AU688819B2 - Viral vector with bovine viral diarrhea virus (BVDV) antigens - Google Patents
Viral vector with bovine viral diarrhea virus (BVDV) antigens Download PDFInfo
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- AU688819B2 AU688819B2 AU10423/95A AU1042395A AU688819B2 AU 688819 B2 AU688819 B2 AU 688819B2 AU 10423/95 A AU10423/95 A AU 10423/95A AU 1042395 A AU1042395 A AU 1042395A AU 688819 B2 AU688819 B2 AU 688819B2
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- virus
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- bvdv
- bhv
- bovine
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- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16641—Use of virus, viral particle or viral elements as a vector
- C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Description
WO 95/12682 PCT/US94/12198 VIRAL VWR WrIT BOVINE VIRAL DIARRHEA VIRUS (BVDV) ANTIGENS BACKGROUND OF THE INVENTION Field of the Invention This invention relates to the field of Bovine Viral Diarrhea Virus (BVDV), and vaccines for the treatment thereof.
Information Disclosure van Zijl, M. et al. Live Attenuated Pseudorabies Virus Expressing Envelope Glycoprotein El of Hog Cholera Virus Protects Swine against both Pseudorabies and Hog Cholera, Journal of Virology, Vol. 65, No. 5, pp. 2761-2765 (1991). U.S. patent 4,703,011, Kit, and Kit, Thymicine Kinase Deletion Mutants of Bovine Herpesvirus-1, issued 27 October 1987. U.S. patent 4,824,667, Kit, and Kit, S.
Thymidine Kinase Deletion Mutants of Bovine Herpesvirus-1, Vaccines Against Infectious Bovine Rhinotracheitis Containing Same and Methods for the Production and Use of Same, issued 25 April, 1989. Collett, et al., Proteins Encoded by Bovine Viral Diarrhea Virus: The Genomic Organization of a Pestivirus, Virology, Vol. 165 pp. 200-208 (1988). Collett, et al., Molecular Cloning and Nucleotide Sequence of the Pestivirus Bovine Viral Diarrhea Virus, Virology, Vol. 165 pp. 191- 199 (1988).
Background Bovine viral diarrhea virus (BVDV) i& a Pestivirus belonging 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 disea6, 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 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 immune system is fully competent. The possible outcome of infections include fetal SWO 95/12682 PCTUS94/12198 reabsorption, abortion, mummification, congenital defects, birth def!)cts, calves born 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 large amounts of virus into their environment which can infect susceptible animals.
Furthermore, even though persistently infected animals are immunotolerant 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 wll not be many pregnant animals infected at the right timi, 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 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 effective as attenuated live vaccines. Inactivated antigen from inactivated vaccine preparation undergoes exogenous processing. After injection into the animal the antigen becomes part of the 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 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 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 causing the immunosuppression normally associated with field isolates. Roth J.A.
-~slac~pl81Cg WO 95/12682 PCT/US94/12198 and Kaeberle 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 creates a serious drawback to the vaccine. An animal owner may be vaccinating animals 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 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 major 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 (BHV/BVDVgp53) is used as a vaccine against BVDV. Donis, R.O. and Dubovi, Glycoproteins of Bovine Viral Diarrhoea- Mucosal Disease Virus in Infected Bovine Cells, Journal of General Virology, Vol. 68, pp. 1607-1616 (1987) and Magar, 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- 314. Cited references are incorporated herein by reference.
-Ul----IIY OF TIL IN ENTION.
A replicating nonpathogenic virus, for preventing disease caus ed Bovine Viral Diarrhea Virus (BVDV), where said replicating nonpa enic virus comprises: a gene or gene combination taken from a BVDV and said replicating nonpathogenic virus functionally expres said gene or gene combination.
Embodiments of this inventio ic ude the following: A virus where said replicating nonpathogenic virus nuated, is selected from attenuated Bovine Herp63 Virus type 1 (B attenuated adenoviruses, attenuated bovine mammillitis virus, tuted bovine papillomavirus, or attenuated pseudorabies virus. A virus where
I
C__I C 3A SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a recombinant attenuated replicating virus for preventing disease caused by Bovine Diarrhoea Virus (BVDV), wherein said virus is rendered non-pathogenic by the insertion of at least one of the BVDV glycoprotein genes gp 53, gp 48 or gp 25 into a pathogenesis related gene of a virus other than BDVD, and wherein said virus expresses at least one of said BVDV glycoprotein genes, said BVDV glycoprotein genes being preceded by a signal peptide gene sequence.
Embodiments of the invention include the following: A virus where said replicating non-pathogenic virus is attenuated, is selected from attenuated Bovine Herpes Virus type 1 (BHV-1), attenuated adenoviruses, attenuated bovine mammillitis virus, attenuated bovine papillomavirus, or attenuated pseudorabies virus. A virus where said replicating nonpathogenic virus is attenuated and contains and expresses any *a a a o 6 a C WINWORDJENNYNODELE E 1042395 DOC Ilh~8k~s~W WO 95/12682 PCTIUS94/12198 combination of the following genes: the genes that cod- for gp48, gp25, p14 capsid protein, p20 N-terminal protease and p1 2 5 /p80 protein. A virus where the attenuation is created by making the thymidine kinase (tk) gene nonfunctional.
A virus where a signal peptide is inserted preceeding the gene or gene 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 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) 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 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, et al., J. Mol. Biol. Vol. 167 pp. 391-409 (1983), incorporated by reference. A virus where said signal peptide gene sequence is taken from Psuedorabies Virus gill gene (PRV) and/or Bovine Growth Hormone (BGH).
A virus where a plasmid is selected from the following plasmids, a) pBHVtkex-l::BGH/p53; b) pBHVtkex-l::gIII/p53; c) pBHVtkex-3::BGH/p53; or d) pBHVtkex-3::gIII/p53. A virus that produces the product of a functionally expressing gene or gene combination is selected from one of the following viruses, T11-3, Tll-6, or T1l-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) 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.
A vaccine as described above for preventing disease caused by Bovine Viral WO 95/12682 PCT/US94/12198 Diarrhea Virus (BDVD) comprising a pharmaceutically effective amount of a virus descr' above and a carrier, said carrier comprising any physiological buffered medi, e. about pH 7.0 to 7.4 containing from about 2.5 to 15% serum which does not contain antibodies to BHV.
A method of immunizing an 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 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 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.
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.
a. EXAMPLE 1. pBHVtkex-3::p53.
b. EXAMPLE 2. pBHVtkex-1::BGH/p53 c. EXAMPLE 3. pBHVtkex-1::glll/p53 d. EXAMPLE 4. pBHVtkex-3::BGH/p53 e. EXAMPLE 5. pBHVtkex-3::glll/p53 Figure 4. Predicted transcript maps of the BHV-1/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 Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is 20 not intended to exclude other additives, components, integers or steps.
**o o SC C WINWORDXSIMONEWORK\U1421C) DOC WO 95/12682 PCT/US94/12198 BREFdESECRIPTION OFM DTuAWiNUS4.---- Figure 1. Construction of the shuttle vectors for inserting foreign genes in BHV-1.
Figure 2. Strategy for appending signal peptide sequencos to the B gp53 gene.
Figure 3. Maps of the five shuttle plasmids for inserting gp53 o BHV-1 a. EXAMPLE 1. pBHVtkex-3::p53.
b. EXAMPLE 2: pBHVtkex-l::BGH c. EXAMPLE 3: pBHVtkex-1:: I p53 d. EXAMPLE 4: pBHVt -3::BGH/p53 e. EXAMPLE 5:pB tkex-3::gIII/p53 Figure 4. Predicted trans ipt maps of the BHV-1/gp53 recombinant viruses.
Figure 5. Northern ots showing transcription of gp53 messenger RNAs in the BHV-1 recom i ants.
Figure mmunoprecipitations showing expression of gp53 protein in the BHV-1 r ombinants.
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 blunt ends," "Tris," chelating buffers such 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 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. et. al., eds., Wiley Interscience, New York.
This invention combines the effectiveness of the attenuated live vaccines with 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 major pathogen of cattle which produces 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 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.
The BHV, a live attenuated virus, will replicate and produce a cell mediated response. As part of that replicative process, the BVDV gp53 gene 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 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 and buffer solutions were made up in glass distilled water unless otherwise indicated.
Compositions and 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 as bovine, sheep and goats.
Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include any physiological buffered medium, about pH 7.0 to 7.4, containing from about 2.5 to 15% serum which does not contain antibodies to BHV, is seronegative for BHV. Serum which does not contain gamma globulin 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 can be employed as a substitute for the serum. However, it is desirable to avoid the 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 106 PFU/ml at -70 0 C to -90 0 C or in a lyophilized state at 2 0
C
to 7°C. The lyophilized virus may be reconstituted for use with sterile distilled water or using an aqueous diluent containing preservatives such as gentamicin and amphotericin B or penicillin and streptomycin.
The useful dosage to be administered will vary depending upon the age, weight and species of the animal vaccinated and the mode of administration. A suitable dosage can be, for example, about 104.5 to 107 PFU/animal, preferably about 104.5 to 10 5 .5 PFU.
The vaccines of the present invention can be administered intranasally, 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 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 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 forstalled, meaning that anticipatory action to prevent or hinder the disease has occurred but the conditions creating the disease have not been eliminated.
The usefulness of 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 BVDV, and one against which the host produces a substantial immune response, the
I
WO 95/12682 PCT/US94/12198 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 major glycoprotein of BVDV, 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 thav produce substantial immune responses can make effective vaccines.
Magar, 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. 303-314. 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 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 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 "T11-3", embodied in Example 3 for vaccine trials. The former virus was deposited to the ATCC under the designation UC VR-58. The latter, "T1l-3" plasmid was also deposited. The virus we labeled "T1l-8" might 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 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 BHV-1. Although this invention shows the utility of BHV-1 as a vector for BVDV WO 95/12682 PCTIUS94/12198 genes many other virusies could fill the same role. Other examples from cattle, sheep and goats would include cow, goat and sheep pox viruses, adenoviruses, 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 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, E.Wimmer, E.A. Emini, and D.C. Diamondand 2) Immunogenicity of Vaccine Products and Neutralizing Antibodies, E Norrby. Both articles are in Edited by Notkins and Oldstone Published by Springer-Verlag New York Inc. 1986.
Since we intended to attenuate BHV-1 by inactivating the viral thymidine kinase Kit, et al., US Patent 4,703,011, (1983 we decided to use the BHV- 1 tk gene for the site of insertion. This approach not only insured the complete inactivation of the viral tk, but also allowed us to select recombinant, tk-negative virus by established methods. M. F. Shih, et al., Proc Natl Acad Sci 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 with plasmid pHAS4 which contains a 2.7kb Sail subfragment of the BHV-1 HindIII-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 Biophys 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 upstream gene homologous to the HSV-1 UL24 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 BgI and XhoI, filling in the ends with the Klenow Fragment of DNA polymerase I (Klenow) and religating the resulting blunt ended fragments. This manipulation restored the BglII recognition site, but not the XhoI site (Fig. 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 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 cloning manipulations, we eliminated the HindIII site in the pUC18 vector by digesting pHAS4ABX with HindIII, filling in the cohesive ends with Klenow, and 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 Gen Virol, 74:471-477 (1993 These gene expression signals are commonly used for high levels of expression of foreign genes in a number of different systems, but other promoter/polyadenylation signal pairs could also be used in this context. The cassette, in vector p3CL-DHFR, is bounded by unique EcoRI and BglII sites and contains, between the promoter and the polyadenylation signal, unique HindIII and Sail restriction sites for cloning of foreign genes. The p3CL-DHFR vector was digested with EcoRI, then filled in and ligated to a BamHI linker (New England Biolabs, Beverly, Massachusetts). This manipulation regenerated the EcoRI site. The construct was then digested with BamHI and BglII and the released cassette was ligated into the BglII site of pHAS4ABX (Fig. The ligations were transformed into E. coli strain DH5a. We isolated recombinant plasmids that contained the p3CL insert in both orientations relative to the BHV-1 tk gene by mapping of asymmetric restriction sites. These two constructs, designated pHAS4ABXex-1 and pHAS4ABXex-3 (Fig. contained then, a strong promoter and polyadenylation signal bounded by the BHV-1 tk gene and flanking regions to allow homologous recombination into the BHV-1 genome.
Figure 1. Construction of shuttle vectors for inserting foreign genes into BHV-1. PHAS4 is a 2.7kb subfragment from the BHV-1 HindIII-A fragment. The BglIIXhoI 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 pHAS4ABX. The CMV immediate early promoter is shown as a light stippled 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 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 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 site 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 C to a G, 11 bases in from 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 The base change was verified by nucleotide sequencing, and the resulting plasmid was called pP53mut. We inserted, into pP53mut sequences, encoding signal peptides from the PRV gIII gene K. Robbins, 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 Sail cohesive ends 5' and BamHI cohesive ends 3' (Fig 2 section These signal peptide cassettes were ligated into pP53mut digested with BamHI and Sail, and transformed into DlH5a. We confirmed the correct insertion of the signal peptide cassettes by nucleotide sequencing.
Complementary oligonucleotides encoding any well characterized signal peptide can be used in this invention. Thirty-nine examples of well characterized signal peptide sequences found in Perlman, 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 gp53 gene. Sectiln A: Synthetic oligonucleotides corresponding to the signal
L-
WO 95/12682 PCT/US94/12198 peptide sequences of Bovine Growth Hormone (BGH), and Pseudorabies virus gIII (PRV gIII). Complementary oligonucleotides were synthesized such that the annealed pairs had Sallsites on the 5' ends and BamHI sites on the 3' ends. The deduced amino acid sequences of the signal peptides are also shown. In each case the predicted cleavage sites for the signal peptides are just after the alanine three amino acids from the ends. Codons for two amino acid residues (F,P in BGH; P,S in gIII) from the original native proteins were left on the signal peptide sequences to ensure correct cleavage.
Section B: Site directed Mutagenesis of the cDNA encoding the BVDV gp53 gene. The first 60 nucleotides 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 acid sequence. The cDNA was then digested with BamHI as shown, allowing in frame ligation to either of the signal peptide sequences shown in 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 include, but are not limited to, the other two BVDV surface glycoproteins, gp48 and (Collett, et al., Virology 165:200-208 (1988)), the p14 capsid protein (Thiel, et al., J. Virol. 65:4705-4712 (1991)), and the p20 N-terminal protease.
Wiskerchen, 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 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, et al., Can. J. Microbiol. 37:815-122 (1991)), which elicits a significant antibody response in infected cows.
Insertion of the BVDV gp53 gene into the BHV-1 expression vectors.
The p 5 3 gene, either with or without added signal peptide sequences, was ligated into the HindIII insertion sites of pHAS4ABXex-1 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 with at least two different signal peptides to ensure that we achieved the most -13- 9~C I I I II r WO 95/12682 PCT/US94/12198 efficient expression. The transformed colonies were screened by colony hybridization using as a probe the p53 insert labelled with Digoxygenin-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 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, whic' are schematically depicted in Figs. 3A-E. Their descriptions are as follows.
EXAMPLE 1. pBHVtkex-3::p53: contains the BVDV gp53 gene inserted 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 preceded by the BGH signal peptide sequence inserted into pHAS4ABXex- 1. See Fig. 3B. This plasmid was used to create the virus T11-6. This virus was deposited.
EXAMPLE 3. pBHVtkex-l::gIII/p53: contains the mutagenized gp53 gene preceded by the PRV gIII signal peptide sequence inserted into pHAS4ABXex-1. See Fig. 3C. This plasmid was used the create the virus Tl.1-3. 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. 3D.
EXAMPLE 5. pBHVtkex-3::gIII/p53: contains the mutagenized gp53 gene preceded by the PRV gIII 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 into BHV-1. The gp53 gene is shown as a solid band, the 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 shown. The various signal peptide sequences are indicated.
1 1~ ~BP--IC I~R IlB c~B~r WO 95/12682 PCT/US94/12198 a. EXAMPLE 1. pBHVtkex-3::p53.
b. EXAMPLE 2. pBHVtkex-1::BGH/p53 c. EXAMPLE 3. pBHVtkex-l::gIII/p53 d. EXAMPLE 4. pBHVtkex-3::BGH/p53 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.
Introduction of the gp53 gene into BHV-1 'Iowa".
The five expression shuttle plasmids carrying gp53 were linearized by XbaI and cotransfected into Bovine Turbinate (BT) cells with unit length DNA from BHV- 1 strain Iowa (tk positive) by the standard CaPO 4 method L. Graham, et al., Virology, 52:456-467 (1973 as modified by Cai Cai, et al., J Virol, 61:714-721 (1987 The cells were obtained from ATCC. The transfections were then subjected to two rounds of selection either on 143tk" cells K. Mittal, et al., J Gen Virol, 70:(1989 or on Rab (BU) cells Kit, et al., Virology, 130:381-389 (1983 in the presence of 100ug/ml 5-Bromo-2'-Deoxyuridine (BDUR, Sigma Chemical Company, St. Louis, Missouri) to isolate virus no longer expressing tk. This is a standard 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 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: 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-3::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 same direction as the BHV-ltk gene.
-r II r WO 95/12682 PCT/US94/12198 EXAMPLE 2. Tll-6 (This virus was submitted to ATCC under 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 CMV promoter and the BGH polyadenlyation signal, with transcriptional orientation in the opposite direction relative to the BHV-1 tk gene.
EXAMPLE 3. T11-3: BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-l::gIII/p53 recombined. Contains the BVDV gp53 gene with the PRV gIII signal peptide sequence situated between the CMV promoter and the BGH polyadenlyation signal, with transcriptional orientation in the opposite direction relative to the BHV-1 tk gene.
EXAMPLE 5. T11-8: BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-3::gIII/p53 recombined. Contains the BVDV gp53 gene with the PRV gIII signal peptide sequence situated between the 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 from cotransfections with "Iowa" DNA and plasmid pBHVtkex3::BGH/p53, EXAMPLE 4, but this prophetic virus, could be easily created, it and any other BHV-1 viruses containing Ithe BVDVgp53 gene inserted 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 gp53 gene and the CMV promoter/BgH polyadenylation cassette as probes (data not shown). All four of the viruses carried the complete promoter/gene/polyadenylation cassettes in the BHV-1 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.
A large number of existing cell lines are pe .sistently infected with noncytopathic 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 of contaminating BVDV. In order to ensure that the BHV-1 viruses carrying the BVDV sequences were not contaminated with noncytopathic BVD virus, we prepared DNA from each of the viruses (including the I ul a I I I ,aL, WO 95/12682 PCT/US94/12198 parent strain Iowa and IowaABX) and subjected the DNA preps to extensive RNAse treatment using a cloned RNAse (RNAse ONE, Promega Corporation, Madison, Wisconsin). Since BVDV has only RNA as its genetic material, this manipulation should eliminate any possible contaminating BVDV sequences from the viral DNA preps. We then transfected these RNAsed viral DNAs into certified BVD-free MDBK cells (ATCC) and picked virus plaques from the transfections to use in further manipulations.
Transcriptional analysis of the gp53 recombinants.
We prepared RNA from each of the recombinant viruses and the parent BHV- 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-1/gp53 recombinant viruses later shown in Figure. 5. The two probes are 1) the gp53 cDNA and 2) the SalI/BglII portion of pHAS4 (shown above the maps). The first map shows the predicted transcripts from viruses Tll-3 and T1l-6, and the second map shows 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 32 P-labelled gp53 probe, the size predicted for transcription initiation at the CMV promoter and termination at the BgH polyadenylation site Fig. 5, probe 1.
The T2-3#3 and T2-2#5 virus are not shown. As additional major bands, T11-3 and Tll-6 made an 8.5kb transcript and Tll-8 and T2-3#3 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 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 the gH gene. A. L. Meyer, et al., Biochim Biophys Acta, 1090:267-9 (1991). All of the viruses made equivalent amounts of the 3.1kb gH message (data not shown).
This probe also hybridized to the longer p53 messages in T11-8 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 a a-LY WO 95/12682 PCT/1US94/12198 used a probe that consisted of the pHAS4 fragment from the upstream Sail site to the BglII site in the tk gene, the beginning of the deletion in the recombinant viruses (probe All of the viruses made a message of approximately 4.4kb which we deduced to be UL24 (Fig. 5, probe This message, however, was smaller than the 5.2kb UL24 message in BHV-1 strain Cooper described by Bello, et al 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 using 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 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-3 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 3.7, 1.8, and 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 pg53 cDNA, and the second panel shows transcripts hybridizing to probe 2, the SalI/BglI subfragment of pHAS4. KEY: M=Mock infected cells, I=BHV- 1 "Iowa" infected cells, 3,6,8=T11-3, Tll-6 and T11-8 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 Detailed procedures for IPs can be found in :.idard references such as "Current Protocols in Molecular Biology", Ausubel, F. et. al., eds., Wiley Interscience, New York. BT cells infected with the BHV-1 recombinants were metabolically labelled with 35 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. VMCRD, Pullman, Washington. Antigen/antibody complexes were precipitated staph A (Immunoprecipitin, Gibco/BRL, Gaithersburg, Maryland,) or protein A sepharose 4B (Pharmacia, Uppsala, Sweden). Immunoreactive proteins were resolved by SDS- Polyacrylamide gel electrophoresis (SDS-PAGE) and flaurography.
Figure 6 shows that all three of the recombinant viruses carrying the gp53 gene preceded by a signal peptide sequence made significant amounts of the protein.
-18- 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 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. Immunoprecipitated proteins showing expression of gp53 in the BHV-1 recombinants. Labelled proteins were precipitated with polyclonal bovineanti-BVDV serum, this serum also had minor reactivity with BHV-1 antigens. KEY: 3,6,8=T11-3, T11-6, and Tll-8 infected cell proteins, IA=BHV-1 "Iowa" infected cell proteins, M=Mock infected cell proteins. MW=approximate protein molecu: .r weight standards, in Kilodaltons.
The gp53 protein bands in T1l-3, Tll-6 and Tll-8 were broad, suggesting 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 Nlinked 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 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 cDNA gp53 clone might not contain the exact amino acids processed from the BVDV 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 those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modification; 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 purposes of description.
~a~a~l WO 95/12682 PCT/US94/12198 All documents cited herein are incorporated by reference.
Deposit of Genetic Materials One skilled in the art should be able to reconstruct all the various embodiments of this invention by utilizing only the written description. However, for the sake of completeness, to ensure enablement, and to provide every opportunity for others to make 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 Amencan Type Culture Coll ction, 12301 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 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, Illinois, zip code 61604, USA. One plasmid was given the Upjohn designation, pUC 1564, E. coli culture UC 15085, referring to pBHVtkexl::gIII\p53, it corresponds to the plasmid used to create the virus described herein as also known as "Example 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-3::gIII\p53, it corresponds to the plasmid used to create the virus described herein as, "T-11-8," also known as "Example This plasmid was given the following number by the depository, NRRL B-21351. Both of the plasmids were received by the Agricultural Research Service Culture Collection depository on 26 October 1994.
~A
1~1_ WO 95/12682 PCT/US94/12198 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: The Upjohn Company INVENTORS (For U.S. Purposes only): Wardley, Richard C. and Haanes, 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: ADDRESSEE: Thomas A Wootton (1920-32-1), The Upjohn Company STREET: 7000 Portage Road CITY: Kalamazoo STATE: Michigan COUNTRY: U.S.A ZIP: 49001-0199 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:
CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: NAME: Wootton, Thomas A.
REGISTRATION NUMBER: 35,004 REFERENCE/DOCKET NUMBER: 4748 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 616 385-7914 TELEFAX: 616 385-6897 TELEX: 224 401 UPJOHN INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 8083 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: 2724 INDIVIDUAL ISOLATE: pBHVtkex-3::p53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: I I WO 95/12682 WO 9512682PCTJUS94/12198 GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA
CGACAGGTTT
CACTCATTAG
TGTGAGCGGA
TTGCATGCCT
GCGGCCGCTG
GTCCGCGTCC
GCGTATAAAA
GCCCAGGCAA
GCCGAAATTT
CTGTTGCCCG
CCGGGGTCGC
GGGGAAGCGG
GCGGCGATCG
CGGCTATTAT
TCACTCTGAG
CCGGGTCTGG
GCAGCCCCCC
ACGTCTTCAG
GTAGGCACAC
CCACGACCTG
GGCGCAGCTT
GGCTGTGGCA
CCGAGCCCGC
GAAAGACAAC
TTTTCCCGGA
TCCTCGCGGC
CCGGCGCACC
GCGGCCCCGT
CCGGGCGGCG
CCCGACTGGI
GCACCCCAG(
TAACAATTTC
GCAGGTCGAC
CCGGCCTGGI
CCAACTCCGC
TTTCGCTCGI
GCALAACTCTP.
CGCCCAGGCA
CGTGCCTGCT
CGGCGGGCGG
CTGCCCCTTC
CCCCGCTCGC
AGCCTCAAGG
CACGCGCATG
GCCCGGGGGG
GAGGCGCTGG
CTCCACGATA
GCAATCGGGC
CTTAAAAACC
CTGCGCGCCA
CCGGATCCCG
GCGCGCTCTC
AACGGGCCGC
GCCGATGGCG
GTCTGCGCGA
GCGCAGACGC
k.CTTAATTTT
".GCTGGTGGA
:GCCCGCTAC
k.TAATAAAAT
CGTCGCGCGA
kAAGCGGGCAC
'CTTTACACTI
'ACACAGGAAM
TTCCGCGCCC
TCCGCGCCCC
GCGAAGACGCG
CCGGTACAAA
AACGCCCGAG
CGCCGCGCCG
GGCCGCCCAC
CCGCGGGGTG
TGCCGCCGCG
CGCGAACGCG
CGCGCCGCGT
CCCCGCTGGG
GCGAGATCGC
TCCATCTTGC
AGACAGACGG
CGCCGGCTTT
TCCGGGGCGC
ACCGCCGCGC
GCGCGCAGGC
CGCGTCGTGC
GCGCTCGCGG
TACTGGCGCA
TGCGCCGCAG
GGACGCGGCG
GCACGCGCGT
CCCTCGTGTT
TGCCTCCGCG
GTGTGTTTGT
TAGTGGTGTT
TGAGCGCAAC
TATGCTTCCG
CAGCTATGAC
GCAATTAATG
GCTCGTATGT
CATGATTACG
TGAGTTAGCT
TGTGTGGAAT
CCAAGCTAGC
CAGGTTGTCC GCGGCGTCTG CCTTCGCCAG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC
GCTCGTCCCA
GACGCGGTCC
CGCCATGGCC
GAAGCGCAGC
GCGACTGCGT
CCGATGCCGC
CCCGACGCGT CTTTAGCGCA CGGCGGCCGC TGTCCCCGGC
CGGCCACAGC
GCCGCGGTTG
CGCGCGCGAA
TGCTAGCGAT
AGACGAACAC
GCACAAGCCG
TGGGCGTGTT
CCCGGGCGTG
GCAGGTTTAC
CAAACTTGCC
GTGCGTCGCA
GCGCACGTCG
GTATCTACCT
CCGCTTCCAC
CGATGTTTGG
CCTCGCACGG
GGCCTGGTTG
GTCCGCC-CTG
CGACCGCCAC
AGATCAACGC
CCGAAATACG
TATCGCCGAT
CGCCCTTTTG
CTCGGCTTTG
TGGGGCGTAC
CGTCTGGGCC
CTGCACCGGC
GGCCGAGTCG
CATCTTCGTT
CCCTGCCTCC
CTCAAAGCTC
CAAAAGCTGG
AGCCAGCGCC
GTCGCGGTCG
GGACGGCGCG
CGCTGGGGAG
TACGGACGCC
GAGCGCACGC
CGTACTACCA
CTGCGCCGCC
CCCGTGGCGC
GGAAGATCCG
CGTTTGAGAT
A.GAGATGGCG
GGAAAGGCCG
GGATGTCCAC
CACAAAGAGC
CCCGCCGGCG
CTCAGCAGGG
CCCGTAGCCA
CGTTTGCCCC
TCGGCGAGCC
GGCAGGCGCG
GCTAGGACCA
CGCAGCTGCC
GAAAAACGGC
GCGACCCGGA
AGAGACACGC
GCGAGGCGCG
TCGTAAAAGC
CGCGCCATGG
CACGGGCAGG
GGCGTGCTCT
TTAAGTGGGA
GCGCGGCGGG
GGCCAGGTTC
TGGGCCGGC3 3CGTGCCTCT k.ATTCCTCGA rTCTGTCCCG kTATTGGAAA 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040
GCTACCCCTT
I CCTGCAGTGA
ACTAAATTCA
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT WO 95/12682 WO 95/ 2682PCT/US94/12 198 GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG ATATCGTTTA CGGGGGATGC CAAATATCGC AGTTTCGAT; GACATCAAGC TGGCACATGC TTAGCCATAT TATTCATTGC ACGTTGTATC CATATCATAP TGTTGACATT GATTATTGAC AGCCCATATA TGGAGTTCCG CCCAACGACC CCCGCCCATT GGGACTTTCC ATTGACGTCA CATCAAGTGT ATCATATGCC GCCTGGCATT ATGCCCAGTA GTATTAGTCA TCGCTATTAC TAGCGGTTTG ACTCACGGGG TTTTGGCACC AAAATCAACG CAAATGGGCG GTAGGCGTGT CGTCAGATCG CCTGGAGACG CGATCCAGCC TCCGCGGCAA GCTACTACTG ATAACAGGGG CATAGCCAGG AATGATAGAA, GGATTACTCA CATGAAATGA TAAGTTTACA TACCTCTCAA AAGAGCCTTG CAGACCAGTG AACATTTGAA ATGGCTGACG AGTAAGAGGG AAGTTCAATA TATAGGATGG ACAGGAACTG AGTAGTGCGT GTGTATAAGA AAGAACTCTG GGGGAGGATC TGGGGACCAG CTACAATACA ATTCCAAAAA AGTGAGGGGT GACTGGCTAC AGATTTGTAG ACAAGGATTG GTAAAGTGTA CAAACTTGGG CCTATGCCTT AAAGACGGCA TGCACCTTCA
CGATAGACGC
TAGGTGACAC
CCAATGCATP
TTATATAGCA
TATGTACATT
TAGTTATTAA
CGTTACATAA
GACGTCAATA
ATGGGTGGAG
AAGTACGCCC
CATGACCTTA
CATGGTGATG
ATTTCCAAGT
GGACTTTCCA
ACGGTGGGAG
CCATCCACGC
GCTGATCCGT
TACAAGGGGA
TTGGCCCATT
AGCTGGAAGA
GGTGCACAAG
TGGTATTCAA
ACTTTGAATT
CAACACTGCT
TGAGCTGTAT
GGTCCAAACC
TCTATAACTG
CAGGAGGCCC
TGCCACACTA
ACGGCACCAC
AGATAGGAGA
GCAAGCCATA
ACTACA-"GAG
CTTTGGT37A(
ACGATATGAC
TCGATCTATI
TAAATCAATI
TATATTGGC9
TAGTAATCAP
CTTACGGTAP,
ATGACGTATG
TATTTACGGT
CCTATTGACG
TGGGACTTTC
CGGTTTTGGC
CTCCACCCCA
AAATGTCGTA
GTCTATATAA
TGTTTTGACC
CAGGGGCCAG
CATTGACTGC
AGGAGCTGAA
CACAATGGTC
AGAAACTAGA
AAAACTTTTC
TGGACTCTGC
AAACGGACCG
GTTAGCTAAT
ATTCCCTTAT
TGATCTTGGA
TGTCGAATCT
CCCCATCGGC
TTGCAACAGA
CACAATCGTA
TGAGATCATA
GACATTAAAA-i
:TTGGGCGATT
;GCTATATCGr
CATTGAATCA
TTGGCTATTG
CATGTCCAAC
TTACGGGGTC
ATGGCCCGCC
TTCCCATAGT
AAkACTGCCCA
TCAATGACGG
CTACTTGGCA
AGTACATCAA
TTGACGTCAA
ACAkACTCCGC
GCAGAGCTCG
TCCATAGAAG
ATGGTACAGG
AAACCTGAAC
GGCCTCACCA
ATAGCTTGGT
TATCTTGCAA
GAGGGGCAAA
CCATGCGATG(
GCCTTCCAGA
AGGGACACCC
AGACAAGGTT
GGGAATTGG1A
TGCAAGTGGTC
AAGTGTAGGT
GAGGGTGTAG C CAGGTCATAG CCAAGTGAGG C AATAAATATT I1
CGATACGCTT
CTGTGTGTCG
'ATAGAGGC
ATATTGGCCA
GCCATTGCAT
ATTACCGCCA
ATTAGTTCAT
TGGCTGACCG
AACGCCAATA
CTTGGCAGTA
TAAATGGCCC
GTACATCTAC
TGGGCGTGGA
TGGGAGTTTG
CCCATTGACG
TTTAGTGAAC
ACACCGGGAC
GCATCCTATG
AC" TCATh CGC
CT('TTTGGAA
GCAAAGACGG
I'TCTGCATTC
GGCAAGGGGA
CCAATCCCGT
TGGTATGCCC
TAGACACAGC
3TATCACCCA 7TTGTGTGAC 3TGGTTATAA
L'GAAGAATGA
CATAGTACC
~TCTTGACAC
;GCCTGTAGA
'TGAGCCCAG
2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 WO 95/12682 WO 5/1682PC'r/US94!1M 1% AGACAGTTAC TTCCAGCAA1 GGTCACTGAC CATCATCGGC ACTGGGTGGA AGATACGTGC GGCCTTGGGG ACCCAATATC TGACAGTATT GAAGTGGTGP TGTAAAGAAG TGGGTCTTAC GC-CGACCTC AGGCATGCAA GTTTGCCCCT CCCCCGTGCC TAATAAAATG AGGAAATTGC GGGGTGGGGC AGGACAGCAA GCGGTGGGCT CTATGGGTAC AAGAAGCAGG CACATCCT CCAGCCCCAC TCATAGGACA AAGTAXCTTGG AGCGGTCTCT GAGTGGGAAG AAATTAAAGC AACATGTGAG GAAGTAATGA ACGTGTCCGC GGCGCCAGAC AGGCCGCTTG CGCGGACGGG CG'.TAGAGAT GGCGGGGGAG CTCGCTCGCT CGCTGCATTT CCCC'.CCCGG CCCCGCCGCG GCGGCGCTAT TGGCAGCGGC GGCGGGGGCG CCGAAGCCAG AATGGTGGTC GGAGCCGGGC GGGCATTGAG CGCGTGGCCC GGTCCACGTT GACGTTGCGC CGCGGCCGTC GACTCTAGAG TACAACGTCG TGACTGGGAA CCCCTTTCGC CAGCTGGCGT TGCGCAGCCT GAATGGCGAA GTATTTCACA CCGCATATGG GCCAGCCCCG ACACCCGCCA CATCCGCTTA CAGACAAGCT
ACATGCTAP.A
ATTACTTCGC
TCTGGTTACT
GGGCAGGGGA
CATATTTCTT
TCTTATACCA
GCTCAGATCC
TTCCTTGACC
ATCGCATTGT
GGGGGAGGAT
CCAGGTGCTG
TCTCTGTGAC
CTCATAGCTC
CCCTCCCTCA
AAGATAGGCT
TAGAAATCAT
GCGTGCGCGG
GCGGCGGGCG
GCCACGGCGG
GCGCCCCGAT
AAGCAGGCCG
TGTCCTCGCG
GGCAGCACAG
CGGCCGTGCA
ACATTGCAAA
GCACCTCTGG
GATCCCCGGG
AACCCTGGCG
AATAGCGAAG
TGGCGCCTGA
TGCACTCTCA
ACACCCGCTG
GTGACCGTCT
AGGAGATTAT
*CGAGTCCATA
*GGTAACATAC
*AGTGGTGATG
GTTGTTATAC
CCTTGATTGA
GCTGTGCCTT
CTGGAAGGTG
CTGAGTAGGT
TGGGAAGACA
AAGAATTGAC
ACACCCTGTC
AGGAGGGCTC
TCAGCCCACC
ATTAAGTGCA
AGAATTGAGA
CCGCCGTACT
(CGACGCTGGC
GCCCTAGGGG
CGCCTTACGG
CCAGACAAAA
CTCGCCGCGG
AGACGCCCGA
CACGTTCACC
CCTCAGCCGG
CCTGCGGGAC
TACCGAGCTC
TTACCCAACT
AGGCCCGCAC
TGCGGTATTT
GTACAATCTG
ACGCGCCCTG
CAATACTGGT
TTGGTGGTGG
ATGGTCCTAT
ATGGGTAACT
CTACTGCTAA
TTGAGGATCA
CTAGTTGCCA
CCACTCCCAC
GTCATTCTAT
ATAGCAGGCA
CCGGTTCCTC
CACGCCCCTG
CGCTTCAATC
AAACCAAACC
GAGGGAGAGA
TCTCGAGGTG
GGACATGCGG
GACCCTGGCG
I4CTATAAAGC
GGACTCGGCG
AAATGCGGCG
GCGCCCCCGC
TACGAAATCG
ATCCGCTGCC
CTGCTGGACG
GCCATGTTTT
'IAATTCACTG
TAATCGCCTT
CGATCGCCCT
TCTCCTTACG
CTCTGATGCC
FCGGGCTTGT
TCGACCTGGA
TGGTAGCTTT
CAGAACAAAA
TGCTAACACA
GAGAGGAGGC
GCTTATCCAG
GCCATCTGTT
TGTCCTTTCC
TCTGGGGGGT
TGCTGGGGAT
CTGGGCCAGA
GTTCTTAGTT
CCACCCGCrA
TAGCCTCCAA
AAATGCCTCC
TTCGTGCTGG
CCCGCCATGC
CGTCAGTTCG
TGCCCCTGCG
CTCGGCGGAT
CCCGCTCTGC
CGCCGCCCGC
A~AGAGTGGGA
TCGGGCCGCG
GGTACATAGC
TCCTGCCGCG
GCCGTCGTTT
GCAGCACATC
TCCCAACAGT
CATCTGTGCG
.,CATAGTTAA
2TGCTCCCGG 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 CCGGGAGCTG CATGTGTCAG AGGTTTTCAC CGTCATCACC GAAACGCGCG AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA WO 95/12682 WO 9512682Pcr/uS94/12198 ATGTCATGAT AATAATGG7T GAACCCCTAT TTGTTTATTT AACCCTGATA AATGCTTCAA GTGTCGCCCT TATTCCCTTT CGCTGGTGAA AGTAAAAGAT TGGATCTCAA CAGCGGTAAG TGAGCACTTT TAAAGTTCTG AGCAACTCGG TCGCCGCATA CAGAAAAGCA TCTTACGGAT TGAGTGATAA CACTGCGGCC CCGCTTTTTT GCACAACATG TGAATGAAGC CATACCAAAC CGTTGCGCAA ACTATTAACT ACTGGATGGA GGCGGATAAA GGTTTATTGC TGATAAATCT TGGGGCCAGA TGGTAAGCCC CTATGGATGA ACGAAATAGA AACTGTCAGA CCAAGTTTAC TTAAAAGGAT CTAGGTGAAG AGTTTTCGTT CCACTGAGCG CTTTTTTTCT GCGCGTAATC TTTGTTTGCC GGATCAAGAG CGCAGATACC AAATACTGTC CTGTAGCACC GCCTACATAC GCGAr' AGTC GTGTCTTACC GGTCGGGCTG AACGGGGGGT AACTGAGATA CCTACAGCGT CGGACAGGTA TCCGGTAAGC GGGGAAACGC CTGGTATCTT GATTTTTGTG ATGCTCGTCA( TTTTACGGTT CCTGGCCTTT CTGATTCTGT GGATAACCGT
TCTTAGACGT
TTCTAAATAC
TAATATTGAA
TTTGCGGCAT
GCTGAAGATC
ATCCTTGAGA
CTATGTGGCG
CACTATTCTC
GGCATGACAG
AACTTACTTC
GGGGATCATG
GACGAGCGTG
GGCGAACTAC
GTTGCAGGAC
GGAGCCGGTG
TCCCGTATCG
CAGATCGCTG
TCATATATAC
ATCCTTTTTG
TCAGACCCCG
TGCTGCTTGC
CTACCAACTC
CTTCTAGTGT
CTCGCTCTGC
GGGTTGGACT(
TCGTGCACAC2
GAGCTATGAG
GGCAGGGTCG IATAGTCCTG 9i ;GGGGGCGGA C LGCTGGCCTT IJ kTTACCGCCT T
CAGGTGGCAC
ATTCAAATAT
AAAGGAAGAG
TTT'OCCTTCC
AGTTGGGTGC
GTTTTCGCCC
CGGTATTATC
AGAATGACTT
TAAGAGAATT
TGACAACGAT
TAACTCGCCT
ACACCACGAT
TTACTCTAGC
CACTTCTGCG
AGCGTGGGTC
TAGTTATCTA
AGATAGGTGC
TTTAGATTGA
ATAATCTCAT
TAGAAAAGAT
A4AACAAAAAA
TTTTTCCGAA
kGCCGTAGTT
L'AATCCTGTT
2AAGACGATA k.GCCCAGCTT kAAGCGCCAC
;AACAGGAGA
'CGGGTTTCG
;CCTATGGAA
'TGCTCACAT
'TGAGTGAGC
TTTTCGGGGA
GTATCCGCTC
TATGAGTATT
TGTTTTTGCT
ACGAGTGGGT
CGAAGAACGT
CCGTATTGAC
GGTTGAGTAC
ATGCAGTGCT
CGGAGGACCG
TGATCGTTGG
GCCTGTAGCA
TTCCCGGCAA
CTCGGCCCTT
TCGCGGTATC
CACGACGGGG
CTCACTGATT
TTTAAAACTT
GACCkAAATC
CAAAGGATCT
ACCACCGCTA
GGTAACTGGC
AGGCCACCAC
ACCAGTGGCT
GTTACCGGAT
GGAGCGAACG
GCTTCCCGAA
GCGCACGAGG
CCACCTCTGA
kAACGCCAGC "TTCTTTCCT PGATACCGCT C
AATGTGCGCG
ATGAGACAAT
CAACATTTCC
CACCCAGAAA
TACATCGAAC
TTTCCAATGA
GCCGGGCAAG
TCACCAGTCA
GCCATAACCA
AAGGAGCTPA
GAACCGGAGC
ATGGCAACAA
CAATTAATAG
CCGGCTGGCT
ATTGCAGCAC
AGTCAGGCAA
AAGCATTGGT
CATTTTTAAT
CCTTAACGTG
TCTTGAGATC
CCAGCGGTGG
TTCAGCAGAG
TTCAAGAACT
GCTGCCAGTG
PAGGCGCAGC
kLCCTACACCG 3GGAGAAAGG 3AGCTTCCAG
,TTGAGCGTC
ACGCGGCCT
;CGTTATCCC
~GCCGCAGCC
6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8083 GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGA WO 95/12682 PcIUS9412 198 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 8149 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: 2724 INDIVIDUAL ISOLATE: pBHVtkex-l::gBGH/p53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCGCCCAATA CGCAAACCGC CGACAGGTTT CCCGACTGGA CACTCATTAG GCACCCCAGC- TGTGAGCGGA TAACAATTTC TTGCATGCCT GCAGGTCGAC GCCGCCGCTG CCGGCCTGGT GTCCGCGTCC CCAACTCCGC GCGTATAAAA TTTCGCTCGT GCCCAGGCAA GCAAACTCTA GCCGAAATTT CGCCCAGGCA CTGTTGCCCG CGTGCCTGCT CCGCGCTCGC CGGCGGGCGG GGGGAAGCGG CTGCCCCTTC GCGGCGATCG CCCCGCTCGC CGGCTATTAT AGCCTCAAGG TCACTCTGAG CACGCGCATG CCGGGTCTGG GCCCGGGGGG GCAGCCCCCC GAGGCGCTGG ACGTCTTCAG CTCCACGATA GTAGGCACAC GCAATCGGGC CCACGACCTG CTTAAAAACC GGCGCAGCTT CTGCGCGCCA GGCTGTGGCA CCGGATCCCG
CTCTCCCCGC
AAGCGGGCAG
CTTTACACTT
ACACAGGAAA
TTCCGCGCCC
TCCGCGCCCG
GCGAAGACGG
CCGGTACAAA
AACGCCCGAG
CGCCGCGCCG
GGCCGCCCAC
CCGCGGGGTG
TGCCGCCGCG
CGCGAACGCG
CGCGCCGCGT
CCCCGCTGGG
GCGAGATCGC
2.CATCTTGC
AGACAGACGG
CGCCGGCTTT
TCCGGGGCGC
GCGTTGGCCG
TGAGCGCAAC
TATGCTTCCG
CAGCTATGAC
GCGGCGTCTG
CCGCCTCGCG
GCTCGTCCCA
GACGCGGTCC
CGCCAIGGCC
CCCGACGCGT
CGGCGGCCGC
CGGCCACAGC
GCCCCGGTTG
CGCGCGCGAA
TGCTAGCGAT
AGACGAACAC
GCACAAGCCG
TGGGCGTGTT
CCCGGGCGTG
GCAGGTTTAC
CAAACTTGCC
ATTCATTAAT
GCAATTAATG
GCTCGTATGT
CATGATTACG
CCTTCGCCAG
GCCAGCTCCC
GAAGCGCAGC
C-GACTGCGT
CCGATGCCGC
CTTTAGCGCA
TGTCCCCGGC
CGCCCTTTTG
CTCGGCTTTG
TGGGGCGTAC
CGTCTGGGCC
CTGCACCGGC
GGCCGAGTCG
CATGTTCGTT
CCCTGCCTCC
CTCAAAGCTC
CAAAAGCTGG
AGCCAGCGCC
GTCGCGGTCG
GCAGCTGGCA
TGAGTTAGCT
TGTGTGGAAT
CCAAGCTAGC
CAGGTTGTCC
GCGCGGGCGC
GGAAAGGCCG
GGATGTCCAC
CACAAAGAGC
CCCGCCGGCG
CTCAGCAGGG
CCCGTAGCCA
CGTTTGCCCC
TCGGCGAGCC
GGCAGGCGCG
GCTAGGACCA
CGCAGCTGCC
GAAAAACGGC
GCGACCCGGA
AGAGACACGC
GCGAGGCGCG
TCGTAAAAGC
CGCGCCATGG
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 ACCGCCGCGC GTGCGTCGCA GCGCGCAGGC GCGCACGTCG WO 95/12682 WO 9512682PCIIUS941 12198 CCGAGCCCGC GCGCGCTCTC GAAAGACAAC AACGGGCCGC TTTTCCCGGA GCCGATGGCC TCCTCGCGGC GTCTGCGCGA CCGGCGCACC GCGCAGACGC GCGGCCCCGT ACTTAATTTT CCGGGCGGCG AGCTGGTGGA GCTACCCCTT CGCCCGCTAC TTTCTATCAT TACTTCCTCA TATCTTGCTT TAATTTCTTC GGAGGGAGAG ACCGCTCCAA TATGAGTGTC CTATGAGTGG CAGAGAAGGG GATGTGCCTG ACCTGGGTAC CCATAGAGCC TCCCCCTTGC TGTCCTGCCC TGCGATGCAA TTTCCTCATT AAGGAAGGCA CGGGGGAGGG CTGAGCTTGC ATGCCTGAGG ATAAGAGTAA GACCCACTTC AATATGTCAC CACTTCAATA CTGCCCCATA TTGGGTCCCC ACCAGAGCAC GTATC-TTCCA AGTAATCCCG ATGATGGTCA GCATGTATTG CTGGAAGTAA TGTAGTTGAA GGTGCATGCC GCTTGCAAGG CATAGGCCCA CTATCTTACA CTTTACCAAT TGCCGTCTAC AAATCTGTAG GTGGCAACCC CTCACTTTTT CTCCTGTGTA TTGTAGCTGG TATAGAGATC CTCCCCCAGA TGGACCTCTT ATACACACGC AGCTCACAGT TCCTGTCCAT GTGTTGTATT GAACPTCCCT
CGCGTCGTGC
GCGCTCGCGG
TACTGGCGCA
TGCGCCGCAG
GGACGCGGCG
GCACGCGCGT
CCCTCGTGTT
TGCCTCCGCG
CATGTTGGAG
CCACTCTTGG
GTACTTTAGC
GGCTGGAACT
CTTCTTTCTG
CACCGCATCC
CACCCCACCC
TTATTAGGAA
GCAAACAACA
TCGACCCTGG
TTTACAGCCT
CTGTCATGTG
AAGGCCTTTT
CCCAGTAAAG
GTGACCTCCA
CTGTCTCTGG
GTCTTTTCTA
AGTTTGGTGT
CCTTGTGGTA
CCAGTCTCAT
TGGAATTTAT
TCCCCAGTCA
GTTCTTTGGG
ACTACTGCTG
CCTATAGGGC
CTTACTACGG
GTATCTACCT
CCGCTTCCAC
CGATGTTTGG
CCTCGCACGG
GGCCTGGTTG
GTCCGCGCTG
CGACCGCCAC
AGATCAACGC
GCATTTTCTC
AGGCTAGGTT
GGGTGGGATT
AAGAACCAGG
GCCCAGGAGG
CCAGCATGCC
CCCAGAATAG
AGGACAGTGG
GATGGCTGGC
ATAAGCTGAT
CCTCTCTTAG
TTAGCAAGTT
GTTCTGATAG
CTACCACCAC
GGTCGAACCA
GCTCAAAATA
CAGGCCCCTC
CAAGAGCTAT
CTATGGCTAC
TCTTCAACCT
!LACCACACCAC
CACMAGTCCA
TGATACAACC
rGTCTAGGGT kTACCATCTG 3ATTGGCATC
GGACGGCGCG
CGCTGGGGAG
TACGGACGCC
GAGCGCACGC
CGTACTACCA
CTGCGCCGCC
CCCGTGGCGC
GGAAGATCTC
TCCCTCTGCA
TGGTTTGGTG
GAA('CGGAGC
GGCGTGGACA
AACCGGGTCA
TGCTATTGTC
AATGACACCT
GAGTGGCACC
AACTAGAAGG
CCTCAATCAA
CAGTAGGTAT
ACCCATCATC
GACCATGTAT
CACCAATATG
GTATTGATAA
TTTATTTTTT
PCTTGGTATG
GACCTGTACG
PCCCTCTCTG
PCACTTGCCG
CTTGCAAGAT
),TTCCCTCCA I TTGTCTAT7.A 'TCCCTATTA C 3AAGGCCGGT C 3CATGGGCAG I
CACGGGCAGG
GGCGTGCTCT
TTAAGTGGGA
GCGCGGCGGG
GGCCAGGTTC
T'GGGCCGGCG
GCGTGCCTCT
AATTCTATGA
CTTAATAGCC
GGCTGATGAG
CCTCCTGAGC
GGGTGTGTCA
ATTCTTCAGC
TTCCCAATCC
ACTCAGACAA
TTCCAGGGTC
CACAGCGGAT
TCAAGGTGGT
AACAACAAGA
ACCACTTCCC
GTTACCAGTA
GACTCGGCGA
TCTCCTTTTA
PATGTCCTCG
ATCTCATATG
%TTGTGTCTC
rTGCAAGTGG
%TGGGGTAGT
CCGACAGGGC
\GATCACAGT
;GGAATGGTT
CTAACATAC
~CGTTTAGCA
~GTCCAAATT
1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 -27- WO 95/12682 WO 9512682PCT/1JS94/12198 CAAAGTCGTC AGCCATTTCI ATACCACACT GGTCTGCAAC TGCACCTTGA GAGGTATGTI CCAGCTTCAT TTCATGTGAC GGCCAATTCT ATCATTCCTC CTTGTACCCC TGTTATCAGI TCCAGGGCAG GCAGAGCAGC TCATGTCGAA GCTTGCCGCC CAGCGTGGAT GGCGTCTCCP ACCTCCCACC GTACACGCCI TTTGGAAAGT CCCGTTGATT AGACTTGGMA ATCCCCGTGA CGCATCACCA TGGTAATAGC CATAAGGTCA TGTACTGGGC GGGGGCGTAC TTGGCATATG TACTCCACCC ATTGACGTCA ATTATTGACG TCAATGGGCG AGTTATGTAA CGCGGAACTC TATTAATAAC TAGTCAATAA TAAATGTACA TATTATGATA TATGCTATAT AACCAATGAA GATATGCATT GGCCATGTGC GTCTGTCACC TATATCGAAA AGGCGTCTAT CGCCATCCCC CAAAAATCAA CTTTTGGAAA TTTTCAATAT GCCATATTTT TAAACACCAC TATCGCGCGA GGACAAACAC ACATTTTATT TGCTGGACGT GTCCGCGGCG CCATGCAGGC CGCTTGCGCG AGTTCGCGCT AGAGATGGCG CCTGCGCTCG CTCGCTCGCT GCGGATCCCC TCCCGGCCCC CTCTGCGCGG CGCTATTGGC k. AATGTTTCCC 3 GCTCTTGAA]
SAACTTACCGI
;TAATCCTTCC
;GCTATGGCGI
AGTAGCCATA
GCGAAAGCCA
GAGC-CTGGAT
GGCGATCTGA
ACCGCCCATT
TTGGTGCCAA
GTCAAACCGC
GATGACTAAT
ATAATGCCAG
ATACACTTGA
ATGGAAAGTC
GGGGTCGTTG
CATATATGGG
TCAATGTCAA
TGGATACAAC
TAATATGGCT
CAGCTTGATG
CTGCGATATT
CGTAAACGAT
AATGGCGATA
CAAATATCGA
CATGAATTTA
ATTCACTGCA
CCAGACGCGT
GACGGGGCGG
GGGGAGGCCA
GCATTTGCGC
GCCGCGAAGC
AGCGGCTGTC
-CTTGCCTTTC.
*GCAGAATTGC
CTTTGCACCP
AAACAGTGGI
ATGAGTGTTC
GGATCCCTGG
*GGAGCAGGGA
CGGTCCCGGT
CGGTTCACTA
TGCGTCAATG
AACAAACTCC
TATCCACGCC
ACGTAGATGT
GCGGGCCATT
TGTACTGCCA
CCTATTGGCG
GGCGGTCAGC
CTATGAACTA
CATGGCGGTA
GTATGCAATG
AATGGCCAAT
TCGCCTCTAT
TGCGACACAC
ATAAGCGTAT
TCAGTTACAC
TTTTTCCAAT
GTCGGGACAG
GGTCGAGGAA
GCGCGGCCGC
CGGGCGCGAC
CGGCGGGCCC
CCCGATCGCC
AGGCCGCCAG
CTCGCGCTCG
CCCCTCGAA2
AAGATATCTI
AGCTATGACC
GAGGCCTTCI
AGGTTTGCAC
GAAGGCGCCC
GGTCCGGGGC
GTCTTCTATC
AACGAGCTCI
GGGCGGAGTI
CATTGACGTC
CATTGATGTA
ACTGCCAAGT
TACCGTCATT
AGTGGGCAGT
TTACTATGGG
CAGGCGGGCC
ATGACCCCGT
ATGTTGGACA
GCCAATAGCC
ATTGAt.,TCAA
CGGCGATATA
AGAATCGCCC
CGCCAGATAT
AGAAACTCAC
ATCGCCATCT
AAATCTCAAA
TTCGGATCTC
CGTACTGGAC
GCTGGCGACC
TAGGGGACTA
TTACGGGGAC
ACAAAAAAAT
CCGCGGGCGC
k. AGTTTTTTGA k. GTTTCTCTTG
ATTGTGTCTT
GCTCCTAATG
TCAATGTCCC
ACCACCTGAG
CCTGCAGCCA
GAGGTCAAAA
GCTTATATAG
GTTACGACAT
AATGGGGTGG
CTGCCAAAAC
AGGAAAGTCC
GACGTCAATA
TTACCGTAAA
AACATACGTC
ATTTACCGTA
AATTGATTAC
TGAGCCAATA
AATATTGATT
TGTATAGATC
GCCTCATATC
AAGTCACCAA
CGCGTATGCC
ATCGGCGACA
CTATCGGCGA
CGCGTATTTC
GAGGTGTTCG
ATGCGGCCCG
CTGGCGCGTC
TAAAGCTGCC
TCGGCGCTCG
GCGGCGCCCG
CCCCGCCGCC
3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 -28- WO 95/12682 WO 9512682PCTIUS94/12198 GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG GTGGGAAATG GTGGTCGGAC GCCGCGGGGC ATTGAGCGCC CATAGCGGTC CACGTTGACC GCCGCGCGCG GCCGTCGACUI TCGTTTTACA ACGTCGTGAC CACATCCCCC TTTCGCCAGC AACAGTTGCG CAGCCTGAAT TGTGCGGTAT TTCACACCGC AGTTAAGCCA GCCCCGACAC TCCCGGCATC CGCTTACAGA TTTCACCGTC ATCACCGAAA AGGTTAATGT CATGATAATA TGCGCGGAAC CCCTATTTGT GACAATAACC CTGATAAATG ATTTCCGTGT CGCCCTTATT CAGAAACGCT GGTGAAAG..
TCGAACTGGA TCTCAACAGC CAATGATGAG CACTTTTAAA GGCAAGAGCA ACTCGGTCGC CAGTCACAGA AAAGCATCTT TAACCATGAG TGATAACACT AGCTAACCGC TTTTTTGCAC CGGAGCTGAA TGAAGCCATA CAACAACGTT GCGCAAACTA TAATAGACTG GATGGAGGCG CTGGCTGGTT TATTGCTGAT CAGCACTGGG GCCAGATGGT AGGCAACTAT GGATGAACGA ATTGGTAACT GTCAGACCAA TTTAATTTAA AAGGATCTAG AACGTGAGTT TTCGTTCCAC GAGATCCTTT TTTTCTGCGC CGGTGGTTTG TTTGCCGGAT
CCGGGCCGGC
TGGCCCACAT
TTGCGCGCAC
CTAGAGGATC
TGGGAAAACC
TGGCGTAATA
GGCGAATGGC
ATATGGTGCA
CCGCCAACAC
CAAGCTGTGA
CGCGCGAGAC
ATGGTTTCTT
TTATTTTTCT
CTTCAATAAT
CCCTTTTTTG
AAAGATGCTG
GGTAAGATCC
GTTCTGCTAT
CGCATACACT
ACGGATGGCA
GCGGCCXXCT
AACATGGG'3G
CCAAACGACG
TTAACTGGCG
GATAAAGTTG
AAATCTGGAG
AAGCCCTCCC
AATAGACAGA
GTTTACTCAT
GTGAAGATCC
TGAGCGTCAG
GT1PATCTGCT
CAAGAGCTAC
*CGTGCACACG
TGCAAACCTC
CTCTGGCCTG
CCCGGGTACC
CTGGCGTTAC
GCGAAGAGGC
GCCTGATGCG
CTCTCAGTAC
CCGCTGACGC
CCGTCTCCGG
GAAAGGGCCT
AGACGTCAGG
AAATACATTC
ATTGAAAAAG
CGGCATTTTG
AAGATCAGTT
TTGAGAGTTT
GTGGCGCGGT
ATTCTCAGAA
TGACAGTAAG
TACTTCTGAC
ATCATGTAAC
AGCGTGACAC
AACTACTTAC
CAGGACCACT
CCGGTGAGCG
GTATCGTAGT
TCGCTGAGAT
ATATACTTTA
TTTTTGATAA
ACCCCGTAGA
GCTTGCAAAC
CAACTCTTTT
TTCACCATCC
AGCCGGCTGC
CGGGACGCCA
GAGCTCGAAT
CCAACTTAAT
CCGCACCGAT
GTATTTTCTC
AATCTGCTCT
GCCCTGACGG
GAGCTGCATG
CGTGATACGC
TGGCACTTTT
AAATATGTAT
GAAGAGTATG
CCTTCCTGTT
GGGTGCACGA
TCGCCCCGAA
ATTATCCCGT
TGACTTGGTT
AGAATTATGC
AACGATCGGA
TCGCCTTGAT
CACGATGCCT
TCTAGCTTCC
TCTGCGCTCG
TGGGTCTCGC
TATCTACACG
A.GGTGCCTCA
GATTGATTTA
TCTCATGACC
PAAAGATCAAA
A~AAAAAACCA(
TCCGAAGGTA
AAATCGAAGA
GCTGCCTCGG
TGGACGGGTA
TGTTTTTCCT
TCACTGGCCG
CGCCTTGCAG
CGCCCTTCCC
CTTACGCATC
GATGCCGCAT
GCTTGTCTGC
TGTCAGAGGT
CTATTTTTAT
CGGGGAAATG
CCGCTCATGA
AGTATTCAAC
TTTGCTCACC
GTGGGTTACA
GAACGTTTTC
ATTGACGCCG
GAGTACTCAC
AGTGCTGCCA
GGACCGAAGG
CGTTGGGAAC
GTAGCAATGG
CGGCAACAAT
GCCCTTCCGG
GGTATCATTG
ACGGGGAGTC
CTGATTAAGC
!AACTTCATT
k4AAATCCCTT 3GATCTTCTT 2CGCTACCAG
%CTGGCTTCA
5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 WO 95/12682 WO 95/2682PW'/S94/ 12198 GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC AGAACTCTGT AGCACCGCCT CCAGTGGCGA TAAGTCGTGT CGCAGCGGTC GGGCTGAACG ACACCGAACT GAGATACCTA GAAAGGCGGA CAGGTATCCG TTCCAGGGGG AAACGCCTGG AGCGTCGATT TTTGTGATGC CGGCCTTTTT ACGGTTCCTG TA"TCCCCTGA TTCTGTGGAT GCAGCCGAAC GACC-GAGCGC ACATACCTCG CTCTGCTAAT CTTACCGGGT TGGACTCAAG GGGGGTTCGT GCACACAGCC CAGCGTGAGC TATGAGAAAG GTAAGCGGCA GGGTCGGAAC TATCTTTATA GTCCTGTCGG TCGTCAGGGG GGCGGAGCCT GCCTTTTGCT GGCCTTTTGC AAC-ZGTATTA CCGCCTTTGA AGCGAGTCAG TGAGCGAGGA
CCTGTTACCA
ACGATAGTTA
CAGCTTGGAG
CGCCACGCTT
CACCACTTCA
GTGGCTGCTG
CCGGATAAGG
CGAACGACCT
CCCGAAGGGA
AGGAGAGCGC ACGAGGGAGC 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8149
GTTTCGCCAC
ATGGAAAAAC
TCACATGTTC
GTGAGCTGAT
AGCGGAAGA
CTCTGACTTG
GCCAGCA.ACG
TTTCCTGCGT
ACC~rTCGCC INFORMATION FOR SEQ ID NO:3: S7QUENCE CHARACTERISTICS: LENGTH: 8135 base pa.irs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: 27.24 INDIVIDUAL ISOLATE: pBHVtkex-l::gIII/p53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCGCCCAATA CGCAAACCGC CGACAGGTTT CCCGACTGGA CACTCATTAG GCACCCCAGG TGTGAGCGGA TAACAATTTC TTGCATGCCT GCAGGTCGAC GCGGCCGCTG CCGGCCTGGT GTCCGCGTCC CCAACTCCGC GCGTATAAAA TTTCGCTCGT GCCCAGGCAA GCAAACTCTA GCCGAAATTT CGCCCAGGCA CTGTTGCCCG CGTGCCTGCT
CTCTCCCCGC
AAGCGGGCAG
CTTTACACTT
ACACAGGAAA
TTCCG'CGCCC
TCCGCGCCCG
GCGAAGACGG
CCGGTACAAA
AACGCCCGAG
CGCCGCGCCG
GGCCGCCCAC
GCGTTGGCCG
TGAGCGCAAC
TATGCTTCCG
CAGC'ATGAC
GCGGYCGTCTG
CCGCCTCGCG
GCTCGTCCCA
GACGCGGTCC
CGCCATGGCC
CCCGACGCGT
CGGCGGCCGC
ATTCATUAAT
GCAAT-TAATG
GCTCGTATGT
CATGATTACG
CCTTCGCCAG
GCCAGCTCCC
GAAGCGCAGC
GCGACTGCGT
CCGATGCCGC
CTTTAGCGCA
TGTCCCCGGC
GCAGCTGGCA
TGAGTTAGCT
TGTGTGGAAT
CCAAGCTAGC
CAGGTTGTCC
GCGCGGGCGC
GG-AAAGGCCG
GGATGTCCAC
CI.CAAAGAGC
CCCGCCGGCG
CTCAGCAGGG
120 180 240 300 360 420 480 540 600 660 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA WO 95/12682 WO 95/ 2682PCT/US94/12198 GGGGJAAGCGG CTGCCCCTTC GCGGCGATCG CCCCGCTCG( C GCTATTAT AGCCTCAAGC 'ICACTCTGAG CACGCGCATC CCGGGTCTGG GCCCGGGGGC GCAGCCCCCC GAGGCGCTGC ACGTCTTCAG CTCCACGATA GTAGGCACAC CiCAATCGGGC CCACGACCTG CTTAAAAACC GGCGCAGCTT CTGCGCGCCA GGCTGTGGCA CCGGA\TCCCG CCGAGCCCGC GCGCGCTCTC GAAAGACAAC AACGGGCCGC TTTTCCCGGA GCCGATGGCG TCCTCGCGGC GTCTGCGCGA CCGGCGCACC GCGCAGACGC GCGGCCCCGT ACTTAATTTT CCGGGCGGCG AGCTGGTGGA GCTACCCCTT CGCCCGCIAC TTTCTATCAT TACTTCCTCA TATCTTGCTT TAATTTCTTC GGACGGAGAG ACCGCTCCAA TATGAGTGTC CTATGAGTGG CAGAGAAGGG GATGTGCCTG ACCTGGGTAC CCATAGAGCC TCCCCCTTGC TGTCCTGCCC TGCGATGCAA TTTCCTCATT AAGGA.AGGCA CGGGGGAGGG CTGAGCTTGC ATGCCTGAC- ATAAGAGTAA GACCCACTTC AATATGTCAC CACTTCAATA CTGCCCCATA TTGGGTCCCC ACCAGAGCAC GTATCTTCCA
TGCCGCCGCG
CGCGAACGCG
CGCGCCGCGT
CCCCGCTGGG
GCGAGATCGC
TCCATCTTGC
AGACAGACGG
CGCCGGCTTT
TCCGGGGCGC
ACCGCCGCGC
GCGCGCAGGC
CGCGTCGTGC
GCGCTCGCGG
TACTGGCGCA
TGCGCCGCAG
GGACGCGGCG
GCAiCGCGCGT
CCCTCGTGTT
TNGCCTCCGCG
CATGTTGGAG
CCACTCTTGG
GTACTTTAGC
GGCTGGAACT
CTTCTTTCTG
CACCGCATCC
CACCCCACCC
GCCGCGGTTG
CGCGCGCGAA
TGCTAGCGAT
AGACGAACAC
GCACAAGCCG
TGGGCGTGTT
CCCGGGCGTG
GCAGGTTTAC
CAAACTTGCC
GTGCGTCGCA
GCGCACGTCG
GTATCTACCT
CCGCTTCCAC
CGATGTTT("G
CCTCGCACGG
GGCCTGGTTG
GTCCGCCACTG
CGACCGCCAC
AGATCAACGC
GCATTTTCTC
AGGCTAGGTT
GGGTGGGATT
AAGAACCAGG
GCCCAGGAGG
CCAGCATGr
CCCAGAATAG
CTCGGCTTTG
TGGGGCGTAC
CGTCTGGGCC
CTGCACCGGC
GGCCGAGTCG
CATGTTCGTT
CCCTGCCTCC
CTCA.AAGCTC
CAAAAGCTGG
CGTTTGCCCC
TCGGCGAGCC
GGCAGGCGCG
GCTAGGACCA
CGCAGCTGCC
GAAAAACGGC
G CGP.CCCGGA
AGAGACACGC
GCGAGGCGCG
TCGTAAAAGC AGCCAGCGCC
GTCGCGGTCG
OGACGGCGCG
CGCTGGGGAG
TACGGACGCC
GAGCGCACGC
CGTACTACCA
CTGCGCCGCC
CCCGTGGCGC
GGAAGATCTC
TCCrTCTGCA
TGGTTTGGTG
GAAGCGGAGC
GGCCTGGACA
AACCGGGTCA
TGCTATTGTC
AATGACACCT
CGCGCCATGG
CACGGGCAGG
GGCGTGCTCT
TTAAGTGGGA
GCGCGGCGGG
GGCCAGGTTC
TGGGCCGGCG
GCGTGCCTCT
AATTCTATGA
CTTAATAGCC
GGCTGATGAG
CCTCCTGAGC
GGGTGTGTCA
ATTCTTCAGC
TTCCCAATCC
ACTCAGACAA
780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700
TTATTAGGAA
GCAAACAACA
!iCGACCCTGG
TTTACAGCCT
CTGTCATGTG
AAGGCCTTTT
CCCAGTAAAG
AGGACAGTGG
GATGGCTGGC
ATAAGCTGAT
CCTCTCTTAG
TTAGCAAGTT
GTTCTGATAG
CTACCACCAC
GAGTGGCACC
AACTAGAAGG
CCTCAATCAA
CAGTAGGTAT
ACCCATCATC
GACCATGTAT
CACCAATATG
TTCCAGGGTC
CACAGCGGAT
TCAAGGTGGT
AACAACAAGA
ACCACTTCCC
GTTACCAGTA
GACTCGGCGA
AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCGAACCA GTATTGATAA TCTCCTTTTA 2760 -31- WO 95/12682 PICrIYUS94I 12198 GCATGTATTG CTGGAAGTAA TGTAGTTGAA GGTGCATGCC GCTTGCAAGG CATAGGCCCA CTATCTTACA CTTTACCAAT TGCCGTCTAC AAATCTGTAG GTGGCAACCC CTCACTTTTT CTCCTGTGTA TTGTAGCTGG TATAGAGATC CTCCCCCAGA TGGACCTCTT ATACACACGC AGCTCACAGT TCCTGTCCAT GTGTTGTATT GAACTTCCCT CAAAGTCGTC AGCCATTTCA ATACCACACT GGTCTGCAAG TGCACCTTGA GAGGTATGTA CCAGCTTCAT TTCATGTGAG GGCCAATTCT ATCATTCCTG CTTGTACCCC TGTTATCAGT Mi CCGCGTAGAG CGCCAGCAGA GCCGCGGAGG CTGGATCGGT TCTCCAGGCG ATCTGACGGT ACGCCTACCG CCCATTTGCG TTGATTTTGG TGCCAAAACA CCGTGAGTCA AACCGCTATC AATAGCGATG ACTAATACGT CTGGGCATAA TGCCAGGCGG CATATGATAC ACTTGATGTA ACGTCAATGG AAAGTCCCTA TGGGCGGGGG TCGTTCGGCG GAACTCCATA TATGGGCTAT CAATAATCAA TGTCAACATG ATGATATGGA TACAACGTAT C AATGAADTAAT ATGGCTAATG C ATGTGCCAGC TTGATGTCGC C
CTGTCTCTGG
GTCTTTTCTA
AGTTTGGTGT
CCTTGTGGTA
CCAGTCTCAT
TGGAATTTAT
TCCCCAGTCA
GTTCTTTGGG
ACTACTGCTG
CCTATAGGGC
CTTACTACGG
AATGTTTCCC
GCTCTTGAAT
AACTTACCGT
TAATCCTTCC
GCTATGGCGT
AGTAGCCATA
GCGAGCATCG
CCCGGTGTCT
TCACTAAACG
2'CAATGGGGC
A.ACTCCCATT
CACG27CCATT
AGATGTACTG
GCCATTTACC
CTGCCAAGTG
TTGGCGTTAC
3TCAGCCAGG 3AACTAATGA C ;CGGTAATGT I CAATGGCCA T CCAATATTG P ~TCTATCGGC G
GCTCAAAATA
CAGGCCCCTC
CAAGAGCIAT
CTATGGCTAC
TCTTCAACCT
AACCACACCA
CACAAGTCCA
TGATACAACC
TGTCTAGGGT
ATACCATCTG
GATTGGCATC
CTTGCCTTTG
GCAGAATTGC
CTTTGCACCA
AAACAGTGGT
ATGAGTGTTC
GGATCCCCGA
CACGCGCGAG
TCTATGGAGG
AGCTCTGCTT
GGAGTTGTTA
GACGTCAATG
GATGTACTGC
:CAAGTAGGA
3TCATTGACG
;GCAGTTTAC
M~TGGGAACA
GGGCCATTT
~CCCGTAATT
~GGACATGAG
TAGCCAATA
LTTCAATGTA
;ATATAGCCT C
TTTATTTTTT
ACTTGGTATG
GACCTGTACG
ACCCTCTCTG
ACACTTGCCG
CTTGCAAGAT
ATTCCCTCCA
!IGCTCTATAA
GTCCCTATTA
GAAGGCCGGT
GCATGGGCAG
CCCCTCGAAA
AAGATATCTA
AGCTATGACC
GAGGCCTTCA
AGGTTTGCAG
CGGCGCCGCG
CGAGGCCATG
TCAAALACAGC
ATATAGACCT
CGACATTTTG
GGGTGGAGAC
C.AAAACCGCA
A.AGTCCCATA
TCAATAGGGG
CGTAAATACT
TACGTCATTA
ACCGTAAGTT
GATTACTATT
,CAATATAAA9 CTGATTTATG C rAGATCGATA I3 ATATCGTCT C
AATGTCCTCG
ATCTCATATG
ATTGTGTCTC
TTGCAAGTGG
ATGGGGTAGT
TCGACAGGGC
AGATCACAGT
GGGAATGGTT
GCTAACATAC
CCGTTTAGCA
AGTCCAAATT
AGTTTTTTGA
GTTTCTCTTG
ATTGTGTCTT
GCTCCTAATG
TCAATGTCCC
GCGATGGCCG
GTCGAAGCTT
GTGGATGGCG
CCCACCGTAC
GAAAGTCCCG
TTGGAAATCC
TCACCATGGT
AGGTCATGTA
GCGTACTTGG
CCACCCATTG
TTGACGTCAA
%TGTAACGCG
k.ATAACTAGT
~GTACATATT
TATATAACC
'GCATTGGCC
;TCACCTATA
2820 2880 2940 3000 3060 3120 3180 J3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 TCGAAACTGC GATATTTGCG ACACACAGIAA TCGCCCAAGT CACCAAAGGC GTCTATCGCC -32- WO 95/12682 WO 9512682PCTJUS94/1 2198 ATCCCCCGTA AACGATATAA GCGTATCGCC AGATATCGCG TATGCCCAAA TGGAAAAATG GCGATATCAC TATTTTCAAA TATCGATTT~I GCGCGACATG AATTTAGTCC TTTATTATTC ACTGCAGGTC GCGGCGCCAG ACGCCGTGCGC TGCGCGGACG GGGCGGCGGG ATGGCGGGGG AGGCCACGGC CTCGCTGCAT TTGCGCCCCG GGCCCCGCCG CGAAGCAGGC ATTGGCAGCG GCTGTCCTCG CGCCGAAGCC AGGGCAGCAC TCGGAGCCGG GCCGGCCGTG AGCGCGTGGC CCACATTGCA TTGACGTTGC GCGCACCTCT TCGACTCTAG AGGATCCCCG CGTGACTGG, AAAACCCTGG GCCAGCTGGC GTAATAGCGA CTGAATGGCG AATGGCGCCT CACCGCATAT GGTGCACTCT CGACACCCGC CAACACCCGC TACAGACAAG CTGTGACCGT CCGAAACGCG CGAGACGAAA ATAATAATGG TTTCTTAGAC ATTTGTTTAT TTTTCTAAAT TAAATGCTTC AATAATATTG CTTATTCCCT TTTTTGCGGC AAAGTAAAAG ATGCTGAAGA AACAGCGGTA AGATCCTTGA TTTAAAGTTC TGCTATGTGG GGTCGCCGCA TACACTATTC CATCTTACGG ATGGCATGAC AACACTGCGG CCAACTTACT TTGfIACAACA TGGGGGATCA
TTACACAGAA
TCCAATATCG
GGACAGAAAT
GAGGAATTCG
GGCCGCCGTA
1CGCGACGCTG
GGGCCCTAGG
ATCGCCTTAC
CGCCAGACAA
CGCTCGCCGC
AGAGACGCCC
CACACGTTCA
AACCTCAGCC
GGCCTGCGGG
GGTACCGAGC
CGTTACCCAA
AGAGGCCCGC
GATGCGGTAT
CAGTACAATC
TGACGCGCCC
CTCCGGGAGC
GGGCCTCGTG
GTCAGGTGGC
ACATTCAAAT
AAAAAGGAAG
ATTTTGCCTT
TCAGTTGGGT
GAGTTTTCGC
CGCGGTATTA
TCAGAATGAC
AGTAAGAGAA
TCTGACAACG
TGTAACTCGC
*ACTCACATC(
CCATCTCTAI
CTCAAACGCC
GATCTCGAGC
CTGGACATGC
uCGACCCTGC
GGACTATA-PA
GGGGACTCGG
AAAAATGCGG
GGGCGCCCCC
GATACGAAAT
CCATCCGCTG
GGCTGCTGGA
ACGCCATGTT
TCGAATTCAC
CTTAATCGCC
ACCGATCGCC
TTTCTCCTTA
TGCTCTGATG
TGACGGGCTT
TGCATGTGTC
ATACGCCTAT
ACTTTTCGGG
ATGTATCCGC
AGTATGAGTA
CCTGTTTTTG
GCACC-AGTGG
CCCGAAGAAC
TCCCGTATTG
TTGGTTGAGT
TTATGCAGTG
ATCGGAGGAC
CTTGATCGTT
3GCGACATTTT
'CGGCGATAAA
TATTTCGGAC
TGTTCGTGCT
GGCCCGCCAT
CGCGTCAGTT
GCTGCCCCTG
CGCTCGGCGG
CGCCCGCTCT
GCCGCCGCCC
CGAAGAGTGG
CCTCGGGCCG
CGGGTACATA
TTTCCTGCCG
TGGCCGTCGT
TTGCAGCACA
CTTCCCAACA
CGCATCTGTG
CCGCATAGTT
GTCTGCTCCC
AGAGGTTTTC
TTTTATAGGT
GAAATGTGCG
TCATGAGACA
TTCAACATTT
CTCACCCAGA
GTTACATCGA
GTTTTCCAAT
ACGCCGGGCA
ACTCACCAGT(
CTGCCATAAC
CGAAGGAGCT
GGGAACCGGA
AATCAACTTT
CAATATGCCA
CACCACTATC
AAACACACAT
GGACGTGTCC
GCAGGCCGCT
CGCqCTAGAG
CGCTCGCTCG
ATCCCCTCCC
GCGCGGCGCT
GCGGCGGGGG
GAAATGGTGG
CGGGGCATTG
GCGGTCCACG
CGCGCGGCCG
TTTACAACGT
TCCCCCTTTC
GTTGCGCAGC
CGGTATTTCA
AAGCCAGCCC
GGCATCCGCT
ACCGTCATCA
TAATGTCATG
CGGAACCCCT
ATAACCCTGA
CCGTGTCGCC
PLACGCTGGTG
PCTGGATCTC
GATGAGCACT
kGAGCAACTC
CACAGAAAAG
CATGAGTGAT
LACCGCTTT
3CTGAATGAA 4860 4920 4980 5040 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 61.20 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 WO 95/12682 WO 9512682PCT/US94/ 12198 GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AAACTATTAA CTGGCGAACT GAGGCGGATA AAGTTGCAGG GCTGATAAAT CTGGAGCCGG GATGGTAAGC CCTCCCGTAT GAACGAAATA GACAGATCGC GACCAAGTTT ACTCATATAT ATCTAGGTGA AGATCCTTTT TTCCACTGAG CGTCAGrACCC CTGCGCGTAA TCTGCTGCTT CCGGATCAAG AGCTACCAAC CCAAATACTG TCCTTCTAGT CCGCCTACAT ACCTCGCTCT TCGTGTCTTA CCGGGTTGGA TGAACGGGGG GTTCGTGCAC TACCTACAGC GTGAGCTATG TATCCGGTAA GCGGCAGGG2 GCCTGGTATC TTTATAGTCC TGATGCTCGT CAGGGGGGCG TTCCTGGCCT TTTGCTGGCC GTGGATAACC GTATTACCGC GAGCGCAGCG AGTCAGTGAG
ACTTACTCTA
ACCACTTCTG
TGAGCGTGGG
CGTAGTTATC
TGAGATAGGT
ACTTTAGATT
TGATAATCTC
CGTAGAAAAG
GCAAACAAAA
TCTTTTTCCG
GTAGCCGTAG
GCTAATCCTG
CTCAAGACGA
ACAGCCCAGC
AGAAAGCGCC
CGGAACAGGA
TGTCGGGTTT
GAGCCTATGG
TTTTGCTCAC
CTTTGAGTGA
CGAGGAAGCG
GCTTCCCGGC
CGCTCGGCCC
TCTCGCGGTA
TACACGACGG
GCCTCACTGA
GATTTAAAAC
ATGACCAAAA
ATCAAAGGAT
AAACCACCGC
AAGGTAACTG
TTAGGCCACC
TTACCAGTGG
TAGTTACCGG
TTGGAGCGAA
ACGCTTCCCG
GAGCGCACGA
CGCCACCTCT
AAAAACGCCA
PLTGTTCTTTC
GCTGATACCG
GAAGA
AACAATTAAT
TTCCGGCTGG
TCATTGCAGC
GGAGTCAGGC
TTAAGCATTG
TTCATTTTTA
TCCCTTAACG
CTTCTTGAGA
TACCAGCGGT
GCTTCAGCAG
ACTTCAAGAA
CTGCTGCCAG
ATAAGGCGCA
CGACCTACAC
AAGGGAGAAA
GGGAGCTTCC
GACTTGAGCG
GCAACGCGGC
CTGCGTTATC
AACGTTGCGC
AGACTGGATG
CTGGTTTATT
ACTGGGGCCA
AACTATGGAT
GTAACTGTCA
ATTTAAAAGG
TGAGTTTTCG
TCCTTTTTTT
GGTTTGTTTG
AGCGCAGATA
CTCTGTAGCA
TGGCGATAAG
GCGGTCGGGC
CGAACTGAGA
GGCGGACAGG
AGGGGGAAAC
TCGATTTTTG
CTTTTTACGG
CCCTGATTCT
6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8135 CTCGCCGCAG CCGAACGACC INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 8149 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: 2724 INDIVIDUAL ISOLATE: pBHVtkex- BGH/p53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -34- WO 95/12682 WO 95/12682P CT/US94/12 198
GCGCCCAATA
CGACAGGTTT
CACTCATTAG
TGTGAGCGGA
TTGCATGCCT
GCGGCCGCTG
GTCCGCGTCC
GCGTATAAAA
GCCCAGGCAA
GCCGAAATTT
CTGTTGCCCG
CCGGGGTCGC
GGGGAAGCGG
GCGGCGATCG
CGGCTATTAT
TCACTCTGAG
CCGGGTCTGG
GCAGCCCCCC
ACGTCTTCAG
GTAGGCACAC
CCACGACCTG
GGCGCAGCTT
GGCTGTGGCA
CCGAGCCCGC
GAAAGACAAC
TTTTCCCGGA
TCCTCGCGGC
CCGGCGCACC C GCGGCCCCGT I CCGGGCGGCG .Z GCTACCCCTT C CCTGCAGTGA A ACTAAATTCA Ti AATCGATATT T CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA
CCCGACTGGI
GCACCCCAGC
TAACAATTTC
GCAGGTCGAC
CCGGCCTGGU
CCAACTCCGC
TTTCGCTCGI
GCAAACTCTA
CGCCCAGGCA
CGTGCCTGCT
CGGCGGGCGG
CTGCCCCTTC
CCC CGCTCGC
AGCCTCAAGG
CACGCGCATG
GCCCGGGGGG
GAGGCGCTGG
CTCCACGATA
GCAATCGGGC
CTTAAAAACC
CTG3CGCGCCA
CCGGATCCCG
GCGCGCTCTC
%.ACGGGCCGC
3CCGATGGCG
;TCTGCGCGA
;CGCAGACGC
~CTTAATTTT
~GCTGGTGGA
GCCCGCTAC
LTAATAAAAT
'GTCGCGCGA
GAAAATATG
AAGCGG.GCAG
;CTTTACACTT
!ACACAGGAAA
TTCCGCGCCC
TCCGCGCCCG
GCGAAGACGG
CCGGTACAAA
*AACGCCCGAG
*CGCCGCGCCG
GGCCGCCCAC
CCGCGGGGTG
TGCCGCCGCG
CGCGAACGCG
CGCGCCGCGT
CCCCGCTGGG
GCGAGATCGC
TCCATCTTGC
AGACAGACGG
CGCCGGCTTT
TCCGGGGCGC
ACCGCCGCGC
GCGCGCAGGC
CGCGTCGTGC
GCGCTCGCGG
TACTGGCGCA
TGCGCCGCAG
GGACGCGGCG
GCACGCGCGT
CCCTCGTGTT
TGCCTCCGCG
GTGTGTTTGT C TAGTGGTGTT 'I
GCATATTGAA
TGAGCGCAAC
TATGCTTCCC
CAGCTATGAC
GCGGCGTCTG
CCGCCTCGCG
GCTCGTCCCA
GACGCGGTCC
CG3CCATGGCC
CCCGACGCGT
CGGCGGCCGC
CGGCCACAGC
GCCGCGGTTG
CGCGCGCGAA
TGCTAGCGAT
AGACGAACAC
GCACAAGCCG
TGGGCGTGTT
CCCGGGCGTG
GCAGGTTTAC
CAAACTTGC
GTGCGTCGCA
GCGCACGTCG
GTATCTACCT
CCGCTTCCAC
CGATGTTTGG
-CTCGCACGG
GGCCTGGTTG
3TCCGCGCTG
IGACCGCCAC
W.ATCAACGC
CGAAATACG
~ATCGCCGAT
ATGTCGCCG
GCAATTAATG
GCTCGTATGT
CATGATTACG
CCTTCGCCAC
GCCAGCTCCC
GAAGCGCAGC
GCGACTGCGT
CCGATGCCGC
CTTTAGCGCA
TGTCCCCGGC
CGCCCTTTTG
CTCGGCTTTG
TGGGGCGTAC
CGTCTGGGCC
CTGCACCGGC
GGCCGAGTCG
CATGTTCGTT
CCCTGCCTCC
CTCAAAGCTC
CAAAAGCTGG
TGAGTTAGCT
TGTGTGGAAT
CCAAGCTAGC
CAGGTTGTCC
GCGCGGCGC
GGAAAGGCCG
GGATGTCCAC
CACAAAGAGC
CCCGCCGGCG
CTCAGCAGGG
CCCGTAGCCA
CGTTTGCCCC
TCGGCGAGCC
GGCAGGCGCG
GCTAGGACCA
CGCAGCTGCC
GAAAAACGGC
GCGACCCGGA
AGAGACACGC
GCGAGGCGCG
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040
AGCCAGCGCC
GTCGCGGTCG
GGACGGCC 2G
CGCTGGGGAG
TACGGACGCC
GAGCGCACGC
CGTACTACCA
CTGCGCCGCC
CCCGTGGCGC
GGAAGATCCG
CGTTTGAGAT
1AGAGATGGCG kTGTGAGTTT
TCGTAAAAGC
CGCGCCATGG
CACGGGCAGG
GGCGTGCTCT
TTAAGTGGGA
GCGCGGCGGG
GGCCAGGTTC
TGGGCCGGCG
GCGTGCCTCT
AATTCCTCGA
TTCTGTCCCG
ATATTGGAAA
CTGTGTAPACT
WO 95/12682 WO 9512682PCII/US94/1 2198 GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG ATATCGTTTA CGGGGGATGC CAAATATCGC AGTTTCGATI GACATCAAGC TGGCACATGC TTAGCCATAT TATTCATTGC ACGTTGTATC CATATCATAI TGTTGACATT GATTATTGAC AGCCCATATA TGGAGTTCCC CCCAACGACC CCCGCCCAT'I GGGACTTTCC ATTGACGTCP CATCAAGTGT ATCATATGCC GCCTGGCATT ATGCCCAGTA GTATTAGTCA TCGCTATTAC TAGCGGTTTG ACTCACGGGG TTTTGGCACC AAAATCAACG CAAATGGGCG GTAGGCGTGT CGTCAGATCG CCTGGAGACG CGATCCAGCC TCCGCGGCAA CCTGGCTTTC GCCC'IGCTCT CCTATGGCTA CTACTGATAA ATACGCCATA GCCAGGAATG TTGGAAGGAT TACTCACATG AGACGGTAAG TTTACATACC GCATTCAAGA GCCTTGCAGA AGGGGAAACA TTTGAAATGG TCCCGTAGTA AGAGGGAAGT ATG'CCCTATP. GGATGGACAG CACA(GCAGTA GTGCGTGTGT CACCCAAAGA ACTCTGGGGG TGTGACTGGG GACCAGCTAC TTATAAATTC CAAAAAGTG GAATGAGACT GGCTACAGAT AGTACCACAA GGATTGGTAA TGACACCAAA CTTGGGCCTA 3CGATAGACGC
~TAGGTGACAG
'CCAATGCATA
;TTATATAGCA
TATGTACATT
TAGTTATTAA
CGTTACATAA
GACGTCAATA
ATGGGTGGAG
AAGTACGCCC
CATGACCTTA
CATGGTGATG
ATTTCCAAGT
GGACTTTCCA
ACGGTGGGAG
CCATCCACGC
GCTTCGACAT
GCCTGCCCTG
CAGGGGTACA
ATAGAATTGG
AAATGAAGCT
TCTCAAGGTG
CCAGTGTGGT
CTGACGACTT
TCAATACAAC
GAACTGTGAG
ATAAGAGGTC
AGGATCTCTA
AATACACAGG
AGGGGTTGCC
TTGTAGACGG
AGTGTAAGAT I TGCCTTGCAA C
CTTTGGTGAC
ACGATATGAG
TCGATCTATA
TAAATCAATA
TATATTGGCT
TAG TAATCPA
CTTACGGTAA
ATGACGTATG
TATTTACGGT
CCTATTGACG
TGGGACTTTC
CGGTTTTGGC
CTCCACCCCA
AAATGTCGTA
GTCTATATAA
TGTTTTGACC
GATGGCTGCA
GACTCAGGTG
AGGGGACATT
CCCATTAGGA
GGAAGACACA
CACAAGAGAA
ATTCAAAAAA
TGAATTTGGA
PCTGCTAAAC
CTGTATGTTA
CAAACCATTC
TAACTGTGAT
%GGCCCTGTC
k.CACTACCCC
:ACCACTTGC
~GGAGACACA
,CCATATGAG
TTGGGCGAT'I
GCTATATCGC
CATTGAATCA
TTCGCTATTG
CATGTCCAAC
TTACGGGGTC
ATGGCCCC-CC
TTCCCATAGT
AAACTGCCCA
TCAATGACGG
CTACTTGGCA
AGTACATCAA
TTGACGTCAA
ACAACTCCGC
GCAGAGCTCG
TCCATAGAAG
GGCCCCCGGA
GTGGGCGCCT
GACTGCAAAC
GCTGAAGGCC
ATGGTCATAG
ACTAGATATC
CTTTTCGAGG
CTCTGCCCAT
GGACCGGCCT
GCTAATAGGG
CCTTATAGAC
CTTGGAGGGA
GAATCTTGCA
ATCGGCAAGT
AACAGAGAGG
ATCGTACAGG
ATCATACCAA
CGATACGCTT
CTGTGTGTCG
CGATAGAGGC
ATATTGGCCA
GCCATTGCAT
ATTACCGCCA
ATTAGTTCAT
TGGCTGACCG
AACGCCAATA
CTTGGCAGTA
TAAATGGCCC
GTACATCTAC
TGGGCGTGGA
TGGGAGTTTG
CCCATTGACG
TTTAGTGAAC
ACACCGGGAC
CCTCCCTGCT
TCCCAGGGAT
CTGAACACTC
TCACCACTGT
CTTGGTGCAA
TTGCAATTCT
GGCAAAGGCA
GCGATGCCAA
TCCAGATGGT
ACACCCTAGA
AAGGTTGTAT
ATTGGACTTG
AGTGGTGTGG
GTAGGTTGAA
GTGTAGCCAT
TCATAGCTCT
GTGAGG'3GCC 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 WO 95112682 WO 9512682PCIUS94I12 198 TGTAGAAAAG ACGGCATGCA CCTTCAACTA CACGAGGACA TTAAAAAATA AATATTTTGA GCCCAGAGAC AGTTACTTCC CCTGGAGGTC ACTGACCATC AGCTTTACTG GGTGGAAGA'I ACAAAAGGCC TTGGIGACCC AACACATGAC AGTATTGAAG GGAGGCTGTA AAGAAGTGGG ATCCAGGGTC GACCTCAGGC TCTGTTGTTT GCCCCTCCCC CTTTCCTAAT AAAATGAGGA GGGGGTGGGG TGGGGCAGGA GGGGATGCGG TGGGCTCTAT GCCAGAAAGA AGCAGGCACA TTAGTTCCAG CCCCACTCAT CCGCTAAAGT ACTTGGAGCG CTCCAAGAGT GGGAAGAAAT GCCTCCAACA TGTGAGGAAG TGCTGGACGT GTCCGCGGCG CCATGCAGGC CGCTTGCGCG ACTTCGCGCT AGAGATGGCG CCTGCGCTCG CTCGCTCGCT GCGGATCCCC TCCCGGCCCC CTCTGCGCGG CGCTATTGGC GCCCGCGGCG GGGGCGCCGA GTGGGAAATG GTGGTCGGAG GCCGCGGGGC ATTGAGCGCG CATAGCGGTC CACGTTGACG GCCGCGCGCG GCCGTCGACT Tr TTTACA ACGTCGTGAC CACATCCCCC TTTCGCCAGC AACAGTTGCG CAGCCTGAAT TGTGCGGTAT TTCACACCGC AGTTAAGCCA GCCCCGACAC TCCCGGCATC CGCTTACAGA
AGCAATACA'I
ATCGGGATTAi
ACGTGCTCTCG
AATATGGGGC
TGGTGACATA
TCTTACTCTT
ATGCAAGCTC
CGTGCCTTCC
AATTGCATCG
CAGCAAGGGG
GGGTACCCAG
TCCCCTTCTC
AGGACACTCA
GTCTCTCCCT
TAAAGCAAGA
TAATGATAGA
CCAGACGCGT
GACGGGGCGG
GGGGAGGCCA
GCATTTGCGC
GCCGCGAAGC
AGCGGCTGTC'
AGCCAGGGCA
CCGGGCCGGC
TGGCCCACAT
TTGCGCGCAC
CTAGAGGATC
TGGGAAAACC
TGGCGTAATA
GGCGAATGGC
ATATGGTGCA
CCGCCAACAC
CAAGCTGTGA
GCTAAAAGGI
CTTCGCCGAC
*GTTACTGGTI
*AGGGGAAGTC
TTTCTTGTTC
ATACCACCTU
AGATCCGCTC
TTGACCCTGC
CATTGTCTGA
GAGGATTGGG
GTGCTGAAGA
TGTGACACAC
TAGCTCAGGA
CCCTCATCAG
TAGGCTATTA
AATCATAGAA
GCGCGGCCGC
CGGGCGCGAC
CGGCGGGCCC
CCCGATCGCC
AGGCCGCCAG
CTCGCGCTCG
GCACAGAGAC
CGTGCACACG
TGCAAACCTC
CTCTGGCCTG
CCCGGGTACC
CTGGCGTTAC
GCGAAGAGGC
GCCTGATGCG
CTCTCAGTAC
CCGCTGACGC
CCGTCTCCGG
k. GATTATCPA
TCCATATTGC.
ACATACATGC
GTGATGATGC
TTATACCTAC
GATTGATTGA
TGCCTTCTAG
AAGGTGCCAC
GTAGGTGTCA
AAGACAATAG
ATTGACCCGG
CCTGTCCACG
GGGCTCCGCT
CCCACCAAAC
AGTGCAGAGG
TTGAGATCTC
CGTACTC-GAC
GCTGGCGACC
TAGGGGACTA
TTACGGGGAC
ACAAAAAAAT
CCGCGGGCGC
GCCCGATACG
TTCACCATCC
AGCCGGCTGC
CGGGACGCCA
GAGCTCGAAT
CCAACTTAAT
CCGCACCGAT
GTATTTTCTC
AATCTGCTCT
GCCCTGACGG
GAGCTGCATG
ACTGGTTCGA
TGGTGGTGGT
TCCTATCAGA
GTAACTTGCT
TGCTAAGAGA
GGATCAGCTT
TTGCCAGCCA
TCCCACTGTC
TTCTATTCTG
CAGGCATGCT
TTCCTCCTGG
CCCCTGGTTC
TCAATCCCAC
CAAACCTAGC
GAGAGAAAAT
GAGGTGTTCG
ATGCGGCCCG
CTGGCGCGTC
TAAAGCTGCC
TCGGCGCTCG
GCGGCGCCCG
CCCCGCCGCC
AAATCGAAGA
GCTGCCTCGG
TGGACGGGTA
TGTTTTTCCT
TCACTGGCCG
CGCCTTGCAG
CGCCCTI :CC
CTTACGCATC
GATGCCGCAT
GCTTGTCTGC
TGTCAGAGGT
4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 -37- WO 95112682 IIC'IYUS94/12198 TTTCACCGTC ATCACCGAAA AGGTTAATGT CATGATAATA TGCGCGGAAC CCCTATTTGT GACAATAACC CTGATAAATG ATTTC.CGTGT CGCCCTTATT CAGAAACGCT GGTGAAAGTA TCGAACTGGA TCTCAACAGC CAATGATGAG CACTTTTAAA GGCAAGAGCA ACTCGGTCGC CAGTCACAGA AAAGCATCTT TAACCATGAG TGATAACACT AGCTAACCGC TTTTTTGCAC CGGAGCTGAA TGAAGCCATA CAACAACGTT GCGCAAACTA TAATAGACTG GATGGAGGCG CTGGCTGGTT TATTGCTGAT CAGCACTGGG GCCAGATGGT AGGCAACTAT GGATGAACGA ATTGGTAACT GTCAGACCAA TTTAJATTTAA AAGGATCTAG AACGTGAGTT TTCGTTCCAC GAGATCCTTT TTTTCTGCGC CGGTGGTTTG TTTGCCGGAT GCAGAGCGCA GATACCAAAT AGAACTCTGT AGCACCGCCT CCAGTGGCGA TAAGTCGTGT CGCAGCGGTC GGGCTGAACG ACACCGAACT GAGATACCTA GAAAGGCGGA CAGGTATCCG TTCCAGGGGG AAACGCCTGG AGCGTCGATT TTTGTGATGC CGGCCTTTTT ACGGTTCCTG TATCCCCTGA TTCTGTGGAT GCAGCCGAAC GACCGAGCGC I
CGCGCGAGAC
ATGGTTTCTT
TTATTTTTCT
CTTCAATAAT
CCCTTTTTTG
AAAGATGCTG
GGTAAGATCC
GTTCTGCTAT
CGCATACACT
ACGGATGGCA
GCGGCCAACT
AACATGGGGG
CCAALACGACG
TTAACTGGCG
GATAAAGTTG
AAATCTGGAG
AAGCCCTCCC
ANAGACAGA
GTTTACTCAT
GTGAAGATCC
TGAGCGTCAG
GTAATCTGCT
CAAGAGCTAC
PCTGTCCTTC
ACATACCTCG
CTTACCGGGT
GGGGGTTCGT
CAGCGTGAGC
GTA.AGCGGCA ['ATCTTTATA C LCGTCAGGGG C 3CCTTTTGCT C ACCGTATTA C ~GCGAGTCAG T
GAAAGGGCCT
AGACGTCAGG
AAATACATTC
ATTGAAAAAG
CGGCATTTTG
AAGATCAGTT
TTGAGAGTTT
GTGGCGCGGT
ATTCTCAGAA
TGACAGTAAG
TACTTCTGAC
ATCATGTAAC
AGCGTGACAC
AACTACTTAC
CAGGACCACT
CCGGTGAGCG
GTATCGTAGT
TCGCTGAGAT
P.TATACTTTA
TTTTTGATAA
PCCCCGTAGA
GCTTGCAAAC
AACTCTTTT
LAGTGTAGCC4 TCTGCTAAT4
LGGACTCAAG
,CACACAGCC
L'ATGAGAAAG
~GGTCGGAAC
,TCCTGTCGG
,GCGGAGCCT2
,GCCTTTTGC
.CGCCTTTGAC
'GAGCGAGGA I
CGTGATACGC
TGGCACTTTT
AAATATGTAT
GAAGAGTATG
CCTTCCTGTT
GGGTGCACGA
TCGCCCCGAA
ATTATCCCGT
TGACTTGGTT
AGAATTATGC
AACGATCGGA
TCGCCTTGAT
CACGATGCCT
TCTAGCTTCC
TCTGCGCTCG
TLGGGTCTCGC
TATCTACACG
AGGTGCCTCA
GATTGATTTA
TCTCATGACC
AAAGATCAAA
AAAAAA\CCA
I'CCGAAGGTA
GTAGTTAGGC
CCTGTTACCA
PLCGATAGTTA
CAGCTTGGAG
CGCCACGCTT
%.GGAGAGCGC
'TTTCGCCAC C kTGGAAAAAC C CCACATGTTC 9i ;TGAGCTGAT PZ
.GCGGAAGA
CTATTTTTAT
CGGGGAAATG
CCGCTCATGA
AGTATTCAAC
TTTGCTCACC
GTGGGTTACA
GAACGTTTTC
ATTGACGCCG
GAGTACTCAC
AGTGCTGCCA
GGACCGAAGG
CGTTGGGAAC
GTAGCALATGG
CGGCAACAAT
GCCCTTCCGG
GGTATCATTG
ACGGGGAGTC
CTGATTAAGC
AAACTTCATT
AAAATCCCTT
GGATCTTCTT
CCGCTACCAG
ACTGGCTTCA
CACCACTTCA
GTGGCTGCTG
XCGGATAAGG
GAACGACCT
'CCGAAGGGA
kCGAGGGAGC
~TCTGACTTG
CCAGCAACG
~TTCCTGCGT
~CCGCTCGCC
6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8149 -38- WO 9512682 W 9 8CITUS94/ 12198 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8135 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bovine viral diarrhea virus STRAIN: 2724 INDIVIDUAL ISOLATE: pBHVtkex-3::gIII/p53 (xi) SEQUENCE DESCRIPTION: SEQ ID GCGCCCAATA CGCAAACCGC CGACAGGTTT CCCGACTGGA CACTCATTAG GCACCCCAGG TGTGAGCGGA TAACAATTTC TTGCATGCCT GCAGGTCGAC GCGGCCGCTG CCGGCCTGGT GTCCGCGTCC CCAACTCCGC GCGTATAAAA TTTCGCTCGT GCCCAGGCAA GCAAACTCTA GCCGAAATTT CGCCCAGGCA CTGTTGCCCG CGTGCCTGCT CCGGGGTCGC CGGCGGGCGG GGGGAAGCGG CTGCCCCTTC GCGGCGATCG CCCCGCTCGC CGGCTATTAT AGCCTCAAGG TCACTCTGAG CACGCGCATG CCGGGTCTGG GCCCGGGGGG GCAGCCCCCC GAGGCGCTGG ACGTCTTCAG CTCCACGATA GTAGGCACAC GCAATCGGGC CCACGACCTG CTTAAAAACC
CTCTCCCCGC
AAGCGGGCAG
CTTTACACTT
ACACAGGAAA
TTCCGCGCCC
TCCGCGCCCG
GCGAAGACGG
CCGGTACAAA
AACGCCCGAG
CGCCGCGCCG
GGCCGCCCAC
CCGCGGGGTG
TGCCGCCGCG
CGCGAACGCG
CGCGCCGCGT
CCCCGCTGGG
GCGAGATCGC
TCCATCTTGC
AGACAGACGG
CGCCGGCTTT
TCCGGGGCGC
GCGTTGGCCG
TGAGCGCAAC
TATGCTTCCG
CAGCTATGAC
GCGGCGTCTG
CCGCCTCGCG
GCTCGTCCCA
GACGCGGTCC
CGCCATGGCC
CCCGACGCGT
CGGCGGCCGC
CGGCCACAGC
GCCGCGGTTG
CGCGCGCGAA
TGCTAGCGAT
AGACGAACAC
GCACAAGCCG
TGGGCGTGTT
CCCGGGCGTG
GCAGGTTTAC
CAAACTTGCC
ATTCATTAAT
GCAATTAATG
GCTCGTATGT
CATGATTACG
CCTTCGCCAG
GCCAGCTCCC
GAAGCGCAGC
GCGACTGCGT
CCGATGCCGC
CTTTAGCGCA
TGTCCCCGGC
CGCCCTTTTG
CTCGGCTTTG
TGGGGCGTAC
CGTCTGGGCC
CTGCACCGGC
GGCCGAGTCG
CATGTTCGTT
CCCTGCCTCC
CTCAAAGCTC
CAAAAGCTGG
GCAGCTGGCA
TGAGTTAGCT
TGTGTGGAAT
CCAAGCTAGC
CAGGTTGTCC
GCGCGGGCGC
GGAAAGGCCG
GGATGTCCAC
CACAAAGAGC
CCCGCCGGCG
CTCAGCAGGG
CCCGTAGCCA
CGTTTGCCCC
TCGGCGAGCC
GGCAGGCGCG
GCTAGGACCA
CGCAGCTGCC
GAAAAACGGC
GCGACCCGGA
AGAGACACGC
GCGAGGCGCG
TCGTAAAAGC
'CCATGG
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GTGCGTCGCA AGCCAGCGCC GCGCACGTCG GTCGCGGTCG I WO 95/12682 W095/12682PC'rUS94/I 2198 CCGAGCCCGC GCGCGCTCTC GAAAGACAAC AACGGGCCGC TTTTCCCGGA GCCGATGGCG TCCTCGCGGC GTCTGCGCGA CCGGCGCACC GCGCAGACGC GCGGCCCCGT ACTTAATTTT CCGGGCGGCG AGCTGGTGGA GCTACCCCTT CGCCCGCTAC CCTGCAGTGA ATAATAAAAT ACTAAATTCA TGTCGCGCGA AATCGATATT TGAAAATATG GATATCGCCA TTTTTCCAAA ATATCGTTTA CGGGGGATGG CAAATATCGC AGTTTCGATA GACATCAAGC TGGCACATGG TTAGCCATAT TATTCATTGG ACGTTGTATC CATATCATAA TGTTGACATT GATTATTGAC AGCCCATATA TGGAGTTCCG CCCAACGACC CCCGCCCATT GGGACTTTCC ATTGACGTCA CATCAAGTGT ATCATATGCC GCCTGGCATT ATGCCCAGTA GTATTAGTCA TCGCTATTAC TAGCGGTTTG ACTCACGGGG TTTTGGCACC AAAATCAAG CAAATGGGCG GTAGGCGTGT CGTCAGATCG CCTGGAGACG CGATCCAGCC TCCGCGGCAA TGCTGGCGCT CTACGCGGCG TGATAACAGG GGTACAAGGG GGAATGATAG AATTGGCCCA CACATGAAAT GAAGCTGGAA CATACCTCTC AAGGTGCACA
CGCGTCGTGC
GCGCTCGCGG
TACTGGCGCA
TGCGCCGCAG
GGACGCGGCG
GCACGCGCGT
CCCTCGTGTT
TGCCTCCGCG
GTGTGTTTGT
TAGTGGTGTT
GCATATTGAA
AGTTGATTTT
CGATAGACGC
TAGGTGACAG
CCAATGCATA
TTATATAGCA
TATGTACATT
TAGTTATTAA
CGTTACATAA
GACGTCAATA
ATGGGTGGAG
AAGTACGCCC
CATGACCTTA
ClkTGGTGATG
ATTTCCAAGT
GGACTTTCCA
ACGGTGGGAG
CCATCCACGC
GCTTCGACCA
GCCATCGCCG
GACATTGACT
TTAGGAGCTG
GACACAATGG
AGAGAAACTA
GTATCTACCT
CCGCTTCCAC
CGATGTTTGG
CCTCGCACGG
GGCCTGGTTG
GTCCGCGCTG
CGACCGCCAC
AGATCAACGC
CCGAAATACG
TATCGCCGAT
AATGTCGCCG
TGGGCATACG
CTTTGGTGAC
ACGATATGAG
TCGATCTATA
TAAATCAATA
TATATTGGCT
TAG TAATCAA
CTTACGGTAA
ATGACGTATG
TATTTACGGT
CCTATTGACG
TGGGACTTTC
CGGTTTTGGC
CTCCACCCCA
AAATGTCGTA
GTCTATATAA
TGTTTTGACC
TGGCCTCGCT
CGGCGCCGTC
.CAAACCTGA2 kAGGCCTCAC
VCATAGCTTG
3ATATCTTGC
GGACGGCGCG
CGCTGGGGAG
TACGGACGCC
GAGCGCACGC
CGTACTACCA
CTGCGCCGCC
CCCGTGGCGC
GGAAGATCCG
CGTTTGAGAT
AGAGATGGCG
ATGTGAGTTT
CGATATCTGG
TTGGGCGATT
GCTATATCGC
CATTGAATCA
TTGGCTATTG
CATGTCCAAC
TTACGGGGTC
ATGGCCCGCC
TTCCCATAGT
AAACTGCCCA
TCAATGACGG
CTACTTGGCA
AGTACATCAA
ITGACGTCAA
ACAACTCCGC
GCAGAGCTCG
TCCATAGAAG2 CGCGCGTGCG2
'GGGATCCTA
kCACTCATAC
'ACTGTTTGG
3TGCAAAGAC C ATTCTGCAT ~I
CACGGGCAGG
GGCGTGCTCT
TTAAGTGGGA
GCGCGGCGGG
GGCCAGGTTC
TGGGCCGGCG
GCGTGCCTCT
AATTCCTCGA
TTCTGTCCCG
ATATTGGAAA
CTGTGTAACT
CGATACGCTT
CTGTGTGTCG
CC-ATAGAGGC
ATATTGGCCA
GCCATTGCAT
ATTACCGCCA
ATTAGTTCAT
TGGCTGACCG
A.ACGCCAATA
CTTGGCAGTA
TAAATGGCCC
GTACATCTAC
TGGGCGTGGA
TGGGAGTTTG
CCCATTGACG
TTTAGTGAAC
%CACCGGGAC
kTGCTCGCTC
UGGCTACTAC
;CCATAGCCA
AGGATTACT
GTAAGTTTA
'CAAGAGCCT
1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 WO 95/12682 WO 9512682I'CT/S94/12 198 TGCAGACCAG TGTGGTATTC AAATGGCI'GA CGACTTTGAP GGAAGTTCAA TACAACACTC GGACAGGAAC TGTGAGCTGI GTGTGTATAA GAGGTCCAAA TGGGGGAGGA TCTCTATAAC AGCTACAATA CACAGGAGGC AAAGTGAGGG GTTGCCACAC ACAGATTTGT AGACGGCACC TGGTAAAGTG TAAGATAGGA GGCCTATGCC TTGCAAGCCA CATGCACCTT CAACTACACG ACTTCCAGCA ATACATGCTA ACCATCATCG GGATTACTTC GAAGATACGT GCTCTGGTTA GGACCCAATA TGGGGCAGGG TTGAAGTGGT GACATATTTC AGTGGGTCTT ACTCTTATAC TCAGGCATGC AAGCTCAGAT CTCCCCCGTG CCTTCCTTGA TGAGGAAATT GCATCGCATT GCAGGACAGC AAGGGGGAGG CTCTATGGGT ACCCAGGTGC GGCACATCCC CTTCTCTGTG ACTCATAGGA CACTCATAGC GGAGCGGTCT CTCCCTCCCT AGAAATTAAA GCIAGATAGG AGGAAGTAAT GATAGAAATC GCGGCGCCAG ACGCGTGCGC TGCGCGGACG GGGCGGCGGG ATGGCGGGGG AGGCCACGGC CTCGCTGCAT TTGCGCCCCG GGCCCCC-CCG CGAAGCAGGC ATTGGCAGCG GCTGTCCTCG
-AAAAAACTTT
TTTGGACTCT
CTAAACGGAC
*ATGTTAGCTA
*CCATTCCCTT
TGTGATCTTG
CCTGTCGAAT
TACCCCATCG
ACTTGCAACA
GACACAATCG
TATGAGATCA
AGGACATTAA
AAAGGAGATT
GCCGAGTCCA
CTGGTAACAT
GAAGTGGTGA
TTGTTGTTAT
CACCTTGATT
CCGCTGTGCC
CCCTGGAAGG
GTCTGAGTAG
ATTGGGAAGA
TGAAGAATTG
ACACACCCTG
TCAGGAGGGC
CATCAGCCCA
CTATTAAGTG
ATAGAATTGA
GGCCGCCGTA
CGCGACGCTG
GGGCCCTAGG
ATCGCCTTAC
CGCCAGACAA
CGCTCGCCGC
TCGAGGGGC7
GCCCATGCGI
CGGCCTTCCI
ATAGGGACAC
ATAGACAAGC
GAGGGAATTC
CTTGCAAGTC
GCAAGTGTAG
GAGAG GGTGT
TACAGGTCAT
TACCAAGTGA
AAAATAAATA
ATCAATACTG
TATTGGTGGT
ACATGGTCCT
TGt.TGGGTAA
ACCTACTGCT
GATTGAGGAT
TTCTAGTTGC
TGCCACTCCC
GTGTCATTCT
CAATAGCAGG
ACCCGGTTCC
TCCACGCCCC
TCCGCTTCAA
CCAAACCAAA
CAGAGGGAGA
GATCTCGAGG
CTGGACATGC
GCGACCCTGG
GGACTATAAA
GGGGACTCGG
AAAAATGCGG
GGGCGCCCCC
AAGGCAAGGC
TGCCAATCCC
GATGGTATGC
CCTAGACACA
TTGTATCACC
GACTTGTGTG
GTGTGGTTAT
GTTGAAGAAT
AGCCATAGTA
AGCTC :TGAc
GGGGCCTGTA
TTTTGAGCCC
GTTCGACCTG
GGTGGTAGCT
ATCAGAACAA
CTTGCTAACA
AAGAGAGGAG
CAGCTTATCC
CA ICCATCTG
ACTGTCCTTT
ATTCTGGGGG
CATGCTGGGG
TCCTGGGCCA
TGGTTCTTAG
TCC'CACCCGC
CCTAGCCTC
GAAAATGCCT
TGTTCGTGCT
GGCCCGCCAT
CGCGTCAGTT
GCTGCCCCTG
CGCTCGGCGG
CGCCCGCTCT
GCCGCCGCCC
GAAACATTTG
GTAGTAAGAG
CCTATAGGAT
GCAGTAGTGC
CAAAGAACTC
ACTGGGGACC
AAATTCCAAA
GAGACTGGCT
CCACAAGGAT
ACCAAACTTG
GAAAAGACGG
AGAGACAGTT
GAGGTCACTG
TTACTGGGTG
AAGGCCTTGG
CATGACAGTA
GCTGTAAAGA
AGGGTCGACC
TTGTTTGCCC
CCTAATAAAA
GTGGGGTGGG
ATGCGGTGGG
GAAAGAAGCA
TTCCAGCCCC
TAAAGTACTT
AAGAGTGGGA
CCAACATGTG
GGACGTGTCC
GCAGGCCGCT
CGCGCTAGAG
CGCTCGCTCG
ATCCCCTCCC
GCGCGGCGCT
GCGGCGGGGG
3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 -41- WO 95/12682 WO 95/12682 '/US94/1 2198 CGCCGAA.GCC AGGGCAGCAC TCGGAGCCGG GCCGGCCGTC AGCGCGTGGC CCACATTGCT TTGACGTTGC GCGCACCTC I TCGACTCTAG AGGATCCCC%' CGTGACTGGG AAAACCCTGC GCCAGCTGGC GTAATAGCGP CTGAATGGCG AATGGCGCCT CACCGCATAT GGTGCACTCT CGACACCCGC CAACACCCGC TACAGACAAG CTGTGACCGT CCGAAACGCG CGAGACGAAA ATAATAATGG TTTCTTAGAC ATTTGTTTAT TTTTCTAAAT TAAATGCTTC AATAATATTG CTTATTCCCT TTTTTGCGGC AAAGTAAAAG ATGCTGAAGA AACAGCGGTA AGATCCTTGA TTTAAAGTTC TGCTATGTGG GGTCGCCGCA TACACTATTC CATCTTACGG ATGGCATGAC AACACTGCGG CCAACTTACT TTGCACAACA TGGGGGATCA GCCATACCAA ACGACGAGCG AAACTATTAA CTGGCGAACT GAGGCGGATA AAGTTGCAGG GCTGATAAAT CTGGAGCCGG GATGGTAAGC CCTCCCGTAT GAACGAAATA GACAGATCGC GACCAAGTTT ACTCATATAT ATCTAGGTGA AGATCCTTTT TTCCACTGAG CGTCAGACCC CTGCGCGTAA TCTGCTGCTT CCGGATCAAG AGCTACCAAC
:AGAGACGCCC
;CACACGTTCP
AACCTCAGCC
GGCCTGCGGG
GGTACCGAGC
CGTTACCCAA
AGAGGCCCGC
G.ATGCGGTAT
CAGTACAATC
TGACGCGCCC
CTCCGGGAGC
GGGCCTCGTG
GTCAGGTGGC
ACATTCAAAT
AAAAAGGAAG
ATTTTGCCTT
TCAGTTGGGT
GAGTTTTCGC
CGCGGTATTA
TCAGAATGAC
AGTAAGAGAA
TCTGACAACG
TGTAACTCGC
TGACACCACG
ACTTACTCTA
ACCACTTCTG
TGAGCGTGGG
CGTAGTTATC
TGAGATAGGT
ACTTTAGATT
TGATAATCTC
CGTAGAAAAG
GCAAACAAAA
TCTTTTTCCG
*GATACGAAAT
LCCATCCGCTG
*GGCTGCTGGA
ACGCCATGTT
TCGAATTCAC
CTTAATCGCC
ACCGATCGCC
TTTCTCCTTA
TGCTCTGATG
TGACGGGCTT
TGCATGTGTC
ATACGCCTAT
ACTTTTCGGG
ATGTATCCGC
AGTATGAGTA
CCTGTTTTTG
GCACGAGTGG
CCCGAAGAAC
TCCCGTATTG
TTGGTTGAGT
TTATGCAGTG
ATCGGAGGAC
CTTGATCGTT
ATGCCTGTAG
GCTTCCCGGC
CGCTCGGCCC
TCTCGCGGTA
TACACGACGG
GCCTCACTGA
GATTTAAAAC9 ATGACCAAAA I ATCAAAGGAT C AAACCACCGC I1 AAGGTAACTG G
CGAAGAGTGG
CCTCGGGCCG
CGGGTACATA
TTTCCTGCCG
TGGCCGTCGT
TTGCAGCACA
CTTCCCAACA
CGCATCTGTG
CCGCATAGTT
GTCTGCTCCC
AGAGGTTTTC
TTTTATAGGT
GAAATGTGCG
TCATGAGACA
TTCAACATTT
CTCACCCAGA
GTTACATCGA
GTTTTCCAAT
ACGCCGGGCA
P.CTCACCAGT
CTGCCATAAC
CGAAGGAGCT
GGGAACCGGA
CAATGGCAAC
k.ACAATTAAT
L'TCCGGCTGGC
['CATTGCAGC I 3GAGTCAGGC UTAAGCATTG C ETCATTTTTA P~ 'CCCTTAACG T ~TTCTTGAGA T 'ACCAGCGGT G CTTCAGCAG A~ GAAAT.JGTGG 5520 CGGGGCATTG 5580 GCGGTCCACG 5640 CGCGCGGCCG 5700 TTTP.CAACGT 5760 TCCCCCTTTC 5820 GTTGCGCAGC 5880 CGGTATTTCA 5940 AAGCCAGCCC 6000 GGCATCCGCT 6060 ACCGTCATCA 6120 TAATGTCATG 6180 CGGAACCCCT 6240 ATAACCCTGA 6300 CCGTGTCGCC 6360 AACGCTGGTG 6420 1CTGGATCTC 6480 GATGAGCACT 6540 AGAGCAACTC 6600 CACAGAAAAG 6660 7ATGAGTGAT 6720 ACCGCTTTT 6780 3CTGAATGAA 6840 kACGTTGCGC 6900 kGACTGGATG 6960 TGGTTTATT 7020 ~CTGGGGCCA 7080 ACTATGGAT 7140 ;TAACTGTCA 7200 LTTTAAAAGG 7260 'GAGTTTTCG 7320 'CCTTTTTTT 7380 GTTTGTTTG 7411~0 .GCGCAGATA 7500 -42- WO 95/12682 WO 95/12682 IV'US94/112198 CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA
CCGCCTACAT
TCGTGTCTTA
TGAACGGGGG
TACCTACAGC
TATCCGGTAA
GCCTGGTATC
TGATGCTCGT
TTCCTGGCCT
GTGGATAACC
GAGCGCAGCG
ACCTCGCTCT
CCGGGTTGGA
GTTCGTGCAC
GTGAGCTATG
GCGGCAGGGT
TTTATAGTCC
CAGGGGGGCG
TTTGCTGGCC
GTATTACCGC
AGTCAGTGAG
GCThkATCCTG
CTCAAGACGA
ACAGCCCAC
AGAAAGCGCC
CGGAACAGGA
TGTCGGGTTT
GAGCCTATGG
TTTTGCTCAC
CTTTGAGTGA
CGAGGAAGCG
TTACCAGTGG
TAGTTACCGG
TTGGAGCGAA
ACGCTTCCCG
GAGCGCACGA
CGCCACCTCT
AAAAACGCCA
ATGTTCTTTC
GCTGATACCG
GAAGA
CTGCTGCCAG
ATAAGGCGCA
CGACCTACAC
AAGGGAGAAA
GGGAGCTTCC
GACTTGAGCG
GCAACXCCGGC
CTGCGTTATC
CTCGCCGCAG
TGGCGATAAG
GCGGTCGGGC
CGAACTGAGA
GGCGGACAGG
AGGGGGAAAC
TCGATTTTTG
CTTTTTACGG
CCCTGATTCT
CCGAACGACC
7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8135 -43-
Claims (12)
1. A recombinant attenuated replicating virus for preventing disease caused by Bovine Diarrhoea Virus (BVDV), wherein said virus is rendered non-pathogenic by the insertion of at least one of the BVDV glycoprotein genes gp 53, gp 48 or gp 25 into a pathogenesis related gene of a virus other than BVDV, and wherein said virus expresses at least one of said BVDV glycoprotein genes, said BVDV glycoprotein genes being preceded by a signal peptide gene sequence. .10 S.. 15 *e o o
2. A virus of claim 1, wherein said virus is selected from Bovine Herpes Virus type 1 (BHV-1), adenoviruses, bovine mammillitis virus, bovine papillomavirus or pseudorabies virus.
3. A virus of claim 1 or claim 2, wherein said virus is attenuated by insertion of at least one of said BVDV glycoprotein genes into the thymidine kinase (tk) gene or into a thymidine kinase (tk) gene having a deletion.
4. A virus of claim 3, wherein said virus is BHV-1.
5. A virus of claim 4, wherein said virus contains and expresses the BVDV gp 53 gene.
6. A virus of claim 1, wherein said signal peptide gene sequence is taken from Pseudorabies Virus gill gene and/or Bovine Growth Hormone gene.
7. A virus of claim 1, wherein said virus is selected from T11-3, T11-6 and T11-8.
8. A vaccine for preventing disease caused by BVDV, comprising a virus of any of claims 1 to 7 and a carrier.
9. A vaccine of claim 8, wherein the carrier comprises a physiological buffered medium, pH 7.0 to 7.4, containing 2.5 to 15 serum which does not contain F S T antibodies to BHV. Use of a virus of any of claims 1 to 7, for the manufacture of a medicament for use in immunisation against a disease caused by BVDV.
11. A method of immunising an animal against infectious disease caused by BVDV comprising administering to the animal a pharmaceutically-effective amount of a virus of any of claims 1 to 7.
12. A virus according to claim 1 substantially as hereinbefore described with reference to any of the figures and/or examples.
13. A method according to claim 11 substantially as hereinbefore described. DATED: 13 January, 1998 15 BAYER AKTIENGESELLSCHAFT By their Patent Attorneys PHILLIPS ORMONDE FITZPATRICK o° oo C WINWORDOJENNY\NODELETE 10423-95 DOC
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14781093A | 1993-11-05 | 1993-11-05 | |
| US147810 | 1993-11-05 | ||
| PCT/US1994/012198 WO1995012682A2 (en) | 1993-11-05 | 1994-10-31 | Viral vector with bovine viral diarrhea virus (bvdv) antigens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU1042395A AU1042395A (en) | 1995-05-23 |
| AU688819B2 true AU688819B2 (en) | 1998-03-19 |
Family
ID=22522990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU10423/95A Ceased AU688819B2 (en) | 1993-11-05 | 1994-10-31 | Viral vector with bovine viral diarrhea virus (BVDV) antigens |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0725831A1 (en) |
| JP (1) | JPH09504435A (en) |
| KR (1) | KR960705944A (en) |
| CN (1) | CN1134175A (en) |
| AU (1) | AU688819B2 (en) |
| CA (1) | CA2172815A1 (en) |
| MX (1) | MXPA94008605A (en) |
| NZ (1) | NZ276234A (en) |
| WO (1) | WO1995012682A2 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6060457A (en) * | 1996-06-20 | 2000-05-09 | Universite De Montreal | DNA plasmid vaccine for immunization of animals against BVDV |
| EP1026252A1 (en) * | 1999-02-02 | 2000-08-09 | Akzo Nobel N.V. | Synthetic gene of bovine viral diarrhoea virus |
| EP1104676A1 (en) * | 1999-11-30 | 2001-06-06 | Boehringer Ingelheim Vetmedica Gmbh | Safe attenuated bovine viral diarrhea viruses for use in pregnant cows |
| KR100331176B1 (en) * | 1999-12-15 | 2002-04-06 | 대한민국(관리청:특허청장, 승계청:국립수의과학검역원장) | A diagnostic method of bovine viral diarrhea using recombination protein as an antigen |
| EP2365082A1 (en) * | 2000-06-27 | 2011-09-14 | Pfizer Animal Health S.A. | BVDV virus-like particles |
| ATE533506T1 (en) | 2002-08-26 | 2011-12-15 | Pfizer Prod Inc | VACCINE AGAINST REPRODUCTIVE AND RESPIRATORY SYNDROME IN CATTLES |
| AU2004315509A1 (en) * | 2003-12-05 | 2005-08-18 | Becton, Dickinson And Company | Methods of enhancing immune response in the intradermal compartment and compounds useful in the methods |
| KR101876535B1 (en) * | 2012-06-14 | 2018-07-09 | 베트올 (주) | Antibody for detecting of bovine viral diarrhea virus(bvdv), bvdv antigen detecting method and test kit using thereof |
| EP2983703A1 (en) | 2013-03-15 | 2016-02-17 | Zoetis Services LLC | Cross-protection of bovines against b. trehalosi infection by a multi-valent vaccine |
| CN113913461A (en) * | 2021-11-15 | 2022-01-11 | 贵州大学 | Construction method of bovine viral diarrhea E0-E2 gene recombinant adenovirus vaccine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990001337A1 (en) * | 1988-08-03 | 1990-02-22 | Institute For Animal Health Limited | Vaccine |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1237668A (en) * | 1983-02-25 | 1988-06-07 | Novagene Inc. | Thymidine kinase-negative temperature resistant bovine herpes-virus-1 mutant as vaccine |
| FR2693472B1 (en) * | 1992-06-26 | 1994-12-23 | Rhone Merieux | Mutants of the infectious bovine rhinotracheitis virus, deleted in one of the genes of minor glycoproteins, vaccines prepared from these strains, production methods and methods of use. |
| SE9002060D0 (en) * | 1990-06-08 | 1990-06-08 | Statens Veterinaermedicinska A | A METHOD OF DETECTING AN INFECTION CAUSED BY A SPECIFIC TYPE OF VIRUSES, PRIMERS, TESTS AND A TEST KIT |
-
1994
- 1994-10-31 CN CN94193978A patent/CN1134175A/en active Pending
- 1994-10-31 AU AU10423/95A patent/AU688819B2/en not_active Ceased
- 1994-10-31 CA CA002172815A patent/CA2172815A1/en not_active Abandoned
- 1994-10-31 NZ NZ276234A patent/NZ276234A/en unknown
- 1994-10-31 EP EP95901037A patent/EP0725831A1/en not_active Withdrawn
- 1994-10-31 JP JP7513263A patent/JPH09504435A/en active Pending
- 1994-10-31 WO PCT/US1994/012198 patent/WO1995012682A2/en not_active Application Discontinuation
- 1994-10-31 KR KR1019960702343A patent/KR960705944A/en not_active Application Discontinuation
- 1994-11-07 MX MXPA94008605A patent/MXPA94008605A/en unknown
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990001337A1 (en) * | 1988-08-03 | 1990-02-22 | Institute For Animal Health Limited | Vaccine |
Non-Patent Citations (1)
| Title |
|---|
| VIROLOGY (1992) 190, 666-73 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1134175A (en) | 1996-10-23 |
| CA2172815A1 (en) | 1995-05-11 |
| KR960705944A (en) | 1996-11-08 |
| MXPA94008605A (en) | 2004-11-11 |
| WO1995012682A3 (en) | 1995-07-06 |
| AU1042395A (en) | 1995-05-23 |
| JPH09504435A (en) | 1997-05-06 |
| EP0725831A1 (en) | 1996-08-14 |
| NZ276234A (en) | 1998-01-26 |
| WO1995012682A2 (en) | 1995-05-11 |
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| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |