AU1042395A - Viral vector with bovine viral diarrhea virus (bvdv) antigens - Google Patents
Viral vector with bovine viral diarrhea virus (bvdv) antigensInfo
- Publication number
- AU1042395A AU1042395A AU10423/95A AU1042395A AU1042395A AU 1042395 A AU1042395 A AU 1042395A AU 10423/95 A AU10423/95 A AU 10423/95A AU 1042395 A AU1042395 A AU 1042395A AU 1042395 A AU1042395 A AU 1042395A
- Authority
- AU
- Australia
- Prior art keywords
- gene
- virus
- plasmid
- thymidine kinase
- combination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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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
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- 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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
VIRAL VECTOR WITH 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 Zyl, 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, M., and Kit, S., Thymidine Kinase Deletion Mutants of Bovine Herpesvirus-1, issued 27 October 1987. U.S. patent 4,824,667, Kit, M., and Kit, S. Thymidine Kinase Deletion Mutants of Bovine Herpesvirus-1, Vaccines Against Infectious Bovine Khinotracheitis Containing Same and Methods for the Production and Use of Same, issued 25 April, 1989. Collett, M.S., et al., Proteins Encoded by Bovine Viral Diarrhea Virus: The Genomic Organization of a Pestivirus, Virology, Vol. 165 pp. 200-208 (1988). Collett, M.S., et al, Molecular Cloning and Nucleotide Sequence of the Pestivirus Bovine Viral Diarrhea Virus, Virology, Vol. 165 pp. 191- 199 (1988). Background
Bovine viral diarrhea virus (BVDV) is 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 disease which is characterized by high morbidity and low mortality. The symptoms can include fever, depression, occulo-nasal discharges, diarrhea and occasionally oral ulcerations. Apart from these primary effects the virus also causes immunosuppression. Although primary BVDV infections are normally relatively mild, the virus may potentiate or enhance the pathogenicity of other co-infecting microorganisms.
In older or susceptible animals, BVDV causes similar symptoms to those described above for younger susceptible calves. In addition, in pregnant a*n*iτnalp 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
reabsorption, abortion, mummification, congenital defects, birth defects, 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 will not be many pregnant animals infected at the right time 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.
and Kaeberle M.L., Suppression of Neutrophil and Lymphocyte Function Induced by a Vaccinal Strain of Bovine Viral Diarrhea Virus With or Without the Administration of ACTH, American Journal of Veterinary Research, Vol. 44 pp. 2366- 2372 (1983). The failure of the vaccine to stop the immunosuppression response 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 l(BHV-l) 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, E.J., Glycoproteins of Bovine Viral Diarrhoea-
Mucosal Disease Virus in Infected Bovine Cells, Journal of General Virology, Vol. 68, pp. 1607-1616 (1987) and Magar, R., et al., Bovine Viral Diarrhea Virus Proteins: Heterogeneity of Cytopathogenic and Noncytopathogenic Strains and Evidence of 53K Glycoprotein Neutralization Epitope, Veterinary Microbiology, Vol. 16, pp. 303- 314. Cited references are incorporated herein by reference.
SUMMARY OF THE INVENTION. A replicating nonpathogenic virus, for preventing disease caused by Bovine Viral Diarrhea Virus (BVDV), where said replicating nonpathogenic virus comprises: a gene or gene combination taken from a BVDV virus, and said replicating nonpathogenic virus functionally expresses said gene or gene combination.
Embodiments of this invention include the following: A virus where said replicating nonpathogenic virus is attenuated, is selected from attenuated Bovine Herpes Virus type 1 (BHV-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
combination of the following genes: the genes that code for gp48, gp25, pl4 capsid protein, p20 N-terminal protease and pl25/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, D., 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::gHI/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, Tll-3, Tll-6, or Tll-8. A virus where the functionally expressing gene or gene combination, used to create the virus, comprises a recombined plasmid with intact viral DNA said plasmid comprising: a) a BHV-1 genomic DNA fragment containing the thymidine kinase (tk) gene and having a deletion to the thymidine kinase (tk) gene, b) a promoter/polyadenylation signal inserted in the thymidine kinase (tk) 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
Diarrhea Virus (BDVD) comprising a pharmaceutically effective amount of a virus described above and a carrier, said carrier comprising any physiological buffered medium, i.e. about pH 7.0 to 7.4 containing 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-l::BGH/p53 c. EXAMPLE 3: pBHVtkex-l::gIII/p53 d. EXAMPLE 4: pBHVtkex-3::BGH/p53 e. EXAMPLE 5:pBHVtkex-3::gIII/p53 Figure 4. Predicted transcript maps of the BHV-l/gp53 recombinant viruses. Figure 5. Northern blots showing transcription of gp53 messenger RNAs in the BHV-1 recombinants.
Figure 6. Immunoprecipitations showing expression of gp53 protein in the BHV-1 recombinants. DESCRIPTION OF THE PREFERRED EMBODIMENTS.
All of the terms used below will be readily understood by one skilled in the art. In many places the name of the manufacturer of equipment or reagents are provided in parenthesis after the equipment or reagent is named. Commonly used terms, reagents and buffers such as "plasmids," "Klenow Fragments," "religating 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. M., 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 respiratoiy disease. Thus, in common with BVDV, BHV also replicates at a mucosal
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, i.e., about pH 7.0 to 7.4, containing from about 2.5 to 15% serum which does not contain antibodies to BHV, i.e., 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 3.0% 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
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 IO5 to IO6 PFU/ml at -70°C to -90°C or in a lyophilized state at 2°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 IO4,5 to IO7 PFU/animal, preferably about IO4-5 to IO5 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
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 that produce substantial immune responses can make effective vaccines. Magar, R., et al., Bovine Viral Diarrhea Virus Proteins: Heterogeneity of
Cytopathogenic and Noncytopathogenic Strains and Evidence of 53K Glycoprotein Neutralization Epitope, Veterinary Microbiology, Vol. 16, pp. 303-314. 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 "Tll-3", embodied in Example 3 for vaccine trials. The former virus was deposited to the ATCC under the designation UC VR-58. The latter, "Tll-3" plasmid was also deposited. The virus we labeled "Tll-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
genes many other viruses 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 non¬ pathogenic 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 non¬ pathogenic viruses might arise from pathogenic parent viruses by natural mutation, might be mutagenized by, for instance, chemicals or light to produce a non¬ pathogenic 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 (tk)(M. 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
Hindlll-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 Ada, 1090:267-9 (1991).
A 424bp deletion was introduced into the tk gene by digesting pHAS4 with Bglll and Xhol, filling in the ends with the Klenow Fragment of DNA polymerase I (Klenow) and religating the resulting blunt ended fragments. This manipulation restored the Bglll recognition site, but not the Xhol site (Fig. 1). The resulting plasmid was named pHAS4ΔBX. This deletion was chosen because it does not impede on the previously identified transcription initiation sites for the UL24 homolog which overlaps the 5' end of the tk gene. L. J. Bello, et al., Virology,
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 Hindlll site in the pUC18 vector by digesting pHAS4ΔBX with Hindlll, 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 Bglll sites and contains, between the promoter and the polyadenylation signal, unique Hindlll 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 Bglll and the released cassette was ligated into the Bglll site of pHAS4ΔBX (Fig. 1). The ligations were transformed into E. coli strain DH5α. 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 pHAS4ΔBXex-l and pHAS4ΔBXex-3 (Fig. 1), 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 Hindm-A fragment. The BglH/XhoI subfragment to be deleted is shown. The deletion derivative of pHAS4 is pHAS4ΔBX. 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 pHAS4ΔBX. 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, pHAS4ΔBXexl and pHAS4ΔBXex3 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
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 the initiation codon used by the cDNA, using a synthetic oligonucleotide and the "Double Take" site directed mutagenesis kit (Stratagene, La Jolla CA) according to the manufacturer's instructions. This base change introduced a unique BamHI site into the gene without altering the gp53 amino acid sequence (Fig. 2 section B). The base change was verified by nucleotide sequencing, and the resulting plasmid was called pP53mut. We inserted, into pP53mut sequences, encoding signal peptides from the PRV gill gene (A 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 A). These signal peptide cassettes were ligated into pP53mut digested with BamHI and Sail, and transformed into DH5α. 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, D., et al., J. Mol . Biol. Vol. 167 pp. 391-409 (1983). Incorporated by reference. These and any other well characterized signal peptides should be suitable for use as embodiments of this invention.
