ctggcgcgtc 5220 agttcgcgct agagatggcg ggggaggcca cggcgggccc taggggacta taaagctgcc 5280 cctgcgctcg ctcgctcgct gcatttgcgc cccgatcgcc ttacggggac tcggcgctcg 5340 gcggatcccc tcccggcccc gccgcgfcagc aggccgccag acaaaaaaat gcggcgcccg 5400 ctctgcgcgg cgctattggc agcggctgtc ctcgcgctcg ccgcgggcgc ccccgccgcc 5460 WO 95/12682 PCT/US94/12198 GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG AAATCGAAGA 5520 GTGGGAAATG GTGGTCGGAG CCGGGCCGGC CGTGCACACG TTCACCATCC GCTGCCTCGG 5580 GCCGCGGGGC ATTGAGCGCG TGGCCCACAT TGCAAACCTC AGCCGGCTGC TGGACGGGTA 5640 CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA TGTTTTTCCT 5700 GCCGCGCGCG GCCGTCGACT CTAGAGGATC CCCGGGTACC GAGCTCGAAT TCACTGGCCG 5760 TCGTTTTACA ACGTCGTGAC TGGGA&AACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820 CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC 5880 AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC 5940 TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 6000 AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC 6060 TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 6120 TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC CTATTTTTAT 6180 AGGTTAATGT CATGATAATA ATGGTTTCTT AGACGTCAGG TGGCACTTTT CGGGGAAATG 6240 TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCXCATGA 6300 GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC 6360 ATTTCCGTGX CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 6420 CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6480 TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC 6540 CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG 6600 GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC 6660 40 CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 6720 TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG 6780 45 AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC 6840 CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GTAGCAATGG 6900 CAACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT 6960 50 TAATAGACTG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 7020 CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 7080 55 CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC 7140 AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 7200 ATTGGTAACT GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 7260 60 TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 7320 AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT 7380 65 GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG 7440 CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA 7500 PCTAJS94/12198 GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA 75uo AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 7620 CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG 7680 CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT 7740 ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA 7800 GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC 7860 TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 7920 AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG 7980 CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT 8040 TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC 8100 GCAGCCGAAC GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAGA 8149 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8135 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 35 (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: Bovine viral diarrhea virus (B) STRAIN: 2724 40 <C) INDIVIDUAL ISOLATE: pBHVtkex-1::gIII/p53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 45 GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60 CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180 50 TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240 TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300 55 GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360 GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480 60 GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540 GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 65 CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720 WO 95/12682 PCT/TJS94/12198 GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780 GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840 CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900 TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960 CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080 ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140 lb GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200 CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320 GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380 CCGAGCCCGC GCGCGCtCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440 GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500 TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560 TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620 CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680 GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740 CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800 GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCTC AATTCTATGA 1860 TTTCTATCAT TACTTCCTCA CATGTTGGAG GCATTTTCTC TCCCTCTGCA CTTAATAGCC 1920 40 TATCTTGC2T TAATTTCTTC CCACTCTTGG AGGCTAGGTT TGGTTTGGTG GGCTGATGAG 1980 GGAGGGAGAG ACCGCTCCAA GTACTTTAGC GGGTGGGATT GAAGCGGAGC CCTCCTGAGC 2040 45 TATGAGTGTC CTATGAGTGG GGCTGGAACT AAGAACCAGG GGCGTGGACA GGGTGTGTCA 2100 CAGAGAAGGG GATGTGCCTG CTTCTTTCTG GCCCAGGAGG AACCGGGTCA ATTCTTCAGC 2160 ACCTGGGTAC CCATAGAGCC CACCGCATCC CCAGCATGCC TGCTATTGTC TTCCCAATCC 2220 50 TCCCCCTTGC TGTCCTGCCC CACCCCACCC CCCAGAATAG AATGACACCT ACTCAGACAA 2280 TGCGATGCAA TTTCCTCATT TTATTAGGAA AGGACAGTGG GAGTGGCACC TTCCAGGGTC 2340 55 AAGGAAGGCA CGGGGGAGGG GCAAACAACA GATGGCTGGC AACTAGAAGG CACAGCGGAT 2400 CTGAGCTTGC ATGCCTGAGG TCGACCCTGG ATAAGCTGAT CCTCAATCAA TCAAGGTGGT 2460 ATAAGAGTAA GACCCACTTC TTTACAGCCT CCTCTCTTAG CAGTAGGTAT AACAACAAGA 2520 60 AATATGTCAC CACTTCAATA CTGTCATGTG TTAGCAAGTT ACCCATCATC ACCACTTCCC 2580 CTGCCCCATA TTGGGTCCCC AAGGCCTTTT GTTCTGATAG GACCATGTAT GTTACCAGTA 2640 65 ACCAGAGCAC GTATCTTCCA CCCAGTAAAG CTACCACCAC CACCAATATG GACTCGGCGA 2700 AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCGAACCA GTATTGATAA TCTCCTTTTA 2760 GCATGTATTG CTGGAAGTAA CTGTCTCTGG GCTCAAAATA TTTATTTTTT AATGTCCTCG 28^0 TGTAGTTGAA GGTGCATGCC GTCTTTTCTA CAGGCCCCTC ACTTGGTATG ATCTCATATG 2880 GCTTGCAAGG CATAGGCCCA AGTTTGGTGT CAAGAGCTAT GACCTGTACG ATTGTGTCTC 2940 CTATCTTACA CTTTACCAAT CCTTGTGGTA CTATGGCTAC ACCCTCTCTG TTGCAAGTGG 3000 TGCCGTCTAC AAATCTGTAG CCAGTCTCAT TCTTCAACCT ACACTTGCCG ATGGGGTAGT 3060 GTGGCAACCC CTCACTTTTT TGGAATTTAT AACCACACCA CTTGCAAGAT TCGACAGGGC 3120 CTCCTGTGTA TTGTAGCTGG TCCCCAGTCA CACAAGTCCA ATTCCCTCCA AGATCACAGT 3180 TATAGAGATC CTCCCCCAGA GTTCTTTGGG TGATACAACC TTGTCTATAA GGGAATGGTT 3240 TGGACCTCTT ATACACACGC ACTACTGCTG TGTCTAGGGT GTCCCTATTA GCTAACATAC 3300 AGCTCACAGT TCCTGTCCAT CCTATAGGGC ATACCATCTG GAAGGCCGGT CCGTTTAGCA 3360 GTGTTGTATT GAACTTCCCT CTTACTACGG GATTGGCATC GCATGGGCAG AGTCCAAATT 3420 CAAAGTCGTC AGCCATTTCA AATGTTTCCC CTTGCCTTTG CCCCTCGAAA AGTTTTTTGA 3480 ATACCACACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAGATATCTA GTTTCTCTTG 3540 TGCACCTTGA GAGGTATGTA AACTTACCGT CTTTGCACCA AGCTATGACC ATTGTGTCTT 3600 CCAGCTTCAT TTCATGTGAG TAATCCTTCC AAACAGTGGT GAGGCCTTCA GCTCCTAATG 3660 GGCCAATTCT ATCATTCCTG GCTATGGCGT ATGAGTGTTC AGGTTTGCAG TCAATGTCCC 3720 CTTGTACCCC TGTTATCAGT AGTAGCCATA GGATCCCCGA CGGCGCCGCG GCGATGGCCG 3780 CCGCGTAGAG CGCCAGCAGA GCGAGCATCG CACGCGCGAG CGAGGCCATG GTCGAAGCTT 3840 GCCGCGGAGG CTGGATCGGT CCCGGTGTCT TCTATGGAGG TCAAAACAGC GTGGATGGCG 3900 TCTCCAGGCG ATCTGACGGT TCACTAAACG AGCTCTGCTT ATATAGACCT CCCACCGTAC 3960 40 ACGCCTACCG CCCATTTGCG TCAATGGGGC GGAGTTGTTA CGACATTTTG GAAAGTCCCG 4020 TTGATTTTGG TGCCAAAACA AACTCCCATT GACGTCAATG GGGTGGAGAC TTGGAAATCC 4080 45 CCGTGAGTCA AACCGCTATC CACGCCCATT GATGTACTGC CAAAACCGCA TCACCATGGT 4140 AATAGCGATG ACTAATACGT AGATGTACTG CCAAGTAGGA AAGTCCCATA AGGTCATGTA 4200 CTGGGCATAA TGCCAGGCGG GCCATTTACC GTCATTGACG TCAATAGGGG GCGTACTTGG 4260 50 CATATGATAC ACTTGATGTA CTGCCAAGTG GGCAGTTTAC CGTAAATACT CCACCCATTG 4320 ACGTCAATGG AAAGTCCCTA TTGGCGTTAC TATGGGAACA TACGTCATTA TTGACGTCAA 4380 55 TGGGCGGGGG TCGTTGGGCG GTCAGCCAGG CGGGCCATTT ACCGTAAGTT ATGTAACGCG 4440 GAACTCCATA TAl-GGGCTAT GAACTAATGA CCCCGTAATT GATTACTATT AATAACTAGT 4500 CAATAATCAA TGTCAACATG GCGGTAATGT TGGACATGAG CCAATATAAA TGTACATATT 4560 60 ATGATATGGA TACAACGTAT GCAATGGCCA ATAGCCAATA TTGATTTATG CTATATAACC 4620 AATGAATAAT ATGGCTAATG GCCAATATTG ATTCAATGTA TAGATCGATA TGCATTGGCC 4680 65 ATGTGCCAGC TTGATGTCGC CTCTATCGGC GATATAGCCT CATATCGTCT GTCACCTATA 4740 TCGAAACTGC GATATTTGCG ACACACAGAA TCGCCCAAGT CACCAAAGGC GTCTATCGCC 4800 atcccccgta aacgatataa tggaaaaatg gcgatatcag 5 tattttcaaa tatcgatttt gcgcgacatg aatttagtcg tttattattc actgcaggtc gcggcgccag acgcgxgcgc tgcgcggacg gggcggcggg 15 atggcggggg aggccacggc ctcgctgcat ttgcgccccg ggccccgccg cgaagcaggc attggcagcg gctgtcctcg cgccgaagcc agggcagcac 25 tcggagccgg gccggccgtg agcgcgtggc ccacattgca ttgacgttgc gcgcacctct tcgactctag aggatccccg cgtgactggg aaaaccctgg 35 gccagctggc gtaatagcga ctgaatggcg aatggcgcct caccgcatat ggtgcactct 40 cgacacccgc caacacccgc tacagacaag ctgtgaccgt 45 ccgaaacgcg cgagacgaaa ataataatgg tttcttagac atttgtttat ttttctaaat 50 taaatgcttc aataatattg cttattccct tttttgcggc 55 aaagtaaaag atgctgaaga aacagcggta agatccttga tttaaagttc tgctatgtgg 60 ggtcgccgca tacactattc catcttacgg atggcatgac 65 aacactgcgg ccaacttact ttgcacaaca tgggggatca GCGTATCGCC AGATATCGCG TTACACAGAA ACTCACATCG TCCAATATCG CCATCTCTAT GGACAGAAAT CTCAAACGCG GAGGAATTCG GATCTCGAGG GGCCGCCGTA CTGGACATGC CGCGACGCTG GCGACCCTGG GGGCCCTAGG GGACTATAAA ATCGCCTTAC GGGGACTCGG CGCCAGACAA AAAAATGCGG CGCTCGCCGC GGGCGCCCCC AGAGACGCCC GATACGAAAT CACACGTTCA CCATCCGCTG AACCTCAGCC GGCTGCTGGA GGCCTGCGGG ACGCCATGTT GGTACCGAGC TCGAATTCAC CGTTACCCAA CTTAATCGCC AGAGGCCCGC ACCGATCGCC GATGCGGTAT TTTCTCCTTA CAGTACAATC TGCTCTGATG TGACGCGCCC TGACGGGCTT CTCCGGGAGC TGCATGTGTC GGGCCTCGTG ATACGCCTAT GTCAGGTGGC AC1TTTCGGG ACATTCAAAT ATGTATCCGC AAAAAGGAAG AGTATGAGTA ATTTTGCCTT CCTGTTTTTG TCAGTTGGGT GCACGAGTGG GAGTTTTCGC CCCGAAGAAC CGCGGTATTA TCCCGTATTG TCAGAATGAC TTGGTTGAGT AGTAAGAGAA TTATGCAGTG TCTGACAACG ATCGGAGGAC TGTAACTCGC CTTGATCGTT TATGCCCAAA AATCAACTTT 4860 GCGACATTTT CAATATGCCA 4920 CGGCGATAAA CACCACTATC 4980 TATTTCGGAC AAACACACAT 5040 TGTTCGTGCT GGACGTGTCC 5100 GGCCCGCCAT GCAGGCCGCT 5160 CGCGTCAGTT CGCGCTAGAG 5220 GCTGCCCCTG CGCTCGCTCG 5280 CGCTCGGCGG ATCCCCTCCC 5340 CGCCCGCTCT GCGCGGCGCT 5400 GCCGCCGCCC GCGGCGGGGG 5460 CGAAGAGTGG GAAATGGTGG 5520 CCTCGGGCCG CGGGGCATTG 5580 CGGGTACATA GCGGTCCACG 5640 TTTCCTGCCG CGCGCGGCCG 5700 TGGCCGTCGT TTTACAACGT 5760 TTGCAGCACA TCCCCCTTTC 5820 CTTCCCAACA GTTGCGCAGC 5880 CGCATCTGTG CGGTATTTCA 5940 CCGCATAGTT AAGCCAGCCC 6000 GTCTGCTCCC GGCATCCGCT 6060 AGAGGTTTTC ACCGTCATCA 6120 TTTTATAGGT TAATGTCATG 6180 GAAATGTGCG CGGAACCCCT 6240 TCATGAGACA ATAACCCTGA 6300 TTCAACATTT CCGTGTCGCC 6360 CTCACCCAGA AACGCTGGTG 6420 GTTACATCGA ACTGGATCTC 6480 GTTTTCCAAT GATGAGCACT 6540 ACGCCGGGCA AGAGCAACTC 6600 ACTCACCAGT CACAGAAAAG 6650 CTGCCATAAC CATGAGTGAT 6720 CGAAGGAGCT AACCGCTTTT 6780 GGGAACCGGA GCTGAATGAA 6840 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 690u aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 6960 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 7020 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 7080 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 7140 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 7200 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 7260 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 7320 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 7380 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 7440 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 7500 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 7560 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 7620 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 7680 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 7740 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 7800 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 7860 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 7920 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 7980 40 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 8040 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 8100 gagcgcagcg agtcagtgag cgaggaagcg gaaga 8135 45 (2) information for seq id no:4: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8149 base pairs (B) TYPE: nucleic acid 50 (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 55 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: 60 (A) ORGANISM: Bovine viral diarrhea virus (B) STRAIN: 2724 (C) INDIVIDUAL ISOLATE: pBHVtkex-3: :BGH/p53 65 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 40 45 50 55 60 65 GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60 CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180 TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240 TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300 GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360 GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480 GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540 GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720 GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780 GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840 CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900 TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960 CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080 ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140 GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200 CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320 GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380 CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440 GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500 TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560 TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620 CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680 GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740 CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800 GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860 CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920 ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980 AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040 GATATCGCCA TTTTTCCAAA AXATCGTTTA CGGGGGATGG 5 CAAATATCGC AGTTTCGATA GACATCAAGC TGGCACATGG TTAGCCATAT TATTCATTGG ACGTTGTATC CATATCATAA TGTTGACATT GATTATTGAC 15 AGCCCATATA TGGAGTTCCG CCCAACGACC CCCGCCCATT GGGACTTTCC ATTGACGTCA CATCAAGTGT ATCATATGCC GCCTGGCATT ATGCCCAGTA 25 GTATTAGTCA TCGCTATTAC TAGCGGTTTG ACTCACGGGG TTTTGGCACC AAAATCAACG CAAATGGGCG GTAGGCGTGT CGTCAGATCG CCTGGAGACG 35 CGATCCAGCC TCCGCGGCAA CCTGGCTTTC GCCCTGCTCT CCTATGGCTA CTACTGATAA 40 ATACGCCATA GCCAGGAATG TTGGAAGGAT TACTCACATG 45 AGACGGTAAG TTTACATACC GCATTCAAGA GCCTTGCAGA AGGGGAAACA TTTGAAATGG 50 TCCCGTAGTA AGAGGGAAGT ATGCCCTATA GGATGGACAG 55 CACAGCAGTA GTGCGTGTGT CACCCAAAGA ACTCTGGGGG TGTGACTGGG GACCAGCTAC 60 TTATAAATTC CAAAAAAGTG GAATGAGACT GGCTACAGAT 65 AGTACCACAA GGATTGGTAA TGACACCAAA CTTGGGCCTA AGTTGATTTT TGGGCATACG CGATAGACGC CTTTGGTGAC TAGGTGACAG ACGATATGAG CCAATGCATA TCGATCTATA TTATATAGCA TAAATCAATA TATGTACATT TATATTGGCT TAGTTATTAA TAGTAATCAA CGTTACATAA CTTACGGTAA GACGTCAATA ATGACGTATG ATGGGTGGAG TATTTACGGT AAGTACGCCC CCTATTGACG CATGACCTTA TGGGACTTTC CATGGTGATG CGGTTTTGGC ATTTCCAAGT CTCCACCCCA GGACTTTCCA AAATGTCGTA ACGGTGGGAG GTCTATATAA CCATCCACGC TGTTTTGACC GCTTCGACAT GATGGCTGCA GCCTGCCCTG GACTCAGGTG CAGGGGTACA AGGGGACATT ATAGAATTGG CCCATTAGGA AAATGAAGCT GGAAGACACA TCTCAAGGTG CACAAGAGAA CCAGTGTQGT ATTCAAAAAA CTGACGACTT TGAATTTGGA TCAATACAAC ACTGCTAAAC GAACTGTGAG CTGTATGTTA ATAAGAGGTC CAAACCATTC AGGATCTCTA TAACTGTGAT AATACACAGG AGGCCCTGTC AGGGGTTGCC ACACTACCCC TTGTAGACGG CACCACTTGC AGTGTAAGAT AGGAGACACA TGCCTTGCAA GCCATATGAG CGATATCTGG CGATACGCTT 2100 TTGGGCGATT CTGTGTGTCG 2160 GCTATATCGC CGATAGAGGC 2220 CATTGAATCA ATATTGGCCA 2280 TTGGCTATTG GCCATTGCAT 2340 CATGTCCAAC ATTACCGCCA 2400 TTACGGGGTC ATTAGTTCAT 2460 ATGGCCCGCC TGGCTGACCG 2520 TTCCCATAGT AACGCCAATA 2580 AAACTGCCCA CTTGGCAGTA 2640 TCAATGACGG TAAATGGCCC 2700 CTACTTGGCA GTACATCTAC 2760 AGTACATCAA TGGGCGTGGA 2820 TTGACGTCAA TGGGAGTTTG 2880 ACAACTCCGC CCCATTGACG 2940 GCAGAGCTCG TTTAGTGAAC 3000 TCCATAGAAG ACACCGGGAC 3060 GGCCCCCGGA CCTCCCTGCT 3120 GTGGGCGCCT TCCCAGGGAT 3180 GACTGCAAAC CTGAACACTC 3240 GCTGAAGGCC TCACCACTGT 3300 ATGGTCATAG CTTGGTGCAA 3360 ACTAGATATC TTGCAATTCT 3420 CTTTTCGAGG GGCAAAGGCA 3480 CTCTGCCCAT GCGATGCCAA 3540 GGACCGGCCT TCCAGATGGT 3600 GCTAATAGGG ACACCCTAGA 3660 CCTTATAGAC AAGGTTGTAT 3720 CTTGGAGGGA ATTGGACTTG 3780 GAATCTTGCA AGTGGTGTGG 3840 ATCGGCAAGT GTAGGTTGAA 3900 AACAGAGAGG GTGTAGCCAT 3960 ATCGTACAGG TCATAGCTCT 4020 ATCATACCAA GTGAGGGGCC 4080 TGTAGAAAAG ACGGCATGCA CCTTCAACTA CACGAGGACA TTAAAAAATA AATATTTTGA 4140 GCCCAGAGAC AGTTACTTCC AGCAATACAT GCTAAAAGGA GATTATCAAT ACTGGTTCGA 4200 CCTGGAGGTC ACTGACCATC ATCGGGATTA CTTCGCCGAG TCCATATTGG TGGTGGTGGT 4260 AGCTTTACTG GGTGGAAGAT ACGTGCTCTG GTTACTGGTA ACATACATGG TCCTATCAGA 4320 ACAAAAGGCC TTGGGGACCC AATATGGGGC AGGGGAAGTG GTGATGATGG GTAACTTGCT 4380 AACACATGAC AGTATTGAAG TGGTGACATA TTTCTTGTTG TTATACCTAC TGCTAAGAGA 4440 GGAGGCTGTA AAGAAGTGGG TCTTACTCTT ATACCACCTT GATTGATTGA GGATCAGCTT 4500 ATCCAGGGTC GACCTCAGGC ATGCAAGCTC AGATCCGCTG TGCCTTCTAG TTGCCAGCCA 4560 TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC 4620 CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG 4680 GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT 4740 GGGGATGCGG TGGGCTCTAT GGGTACCCAG GTGCTGAAGA ATTGACCCGG TTCCTCCXGG 4800 GCCAGAAAGA AGCAGGCACA TCCCCTTCTC XGXGACACAC CCTGTCCACG CCCCTGGXXC 4860 XXAGXICCAG CCCCACICAT AGGACACTCA XAGCICAGGA GGGCICCGCI TCAAICCCAC 4920 CCGCXAAAGI ACXXGGAGCG GTCTCTCCCT CCCICATCAG CCCACCAAAC CAAACCIAGC 4980 CXCCAAGAGX GGGAAGAAAX XAAAGCAAGA XAGGCTAXXA AGIGCAGAGG GAGAGAAAAT 5040 GCCTCCAACA XGXGAGGAAG TAATGATAGA AAXCATAGAA XTGAGAXCXC GAGGIGXICG 5100 XGCXGGACGX GTCCGCGGCG CCAGACGCGX GCGCGGCCGC CGIACXGGAC AIGCGGCCCG 5160 CCAXGCAGGC CGCXXGCGCG GACGGGGCGG CGGGCGCGAC GCXGGCGACC CXGGCGCGXC 5220 AGXXCGCGCX AGAGATGGCG GGGGAGGCCA CGGCGGGCCC XAGGGGACXA TAAAGCTGCC 5280 40 CCTGCGCTCG CTCGCTCGCT GCATTTGCGC CCCGATCGCC TTACGGGGAC TCGGCGCTCG 5340 GCGGATCCCC TCCCGGCCCC GCCGCGAAGC AGGCCGCCAG ACAAAAAAAT GCGGCGCCCG 5400 45 CTCXGCGCGG CGCTATTGGC AGCGGCTGTC CTCGCGCTCG CCGCGGGCGC CCCCGCCGCC 5460 GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG AAATCGAAGA 5520 GTGGGAAATG GTGGTCGGAG CCGGGCCGGC CGTGCACACG TTCACCATCC GCTGCCTCGG 5580 50 GCCGCGGGGC ATXGAGCGCG TGGCCCACAT TGCAAACCTC AGCCGGCTGC TGGACGGGTA 5640 CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA TGTTTTTCCT 5700 55 GCCGCGCGCG GCCGTCGACT CTAGAGGAXC CCCGGGTACC GAGCTCGAAT TCACTGGCCG 5760 TCGTTTTACA ACG7CGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820 CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC 5880 60 AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC 5940 TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 6000 65 AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC 6060 . TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 6120 tttcaccgtc atcaccgaaa aggttaatgt catgataata 5 tgcgcggaac ccctatttgt gacaataacc ctgataaatg atttccgtgt cgcccttatt cagaaacgct ggtgaaagta tcgaactgga tctcaacagc 15 caatgatgag cacttttaaa ggcaagagca actcggtcgc cagtcacaga aaagcatctt taaccatgag tgataacact agctaaccgc ttttttgcac 25 cggagctgaa tgaagccata caacaacgtt gcgcaaacta taatagactg gatggaggcg ctggctggtt tattgctgat cagcactggg gccagatggt 35 aggcaactat ggatgaacga attggtaact gtcagaccaa tttaatttaa aaggatctag 40 aacgtgagtt ttcgttccac gagatccttt ttttctgcgc 45 cggtggtttg tttgccggat gcagagcgca gataccaaat agaactctgt agcaccgcct 50 ccagtggcga taagtcgtgt cgcagcggtc gggctgaacg 55 acaccgaact gagataccta gaaaggcgga caggtatccg ttccaggggg aaacgccvgg 60 agcgtcgatt tttgtgatgc cggccttttt acggttcctg 65 tatcccctga ttctgtggat gcagccgaac- gaccgagcgc cgcgcgagac gaaagggcct atggtttctt agacgtcagg ttatttttct aaatacattc cttcaataat attgaaaaag cccttttttg cggcattttg aaagatgctg aagatcagtt ggtaagatcc ttgagagttt gttctgctat gtggcgcggt cgcatacact attctcagaa acggatggca tgacagtaag gcggccaact tacttctgac aacatggggg atcatgtaac ccaaacgacg agcgtgacac ttaactggcg aactacttac gataaagttg caggaccact aaatctggag ccggtgagcg aagccctccc gtatcgtagt aatagacaga tcgctgagat gtttactcat atatacttta gtgaagatcc tttttgataa tgagcgtcag accccgtaga gtaatctgct gcttgcaaac caagagctac caactctttt actgtccttc tagtgtagcc I o % ctctgctaat cttaccgggt tggactcaag gggggttcgt gcacacagcc cagcgtgagc tatgagaaag gtaagcggca gggtcggaac tatctttata gtcctgtcgg tcgtcagggg ggcggagcct gccttttgct ggccttttgc aaccgtatta ccgcctttga agcgagtcag tgagcgagga PCX/US94/12198 cgtgatacgc ctatttttat 61fa. tggcactttt cggggaaatg 6240 aaatatgtat ccgctcatga 6300 gaagagtatg agtattcaac 6360 ccttcctgtt tttgctcacc 6420 gggtgcacga gtgggttaca 6480 tcgccccgaa gaacgttttc 6540 attatcccgt attgacgccg 6600 tgacttggtt gagtactcac 6660 agaattatgc agtgctgcca 6720 aacgatcgga ggaccgaagg 6780 tcgccttgat cgttgggaac 6840 cacgatgcct gtagcaatgg 6900 tctagcttcc cggcaacaat 6960 tctgcgctcg gcccttccgg 7020 tgggtc'fcgc ggtatcattg 7080 tatctacacg acggggagtc 7140 aggtgcctca ctgattaagc 7200 gattgattta aaacttcatt 7260 tctcatgacc aaaatccctt 7320 aaagatcaaa ggatcttctt 7380 aaaaaaacca ccgctaccag 7440 tccgaaggta actggcttca 7500 gtagttaggc caccacttca 7560 cctgttacca gtggctgctg 7620 acgatagtta ccggataagg 7680 cagcttggag cgaacgacct 7740 cgccacgctt cccgaaggga 7800 aggagagcgc acgagggagc 7860 gtttcgccac ctctgacttg 7920 atggaaaaac gccagcaacg 7980 tcacatgttc tttcctgcgt 8040 gtgagctgat accgctcgcc 8100 agcggaaga 8149 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8135 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bovine viral diarrhea virus (B) STRAIN: 2724 (C) INDIVIDUAL ISOLATE: pBHVtkex-3::gIII/p53 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60 CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180 TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240 TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300 GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360 GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480 GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540 40 GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 66D 45 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720 GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780 GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840 50 CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900 TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960 55 CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080 ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140 60 GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200 CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260 65 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320 GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380 CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GAAAGACAAC AACGGGCCGC GCGCTCGCGG 5 TTTTCCCGGA GCCGATGGCG TACTGGCGCA TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCGG-GCACC GCGCAGACGC GGACGCGGCG GCGGCCCCGT ACTTAATTTT GCACGCGCGT CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT 15 GCTACCCCTT CGCCCGCTAC TGCCTCCGCG CCTGCAGTGA ATAATAAAAT GTGTGTTTGT ACTAAATTCA TGTCGCGCGA TAGTGGTGTT AATCGATATT TGAAAATATG GCATATTGAA GATATCGCCA TTTTTCCAAA AGTTGATTTT 25 ATATCGTTTA CGGGGGATGG CGATAGACGC CAAATATCGC AGTTTCGATA TAGGTGACAG GACATCAAGC TGGCACATGG CCAATGCATA TTAGCCATAT TATTCATTGG TTATATAGCA ACGTTGTATC CATATCATAA TATGTACATT 35 TGTTGACATT GATTATTGAC TAGTTATTAA AGCCCATATA TGGAGTTCCG CGTTACATAA CCCAACGACC CCCGCCCATT GACGTCAATA 40 GGGACTTTCC ATTGACGTCA ATGGGTGGAG CATCAAGTGT ATCATATGCC AAGTACGCCC 45 GCCTGGCATT ATGCCCAGTA CATGACCTTA GTATTAGTCA TCGCTATTAC CATGGTGATG TAGCGGTTTG ACTCACGGGG ATTTCCAAGT 50 TTTTGGCACC AAAATCAACG GGACTTTCCA CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 55 CGTCAGATCG CCTGGAGACG CCATCCACGC CGATCCAGCC TCCGCGGCAA GCTTCGACCA TGCTGGCGCT CTACGCGGCG GCCATCGCCG 60 TGATAACAGG GGTACAAGGG GACATTGACT GGAATGATAG AATTGGCCCA TTAGGAGCTG 65 CACATGAAAT GAAGCTGGAA GACACAATGG CATACCTCTC AAGGTGCACA AGAGAAACTA GTATCTACCT GGACGGCGCG CACGGGCAGG 144u CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500 CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560 CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620 GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680 GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740 CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800 AGATCAACGC GGAAGATCCG AATTCCTCGA 1860 CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920 TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980 AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040 TGGGCATACG CGATATCTGG CGATACGCTT 2100 CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160 ACGATATGAG GCTATATCGC CGATAGAGGC 2220 TCGATCTATA CATTGAATCA ATATTGGCCA 2280 TAAATCAATA TTGGCTATTG GCCATTGCAT 2340 TATATTGGCT CATGTCCAAC ATTACCGCCA 2400 TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460 CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520 ATGACGTATG TTCCCATAGT AACGCCAATA 2580 TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640 CCTATTGACG TCAATGACGG TAAATGGCCC 2700 TGGGACTTTC CTACTTGGCA GTACATCTAC 2760 CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820 CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2880 AAATGTCGTA ACAACTCCGC CCCATTGACG 2940 GTCTATATAA GCAGAGCTCG TTTAGTGAAC 3000 TGTTTTGACC TCCATAGAAG ACACCGGGAC 3060 TGGCCTCGCT CGCGCGTGCG ATGCTCGCTC 3120 CGGCGCCGTC GGGGATCCTA TGGCTACTAC 3180 GCAAACCTGA ACACTCATAC GCCATAGCCA 3240 AAGGCCTCAC CACTGTTTGG AAGGATTACT 3300 TCATAGCTTG GTGCAAAGAC GGTAAGTTTA 3360 GATATCTTGC AATTCTGCAT TCAAGAGCCT 3420 PCTAJS94/12198 TGCAGACCAG TGTGGTATTC AAAAAACTTT TCGAGGGGCA AAGGCAAGGG GAAACATTTG 3480 AAATGGCTGA CGACTTTGAA TTTGGACTCT GCCCATGCGA TGCCAATCCC GTAGTAAGAG 3540 GGAAGTTCAA TACAACACTG CTAAACGGAC CGGCCTTCCA GATGGTATGC CCTATAGGAT 3600 GGACAGGAAC TGTGAGCTGT ATGXTAGCTA ATAGGGACAC CCTAGACACA GCAGTAGTGC 3660 GTGTGTATAA GAGGTCCAAA CCAITCCCTT ATAGACAAGG TTGTATCACC CAAAGAACTC 3720 TGGGGGAGGA TCTCTATAAC TGTGATCTTG GAGGGAATTG GACTTGTGTG ACTGGGGACC 3780 AGCTACAATA CACAGGAGGC CCTGTCGAAT CTTGCAAGTG GTGTGGTTAT AAATTCCAAA 3840 AAAGTGAGGG GTTGCCACAC TACCCCATCG GCAAGTGTAG GTTGAAGAAT GAGACTGGCT 3900 ACAGATTTGT AGACGGCACC ACTTGCAACA GAGAGGGTGT AGCCATAGTA CCACAAGGAT 3960 TGGTAAAGTG TAAGATAGGA GACACAATCG TACAGGTCAT AGCTCTTGAC ACCAAACTTG 4020 GGCCTATGCC TTGCAAGCCA TATGAGATCA TACCAAGTGA GGGGCCTGTA GAAAAGACGG 4080 CATGCACCTT CAACTACACG AGGACATTAA AAAATAAATA TTTTGAGCCC AGAGACAGXT 4140 ACTTCCAGCA ATACATGCTA AAAGGAGATT ATCAATACTG GTTCGACCTG GAGGTCACTG 4200 ACCATCATCG GGATTACTTC GCCGAGTCCA TATTGGTGGT GGTGGTAGCT TTACTGGGTG 4260 GAAGATACGT GCTCTGGTTA CTGGTAACAT ACATGGTCCT ATCAGAACAA AAGGCCTTGG 4320 GGACCCAATA TGGGGCAGGG GAAGTGGTGA TGATGGGTAA CTTGCTAACA CATGACAGTA 4380 TTGAAGTGGT GACATATTTC TXGTTGTTAT ACCTACTGCT AAGAGAGGAG GCTGTAAAGA 4440 AGTGGGTCTX ACTCTTATAC CACCTTGATT GATTGAGGAT CAGCTTATCC AGGGTCGACC 4500 TCAGGCATGC AAGCTCAGAT CCGCTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC 4560 CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACXCCC ACXGTCCTTT CCTAATAAAA 4620 40 TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG 4680 GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG 4740 45 CTCTATGGGT ACCCAGGTGC TGAAGAATTG ACCCGGTTCC TCCTGGGCCA GAAAGAAGCA 4800 GGCACATCCC CTTCTCTGTG ACACACCCTG TCCACGCCCC TGGTTCTTAG TTCCAGCCCC 4860 ACTCATAGGA CACTCATAGC TCAGGAGGGC TCCGCTTCAA TCCCACCCGC TAAAGTACTT 4920 50 GGAGCGGTCT CTCCCTCCCT CAXCAGCCCA CCAAACCAAA CCTAGCCTCC AAGAGTGGGA 4980 AGAAATTAAA GCAAGATAGG CTATTAAGTG CAGAGGGAGA GAAAATGCCT CCAACATGTG 5040 55 AGGAAGTAAT GATAGAAATC ATAGAATTGA GATCTCGAGG TGTTCGTGCT GGACGTGTCC 5100 GCGGCGCCAG ACGCGTGCGC GGCCGCCGTA CTGGACATGC GGCCCGCCAT GCAGGCCGCT 5160 TGCGCGGACG GGGCGGCGGG CGCGACGCTG GCGACCCTGG CGCGTCAGTT CGCGCTAC AG 5220 60 ATGGCGGGGG AGGCCACGGC GGGCCCTAGG GGACTATAAA GCTGCCCCTG CGCTCGCTCG 5280 CTCGCTGCAT TTGCGCCCCG ATCGCCTTAC GGGGACTCGG CGCTCGGCGG ATCCCCTCCC 5340 65 GGCCCCGCCG CGAAGCAGGC CGCCAGACAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400 ATTGGCAGCG GCTGTCCTCG CGCTCGCCGC GGGCGCCCCC GCCGCCGCCC GCGGCGGGGG 5460 40 45 50 55 60 65 CGCCGAAGCC AGGGCAGCAC AGAGACGCCC GATACGAAAT CGAAGAGTGG GAAATGGTGG 55^0 TCGGAGCCGG GCCGGCCGTG CACACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580 AGCGCGTGGC Cf „„*TGCA AACCTCAQCC GGCTGCTGGA CGGGTACATA GCGGTCCACG 5640 TTGACGTTGC GCGCACCTCT GGCCTGCQGG ACGCCATGTT TTTCCTGCCG CGCGCGGCCG 5700 TCGACTCTAG AGGATCCCCG GGTACCGAGC TCGAATTCAC TGGCCGTCGT TTTACAACGT 5760 CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA TCCCCCTTTC 5820 GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC 5880 CTGAATGGCG AATGGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA 5940 CACCGCATAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGCCC 6000 CGACACCCGC CAACACCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC GGCATCCGCT 6060 TACAGACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA 6120 CCGAAACGCG CGAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 6180 ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 6240 ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 6300 TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC 6360 CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG 6420 AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 6480 AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT 6540 TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACIC 6600 GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG 6660 CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT 6720 AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 6780 TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA 6840 GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 6900 AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG 6960 GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT 7020 GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 7080 GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT 7140 GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA 7200 GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 7260 ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG 7320 TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTT3TTTT 7380 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG 7440 CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AC*3CAGATA 7500 WO 95/12682 PCT/US94/12198 i "■ i ' CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 7560 CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG 7620 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 7680 TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 7740 TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 7800 TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 7860 GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 7920 TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 7980 TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT 8040 GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC 8100 GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAGA 8135 » • • • t • • • • * • • • • ♦ • • • • • «• - * * • « • • • • • t • ■ »• w •• ••* 276234 • • • • • • ii 44

Claims (24)

1. An attenuated replicating non-pathogenic virus, for preventing disease caused by Bovine Viral L-arrhoea Virus (BVDV), which comprises a gene or gene combination from a 5 BVDV virus, which functionally expresses the BVDV glycoprotein gp53, gp48 or gp25, or any combination thereof.

2. A virus of claim 1, which is selected from attenuated Bovine Herpes Virus type 1 (BHV-1), attenuated 10 adenoviruses, attenuated bovine mammillitis virus, attenuated bovine papillomavirus or attenuated pseudorabies virus.

3. A virus of claim 1 or claim 2, which is attenuated because the thymidine kinase (tk) gene is non-functional. 15

4. A virus of claim 3, which is attenuated BHV-1.

5. A virus of claim 4, which contains and expresses the gene that codes for BVDV gp53.

6. A virus of claim 5, where a signal peptide is inserted preceding the gene or gene combination that codes for gp53 20 in BHV-1.

7. A virus of claim 5 or claim 6, where the gene that codes for gp53 is inserted into the inactivated tk gene site.

8. A virus* of claim 7, where the gene or gene combination 25 comprises a recombined plasmid with intact viral DNA, said plasmid comprising: a) a BHV-1 genomic DNA fragment containing the tk gene and having a deletion to the tk gene; b) a promoter/polyadenylation signal inserted in the 30 tk gene deletion; and c) a signal peptide gene sequence preceding a gp53 gene or gene combination, all of which is inserted between the promoter and the polyadenylation signal.

9. A virus of claim 8, where the signal peptide gene 35 sequence is any of the thirty-nine examples of well- characterised.signal peptide sequences found in Perlman et al, J. Mol. Biol., Vol. 167:391-409 (1983). amended sheet N-Z. PATENT OrncE 2 1 MAR 1996 RECElVrn 10 IS 20 25 30 UJ o Ll. u_ O Ir— 2 uj £ N Z 3 I a Cfc" ■Sft C.i, s: u UJ £T «M «» ft % • • • • * • • % » • I 45 • ••• •• «U Mta- || 27623* ♦ • I • I IM • • • • • •

10. A virus of claim 9, where the signal peptide gene sequence is taken from Pseudorabies Virus gill gene and/or Bovine Growth Hormone.

11. A virus of claim 10, where the plasmid is selected from a) pBHVtkex-a::BGH/p53; b) pBHVtkex-1::gIII/p53; c) pBHVtlex-3::BGH/p53; and d) pBHVtkex-3::gIII/p53.

12. A virus of claim 11, selected from Tll-3, Tll-6 and Tll-8, e.g. Tll-6.

13. A virus of claim 7, where the gene or gene combination comprises a recombined plasmid with intact viral DNA, said plasmid comprising: a) a BHV-1 genomic DNA fragment as defined in claim 8; b) a promoter/polyadenylation signal as defined in claim 8; and c) a gp53 gene or gene combination inserted between the promoter and the polyadenylation signal.

14. A virus of claim 13, where the plasmid is made from a plasmid having the characteristics of plasmid pHAS4, e.g. pBHVtkex-3::p53.

15. A virus of claim 14, where the virus is T2-3/3 or T2-2/5.

16. A vaccine for preventing disease caused by BVDV, comprising a virus of claim 1 and a carrier.

17. A vaccine as claimed in claim 16, where the carrier comprises a physiological buffered medium, pH 7.0 to 7.4, containing 2.5 to 15% serum which does not contain antibodies to BHV.

18. A process of preparing a virus of claim 1, comprising: a) isolation of a functionally expressing gene or gene combination that causes BVDV; b) inserting the gene or gene combination into a replicating non-pathogenic virus; and c) selecting a live virus that functionally expresses the product of said gene or gene combination.

19. A method of preparing a virus of claim 8, comprising transfecting cells with the plasmid to produce a Amended sheet 46 27 6 2 3 4 recombinant virus containing the functional gene or gene combination inserted into a live virus th^t does not cause immunosuppression in the usual host and expresses the functional gene or gene combination.

20. An attenuated replicating non-pathogenic virus as claimed in claim 1 substantial]y as herein described with reference to any example thereof.

21. A vaccine for preventing disease caused by BVDV comprising a virus as claimed in claim 20 and a carrier.

22. A process for preparing a virus as claimed in any one of claims 1 to 15 and 20 substantially as herein described with reference to any example thereof.

23. Use of a virus of any one of claims 1 to 15 and 20, for the manufacture of a medicament for use in immunisation against a disease caused by BVDV.

24. A method of immunising a non-human animal against infectious disease caused by BVDV comprising administering to the animal a pharmaceuti ;ally-effective amount of a virus of any one of claims 1 to 15 and 20. end of claims