Figure 2. Strategy for appending signal peptide sequences to the BVDV gp53 gene. Section A: Synthetic oligonucleotides corresponding to the signal
peptide sequences of Bovine Growth Hormone (BGH), and Pseudorabies virus gill (PRV gill). 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 (A), three amino acids from the ends. Codons for two amino acid residues (F,P in BGH; P,S in gill) 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 gp25 (Collett, M.S., et al., Virology 165:200-208 (1988)), the pl4 capsid protein (Thiel, H.J., et al., J. Virol. 65:4705-4712 (1991)), and the p20 N-teπninal protease. Wiskerchen, M., et al., J. Virol. 65:4508-4514 (1991). This group of proteins, along with the gp53 gene, can be expressed together from a single cDNA molecule, the expressed polyprotein will process itself correctly into the separate proteins. Another BVDV protein candidate to express in a vaccine is the nonstructural pl25/p80 protein (Deregt, D., et 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 p53 gene, either with or without added signal peptide sequences, was ligated into the Hindlll insertion sites of pHAS4ΔBXex-l and pHAS4ΔBXex-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 DH5α. 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
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, which 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 pHAS4ΔBXex-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 pHAS4ΔBXex-
1. See Fig. 3B. This plasmid was used to create the virus Tll-6. This virus was deposited. EXAMPLE 3. pBHVtkex-l::gIII/p53: contains the mutagenized gp53 gene preceded by the PRV gill signal peptide sequence inserted into pHAS4ΔBXex-l. See Fig. 3C. This plasmid was used the create the virus
Tll-3. This plasmid was deposited. EXAMPLE 4. pBHVtkex-3::BGH/p53: contains the mutagenized gp53 gene preceded by the BGH signal peptide sequence inserted into pHAS4ΔBXex- 3. See Fig. 3D. EXAMPLE 5. pBHVtkex-3::gIH/p53: contains the mutagenized gp53 gene preceded by the PRV gill signal peptide sequence inserted into pHAS4ΔBXex-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, pUClδ, 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.
a. EXAMPLE 1. PBHVtkex-3::p53. b. EXAMPLE 2. pBHVtkex-l::BGH/p53 c. EXAMPLE 3. pBHVtkex-l::gIII/p53 d. EXAMPLE 4. pBHVtkex-3::BGH/p53 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 Xbal and cotransfected into Bovine Turbinate (BT) cells with unit length DNA from BHV- 1 strain Iowa (tk positive) by the standard CaPO4 method (R. L. Graham, et al., Virology, 52:456-467 (1973 )) as modified by Cai (W. 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 (S. K Mittal, et al., J Gen Virol, 70:(1989 )), or on Rab (BU) cells (S. Kit, et al., Virology, 130:381-389 (1983 )) in the presence of lOOug ml 5-Bromo-2'-Deoxyuridine (BDUR, Sigma Chemical Company, St. Louis, Missouri) to isolate virus no longer expressing tk. This is a standard 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.
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. Tll-3: BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-l::gIII/p53 recombined. Contains the BVDV gp53 gene with the PRV gill 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. Tll-8: BHV-1 "Iowa" into which the insert sequences contained in pBHVtkex-3::gIII/p53 recombined. Contains the BVDV gp53 gene with the PRV gill 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 the 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 pHAS4ΔBX 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 IowaΔBX. All of these viruses were plaque purified twice by limiting dilution on BT cells. 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. In order to ensure that the BHV-1 viruses carrying the BVDV sequences were not contaminated with non- cytopathic BVD virus, we prepared DNA from each of the viruses (including the
parent strain Iowa and IowaΔBX) 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-l/gp53 recombinant viruses later shown in Figure. 5. The two probes are 1) the gp53 cDNA and 2) the Sall/Bglll portion of pHAS4 (shown above the maps). The first map shows the predicted transcripts from viruses Tll-3 and Tll-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 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, Tll-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, IaΔBX 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 Tll-8 and T2-3-3, and to the 4.3kb tk message in Iowa, which is 3' coterminal with the gH transcript. L. J. Bello, et al., Virology, 189:407-414 (1992). To examine the transcription patterns upstream of the gp53 insertions, we
used a probe that consisted of the pHAS4 fragment from the upstream Sail site to the Bglll site in the tk gene, the beginning of the deletion in the recombinant viruses (probe 2). All of the viruses made a message of approximately 4.4kb which we deduced to be UL24 (Fig. 5, probe 2). This message, however, was smaller than the 5.2kb UL24 message in BHN-1 strain Cooper described by Bello, et al (L. J. Bello, et al., Virology, 189:407-414 (1992 )) and comigrated with the tk message in the wild-type strain Iowa. Although we did not evaluate these comigrating messages further by using single stranded probes, we detected a tk transcript of 4.2 kb only in the Iowa DΝA 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 5.0, 3.7, 1.8, and l.Okb.
Figure 5. Northern blots showing transcription of gp53 messenger RNAs in the BHV-1 recombinant viruses. The first panel shows transcripts hybridizing to probe 1, the pg53 cDNA, and the second panel shows transcripts hybridizing to probe 2, the Sall/Bgll subfragment of pHAS4. KEY: M=Mock infected cells, I=BHV- 1 "Iowa" infected cells, 3,6,8=Tll-3, Tll-6 and Tll-8 infected cells. RNA size standards, in kilobases (kB) are given to the left of each panel. Expression of BVDV gp53 protein in BHV-1. We evaluated expression of gp53 protein in the BHV-1 recombinants by immunoprecipitation (IP). Detailed procedures for IPs can be found in standard references such as "Current Protocols in Molecular Biology", Ausubel, F. M., et. al., eds., Wiley Interscience, New York. BT cells infected with the BHV-1 recombinants were metabolically labelled with S-methionine (Amersham, Arlington Heights, Illinois). The viral infected cells were lysed and soluble proteins were reacted with hyperimmune serum from bovine or goat against BVDV. VMRD, Pullman, 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 fluorography.
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.
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 bovine- anti-BVDV serum, this serum also had minor reactivity with BHV-1 antigens. KEY: 3,6,8=Tll-3, Tll-6, and Tll-8 infected cell proteins, IA=BHV-1 "Iowa" infected cell proteins, M=Mock infected cell proteins. MW=approximate protein molecular weight standards, in Kilodaltons.
The gp53 protein bands in Tll-3, Tll-6 and Tll-8 were broad, suggesting that the proteins were processed, and they appeared to be equivalent but not identical in size to the gp53 protein in NADL (data not shown). Removal of the N- linked sugars from the BVDV-NADL and BHV-1 expressed gp53 proteins by digestion with N-glycansase (Genzyme, Cambridge, Massachusetts) did not resolve the size difference in the proteins, but the proportional reduction in size of the 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 modifications are intended to fall within the scope of the appended claims.
It is also to be understood that all base pair and amino acid residue numbers and sizes given for nucleotides and peptides are approximate and used for the purposes of description.
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 American Type Culture Collection, 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 2." This deposit was received by the American Type Culture Collection depository on 28 October 1993. Several plasmids were deposited with the Agricultural Research Service
Culture Collection (NRRL), of the U.S. Department of Agriculture, at 1815 North University Street, Peoria, Illinois, zip code 61604, USA. One plasmid was given the Upjohn designation, pUC 1564, E. coli culture UC 15085, referring to pBHVtkex- l::gIII\p53, it corresponds to the plasmid used to create the virus described herein as "Tll-3," also known as "Example 3." This plasmid was given the following number by the depository, NRRL B-21350. Another deposit was given the Upjohn designation, pUC 1565, E. coli culture UC 15086, referring to pBHVtkex-3::gIII\p53, it corresponds to the plasmid used to create the virus described herein as, "T-ll-8," also known as "Example 5." This plasmid was given the following number by the depository, NRRL B-21351. Both of the plasmids were received by the Agricultural Research Service Culture Collection depository on 26 October 1994.
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION :
(i) 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:
(A) ADDRESSEE: Thomas A Wootton (1920-32-1), The Upjohn
Company
(B) STREET: 7000 Portage Road
(C) CITY: Kalamazoo (D) STATE: Michigan
(E) COUNTRY: U.S.A
(F) ZIP: 49001-0199
(V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Wootton, Thomas A.
(B) REGISTRATION NUMBER: 35,004
(C) REFERENCE/DOCKET NUMBER: 4748 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 616 385-7914
(B) TELEFAX: 616 385-6897
(C) TELEX: 224 401 UPJOHN
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8083 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkex-3: :p53
( i) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720 GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320 GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620 CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860
CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920 ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040
GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT 2100
ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160 CAAATATCGC AGTTTCGATA TAGGTGACAG ACGATATGAG GCTATATCGC CGATAGAGGC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460 AGCCCATAT TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760 GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC 3000
CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC 3060 CGATCCAGCC TCCGCGGCAA GCTGATCCGT CAGGGGCCAG ATGGTACAGG GCATCCTATG 3120
GCTACTACTG ATAACAGGGG TACAAGGGGA CATTGACTGC AAACCTGAAC ACTCATACGC 3180
CATAGCCAGG AATGATAGAA TTGGCCCATT AGGAGCTGAA GGCCTCACCA CTGTTTGGAA 3240
GGATTACTCA CATGAAATGA AGCTGGAAGA CACAATGGTC ATAGCTTGGT GCAAAGACGG 3300
TAAGTTTACA TACCTCTCAA GGTGCACAAG AGAAACTAGA TATCTTGCAA TTCTGCATTC 3360 AAGAGCCTTG CAGACCAGTG TGGTATTCAA AAAACTTTTC GAGGGGCAAA GGCAAGGGGA 3420
AACATTTGAA ATGGCTGACG ACTTTGAATT TGGACTCTGC CCATGCGATG CCAATCCCGT 3480
AGTAAGAGGG AAGTTCAATA CAACACTGCT AAACGGACCG GCCTTCCAGA TGGTATGCCC 3540
TATAGGATGG ACAGGAACTG TGAGCTGTAT GTTAGCTAAT AGGGACACCC TAGACACAGC 3600
AGTAGTGCGT GTGTATAAGA GGTCCAAACC ATTCCCTTAT AGACAAGGTT GTATCACCCA 3660 AAGAACTCTG GGGGAGGATC TCTATAACTG TGATCTTGGA GGGAATTGGA CTTGTGTGAC 3720
TGGGGACCAG CTACAATACA CAGGAGGCCC TGTCGAATCT TGCAAGTGGT GTGGTTATAA 3780
ATTCCAAAAA AGTGAGGGGT TGCCACACTA CCCCATCGGC AAGTGTAGGT TGAAGAATGA 3840
GACTGGCTAC AGATTTGTAG ACGGCACCAC TTGCAACAGA GAGGGTGTAG CCATAGTACC 3900
ACAAGGATTG GTAAAGTGTA AGATAGGAGA CACAATCGTA CAGGTCATAG CTCTTGACAC 3960 CAAACTTGGG CCTATGCCTT GCAAGCCATA TGAGATCATA CCAAGTGAGG GGCCTGTAGA 4020
AAAGACGGCA TGCACCTTCA ACTACACGAG GACATTAAAA AATAAATATT TTGAGCCCAG 4080
AGACAGTTAC TTCCAGCAAT ACATGCTAAA AGGAGATTAT CAATACTGGT TCGACCTGGA 4140
GGTCACTGAC CATCATCGGG ATTACTTCGC CGAGTCCATA TTGGTGGTGG TGGTAGCTTT 4200 ACTGGGTGGA AGATACGTGC TCTGGTTACT GGTAACATAC ATGGTCCTAT CAGAACAAAA 4260
GGCCTTGGGG ACCCAATATG GGGCAGGGGA AGTGGTGATG ATGGGTAACT TGCTAACACA 4320
TGACAGTATT GAAGTGGTGA CATATTTCTT GTTGTTATAC CTACTGCTAA GAGAGGAGGC 4380
TGTAAAGAAG TGGGTCTTAC TCTTATACCA CCTTGATTGA TTGAGGATCA GCTTATCCAG 4440
GGTCGACCTC AGGCATGCAA GCTCAGATCC GCTGTGCCTT CTAGTTGCCA GCCATCTGTT 4500 GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC 4560
TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT 4620
GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT 4680
GCGGTGGGCT CTATGGGTAC CCAGGTGCTG AAGAATTGAC CCGGTTCCTC CTGGGCCAGA 4740
AAGAAGCAGG CACATCCCCT TCTCTGTGAC ACACCCTGTC CACGCCCCTG GTTCTTAGTT 4800 CCAGCCCCAC TCATAGGACA CTCATAGCTC AGGAGGGCTC CGCTTCAATC CCACCCGCTA 4860
AAGTACTTGG AGCGGTCTCT CCCTCCCTCA TCAGCCCACC AAACCAAACC TAGCCTCCAA 4920
GAGTGGGAAG AAATTAAAGC AAGATAGGCT ATTAAGTGCA GAGGGAGAGA AAATGCCTCC 4980
AACATGTGAG GAAGTAATGA TAGAAATCAT AGAATTGAGA TCTCGAGGTG TTCGTGCTGG 5040
ACGTGTCCGC GGCGCCAGAC GCGTGCGCGG CCGCCGTACT GGACATGCGG CCCGCCATGC 5100 AGGCCGCTTG CGCGGACGGG GCGGCGGGCG CGACGCTGGC GACCCTGGCG CGTCAGTTCG 5160
CGCTAGAGAT GGCGGGGGAG GCCACGGCGG GCCCTAGGGG ACTATAAAGC TGCCCCTGCG 5220
CTCGCTCGCT CGCTGCATTT GCGCCCCGAT CGCCTTACGG GGACTCGGCG CTCGGCGGAT 5280
CCCCTCCCGG CCCCGCCGCG AAGCAGGCCG CCAGACAAAA AAATGCGGCG CCCGCTCTGC 5340
GCGGCGCTAT TGGCAGCGGC TGTCCTCGCG CTCGCCGCGG GCGCCCCCGC CGCCGCCCGC 5400 GGCGGGGGCG CCGAAGCCAG GGCAGCACAG AGACGCCCGA TACGAAATCG AAGAGTGGGA 5460
AATGGTGGTC GGAGCCGGGC CGGCCGTGCA CACGTTCACC ATCCGCTGCC TCGGGCCGCG 5520
GGGCATTGAG CGCGTGGCCC ACATTGCAAA CCTCAGCCGG CTGCTGGACG GGTACATAGC 5580
GGTCCACGTT GACGTTGCGC GCACCTCTGG CCTGCGGGAC GCCATGTTTT TCCTGCCGCG 5640
CGCGGCCGTC GACTCTAGAG GATCCCCGGG TACCGAGCTC GAATTCACTG GCCGTCGTTT 5700 TACAACGTCG TGACTGGGAA AACCCTGGCG TTACCCAACT TAATCGCCTT GCAGCACATC 5760
CCCCTTTCGC CAGCTGGCGT AATAGCGAAG AGGCCCGCAC CGATCGCCCT TCCCAACAGT 5820
TGCGCAGCCT GAATGGCGAA TGGCGCCTGA TGCGGTATTT TCTCCTTACG CATCTGTGCG 5880
GTATTTCACA CCGCATATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA 5940
GCCAGCCCCG ACACCCGCCA ACACCCGCTG ACGCGCCCTG ACGGGCTTGT CTGCTCCCGG 6000 CATCCGCTTA CAGACAAGCT GTGACCGTCT CCGGGAGCTG CATGTGTCAG AGGTTTTCAC 6060
CGTCATCACC GAAACGCGCG AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA 6120
ATGTCATGAT AATAATGGTT TCTTAGACGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG 6180
GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT 6240 AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC 6300
GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA 6360
CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 6420
TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT TTTCCAATGA 6480
TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTATTGAC GCCGGGCAAG 6540 AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA 6600
CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA 6660
TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA 6720
CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC 6780
TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT GCCTGTAGCA ATGGCAACAA 6840 CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG 6900
ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT 6960
GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC 7020
TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA 7080
CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT 7140 AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT 7200
TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG 7260
AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC 7320
CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG 7380
TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG 7440 CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT 7500
CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG 7560
GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC 7620
GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG 7680
AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG 7740 CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG 7800
GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC 7860
GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT 7920
TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC 7980
CTGATTCTGT GGAT ACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC 8040 GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGA 8083
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8149 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkeχ-1: :gBGH/p53
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60 CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360 GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960 CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500 TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800 GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCTC AATTCTATGA 1860
TTTCTATCAT TACTTCCTCA CATGTTGGAG GCATTTTCTC TCCCTCTGCA CTTAATAGCC 1920
TATCTTGCTT TAATTTCTTC CCACTCTTGG AGGCTAGGTT TGGTTTGGTG GGCTGATGAG 1980
GGAGGGAGAG ACCGCTCCAA GTACTTTAGC GGGTGGGATT GAAGCGGAGC CCTCCTGAGC 2040
TATGAGTGTC CTATGAGTGG GGCTGGAACT AAGAACCAGG GGCGTGGACA GGGTGTGTCA 2100 CAGAGAAGGG GATGTGCCTG CTTCTTTCTG GCCCAGGAGG AACCGGGTCA ATTCTTCAGC 2160
ACCTGGGTAC CCATAGAGCC CACCGC TCC CCAGCATGCC TGCTATTGTC TTCCCAATCC 2220
TCCCCCTTGC TGTCCTGCCC CACCCCACCC CCCAGAATAG AATGACACCT ACTCAGACAA 2280
TGCGATGCAA TTTCCTCATT TTATTAGGAA AGGACAGTGG GAGTGGCACC TTCCAGGGTC 2340
AAGGAAGGCA CGGGGGAGGG GCAAACAACA GATGGCTGGC AACTAGAAGG CACAGCGGAT 2400 CTGAGCTTGC ATGCCTGAGG TCGACCCTGG ATAAGCTGAT CCTCAATCAA TCAAGGTGGT 2460
ATAAGAGTAA GACCCACTTC TTTACAGCCT CCTCTCTTAG CAGTAGGTAT AACAACAAGA 2520
AATATGTCAC CACTTCAATA CTGTCATGTG TTAGCAAGTT ACCCATCATC ACCACTTCCC 2580
CTGCCCCATA TTGGGTCCCC AAGGCCTTTT GTTCTGATAG GACCATGTAT GTTACCAGTA 2640
ACCAGAGCAC GTATCTTCCA CCCAGTAAAG CTACCACCAC CACCAATATG GACTCGGCGA 2700 AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCGAACCA GTATTGATAA TCTCCTTTTA 2760
GCATGTATTG CTGGAAGTAA CTGTCTCTGG GCTCAAAATA TTTATTTTTT AATGTCCTCG 2820
TGTAGTTGAA GGTGCATGCC GTCTTTTCTA CAGGCCCCTC ACTTGGTATG ATCTCATATG 2880
GCTTGCAAGG CATAGGCCCA AGTTTGGTGT CAAGAGCTAT GACCTGTACG ATTGTGTCTC 2940
CTATCTTACA CTTTACCAAT CCTTGTGGTA CTATGGCTAC ACCCTCTCTG TTGCAAGTGG 3000 TGCCGTCTAC AAATCTGTAG CCAGTCTCAT TCTTCAACCT ACACTTGCCG ATGGGGTAGT 3060
GTGGCAACCC CTCACTTTTT TGGAATTTAT AACCACACCA CTTGCAAGAT TCGACAGGGC 3120
CTCCTGTGTA TTGTAGCTGG TCCCCAGTCA CACAAGTCCA ATTCCCTCCA AGATCACAGT 3180
TATAGAGATC CTCCCCCAGA GTTCTTTGGG TGATACAACC TTGTCTATAA GGGAATGGTT 3240
TGGACCTCTT ATACACACGC ACTACTGCTG TGTCTAGGGT GTCCCTATTA GCTAACAT C 3300 AGCTCACAGT TCCTGTCCAT CCTATAGGGC ATACCATCTG GAAGGCCGGT CCGTTTAGCA 3360
GTGTTGTATT GAACTTCCCT CTTACTACGG GATTGGCATC GCATGGGCAG AGTCCAAATT 3420
CAAAGTCGTC AGCCATTTCA AATGTTTCCC CTTGCCTTTG CCCCTCGAAA AGTTTTTTGA 3480
ATACCACACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAGATATCTA GTTTCTCTTG 3540 TGCACCTTGA GAGGTATGTA AACTTACCGT CTTTGCACCA AGCTATGACC ATTGTGTCTT 3600
CCAGCTTCAT TTCATGTGAG TAATCCTTCC AAACAGTGGT GAGGCCTTCA GCTCCTAATG 3660
GGCCAATTCT ATCATTCCTG GCTATGGCGT ATGAGTGTTC AGGTTTGCAG TCAATGTCCC 3720
CTTGTACCCC TGTTATCAGT AGTAGCCATA GGATCCCTGG GAAGGCGCCC ACCACCTGAG 3780
TCCAGGGCAG GCAGAGCAGG GCGAAAGCCA GGAGCAGGGA GGTCCGGGGG CCTGCAGCCA 3840 TCATGTCGAA GCTTGCCGCG GAGGCTGGAT CGGTCCCGGT GTCTTCTATG GAGGTCAAAA 3900
CAGCGTGGAT GGCGTCTCCA GGCGATCTGA CGGTTCACTA AACGAGCTCT GCTTATATAG 3960
ACCTCCCACC GTACACGCCT ACCGCCCATT TGCGTCAATG GGGCGGAGTT GTTACGACAT 4020
TTTGGAAAGT CCCGTTGATT TTGGTGCCAA AACAAACTCC CATTGACGTC AATGGGGTGG 4080
AGACTTGGAA ATCCCCGTGA GTCAAACCGC TATCCACGCC CATTGATGTA CTGCCAAAAC 4140 CGCATCACCA TGGTAATAGC GATGACTAAT ACGTAGATGT ACTGCCAAGT AGGAAAGTCC 4200
CATAAGGTCA TGTACTGGGC ATAATGCCAG GCGGGCCATT TACCGTCATT GACGTCAATA 4260
GGGGGCGTAC TTGGCATATG ATACACTTGA TGTACTGCCA AGTGGGCAGT TTACCGTAAA 4320
TACTCCACCC ATTGACGTCA ATGGAAAGTC CCTATTGGCG TTACTATGGG AACATACGTC 4380
ATTATTGACG TCAATGGGCG GGGGTCGTTG GGCGGTCAGC CAGGCGGGCC ATTTACCGTA 4440 AGTTATGTAA CGCGGAACTC CATATATGGG CTATGAACTA ATGACCCCGT AATTGATTAC 4500
TATTAATAAC TAGTCAATAA TCAATGTCAA CATGGCGGTA ATGTTGGACA TGAGCCAATA 4560
TAAATGTACA TATTATGATA TGGATACAAC GTATGCAATG GCCAATAGCC AATATTGATT 4620
TATGCTATAT AACCAATGAA TAATATGGCT AATGGCCAAT ATTGATTCAA TGTATAGATC 4680
GATATGCATT GGCCATGTGC CAGCTTGATG TCGCCTCTAT CGGCGATATA GCCTCATATC 4740 GTCTGTCACC TATATCGAAA CTGCGATATT TGCGACACAC AGAATCGCCC AAGTCACCAA 4800
AGGCGTCTAT CGCCATCCCC CGTAAACGAT ATAAGCGTAT CGCCAGATAT CGCGTATGCC 4860
CAAAAATCAA CTTTTGGAAA AATGGCGATA TCAGTTACAC AGAAACTCAC ATCGGCGACA 4920
TTTTCAATAT GCCATATTTT CAAATATCGA TTTTTCCAAT ATCGCCATCT CTATCGGCGA 4980
TAAACACCAC TATCGCGCGA CATGAATTTA GTCGGGACAG AAATCTCAAA CGCGTATTTC 5040 GGACAAACAC ACATTTTATT ATTCACTGCA GGTCGAGGAA TTCGGATCTC GAGGTGTTCG 5100
TGCTGGACGT GTCCGCGGCG CCAGACGCGT GCGCGGCCGC CGTACTGGAC ATGCGGCCCG 5160
CCATGCAGGC CGCTTGCGCG GACGGGGCGG CGGGCGCGAC GCTGGCGACC CTGGCGCGTC 5220
AGTTCGCGCT AGAGATGGCG GGGGAGGCCA CGGCGGGCCC TAGGGGACTA TAAAGCTGCC 5280
CCTGCGCTCG CTCGCTCGCT GCATTTGCGC CCCGATCGCC TTACGGGGAC TCGGCGCTCG 5340 GCGGATCCCC TCCCGGCCCC GCCGCGAAGC AGGCCGCCAG ACAAAAAAAT GCGGCGCCCG 5400
CTCTGCGCGG CGCTATTGGC AGCGGCTGTC CTCGCGCTCG CCGCGGGCGC CCCCGCCGCC 5460
GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG AAATCGAAGA 5520
GTGGGAAATG GTGGTCGGAG CCGGGCCGGC CGTGCACACG TTCACCATCC GCTGCCTCGG 5580 GCCGCGGGGC ATTGAGCGCG TGGCCCACAT TGCAAACCTC AGCCGGCTGC TGGACGGGTA 5640
CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA TGTTTTTCCT 5700
GCCGCGCGCG GCCGTCGACT CTAGAGGATC CCCGGGTACC GAGCTCGAAT TCACTGGCCG 5760
TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820
CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC 5880 AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC 5940
TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 6000
AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC 6060
TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 6120
TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC CTATTTTTAT 6180 AGGTTAATGT CATGATAATA ATGGTTTCTT AGACGTCAGG TGGCACTTTT CGGGGAAATG 6240
TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA 6300
GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC 6360
ATTTCCGTGT CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 6420
CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6480 TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC 6540
CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG 6600
GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC 6660
CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 6720
TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG 6780 AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC 6840
CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GTAGCAATGG 6900
CAACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT 6960
TAATAGACTG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 7020
CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 7080 CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC 7140
AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 7200
ATTGGTAACT GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 7260
TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 7320
AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT 7380 GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG 7440
CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA 7500
GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA 756
AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 762 CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG 768
CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT 774
ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA 780
GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC 786
TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 792 AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG 798
CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT 8040
TATCCCCTG TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC 8100
GCAGCCGAAC GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAGA 8149 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8135 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(Vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine viral diarrhea virus
(B) STRAIN: 2724 (C) INDIVIDUAL ISOLATE: pBHVtkex-1: :gIII/p53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTC TTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATG TTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300 GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600 CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840 CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140 GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440 GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740 CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCTC AATTCTATGA 1860
TTTCTATCAT TACTTCCTCA CATGTTGGAG GCATTTTCTC TCCCTCTGCA CTTAATAGCC 1920
TATCTTGCTT TAATTTCTTC CCACTCTTGG AGGCTAGGTT TGGTTTGGTG GGCTGATGAG 1980
GGAGGGAGAG ACCGCTCCAA GTACTTTAGC GGGTGGGATT GAAGCGGAGC CCTCCTGAGC 2040 TATGAGTGTC CTATGAGTGG GGCTGGAACT AAGAACCAGG GGCGTGGACA GGGTGTGTCA 2100
CAGAGAAGGG GATGTGCCTG CTTCTTTCTG GCCCAGGAGG AACCGGGTCA ATTCTTCAGC 2160
ACCTGGGTAC CCATAGAGCC CACCGCATCC CCAGCATGCC TGCTATTGTC TTCCCAATCC 2220
TCCCCCTTGC TGTCCTGCCC CACCCCACCC CCCAGAATAG AATGACACCT ACTCAGACAA 2280
TGCGATGCAA TTTCCTCATT TTATTAGGAA AGGACAGTGG GAGTGGCACC TTCCAGGGTC 2340 AAGGAAGGCA CGGGGGAGGG GCAAACAACA GATGGCTGGC AACTAGAAGG CACAGCGGAT 2400
CTGAGCTTGC ATGCCTGAGG TCGACCCTGG ATAAGCTGAT CCTCAATCAA TCAAGGTGGT 2460
ATAAGAGTAA GACCCACTTC TTTACAGCCT CCTCTCTTAG CAGTAGGTAT AACAACAAGA 2520
AATATGTCAC CACTTCAATA CTGTCATGTG TTAGCAAGTT ACCCATCATC ACCACTTCCC 2580
CTGCCCCATA TTGGGTCCCC AAGGCCTTTT GTTCTGATAG GACCATGTAT GTTACCAGTA 2640 ACCAGAGCAC GTATCTTCCA CCCAGTAAAG CTACCACCAC CACCAATATG GACTCGGCGA 2700
AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCGAACCA GTATTGATAA TCTCCTTTTA 2760
GCATGTATTG CTGGAAGTAA CTGTCTCTGG GCTCAAAATA TTTATTTTTT AATGTCCTCG 2820
TGTAGTTGAA GGTGCATGCC GTCTTTTCTA CAGGCCCCTC ACTTGGTATG ATCTCATATG 2880 GCTTGCAAGG CATAGGCCCA AGTTTGGTGT CAAGAGCTAT GACCTGTACG ATTGTGTCTC 2940
CTATCTTACA CTTTACCAAT CCTTGTGGTA CTATGGCTAC ACCCTCTCTG TTGCAAGTGG 3000
TGCCGTCTAC AAATCTGTAG CCAGTCTCAT TCTTCAACCT ACACTTGCCG ATGGGGTAGT 3060
GTGGCAACCC CTCACTTTTT TGGAATTTAT AACCACACCA CTTGCAAGAT TCGACAGGGC 3120
CTCCTGTGTA TTGTAGCTGG TCCCCAGTCA CACAAGTCCA ATTCCCTCCA AGATCACAGT 3180 TATAGAGATC CTCCCCCAGA GTTCTTTGGG TGATACAACC TTGTCTATAA GGGAATGGTT 3240
TGGACCTCTT ATACACACGC ACTACTGCTG TGTCTAGGGT GTCCCTATTA GCTAACATAC 3300
AGCTCACAGT TCCTGTCCAT CCTATAGGGC ATACCATCTG GAAGGCCGGT CCGTTTAGCA 3360
GTGTTGTATT GAACTTCCCT CTTACTACGG GATTGGCATC GCATGGGCAG AGTCCAAATT 3420
CAAAGTCGTC AGCCATTTCA AATGTTTCCC CTTGCCTTTG CCCCTCGAAA AGTTTTTTGA 3480 ATACCACACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAGATATCTA GTTTCTCTTG 3540
TGCACCTTGA GAGGTATGTA AACTTACCGT CTTTGCACCA AGCTATGACC ATTGTGTCTT 3600
CCAGCTTCAT TTCATGTGAG TAATCCTTCC AAACAGTGGT GAGGCCTTCA GCTCCTAATG 3660
GGCCAATTCT ATCATTCCTG GCTATGGCGT ATGAGTGTTC AGGTTTGCAG TCAATGTCCC 3720
CTTGTACCCC TGTTATCAGT AGTAGCCATA GGATCCCCGA CGGCGCCGCG GCGATGGCCG 3780 CCGCGTAGAG CGCCAGCAGA GCGAGCATCG CACGCGCGAG CGAGGCCATG GTCGAAGCTT 3840
GCCGCGGAGG CTGGATCGGT CCCGGTGTCT TCTATGGAGG TCAAAACAGC GTGGATGGCG 3900
TCTCCAGGCG ATCTGACGGT TCACTAAACG AGCTCTGCTT ATATAGACCT CCCACCGTAC 3960
ACGCCTACCG CCCATTTGCG TCAATGGGGC GGAGTTGTTA CGACATTTTG GAAAGTCCCG 4020
TTGATTTTGG TGCCAAAACA AACTCCCATT GACGTCAATG GGGTGGAGAC TTGGAAATCC 4080 CCGTGAGTCA AACCGCTATC CACGCCCATT GATGTACTGC CAAAACCGCA TCACCATGGT 4140
AATAGCGATG ACTAATACGT AGATGTACTG CCAAGTAGGA AAGTCCCATA AGGTCATGTA 4200
CTGGGCATAA TGCCAGGCGG GCCATTTACC GTCATTGACG TCAATAGGGG GCGTACTTGG 4260
CATATGATAC ACTTGATGTA CTGCCAAGTG GGCAGTTTAC CGTAAATACT CCACCCATTG 4320
ACGTCAATGG AAAGTCCCTA TTGGCGTTAC TATGGGAACA TACGTCATTA TTGACGTCAA 4380 TGGGCGGGGG TCGTTGGGCG GTCAGCCAGG CGGGCCATTT ACCGTAAGTT ATGTAACGCG 4440
GAACTCCATA TATGGGCTAT GAACTAATGA CCCCGTAATT GATTACTATT AATAACTAGT 4500
CAATAATCAA TGTCAACATG GCGGTAATGT TGGACATGAG CCAATATAAA TGTACATATT 4560
ATGATATGGA TACAACGTAT GCAATGGCCA ATAGCCAATA TTGATTTATG CTATATAACC 4620
AATGAATAAT ATGGCTAATG GCCAATATTG ATTCAATGTA TAGATCGATA TGCATTGGCC 4680 ATGTGCCAGC TTGATGTCGC CTCTATCGGC GATATAGCCT CATATCGTCT GTCACCTATA 4740
TCGAAACTGC GATATTTGCG ACACACAGAA TCGCCCAAGT CACCAAAGGC GTCTATCGCC 4800
ATCCCCCGTA AACGATATAA GCGTATCGCC AGATATCGCG TATGCCCAAA AATCAACTTT 4860
TGGAAAAATG GCGATATCAG TTACACAGAA ACTCACATCG GCGACATTTT CAATATGCCA 4920 TATTTTCAAA TATCGATTTT TCCAATATCG CCATCTCTAT CGGCGATAAA CACCACTATC 4980
GCGCGACATG AATTTAGTCG GGACAGAAAT CTCAAACGCG TATTTCGGAC AAACACACAT 5040
TTTATTATTC ACTGCAGGTC GAGGAATTCG GATCTCGAGG TGTTCGTGCT GGACGTGTCC 5100
GCGGCGCCAG ACGCGTGCGC GGCCGCCGTA CTGGACATGC GGCCCGCCAT GCAGGCCGCT 5160
TGCGCGGACG GGGCGGCGGG CGCGACGCTG GCGACCCTGG CGCGTCAGTT CGCGCTAGAG 5220 ATGGCGGGGG AGGCCACGGC GGGCCCTAGG GGACTATAAA GCTGCCCCTG CGCTCGCTCG 5280
CTCGCTGCAT TTGCGCCCCG ATCGCCTTAC GGGGACTCGG CGCTCGGCGG ATCCCCTCCC 5340
GGCCCCGCCG CGAAGCAGGC CGCCAGACAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400
ATTGGCAGCG GCTGTCCTCG CGCTCGCCGC GGGCGCCCCC GCCGCCGCCC GCGGCGGGGG 5460
CGCCGAAGCC AGGGCAGCAC AGAGACGCCC GATACGAAAT CGAAGAGTGG GAAATGGTGG 5520 TCGGAGCCGG GCCGGCCGTG CACACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580
AGCGCGTGGC CCACATTGCA AACCTCAGCC GGCTGCTGGA CGGGTACATA GCGGTCCACG 5640
TTGACGTTGC GCGCACCTCT GGCCTGCGGG ACGCCATGTT TTTCCTGCCG CGCGCGGCCG 5700
TCGACTCTAG AGGATCCCCG GGTACCGAGC TCGAATTCAC TGGCCGTCGT TTTACAACGT 5760
CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA TCCCCCTTTC 5820 GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC 5880
CTGAATGGCG AATGGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA 5940
CACCGCATAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGCCC 6000
CGACACCCGC CAACACCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC GGCATCCGCT 6060
TACAGACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA 6120 CCGAAACGCG CGAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 6180
ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 6240
ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 6300
TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC 6360
CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG 6420 AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 6480
AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT 6540
TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC 6600
GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG 6660
CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT 6720 AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 6780
TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA 6840
GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 6900
AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG 6960 GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT 7020
GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 7080
GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT 7140
GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA 7200
GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 7260 ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG 7320
TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 7380
CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG 7440
CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA 7500
CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 7560 CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG 7620
TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 7680
TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 7740
TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 7800
TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACG GGGAGCTTCC AGGGGGAAAC 7860 GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 7920
TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCC GCAACGCGGC CTTTTTACGG 7980
TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT 8040
GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC 8100 GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAGA 8135 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8149 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkeχ-3: :BGH/ 53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120 CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420 GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720 GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020 GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320 GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620 CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860
CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920 ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040
GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT 2100
ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160 CAAATATCGC AGTTTCGATA TAGGTGACAG ACGATATGAG GCTATATCGC CGATAGAGGC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
ACGTTGTATC C TATCATAA TATGTACATT T TATTGGCT CATGTCCAAC ATTACCGCCA 2400
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460 AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760 GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC 3000
CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC 3060 CGATCCAGCC TCCGCGGCAA GCTTCGACAT GATGGCTGCA GGCCCCCGGA CCTCCCTGCT 3120
CCTGGCTTTC GCCCTGCTCT GCCTGCCCTG GACTCAGGTG GTGGGCGCCT TCCCAGGGAT 3180
CCTATGGCTA CTACTGATAA CAGGGGTACA AGGGGACATT GACTGCAAAC CTGAACACTC 3240
ATACGCCATA GCCAGGAATG ATAGAATTGG CCCATTAGGA GCTGAAGGCC TCACCACTGT 3300
TTGGAAGGAT TACTCACATG AAATGAAGCT GGAAGACACA ATGGTCATAG CTTGGTGCAA 3360 AGACGGTAAG TTTACATACC TCTCAAGGTG CACAAGAGAA ACTAGATATC TTGCAATTCT 3420
GCATTCAAGA GCCTTGCAGA CCAGTGTGGT ATTCAAAAAA CTTTTCGAGG GGCAAAGGCA 3480
AGGGGAAACA TTTGAAATGG CTGACGACTT TGAATTTGGA CTCTGCCCAT GCGATGCCAA 3540
TCCCGTAGTA AGAGGGAAGT TCAATACAAC ACTGCTAAAC GGACCGGCCT TCCAGATGGT 3600
ATGCCCTATA GGATGGACAG GAACTGTGAG CTGTATGTTA GCTAATAGGG ACACCCTAGA 3660 CACAGCAGTA GTGCGTGTGT ATAAGAGGTC CAAACCATTC CCTTATAGAC AAGGTTGTAT 3720
CACCCAAAGA ACTCTGGGGG AGGATCTCTA TAACTGTGAT CTTGGAGGGA ATTGGACTTG 3780
TGTGACTGGG GACCAGCTAC AATACACAGG AGGCCCTGTC GAATCTTGCA AGTGGTGTGG 3840
TTATAAATTC CAAAAAAGTG AGGGGTTGCC ACACTACCCC ATCGGCAAGT GTAGGTTGAA 3900
GAATGAGACT GGCTACAGAT TTGTAGACGG CACCACTTGC AACAGAGAGG GTGTAGCCAT 3960 AGTACCACAA GGATTGGTAA AGTGTAAGAT AGGAGACACA ATCGTACAGG TCATAGCTCT 4020
TGACACCAAA CTTGGGCCTA TGCCTTGCAA GCCATATGAG ATCATACCAA GTGAGGGGCC 4080
TGTAGAAAAG ACGGCATGCA CCTTCAACTA CACGAGGACA TTAAAAAATA AATATTTTGA 4140
GCCCAGAGAC AGTTACTTCC AGCAATACAT GCTAAAAGGA GATTATCAAT ACTGGTTCGA 4200 CCTGGAGGTC ACTGACCATC ATCGGGATTA CTTCGCCGAG TCCATATTGG TGGTGGTGGT 4260
AGCTTTACTG GGTGGAAGAT ACGTGCTCTG GTTACTGGTA ACATACATGG TCCTATCAGA 4320
ACAAAAGGCC TTGGGGACCC AATATGGGGC AGGGGAAGTG GTGATGATGG GTAACTTGCT 4380
AACACATGAC AGTATTGAAG TGGTGACATA TTTCTTGTTG TTATACCTAC TGCTAAGAGA 4440
GGAGGCTGTA AAGAAGTGGG TCTTACTCTT ATACCACCTT GATTGATTGA GGATCAGCTT 4500 ATCCAGGGTC GACCTCAGGC ATGCAAGCTC AGATCCGCTG TGCCTTCTAG TTGCCAGCCA 4560
TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC 4620
CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG 4680
GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT 4740
GGGGATGCGG TGGGCTCTAT GGGTACCCAG GTGCTGAAGA ATTGACCCGG TTCCTCCTGG 4800 GCCAGAAAGA AGCAGGCACA TCCCCTTCTC TGTGACACAC CCTGTCCACG CCCCTGGTTC 4860
TTAGTTCCAG CCCCACTCAT AGGACACTCA TAGCTCAGGA GGGCTCCGCT TCAATCCCAC 4920
CCGCTAAAGT ACTTGGAGCG GTCTCTCCCT CCCTCATCAG CCCACCAAAC CAAACCTAGC 4980
CTCCAAGAGT GGGAAGAAAT TAAAGCAAGA TAGGCTATTA AGTGCAGAGG GAGAGAAAAT 5040
GCCTCCAACA TGTGAGGAAG TAATGATAGA AATCATAGAA TTGAGATCTC GAGGTGTTCG 5100 TGCTGGACGT GTCCGCGGCG CCAGACGCGT GCGCGGCCGC CGTACTGGAC ATGCGGCCCG 5160
CCATGCAGGC CGCTTGCGCG GACGGGGCGG CGGGCGCGAC GCTGGCGACC CTGGCGCGTC 5220
AGTTCGCGCT AGAGATGGCG GGGGAGGCCA CGGCGGGCCC TAGGGGACTA TAAAGCTGCC 5280
CCTGCGCTCG CTCGCTCGCT GCATTTGCGC CCCGATCGCC TTACGGGGAC TCGGCGCTCG 5340
GCGGATCCCC TCCCGGCCCC GCCGCGAAGC AGGCCGCCAG ACAAAAAAAT GCGGCGCCCG 5400 CTCTGCGCGG CGCTATTGGC AGCGGCTGTC CTCGCGCTCG CCGCGGGCGC CCCCGCCGCC 5460
GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG AAATCGAAGA 5520
GTGGGAAATG GTGGTCGGAG CCGGGCCGGC CGTGCACACG TTCACCATCC GCTGCCTCGG 5580
GCCGCGGGGC ATTGAGCGCG TGGCCCACAT TGCAAACCTC AGCCGGCTGC TGGACGGGTA 5640
CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA TGTTTTTCCT 5700 GCCGCGCGCG GCCGTCGACT CTAGAGGATC CCCGGGTACC GAGCTCGAAT TCACTGGCCG 5760
TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820
CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC 5880
AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC 5940
TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 6000 AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC 6060
TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 6120
TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC CTATTTTTAT 6180
AGGTTAATGT CATGATAATA ATGGTTTCTT AGACGTCAGG TGGCACTTTT CGGGGAAATG 6240 TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA 6300
GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC 6360
ATTTCCGTGT CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 6420
CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6480
TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC 6540 CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG 6600
GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC 6660
CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 6720
TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG 6780
AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC 6840 CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GTAGCAATGG 6900
CAACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT 6960
TAATAGACTG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 7020
CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 7080
CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC 7140 AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 7200
ATTGGTAACT GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 7260
TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 7320
AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT 7380
GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG 7440 CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA 7500
GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA 7560
AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 7620
CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG 7680
CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT 7740 ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA 7800
GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC 7860
TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 7920
AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG 7980
CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT 8040 TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC 8100
GCAGCCGAAC- GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAGA 8149
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8135 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine viral diarrhea virus
(B) STRAIN: 2724
(C) INDIVIDUAL ISOLATE: pBHVtkeχ-3: :gIII/p53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60 CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360 GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660 CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960 CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260 GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500 TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800 GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860
CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920
ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040
GAT TCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT 2100 ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160
CAAATATCGC AGTTTCGATA TAGGTGACAG ACGATATGAG GCT TATCGC CGATAGAGGC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400 TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460
AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700 GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC 3000 CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC 3060
CGATCCAGCC TCCGCGGCAA GCTTCGACCA TGGCCTCGCT CGCGCGTGCG ATGCTCGCTC 3120
TGCTGGCGCT CTACGCGGCG GCCATCGCCG CGGCGCCGTC GGGGATCCTA TGGCTACTAC 3180
TGATAACAGG GGTACAAGGG GACATTGACT GCAAACCTGA ACACTCATAC GCCATAGCCA 3240
GGAATGATAG AATTGGCCCA TTAGGAGCTG AAGGCCTCAC CACTGTTTGG AAGGATTACT 3300 CACATGAAAT GAAGCTGGAA GACACAATGG TCATAGCTTG GTGCAAAGAC GGTAAGTTTA 3360
CATACCTCTC AAGGTGCACA AGAGAAACTA GATATCTTGC AATTCTGCAT TCAAGAGCCT 3420
TGCAGACCAG TGTGGTATTC AAAAAACTTT TCGAGGGGCA AAGGCAAGGG GAAACATTTG 3480
AAATGGCTGA CGACTTTGAA TTTGGACTCT GCCCATGCGA TGCCAATCCC GTAGTAAGAG 3540 GGAAGTTCAA TACAACACTG CTAAACGGAC CGGCCTTCCA GATGGTATGC CCTATAGGAT 3600
GGACAGGAAC TGTGAGCTGT ATGTTAGCTA ATAGGGACAC CCTAGACACA GCAGTAGTGC 3660
GTGTGTATAA GAGGTCCAAA CCATTCCCTT ATAGACAAGG TTGTATCACC CAAAGAACTC 3720
TGGGGGAGGA TCTCTATAAC TGTGATCTTG GAGGGAATTG GACTTGTGTG ACTGGGGACC 3780
AGCTACAATA CACAGGAGGC CCTGTCGAAT CTTGCAAGTG GTGTGGTTAT AAATTCCAAA 3840 AAAGTGAGGG GTTGCCACAC TACCCCATCG GCAAGTGTAG GTTGAAGAAT GAGACTGGCT 3900
ACAGATTTGT AGACGGCACC ACTTGCAACA GAGAGGGTGT AGCCATAGTA CCACAAGGAT 3960
TGGTAAAGTG TAAGATAGGA GACACAATCG TACAGGTCAT AGCTCTTGAC ACCAAACTTG 4020
GGCCTATGCC TTGCAAGCCA TATGAGATCA TACCAAGTGA GGGGCCTGTA GAAAAGACGG 4080
CATGCACCTT CAACTACACG AGGACATTAA AAAATAAATA TTTTGAGCCC AGAGACAGTT 4140 ACTTCCAGCA ATACATGCTA AAAGGAGATT ATCAATACTG GTTCGACCTG GAGGTCACTG 4200
ACCATCATCG GGATTACTTC GCCGAGTCCA TATTGGTGGT GGTGGTAGCT TTACTGGGTG 4260
GAAGATACGT GCTCTGGTTA CTGGTAACAT ACATGGTCCT ATCAGAACAA AAGGCCTTGG 4320
GGACCCAATA TGGGGCAGGG GAAGTGGTGA TGATGGGTAA CTTGCTAACA CATGACAGTA 4380
TTGAAGTGGT GACATATTTC TTGTTGTTAT ACCTACTGCT AAGAGAGGAG GCTGTAAAGA 4440 AGTGGGTCTT ACTCTTATAC CACCTTGATT GATTGAGGAT CAGCTTATCC AGGGTCGACC 4500
TCAGGCATGC AAGCTCAGAT CCGCTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC 4560
CTCCCCCGTG CCTTCCTTG CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA 4620
TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG 4680
GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG 4740 CTCTATGGGT ACCCAGGTGC TGAAGAATTG ACCCGGTTCC TCCTGGGCCA GAAAGAAGCA 4800
GGCACATCCC CTTCTCTGTG ACACACCCTG TCCACGCCCC TGGTTCTTAG TTCCAGCCCC 4860
ACTCATAGGA CACTCATAGC TCAGGAGGGC TCCGCTTCAA TCCCACCCGC TAAAGTACTT 4920
GGAGCGGTCT CTCCCTCCCT CATCAGCCCA CCAAACCAAA CCTAGCCTCC AAGAGTGGGA 4980
AGAAATTAAA GCAAGATAGG CTATTAAGTG CAGAGGGAGA GAAAATGCCT CCAACATGTG 5040 AGGAAGTAAT GATAGAAATC ATAGAATTGA GATCTCGAGG TGTTCGTGCT GGACGTGTCC 5100
GCGGCGCCAG ACGCGTGCGC GGCCGCCGTA CTGGACATGC GGCCCGCCAT GCAGGCCGCT 5160
TGCGCGGACG GGGCGGCGGG CGCGACGCTG GCGACCCTGG CGCGTCAGTT CGCGCTAGAG 5220
ATGGCGGGGG AGGCCACGGC GGGCCCTAGG GGACTATAAA GCTGCCCCTG CGCTCGCTCG 5280
CTCGCTGCAT TTGCGCCCCG ATCGCCTTAC GGGGACTCGG CGCTCGGCGG ATCCCCTCCC 5340 GGCCCCGCCG CGAAGCAGGC CGCCAGACAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400
ATTGGCAGCG GCTGTCCTCG CGCTCGCCGC GGGCGCCCCC GCCGCCGCCC GCGGCGGGGG 5460
-«• « •*• -Ψ
CGCCGAAGCC AGGGCAGCAC AGAGACGCCC GATACGAAAT CGAAGAGTGG GAAATGGTGG 5520
TCGGAGCCGG GCCGGCCGTG CACACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580
AGCGCGTGGC CCACATTGCA AACCTCAGCC GGCTGCTGGA CGGGTACATA GCGGTCCACG 5640
TTGACGTTGC GCGCACCTCT GGCCTGCGGG ACGCCATGTT TTTCCTGCCG CGCGCGGCCG 5700
TCGACTCTAG AGGATCCCCG GGTACCGAGC TCGAATTCAC TGGCCGTCGT TTTACAACGT 5760
CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA TCCCCCTTTC 5820
GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC 5880
CTGAATGGCG AATGGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA 5940
CACCGCATAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGCCC 6000
CGACACCCGC CAACACCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC GGCATCCGCT 6060
TACAGACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA 6120
CCGAAACGCG CGAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 6180
ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 6240
ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 6300
TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC 6360
CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG 6420
AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 6480
AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT 6540
TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC 6600
GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG 6660
CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT 6720
AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 6780
TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA 6840
GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 6900
AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG 6960
GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT 7020
GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 7080
GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT 7140
GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA 7200
GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 7260
ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG 7320
TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 7380
CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG 7440
CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA 7500
CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 7560
CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG 7620 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 7680
TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 7740
TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 7800
TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 7860
GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 7920 TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 7980
TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT 8040
GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC 8100
GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAGA 8135
Claims (30)
1. A replicating nonpathogenic virus, for preventing disease caused by Bovine Viral Diarrhea Virus (BVDV), where said replicating nonpathogenic virus comprises: a gene or gene combination taken from a BVDV virus, and said replicating nonpathogenic virus functionally expresses said gene or gene combination.
2. A virus of claim 1, where said replicating nonpathogenic virus is attenuated.
3. A virus of claim 2, where said replicating nonpathogenic virus is selected from attenuated Bovine Herpes Virus type 1 (BHV-1), attenuated adenoviruses, attenuated bovine mammillitis virus, attenuated bovine papillomavirus, or attenuated pseudorabies virus.
4. A virus of claim 2, where said replicating nonpathogenic virus is attenuated and contains and expresses any combination of the following genes: the genes that code for gp48, gp25, pl4 capsid protein, p20 N-terminal protease and pl25/p80 protein.
5. A virus of claim 3, where said replicating nonpathogenic virus is attenuated and contains and expresses any combination of the following genes: the genes that code for gp48, gp25, pl4 capsid protein, p20 N-terminal protease and pl25/p80 protein.
6. A virus of claim 2, where said attenuation is created by making the thymidine kinase (tk) gene nonfunctional.
7. A virus of claim 3, where said attenuation is created by making the thymidine kinase (tk) gene nonfunctional.
8. A virus of claim 4, where said attenuation is created by making the thymidine kinase (tk) gene nonfunctional.
9. A virus of claim 5, where said attenuation is created by making the thymidine kinase (tk) gene nonfunctional.
10. A virus of claim 9, where said replicating nonpathogenic virus is attenuated Bovine Herpes Virus type 1 (BHV-1).
11. A virus of claim 10, where said replicating nonpathogenic virus contains and 5 expresses the gene that codes for gp53, a glycoprotein of the Bovine Viral Diarrhea
Virus (BVDV).
12. A virus of claim 11, 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).
10
13. A virus of claim 12, where said gene that codes for gp53 is inserted into the inactivated thymidine kinase (tk) gene site.
14. A virus of claim 13, where the functionally expressing gene or gene
15 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) 20 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.
15. A virus of claim 14, where said signal peptide gene sequence is taken from 25 any well characterized signal peptide sequences such as any of the thirty-nine examples of well characterized signal peptide sequences found in Perlman, D., et al., J. Mol . Biol. Vol. 167 pp. 391-409 (1983).
16. A virus of claim 15, where said signal peptide gene sequence is taken from 30 Psuedorabies Virus gill gene (PRV) and/or Bovine Growth Hormone (BGH).
17. A virus of claim 16 where the plasmid is selected from the following plasmids, a) pBHVtkex-l::BGH/p53; •35 b) pBHVtkex-l::gIII/p53; c) pBHVtkex-3::BGH/p53; or d) pBHVtkex-3::gIII/p53.
18. A virus of claim 17, where said virus that produces the product of a functionally expressing gene or gene combination is selected from one of the following viruses,
Tll-3, Tll-6, or Tll-8.
19. A virus of claim 18, where said gene or gene combination is Tll-6.
20. A virus of claim 11, where said gene that codes for gp53 is inserted into the inactivated thymidine kinase (tk) gene site.
21. A virus of claim 20, 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.
22. A virus of claim 21, where said plasmid is made from a plasmid having the characteristics of plasmid pHAS4.
23. A virus of claim 22, where said plasmid is pBHVtkex-3::p53.
24. A virus of claim 23, where said virus is selected from one of the following viruses,
T2-3#3 or T2-2#5.
25. A vaccine for preventing disease caused by Bovine Viral Diarrhea Virus (BDVD) comprising a pharmaceutically effective amount of a virus of claim 1 and a carrier.
26. A vaccine as claimed in claim 25, for preventing disease caused by Bovine Viral Diarrhea Virus (BDVD) comprising a pharmaceutically effective amount of a virus of claim 1 and a carrier, said carrier comprising any physiological buffered medium, i.e. about pH 7.0 to 7.4 containing from about 2.5 to 15% serum which does not contain antibodies to BHV.
27. 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 of claim 1.
28. A process of preparing a virus of claim 1 comprising: a) isolation of a functionally expressing gene or gene combination that causes BVDV, b) inserting 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.
29. A method of preparing a virus of claim 11 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.
30. A method of preparing a virus of claim 12 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.
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 true AU1042395A (en) | 1995-05-23 |
| AU688819B2 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 |
| EP1170367A1 (en) | 2000-06-27 | 2002-01-09 | Bayer Ag | BVDV virus-like particles |
| CA2496750C (en) | 2002-08-26 | 2014-10-21 | Pfizer Products Inc. | Vaccine for respiratory and reproductive system infections in cattle |
| CN1905896A (en) * | 2003-12-05 | 2007-01-31 | 贝克顿·迪金森公司 | Methods of enhancing immune response in the intradermal compartment and compounds useful thereof |
| 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 |
| JP2016512841A (en) | 2013-03-15 | 2016-05-09 | ゾエティス・サービシーズ・エルエルシー | B. Multivalent vaccine Interference effect of cattle on trehaloci infection |
| CN113913461A (en) * | 2021-11-15 | 2022-01-11 | 贵州大学 | Construction method of bovine viral diarrhea E0-E2 gene recombinant adenovirus vaccine |
Family Cites Families (4)
| 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 |
| GB8818415D0 (en) * | 1988-08-03 | 1988-09-07 | Animal Health Inst | 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 EP EP95901037A patent/EP0725831A1/en not_active Withdrawn
- 1994-10-31 AU AU10423/95A patent/AU688819B2/en not_active Ceased
- 1994-10-31 NZ NZ276234A patent/NZ276234A/en unknown
- 1994-10-31 CA CA002172815A patent/CA2172815A1/en not_active Abandoned
- 1994-10-31 WO PCT/US1994/012198 patent/WO1995012682A2/en not_active Application Discontinuation
- 1994-10-31 JP JP7513263A patent/JPH09504435A/en active Pending
- 1994-10-31 CN CN94193978A patent/CN1134175A/en active Pending
- 1994-10-31 KR KR1019960702343A patent/KR960705944A/en not_active Application Discontinuation
- 1994-11-07 MX MXPA94008605A patent/MXPA94008605A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0725831A1 (en) | 1996-08-14 |
| CA2172815A1 (en) | 1995-05-11 |
| WO1995012682A3 (en) | 1995-07-06 |
| JPH09504435A (en) | 1997-05-06 |
| MXPA94008605A (en) | 2004-11-11 |
| NZ276234A (en) | 1998-01-26 |
| WO1995012682A2 (en) | 1995-05-11 |
| KR960705944A (en) | 1996-11-08 |
| AU688819B2 (en) | 1998-03-19 |
| CN1134175A (en) | 1996-10-23 |
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| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |