HK1067383B - Attenuated forms of bovine viral diarrhea virus - Google Patents

Description

Attenuated bovine viral diarrhea virus

The present invention relates to a method for producing an attenuated Bovine Viral Diarrhea (BVD) virus by inactivating a specific gene in the viral genome. The attenuated virus or mutated viral genome may be used to generate antibodies against BVD virus or as a vaccine to prevent viral infection in cattle.

Bovine Viral Diarrhea (BVD) viruses belong to the pestivirus genus and the flaviviruses. It is closely related to the virus that causes the disease of sheep Border and the typical fever of pigs. Infected cattle may exhibit "mucosal disease" characterized by elevated body temperature, diarrhea, cough, and ulceration of the digestive mucosa (Olafson et al, Cornell vet.36: 205-. BVD virus can cross the placenta of pregnant cows, leading to persistent infected newborn calves (Malmquist J.USA veterinary medical Association (J.Am.vet.Med.Assoc.188: 618-464-619 (1986)). these calves are immune-tolerant to the virus and will develop persistent viremia throughout life. these calves are susceptible to outbreak of mucosal disease (Liess et al, Dtsch.Tierartl.wschr.81: 481-487(1974)) and are very susceptible to microbial infections that cause diseases such as pulmonary diseases, inflammatory bowel diseases (Baret al, veterinarian (vet.Rec.) 117: 459-464 (1985)).

BVD viruses were classified as having two different biological types. Viruses with the "cp" organism type induce cytopathic effects in cultured cells, whereas viruses of the "ncp" organism type do not (Gillespie et al, Cornell Vet 50: 73-79 (1960)). In addition, two major genotypes (type I and type II) have been identified, both of which produce a range of clinical symptoms (Pellerin et al Virology (Virology) 203: 260-268 (1994); Ridpath et al Virology 205: 66-74 (1994)).

The BVD genome is about 12.5kb in length and contains an open reading frame located between the 5 'and 3' untranslated regions (NTRs) (Collet et al, virology 165:191-199(1988)). From this framework, a polyprotein of about 438kD can be translated and processed into structural and nonstructural proteins of the virus by the action of cellular and viral proteases (Tautz et al, J.virol 71: 5415-5422 (1977); Xu et al, J.viro., 5312-71: 5322 (1977); Elber et al, J.viro., 70: 4131-4135 (1996); and Wiskerchen et al, virology 184: 341-350 (1991); viral enzyme systems involved in this process are N^proAnd NS3 protease. N is a radical of^proIs the first protein encoded by the open reading frame of the virus, which can cleave itself from the remaining synthetic polyprotein (Stark et al, J. Virol. 67: 7088-7093 (1993); Wiskerchhen et al, Virol. 65: 4508-4514 (1991)).

The viruses in the current BVD vaccines are chemically inactivated (Mcclaukin et al, Arch. Virol.58: 119 (1978); Fernelius et al, J.E.Res.) -33: 1421-.

In addition, Modified Live Viruses (MLV) can be obtained using BVD virus by repeated passages in bovine or porcine cells (Coggins et al, Cornell Vet 51: 539 (1961); and Pillips et al, J.veterinary Res., 36: 135(1975)) or by chemical mutagenesis to produce a temperature sensitive viral phenotype (Lobmann et al, J.veterinary Res., 45: 2498 (1984)) and (Lobmann et al, J.veterinary Res., 47: 557-561(1986)) have demonstrated that a single dose of MLV vaccine is sufficient to produce an immune effect in vaccinated cattle for a period of years (Coria et al, Can.J.Con.Med.42: 239(1978)) and further, it has been reported that MLV-vaccinated calves produce a cross-protective effect (Martin et al, animal disease investigator university paper (Aren. Ex. research) (183. discs 183. 75.),1994), a major problem with these vaccines is safety issues, such as the possibility of transfer of the virus to the matrix (Bolin, vet. Clin. North am. food Amm. practice 11: 615-.

There is clearly a need for new effective vaccines to control the spread of BVD viruses. Considering that the disease caused by the virus is one of the most transmissible diseases in cattle, which also affect the economic value, the vaccine has important significance in animal husbandry. .

The present invention is based on the discovery that N is a member of the group^proDeletion or inactivation of protease gene can result in attenuated BVD virus. These viruses are much less infectious than the corresponding wild-type viruses in bovine cell lines and are suitable for use as bovine vaccines. The entire genomic sequence of one such attenuated virus is disclosed as deposited with the American Type Culture Collection (ATCC) under accession number ATCC203354 as a plasmid encoding the virus, p BVDdN 1.

A. BVDdN1 attenuated virus-based compositions and methods

In a first aspect, the present invention is based on the study of a particular attenuated BVD virus strain. Wild type virus genome mutation deletion N^proThe full-length sequence of the strain is shown in SEQ ID NO: 1 and FIG. 2 from nucleotides 39 to 12116. The invention therefore relates to a virus, the viral genome comprising and preferably essentially consisting of the sequence as shown. Typically, BVDs have a viral genome in the form of RNA. After cloning, the DNA is usually in the form of a DNA. Unless otherwise indicated, the term "nucleic acid" refers to BVD viral DNA and RNA sequences. For convenience, the sequence listing lists only DNA sequences, and it will be readily apparent to those skilled in the art that the corresponding RNA sequences will be accessible. The term "consisting essentially of …" means substantially corresponding toSequences with the same structure and function as the already-defined sequences. Thus, the present invention includes not only the explicitly expressed sequences but also the corresponding sequences with the addition of non-essential sequences or the introduction of non-essential substitution sequences. Specifically, the invention includes nucleic acids encoding a polypeptide substantially similar to SEQ ID NO: 1 identical to the degenerate nucleic acid sequence of the BVD protein. Conveniently, the sequence, i.e. SEQ ID NO: nucleotide numbers 39-12116 of 1, and the corresponding virus encoded by it are referred to as "BVDdN 1" genome and virus. The virus may be part of a relatively crude preparation or may be in substantially purified form, i.e. substantially free of any other virus type.

The invention includes host cells carrying a bvdn 1 nucleic acid molecule of the invention. The term "host cell" includes any prokaryotic cell carrying a bvdn 1 nucleic acid molecule, and any eukaryotic cell infected with a virus or carrying a bvdn 1 nucleic acid molecule. Among prokaryotic cells, the plasmid amplified by STBL2 strain (GibcoBRL) of E.coli (E.coli) was found to be the most effective, and this strain is generally preferred. Among eukaryotic cells, MDBK cells (ATCC CCL-22) and RD cells (stably transformed bovine testis cells) which are mammalian cells are generally preferred. However, other cultured cells may be used. The invention also includes progeny viruses produced by such host cells.

The animal can be infected with BVDdN1 virus to induce antibody production, and the effective dose is enough to stimulate antibody production. Any animal type normally used for this purpose (e.g., mouse, immune, goat, sheep) can produce the antibody, but preferably the antibody is produced in cattle. The term "anti-BVD virus antibody" as used herein refers to an antibody having at least 100-fold higher affinity for a BVD virus strain than any other non-BVD virus strain. Although not preferred, the virus may also be inactivated by chemical treatment prior to injection into the animal, including, for example, formalin, paraformaldehyde, phenol, lactate, psoralen, platinum complexes, ozone, or other miscellaneous viral agents. Antibodies produced by these methods are themselves within the scope of the present invention and can be isolated by methods well known in the art (see, e.g., Harlow et al, antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1988)).

In another aspect, the invention relates to a vaccine comprising bvdn 1 virus and a veterinarily acceptable carrier. The vaccine may comprise any adjuvant and other agents typically used in such preparations. Administration of the vaccine to cattle induces an immune response at a dose sufficient to induce a protective immune response against subsequent BVD virus infection. Typically, the vaccine is administered parenterally, but other modes of administration are also suitable for the present invention. If desired, two or more inoculations are carried out periodically, e.g., every 2 to 8 weeks. The immunization procedure can be optimized using standard methods well known in the art.

BVDdN1 genome nucleic acid-based compositions and methods

Recent work has shown that it is possible to inject nucleic acids encoding immunogens into animals to produce effective vaccines. Methods for preparing and administering these "DNA vaccines" have been described in detail (see, e.g., U.S. Pat. Nos. 5,589,466, 5,580,859, and 5,703,055), and can be used for BVDdN1 genomic nucleic acid. Thus, in another aspect, the invention relates to a nucleic acid molecule, preferably in substantially pure form, comprising the nucleic acid sequence of seq id NO: 1 from nucleotide 39 to nucleotide 12116, or a degenerate variant thereof. In a preferred embodiment, the invention relates to a nucleic acid molecule, preferably in substantially pure form, consisting essentially of the nucleic acid sequence of SEQ ID no: NO: 1 from nucleotide 39 to nucleotide 12116. As used herein, "substantially pure" means that the desired product is substantially free of impurities. For example, a "substantially pure" nucleic acid molecule is one which is substantially free of other contaminating nucleic acid molecules in the sample and generally comprises at least 85% by weight of the nucleic acid molecule, with higher percentages being preferred. One method of determining the purity of nucleic acids is to electrophorese the preparation in a matrix such as polyacrylamide or agarose. After staining, the molecule was pure if only a single band appeared. Other methods of analyzing purity include chromatography and analytical centrifugation.

Bvdn 1 genomic nucleic acid may be ligated into the vector as an independent coding element. The phrase "separate coding element" refers to a portion of the vector that is translatable into a viral polypeptide and ultimately into a virus. It is independent in that it does not comprise any other translational element that can substantially alter the bvdn 1 product. The vector, or bvdn 1 nucleic acid itself, can be used to transfect a host cell to obtain an attenuated progeny virus.

The invention also includes methods of inducing the production of anti-BVD antibodies by injecting bvdn 1 nucleic acid or a vector containing the nucleic acid directly into an animal. Any animal capable of producing antibodies may be used, with bovine generally preferred. The antibodies produced in this way are part of the invention and may be purified from animals and used, for example, in methods for detecting the presence of BVD virus in a culture or biological fluid.

Vaccines for administration to cattle (see references cited above) may be prepared on the basis of bvdn 1 genomic nucleic acid in combination with a veterinarily acceptable vector, and used to induce protective immunity against subsequent viral infection in an optimized immunization protocol.

Mutation method of C wild type BVD genome

In general, the present invention relates to a method to modify the genome from a substantially pure wild-type BVD virus to make it suitable for use as a vaccine. The term "substantially pure" as used herein refers to a virus preparation which preferably consists of a single BVD virus strain without the presence of other types of viruses. The method is mainly characterized in that the genome is mutated to inactivate N^proA protease gene. In this context, a product is considered to be inactivated if it is not produced (e.g., a gene deletion), or if it is no longer capable of normal biological function (e.g., proteolytic cleavage), or if it is produced with a significantly reduced efficiency despite its normal biological function. Available enable N^proAny method of protease inactivation. For example, genomic RNA is isolated from wild-type BVD virus by standard methods, reverse transcribed to form cDNA, and cloned. Using some method to make N^proIntroduction of mutations into protease genes, such as Polymerase Chain Reaction (PCR), site-directed mutagenesis methods,Synthesis and ligation of DNA fragments to make N^proPartial or total elimination, or random mutagenesis techniques include, for example, exposure to chemical mutagens or radiation as known in the art, or a combination of these methods.

Once the BVD viral genome has been modified to N^proThe gene is inactivated and can be cloned into a suitable vector and amplified in large quantities. As mentioned above, the vector should contain BVD sequences which as an independent element comprise or essentially consist of mutated wild type viral sequences. The mutated BVD genome or a vector containing the genome can be transformed or transfected into a host cell for the bulk amplification of the viral nucleic acid or the virus itself.

anti-BVD virus antibodies can be obtained by administering to an animal any wild-type BVD virus genome mutated by the above-described methods, as described above for bvdn 1 genomic DNA. Generally, it is preferred to produce antibodies in cattle, but other animals may be used.

Vaccines comprising the mutated BVD genomic nucleic acid have been obtained and used to induce an immune response in cattle following standard DNA immunization protocols (as described in U.S. Pat. Nos. 5,589,466, 5,580,859, and 5,703,055). Vaccines, antibodies and nucleic acids obtainable by the methods described herein are part of the present invention.

Method for producing attenuated BVD viruses

It has been found that when BVD virus nucleic acid is mutated to make N^proWhen protease gene is inactivated, attenuated viruses having a greatly reduced infectivity on cultured cells can be produced. The relatively slow replication rate of these attenuated viruses may allow the animal to mobilize its immune defense mechanisms in a manner that is not possible in the case of a rapidly proliferating wild-type virus infection. Thus, the method of generating the bvdn 1 mutant virus genome as described above directly results in a general method of generating an attenuated BVD virus, making it suitable for use as a vaccine. In general the method comprises isolating the wild-type BVD virus, cloning its genomic nucleic acid, mutating the cloned nucleic acid so that N is present^proInactivating protease gene, and addingThe mutated nucleic acid is transformed or transfected into a host to produce an attenuated virus. Although any method described above that can generate mutations can be used, it is preferred to make N^proA method of deleting all or part of a protease gene.

The invention encompasses not only methods for producing attenuated viruses, but also the viruses themselves, virally infected host cells, and progeny viruses produced by such host cells. Infection of an animal, preferably a bovine, with an effective dose of an attenuated BVD virus can result in the production of antibodies against the attenuated BVD virus. Antibodies produced by this method are part of the invention and these antibodies can be isolated and used to diagnose or detect the presence of BVD in cell culture.

As described above with respect to BVDdN1, the feature may be N^proAttenuated viruses with inactivated protease genes are incorporated into vaccines for inducing an immune response in cattle. The dosage and immunization regimen can be optimized to generate a protective immune response in the vaccinated animal against subsequent viral infection.

FIG. 1 (groups A and B): panel a is a graphical illustration of plasmid pVVNADL. The plasmid is mutated to introduce the first gene in the open reading frame, N^proProtease, deletion. The resulting mutant plasmid was p BVDdN1, see panel B. Several other gene regions are also shown in FIG. 1. C represents a gene encoding a packaging genomic RNA and a core structural protein forming a virus particle. Followed by genes encoding the three envelope glycoproteins-E0, E1 and E2. P7 encodes a nonstructural protein of unknown function, followed by the region designated "NS 2-insertion-NS 3". NS2 encodes a highly hydrophobic protein containing a zinc finger motif. NS3 is highly hydrophilic and is a hallmark of cytopathic BVD viruses. The ncp virus can be transformed into the cp biotype upon replication in infected animals by genetic recombination, which involves the insertion of additional viral or cellular RNA sequences between the NS2 and NS3 coding regions. The result of this recombination was the release of free NS2 and NS3 proteins. The latter, NS3, is a protease responsible for the processing of most nonstructural proteins. NS4A is next to NS3, whose coding is known to involveAnd a cofactor for the protease activity of NS 3. Next to NS4A were two genes encoding the viral proteins NS4B and NS5A, the functions of which were unknown. The last gene is NS5B, which encodes an RNA-dependent RNA polymerase, responsible for viral replication. The nucleotide sequence shown in group B (SEQ ID NO: 9) is the sequence around the start code of p BVDdN 1.

FIG. 2 shows the entire nucleotide sequence of plasmid pBVDdN 1. The genomic sequence of BVDdN1 is the sequence from nucleotide 39 to nucleotide 12116.

The data in FIG. 3 show in vivo seroconversion in cattle following administration of BVDdN1 virus.

Preparation of A BVDdN1 and nucleic acid encoding the Virus

The present invention relates to N deletion^proBVD viruses with protease genes attenuated accordingly. This virus is designated bvdn 1 and, as the term "attenuated", refers to it replicating at a much slower rate in susceptible cell lines, such as bovine testicular cell line (RD), or bovine kidney cell line (MDBK), than the wild-type virus. In addition, bvdn 1 did not cause a productive infection in fetal bovine tracheal cells (EBTr) or bovine turbinate cells (BT-2), which could lead to a productive infection in contrast to a wild-type viral infection. The slow growth of bvdn 1 virus in different bovine cell lines suggests a broad tissue tropism attenuation in animals. BVDdN1 is stable genetically and can maintain N after 10 generations in bovine RD cell line^proIs absent. Although the viral genome is RNA in nature, it can be cloned after reverse transcription. It will be appreciated that reference herein to nucleic acid and BVD viral sequences includes not only DNA sequences reverse transcribed from viral RNA sequences, but also the corresponding RNAs themselves.

The complete nucleotide sequence of the viral genome of bvdn 1 is shown in SEQ ID NO: nucleotide numbers 39 to 12116 in 1. It is to be understood that the invention encompasses not only the viral genome of the precise sequence shown, but also other sequences that differ substantially in structure or function therefrom, including sequences that encode sequences that differ from the nucleotide sequence of SEQ id no: 1, the same sequence as that of the BVD protein shown in (1). Alternatively, variations can be introduced into the nucleic acid structure, for example, by well-known techniques such as site-directed mutagenesis. Mutations in the nucleic acid sequence of BVD viruses produced by this or similar methods or by random mutagenesis methods known in the art are also encompassed by the present invention, provided that the virus produced retains at least one major biological property which is essentially the same as the virus from which it was derived. In particular, mutations that do not substantially alter the infectious properties of BVDdN1 fall within the scope of the present invention.

Mutated BVDdN1 the mutated nucleic acid was derived from the National Animal Disease Laboratory (NADL) BVD strain, obtained from ATCC (VR-534). This was ligated to a vector and full-length N was ligated by selective PCR and religation as shown in the examples below^proThe protease gene is deleted. Although the viral genome and ultimately the virus itself can be obtained by this method, a plasmid named bvdn 1 containing the complete bvdn 1 genomic sequence has been deposited with the ATCC under accession No. 203354. This plasmid is the preferred source in the isolation process. The plasmid can be amplified and purified by standard methods and transfected into a host cell that can sustain virus production. The prokaryotic host cell used for plasmid amplification is preferably E.coli STBL2 cell (from Gioco BRL), but other cell types may also be used. The virus may be prepared in eukaryotic cells such as RD or MDBK cells and isolated therefrom in its highly purified form by known isolation methods such as sucrose gradient centrifugation for use as a vaccine or for the production of antibodies. Alternatively, the plasmid may be used to isolate bvdnn 1 genomic sequence, which may be used directly for antibody production or for vaccines.

Preparation of other attenuated BVD Virus strains

The basic methods for generating bvdn 1 virus and genomic nucleic acid can be applied equally to other BVD wild virus strains. In each case, the wild-type virus was isolated such that N was^proThe protease gene is inactivated to achieve the aim of attenuation. Preferably, the entire gene is deleted for attenuation by a PCR-based method as used herein for BVDdN 1. However, other methods of gene inactivation may be used, such as deletion of portions of the sequence, or introduction of mutations randomly or at specific positions.In all cases, the aim was to generate mutant viruses that proliferate at a low rate after infection. The infectivity of the virus can be determined in vitro by immunohistochemical methods using monoclonal antibodies specific for BVD virus, as described in the examples section.

Preparation of C-anti-BVD attenuated Virus antibody

anti-BVD virus antibodies can be prepared in any animal commonly used to produce antibodies, including, e.g., mice, immunizations, and the like. But preferably the antibody is produced in cattle. The virus-containing composition is administered to the animal by any route, but the general route is intramuscular injection, subcutaneous injection or intravenous injection into the animal. In general, the viral formulation includes an adjuvant such as Freund's complete or incomplete adjuvant. Suitable formulations for injection, methods of injection, and The like are well known in The art and may be used (see, e.g., Harlow et al, Antibodies: A Laboratory Manual, Coldspring Harbor Lab. N.Y. (1988); Klein, Immunology: self-non-self recognition Science (Immunology; The Science of self-Nonself Discrimation (1982)), Monoclonal Antibodies and hybridomas can also be prepared using standard methods (Kennett et al: Monoclonal Antibodies and hybridomas: New thinking of Biological analysis (Monoclonal Antibodies and hybridomas) (1980); Camcell "Monoclonal Antibody Technology" (Monoclonal Antibody Technology) biochemical and Molecular Biology Laboratory Technology (Laboratory technologies Biology) (1984).

Antibodies or antibody fragments specific for BVD viruses (i.e. having at least 100-fold greater affinity for BVD than other types of viruses) may be used in any immunological assay. For example, in a radioimmunoassay or immunometric Assay (also known as a "two-site" or "sandwich" Assay), BVD virus is detected using the antibody (see Chard "radioimmunoassay and related technical Introduction" (An Introduction to Radioimmune Assay and related techniques), N.Y. (1978) in a typical immunometric Assay, a quantity of unlabelled antibody is bound to a solid support which is insoluble in the liquid to be tested, e.g., blood, lymph, cell extracts, etc. after initial binding of the antigen to the immobilized antibody, a quantity of a detectable second antibody (which may be the same as or different from the first antibody) is added to detect and/or quantify the antigen (see, E & S immunization Methods, rikkam et al, eds 199. page 206. E & S likrienvine et al (1970. many different Methods known in the art), it can be used for detecting BVD virus.

D conventional vaccine (preparation) and vaccination method

Many documents discuss the methods of vaccine preparation and vaccination using BVD virus (see, e.g., Fennelius et al, J.veterinary Res.33: 1421-1431 (1972); Kolar et al, J.veterinary Res.33: 1415-1420 (1972); McClurkin et al, virology (Arch.Virol) 58: 119 (1978); Coggins et al, Cornellt.51: 539 (1961); Pillips et al, J.Res.36: 135 (1975); Lobmann et al, U.S. veterinary Res 45: 2498 (1984); Coria et al, Can.J.Comp.Med.42: 197239 (8); the Proceedings et al of the 1994 research works utensil 75: 183: 4,618,493; and U.S. patent No. 4,618,493). Typically, a vaccine contains about 1X 10⁶To 1X 10⁸Individual viral particles, and a veterinarily acceptable carrier in a volume of 0.5-5 ml. Can be formulated as described in Remington's pharmaceutical sciences (Mack Publishing CO. Easton, Pa 16 th edition 1982). The present invention encompasses a variety of excipients and adjuvants, which may be incorporated into the formulation as desired. For example, the vaccine compositions of the present invention may be formulated conventionally with standard buffer salts, carriers, stabilizers, diluents, preservatives and cosolvents, or may be formulated for convenient sustained release. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic additives may include sodium chloride, dextrose, mannitol, sorbitol, lactose, and others. Stabilizers include albumin and others. Non-limiting examples of adjuvants are RIBI adjuvant System (Ribi Inc), Alum, aluminum hydroxide gel, oil-in-water emulsion, water-in-oil emulsion such as Freund's complete and incomplete adjuvant, Block co-polymer (Cyt)Rx，Atlanta GA)、SAF-M(Chiron，Emery Ville CA)、AMPHIGEN^Adjuvants, saponins, Quil a, QS-21(Cambridge Biotech inc., Cambridge ma) or other saponin components, monophosphoryl lipid A, Avridine lipoamine adjuvant, escherichia coli-derived heat-labile enterotoxin (recombinant or otherwise), cholera toxin, or muramyl dipeptide, and the like. The vaccine may also include one or more other immunomodulators such as interleukins, interferons or other cytokines. Vaccines are typically administered parenterally, but the invention is also applicable to other routes, such as oral, intranasal, intramuscular, intralymphatic, intradermal, intraperitoneal, subcutaneous, rectal or vaginal administration, or administration using a combination of routes. The skilled artisan can formulate the vaccine composition according to the chosen route.

Methods of immunization can be optimized using methods well known in the art. The animals may be administered a single dose or, alternatively, two or more vaccinations at intervals of 2 to 10 weeks. If necessary, sera from vaccinated animals are collected and tested for the presence of anti-BVD virus antibodies.

The term "induction of an immune response" and similar terms, as used herein, broadly includes the induction or increase of any immune-based response to a vaccine in a bovine subject, including antibody or cell-mediated immune responses that protect vaccinated animals against BVD virus, or both. The terms "protective immunity", "protective immune response", "protection" and the like as used herein are not limited to absolute protection of cattle from viral diarrhea, or BVD virus infection in cattle, but are intended to include any reduction in the extent or rate of infection by any pathogen, or any relief in the severity or any symptoms or state of disease caused by infection by a pathogen, as compared to an unvaccinated infected animal.

E DNA vaccine

References to methods of vaccine preparation and vaccination using nucleic acids (DNA or mRNA) include U.S. Pat. Nos. 5,703,055, 5,580,859, 5,589,466, International patent publication No. WO 98/35562, and various scientific publications including Ramsay et al, 1997, immunocytobiology (immunol. cell Biol) 75: 360-363; davis, 1997, current view of biotechnology (cur. opinion Biotech) 8: 635-640; manickan et al, 1997, an immunological reviews (Critical Rev. immunological) 17: 139-154; robinson, 1997, Vaccine (Vaccine)15 (8): 785-787; robinson et al, 1996; AIDS res.hum.retr.12 (5): 455-; lai and Bennet, 1998, immunological reviews 18: 449-; and Vogel and Sarver, 1995, review in clinical microbiology (clin. microbiol, Rev)8 (3): 406, 410, incorporated herein by reference. These methods can be used to generate vaccines against BVD virus, wherein a nucleic acid corresponding to BVDdN1 nucleic acid, or corresponding to N-channel, is administered to cattle^proInactivation of the protease gene results in an attenuated nucleic acid resembling the BVD viral genome or a degenerate variant thereof. Vectors containing these nucleic acid molecules may also be used. Immunogens delivered in this manner will generally elicit both humoral and cell-mediated immune responses.

Either DNA or RNA encoding the attenuated BVD virus genome may be used as a vaccine. The DNA or RNA molecule may be in "naked" form or administered with a component that promotes cellular uptake, such as a liposome or cationic lipid. The mode of administration is generally intramuscular injection of about 0.1 to 5ml of vaccine. The total amount of polynucleotide in the vaccine should generally be between about 0.1. mu.g/ml and 5 mg/ml. The polynucleotide may be in the form of a suspension, solution or emulsion, but aqueous carriers are generally preferred. The immunization process is completed by a single vaccination or multiple vaccinations. If desired, sera from vaccinated animals are collected and tested for the presence of anti-BVD virus antibodies.

The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: analytical and experimental methods

DNA

Infectious full-length pVVNADL clones are shown in figure 1A. The plasmid contains a plasmid derived from pGE₄ColE of (Promega Corp.)₁Replicons of 14,578bp in length (Vassilev et alHuman, journal of virology 71: 471-478(1997)). A T7RNA polymerase promoter is inserted upstream of the BVD viral genome, which promoter directs the virus to synthesize RNA. The BVD virus genome sequence is derived from NADL strain of BVD virus (ATCCVR-534).

pVVNDL amplification in E.coli

In general, it is difficult to amplify full-length pVVNADL clones in e. Amplification of pestivirus cDNA and full-length cloning in E.coli has been reported to produce deleterious effects (Moormann et al, J. Virol 70: 763-34770 (1996); Rugglie et al, J. Virol 70: 3478-3487 (1996)). The stability of pVVNADL was tested in several bacterial hosts including E.coli JM109(stratagene), DH 5 alpha (GibcoBRL) and STBL2 cells (GibcoBRL). After transformation of plasmid DNA into these strains, the clone sizes were examined and the general structure of the plasmids was analyzed by restriction mapping. The best results were obtained with STBL2 cells. The pVVNADL transformation into these cells can produce a relatively single small clonal population, and in the limited growth environment (30 ℃ for no more than 20 hours) no evidence of DNA rearrangement is found, and the DNA yield is reasonable.

In vitro transcription and RNA transfection

RNA transcripts were synthesized in vitro with T7RNA polymerase using MEGAscript reagent (Am bion) as per the manufacturer's instructions. After linearization of the pVVNADL DNA template with SacII, the 3' overhang was removed by treatment with T4DNA polymerase. The transcription reaction products were analyzed by gel electrophoresis. 1 to 5. mu.g of RNA transcript were added to 200. mu.l of Opti-MEM (GibcoBRL) containing 6. mu.g of lipofectin (GibcoBRL), and the RNA/lipid samples were incubated at room temperature for 10 to 15 minutes. During this period, monolayers (50-60% confluent) of MDBK (from Madin Darby bovine kidney cells (clone 6)) or RD (stably transformed bovine testis cell line) cells grown in 6-well plates (35 mm diameter) were washed twice with RNase-free PBS and once with Opti-MEM. After the last wash, the transfection mixture was added to each well and incubated for 10 minutes at room temperature with gentle shaking. 1ml of Opti-MEM was added to each well and incubated at 37 ℃ for 3 hours. 3ml of Opti-MEM containing 5% fetal horse serum (RD cells) or fetal bovine serum (MDBK cells) was added to each well. After incubation at 37 ℃ for 1 to 4 days, the cells were fixed with 80% acetone and BVD plaques were observed by immunohistochemistry.

Example 2: n is a radical of^proConstruction of Gene-deleted BVD Virus clone

To obtain deletion N in the genome^proThe virus of gene is prepared by preparing 3 DNA fragments and then connecting the DNA fragments. The exact procedure is as follows.

Obtaining PCR fragment 1

"PCR fragment 1" contains N^proDeletion of the coding sequence. Three PCR amplification reactions were required to obtain this fragment. First, N was amplified using 5NTR3(+) and 5NTR4(-) as primers^proHalf of the 5' NTR region upstream of the coding sequence. The 5 'positive sense primer 5' NTR3(+) sequence is: 5'-AAAGGTCTCGAGATGCCACG-3' (oligonucleotide 218-237, SEQ ID NO: 2). The 5 'NTR 4(-) sequence of the 3' negative antisense primer is 5'-GTCTGACATGTGCCATGTACAGCAGAGATTTTTAGTAGC-3' (oligonucleotide 895-. Both primers are located in the 5' NTR region of the viral genome, and the 5NTR3(+) primer contains a single Xhol restriction site. The 5 'end of the 5NTR4(-) primer contained 6 additional oligonucleotides homologous to the 5' end of the BVD virus C protein coding sequence. In the PCR amplification reaction, primers were used at a final concentration of 0.5. mu.M, 10ng of DNA template of plasmid pVVNADL, and 2.5U of Pfu DNA polymerase (Stratagene, La Jolla, Calif.). 20 cycles of amplification were performed using the following conditions: denaturation at 94 ℃ for 30 seconds; annealing at 55 ℃ for 1 min and extension at 72 ℃ for 2 min. The 177 base pair fragment (fragment A) produced after agarose gel electrophoresis purification was resuspended in TE buffer.

Another PCR amplification reaction was performed using oligonucleotides NADLC6(+) and Seq23(-) as primers to amplify N^proA downstream fragment of a coding sequence. The 5' sense primer NADLC6(+) sequence is: 5'-CACATGTCAGACACGAAAGAAGAGGGAGC-3' (oligonucleotide 383-388+890-913, SEQ ID NO: 4). The 3' antisense primer Seq23(-) sequence is: 5'-CAGGTTTGCAATCCAAGTGCCC-3' (oligonucleotide 2480-2459, SEQ ID NO: 5). Primer NADLC6 was located at the N-terminus of protein C and included 3 additions homologous to the 3 'terminus of the 5' NTRNucleotide, which has an ATG start code at its 5' end. The primer Seq23(-) is near the N-terminus of the E2 protein. The plasmid pVVNADL was used as template in the amplification reaction, the reaction conditions were as described above. The resulting DNA fragment (fragment B) was purified by agarose gel electrophoresis and was 1596bp in length.

The third amplification reaction used 0.5. mu.M concentration of oligonucleotides 5' NTR3(+) (SEQ ID NO: 2) and Seq23(-) (SEQ ID NO: 5) as primers, fragment A and fragment B as templates, plus 2.5U Pfu DNA polymerase (Stratazone, La fola, CA). The first four cycles of the amplification process were: denaturation at 94 ℃ for 30 seconds; annealing at 40 ℃ for 1 minute; extension at 72 ℃ for 2 min. The conditions immediately following 20 cycles were: denaturation at 94 ℃ for 30 seconds; annealing at 60 ℃ for 1 min and extension at 72 ℃ for 2 min. The final product thus obtained was "PCR fragment 1" and 1767bp long. It was used to cleave the fragment with XhoI and PvuI to form a 1175bp long fragment before ligation.

Obtaining PCR fragment II

"PCR fragment II" was obtained using the oligonucleotides Seq 2(+) and Seq 24(-) as primers. The 5' sense primer Seq 2(+) sequence is as follows: 5'-GGAGCATACGCTGCTTCCCC-3' (oligonucleotide 1865-1884, SEQ ID NO: 6). The 3' antisense primer Seq 24(-) sequence is: 5'-GCCTTGCCTATGAGGGAATGG-3' (oligonucleotide 2963-2942, SEQ ID NO: 7). Oligonucleotide Seq 2(+) near the N-terminus of E1 and oligonucleotide Seq 24(-) near the middle of the E2 region. Amplification was performed using plasmid pVVNADL DNA as template and the reaction conditions were as described above for the amplification reaction using the A and B fragments. The resulting fragment was called "PCR fragment II" and was 1098bp in length. It was cleaved with PvuI and RsrII before ligation to form a 929bp fragment.

Obtaining the vector fragment III

Plasmid pVVDADL of 14579bp was digested with XhoI and RsrII to give a 11974bp long fragment, called "vector fragment III".

Obtaining the plasmid pBVDdN1

PCR fragments I and II and vector fragment III were mixed in a molecular ratio of 2: 1, using 200 units of T4DNA ligase (Boehringer Mann)heim) and incubated overnight at 16 ℃. The ligation products were transformed into E.coli STBL2 cells and heterologous clones were screened by small DNA purification and specific restriction enzyme digestion. The plasmid with the expected length (14079bp) was further subjected to sequence analysis. The resulting p BVDdN1 plasmid, which contains N from the BVD viral genome, is shown in FIG. 1^proDeletion of protease gene. The pBVDdN1 vector background is the same as pVVNAL.

Example 3: n is a radical of^proCharacterization of the Gene deleted BVD Virus clone

N^proInfectivity of Gene deleted BVD Virus clone pBVDdN1

RNA was synthesized in vitro from pBVDdN1 and pVVNADL (positive control) as described before and RNA transfected on RD monolayer cells with lipofectamine. At 48, 72 and 96 hours post-transfection, supernatants of transfected cells were collected and used to re-infect fresh RD monolayers. Transfected cells were fixed with 80% acetone and immunohistochemical analysis was performed using the Vectastain Elite ABC kit (Vector Laboratories). Monoclonal antibodies used to detect BVD-specific viruses were 15C5 (specific for E0) and CA3 (specific for E2) (Pfizer inhibitor), other monoclonal antibodies against these antigens can also be prepared using standard methods and used in the same method. These antibodies were diluted 1: 1000 at the time of use. E0 and E2 envelope proteins and the resulting virus were detected 24 hours after transfection with RNA from the parental virus. In contrast, E0 and E2 proteins were detected in cells 48 hours after transfection with RNA from p BVDdN 1. Bvdnn 1 virus was not produced until 72 hours post-transcription.

Phenotypic analysis

Single layer cells of RD and MDBK were seeded with bvdn 1 strain from early passage (3 RD generation). These cells were compared to control cells inoculated with the parental virus. Monolayer cells were fixed with 80% acetone 20 hours (RD cells) or 24 hours (MDBK cells) after transfection. Fixed cells were immunohistochemically analyzed with the E2-specific monoclonal antibody CA3 diluted 1: 1000 and examined microscopically. The parental viral replication rate was found to be much faster than the bvdn 1 viral replication rate in both cell types.

Genotyping analysis

Using Ultraspec according to the manufacturer's instructions^TMRNA reagent (biotect) RNA of the parental virus and bvdn 1(3 RD generation) was purified from infected RD monolayer cells. RT/PCR reactions were performed using RT-PCR beads (Pharmacia Biotech) and the oligonucleotides NADLE07(-) and 5NTR3 (+). The 5NTR3(+) oligonucleotide sequences and positions are as described above. The sequence of the NADLE07(-) oligonucleotide is: 5'-CACTTGCATCCATCATACC-3' (antisense, oligonucleotide 1379-1361, SEQ ID NO: 8). The oligonucleotide was located approximately 150bp from the N-terminus of E0. RT/PCR was found to give 1162bp sized fragments from parental viral RNA. RT/PCR A661 bp fragment was obtained from BVDdN1 RNA, corresponding to the expected deletion of N^proThe fragments of the protease genes are of the same size. The RT/PCR fragment obtained from the RNA of the parental virus and BVDdN1 was sequenced and in both cases the resulting sequences were as expected, corresponding to the arrangement of elements shown in FIG. 1. The complete sequence of bvdn 1 is shown in SEQ ID NO: 1 from nucleotide 39 to nucleotide 12116.

Example 4: efficacy study of BVDdN1

The objective of this study was to evaluate the ability of bvdn 1 containing vaccines to seroconvert calves. 15 animals were randomly assigned to one room (10 were double-dosed and 5 were used as observation controls). Another 10 animals were randomized to another room (single dose vaccination, no control observed). 2.0ml of BVDdN1 virus in MDBK cell lysate at 10 deg.C⁷TCID₅₀Dose per animal animals were administered subcutaneously. When immunisation was performed, 5 animals designated as observation controls were left from the room. The remaining 10 calves were inoculated with bvdnn 1 virus. Approximately 24 hours after immunization, the observation control animals were returned to the inoculation room. The first 10 animals were vaccinated with the second dose of vaccine in the same manner about 28 days after the first dose.

Rectal temperature was measured on days-1, 0 (pre-inoculation), 1, 2, 3, 4,5, 6, 7, 8, 9, 10 (all groups of animals) and on days 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 and 38 (double dose groups of animals). Blood samples were collected from the double dose group on day 0 and weekly thereafter (i.e., on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91). Blood samples were collected from single dose groups of animals on days 0, 7, 14, 21, 28, 35, 42, 49, 56 and 63.

Serum neutralizing antibodies (BVD virus isolate 5960 for type I and isolate 890 for type II) were tested by SN analysis to monitor for the presence of the expected homologous and heterologous protective effects.

No significant difference was observed in either gross or rectal temperature. No seroconversion was observed in any of the control animals observed during the study (data not shown).

Neutralization analysis in type I sera revealed that all animals vaccinated with bvdn 1 virus had seroconverted. Positive titers of 1: 8 or greater were seen in 60% of the animals and 1: 8 or greater in 90% of the animals on day 35 after single dose vaccination, and were maintained at high titer levels later on (FIG. 3A). Neutralization analysis in type II sera found that 70% of the animals were positive 63 days after inoculation (data not shown). After double-dose inoculation, the type I serum neutralization assay showed that all animals developed a positive titer of 1: 64 or greater on day 7 and later were maintained at similar seroconversion levels (fig. 3A). Neutralization analysis of type II sera revealed positive titers in most animals 7 days after the second inoculation, with at least 60% of the animals appearing to have a positive titer of 1: 8 or higher on day 28 (FIG. 3B). These results indicate that bvdn 1 virus can replicate in cattle and induce positive neutralizing sera against type I and type II viruses, indicating that the virus can be used as a vaccine against BVD virus.

Biological material preservation

Plasmid pBVDdN1 was deposited at 20.10.1998 at Blvd, American Type Culture Collection (ATCC), university 10801, Manassas, VA, 20110, USA, with accession number ATCC 203354.

All patents, patent applications, and publications mentioned above are incorporated herein by reference in their entirety.

The embodiments of the present invention are merely illustrative of one aspect of the present invention, and the scope of the present invention is not limited thereto. Compositions and methods of equivalent function are within the scope of the invention. Indeed, various modifications in addition to those mentioned 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.

Sequence listing

<110> Peui products Co

<120> attenuated bovine viral diarrhea virus

<130>PC10435A

<140>

<141>

<150>60/107,908

<151>1998-11-10

<160>9

<170>PatentIn Ver.2.0

<210>1

<211>14078

<212>DNA

<213> bovine attenuated diarrhea virus

<400>1

cacgcgtatc gatgaattcg ttaatacgac tcactatagt atacgagaat tagaaaaggc 60

actcgtatac gtattgggca attaaaaata ataattaggc ctagggaaca aatccctctc 120

agcgaaggcc gaaaagaggc tagccatgcc cttagtagga ctagcataat gaggggggta 180

gcaacagtgg tgagttcgtt ggatggctta agccctgagt acagggtagt cgtcagtggt 240

tcgacgcctt ggaataaagg tctcgagatg ccacgtggac gagggcatgc ccaaagcaca 300

tcttaacctg agcgggggtc gcccaggtaa aagcagtttt aaccgactgt tacgaataca 360

gcctgatagg gtgctgcaga ggcccactgt attgctacta aaaatctctg ctgtacatgg 420

cacatgtcag acacgaaaga agagggagca acaaaaaaga aaacacagaa acccgacaga 480

ctagaaaggg ggaaaatgaa aatagtgccc aaagaatctg aaaaagacag caaaactaaa 540

cctccggatg ctacaatagt ggtggaagga gtcaaatacc aggtgaggaa gaagggaaaa 600

accaagagta aaaacactca ggacggcttg taccataaca aaaacaaacc tcaggaatca 660

cgcaagaaac tggaaaaagc attgttggcg tgggcaataa tagctatagt tttgtttcaa 720

gttacaatgg gagaaaacat aacacagtgg aacctacaag ataatgggac ggaagggata 780

caacgggcaa tgttccaaag gggtgtgaat agaagtttac atggaatctg gccagagaaa 840

atctgtactg gcgtcccttc ccatctagcc accgatatag aactaaaaac aattcatggt 900

atgatggatg caagtgagaa gaccaactac acgtgttgca gacttcaacg ccatgagtgg 960

aacaagcatg gttggtgcaa ctggtacaat attgaaccct ggattctagt catgaataga 1020

acccaagcca atctcactga gggacaacca ccaagggagt gcgcagtcac ttgtaggtat 1080

gatagggcta gtgacttaaa cgtggtaaca caagctagag atagccccac acccttaaca 1140

ggttgcaaga aaggaaagaa cttctccttt gcaggcatat tgatgcgggg cccctgcaac 1200

tttgaaatag ctgcaagtga tgtattattc aaagaacatg aacgcattag tatgttccag 1260

gataccactc tttaccttgt tgacgggttg accaactcct tagaaggtgc cagacaagga 1320

accgctaaac tgacaacctg gttaggcaag cagctcggga tactaggaaa aaagttggaa 1380

aacaagagta agacgtggtt tggagcatac gctgcttccc cttactgtga tgtcgatcgc 1440

aaaattggct acatatggta tacaaaaaat tgcacccctg cctgcttacc caagaacaca 1500

aaaattgtcg gccctgggaa atttggcacc aatgcagagg acggcaagat attacatgag 1560

atggggggtc acttgtcgga ggtactacta ctttctttag tggtgctgtc cgacttcgca 1620

ccggaaacag ctagtgtaat gtacctaatc ctacattttt ccatcccaca aagtcacgtt 1680

gatgtaatgg attgtgataa gacccagttg aacctcacag tggagctgac aacagctgaa 1740

gtaataccag ggtcggtctg gaatctaggc aaatatgtat gtataagacc aaattggtgg 1800

ccttatgaga caactgtagt gttggcattt gaagaggtga gccaggtggt gaagttagtg 1860

ttgagggcac tcagagattt aacacgcatt tggaacgctg caacaactac tgctttttta 1920

gtatgccttg ttaagatagt cagggggcca gatggtacag ggcattctgt ggctactatt 1980

gataacaggg gtacaagggc acttggattg caaacctgaa ttctcgtatg ccatagcaaa 2040

ggacgaaaga attggtcaac tgggggctga aggccttacc accacttgga aggaatactc 2100

acctggaatg aagctggaag acacaatggt cattgcttgg tgcgaagatg ggaagttaat 2160

gtacctccaa agatgcacga gagaaaccag atatctcgca atcttgcata caagagcctt 2220

gccgaccagt gtggtattca aaaaactctt tgatgggcga aagcaagagg atgtagtcga 2280

aatgaacgac aactttgaat ttggactctg cccatgtgat gccaaaccca tagtaagagg 2340

gaagttcaat acaacgctgc tgaacggacc ggccttccag atggtatgcc ccataggatg 2400

gacagggact gtaagctgta cgtcattcaa tatggacacc ttagccacaa ctgtggtacg 2460

gacatataga aggtctaaac cattccctca taggcaaggc tgtatcaccc aaaagaatct 2520

gggggaggat ctccataact gcatccttgg aggaaattgg acttgtgtgc ctggagacca 2580

actactatac aaagggggct ctattgaatc ttgcaagtgg tgtggctatc aatttaaaga 2640

gagtgaggga ctaccacact accccattgg caagtgtaaa ttggagaacg agactggtta 2700

caggctagta gacagtacct cttgcaatag agaaggtgtg gccatagtac cacaagggac 2760

attaaagtgc aagataggaa aaacaactgt acaggtcata gctatggata ccaaactcgg 2820

acctatgcct tgcagaccat atgaaatcat atcaagtgag gggcctgtag aaaagacagc 2880

gtgtactttc aactacacta agacattaaa aaataagtat tttgagccca gagacagcta 2940

ctttcagcaa tacatgctaa aaggagagta tcaatactgg tttgacctgg aggtgactga 3000

ccatcaccgg gattacttcg ctgagtccat attagtggtg gtagtagccc tcttgggtgg 3060

cagatatgta ctttggttac tggttacata catggtctta tcagaacaga aggccttagg 3120

gattcagtat ggatcagggg aagtggtgat gatgggcaac ttgctaaccc ataacaatat 3180

tgaagtggtg acatacttct tgctgctgta cctactgctg agggaggaga gcgtaaagaa 3240

gtgggtctta ctcttatacc acatcttagt ggtacaccca atcaaatctg taattgtgat 3300

cctactgatg attggggatg tggtaaaggc cgattcaggg ggccaagagt acttggggaa 3360

aatagacctc tgttttacaa cagtagtact aatcgtcata ggtttaatca tagctaggcg 3420

tgacccaact atagtgccac tggtaacaat aatggcagca ctgagggtca ctgaactgac 3480

ccaccagcct ggagttgaca tcgctgtggc ggtcatgact ataaccctac tgatggttag 3540

ctatgtgaca gattatttta gatataaaaa atggttacag tgcattctca gcctggtatc 3600

tgcggtgttc ttgataagaa gcctaatata cctaggtaga atcgagatgc cagaggtaac 3660

tatcccaaac tggagaccac taactttaat actattatat ttgatctcaa caacaattgt 3720

aacgaggtgg aaggttgacg tggctggcct attgttgcaa tgtgtgccta tcttattgct 3780

ggtcacaacc ttgtgggccg acttcttaac cctaatactg atcctgccta cctatgaatt 3840

ggttaaatta tactatctga aaactgttag gactgataca gaaagaagtt ggctaggggg 3900

gatagactat acaagagttg actccatcta cgacgttgat gagagtggag agggcgtata 3960

tctttttcca tcaaggcaga aagcacaggg gaatttttct atactcttgc cccttatcaa 4020

agcaacactg ataagttgcg tcagcagtaa atggcagcta atatacatga gttacttaac 4080

tttggacttt atgtactaca tgcacaggaa agttatagaa gagatctcag gaggtaccaa 4140

cataatatcc aggttagtgg cagcactcat agagctgaac tggtccatgg aagaagagga 4200

gagcaaaggc ttaaagaagt tttatctatt gtctggaagg ttgagaaacc taataataaa 4260

acataaggta aggaatgaga ccgtggcttc ttggtacggg gaggaggaag tctacggtat 4320

gccaaagatc atgactataa tcaaggccag tacactgagt aagagcaggc actgcataat 4380

atgcactgta tgtgagggcc gagagtggaa aggtggcacc tgcccaaaat gtggacgcca 4440

tgggaagccg ataacgtgtg ggatgtcgct agcagatttt gaagaaagac actataaaag 4500

aatctttata agggaaggca actttgaggg tatgtgcagc cgatgccagg gaaagcatag 4560

gaggtttgaa atggaccggg aacctaagag tgccagatac tgtgctgagt gtaataggct 4620

gcatcctgct gaggaaggtg acttttgggc agagtcgagc atgttgggcc tcaaaatcac 4680

ctactttgcg ctgatggatg gaaaggtgta tgatatcaca gagtgggctg gatgccagcg 4740

tgtgggaatc tccccagata cccacagagt cccttgtcac atctcatttg gttcacggat 4800

gcctttcagg caggaataca atggctttgt acaatatacc gctagggggc aactatttct 4860

gagaaacttg cccgtactgg caactaaagt aaaaatgctc atggtaggca accttggaga 4920

agaaattggt aatctggaac atcttgggtg gatcctaagg gggcctgccg tgtgtaagaa 4980

gatcacagag cacgaaaaat gccacattaa tatactggat aaactaaccg catttttcgg 5040

gatcatgcca agggggacta cacccagagc cccggtgagg ttccctacga gcttactaaa 5100

agtgaggagg ggtctggaga ctgcctgggc ttacacacac caaggcggga taagttcagt 5160

cgaccatgta accgccggaa aagatctact ggtctgtgac agcatgggac gaactagagt 5220

ggtttgccaa agcaacaaca ggttgaccga tgagacagag tatggcgtca agactgactc 5280

agggtgccca gacggtgcca gatgttatgt gttaaatcca gaggccgtta acatatcagg 5340

atccaaaggg gcagtcgttc acctccaaaa gacaggtgga gaattcacgt gtgtcaccgc 5400

atcaggcaca ccggctttct tcgacctaaa aaacttgaaa ggatggtcag gcttgcctat 5460

atttgaagcc tccagcggga gggtggttgg cagagtcaaa gtagggaaga atgaagagtc 5520

taaacctaca aaaataatga gtggaatcca gaccgtctca aaaaacagag cagacctgac 5580

cgagatggtc aagaagataa ccagcatgaa caggggagac ttcaagcaga ttactttggc 5640

aacaggggca ggcaaaacca cagaactccc aaaagcagtt atagaggaga taggaagaca 5700

caagagagta ttagttctta taccattaag ggcagcggca gagtcagtct accagtatat 5760

gagattgaaa cacccaagca tctcttttaa cctaaggata ggggacatga aagaggggga 5820

catggcaacc gggataacct atgcatcata cgggtacttc tgccaaatgc ctcaaccaaa 5880

gctcagagct gctatggtag aatactcata catattctta gatgaatacc attgtgccac 5940

tcctgaacaa ctggcaatta tcgggaagat ccacagattt tcagagagta taagggttgt 6000

cgccatgact gccacgccag cagggtcggt gaccacaaca ggtcaaaagc acccaataga 6060

ggaattcata gcccccgagg taatgaaagg ggaggatctt ggtagtcagt tccttgatat 6120

agcagggtta aaaataccag tggatgagat gaaaggcaat atgttggttt ttgtaccaac 6180

gagaaacatg gcagtagagg tagcaaagaa gctaaaagct aagggctata actctggata 6240

ctattacagt ggagaggatc cagccaatct gagagttgtg acatcacaat ccccctatgt 6300

aatcgtggct acaaatgcta ttgaatcagg agtgacacta ccagatttgg acacggttat 6360

agacacgggg ttgaaatgtg aaaagagggt gagggtatca tcaaagatac ccttcatcgt 6420

aacaggcctt aagaggatgg ccgtgactgt gggtgagcag gcgcagcgta ggggcagagt 6480

aggtagagtg aaacccggga ggtattatag gagccaggaa acagcaacag ggtcaaagga 6540

ctaccactat gacctcttgc aggcacaaag atacgggatt gaggatggaa tcaacgtgac 6600

gaaatccttt agggagatga attacgattg gagcctatac gaggaggaca gcctactaat 6660

aacccagctg gaaatactaa ataatctact catctcagaa gacttgccag ccgctgttaa 6720

gaacataatg gccaggactg atcacccaga gccaatccaa cttgcataca acagctatga 6780

agtccaggtc ccggtcctat tcccaaaaat aaggaatgga gaagtcacag acacctacga 6840

aaattactcg tttctaaatg ccagaaagtt aggggaggat gtgcccgtgt atatctacgc 6900

tactgaagat gaggatctgg cagttgacct cttagggcta gactggcctg atcctgggaa 6960

ccagcaggta gtggagactg gtaaagcact gaagcaagtg accgggttgt cctcggctga 7020

aaatgcccta ctagtggctt tatttgggta tgtgggttac caggctctct caaagaggca 7080

tgtcccaatg ataacagaca tatataccat cgaggaccag agactagaag acaccaccca 7140

cctccagtat gcacccaacg ccataaaaac cgatgggaca gagactgaac tgaaagaact 7200

ggcgtcgggt gacgtggaaa aaatcatggg agccatttca gattatgcag ctgggggact 7260

ggagtttgtt aaatcccaag cagaaaagat aaaaacagct cctttgttta aagaaaacgc 7320

agaagccgca aaagggtatg tccaaaaatt cattgactca ttaattgaaa ataaagaaga 7380

aataatcaga tatggtttgt ggggaacaca cacagcacta tacaaaagca tagctgcaag 7440

actggggcat gaaacagcgt ttgccacact agtgttaaag tggctagctt ttggagggga 7500

atcagtgtca gaccacgtca agcaggcggc agttgattta gtggtctatt atgtgatgaa 7560

taagccttcc ttcccaggtg actccgagac acagcaagaa gggaggcgat tcgtcgcaag 7620

cctgttcatc tccgcactgg caacctacac atacaaaact tggaattacc acaatctctc 7680

taaagtggtg gaaccagccc tggcttacct cccctatgct accagcgcat taaaaatgtt 7740

caccccaacg cggctggaga gcgtggtgat actgagcacc acgatatata aaacatacct 7800

ctctataagg aaggggaaga gtgatggatt gctgggtacg gggataagtg cagccatgga 7860

aatcctgtca caaaacccag tatcggtagg tatatctgtg atgttggggg taggggcaat 7920

cgctgcgcac aacgctattg agtccagtga acagaaaagg accctactta tgaaggtgtt 7980

tgtaaagaac ttcttggatc aggctgcaac agatgagctg gtaaaagaaa acccagaaaa 8040

aattataatg gccttatttg aagcagtcca gacaattggt aaccccctga gactaatata 8100

ccacctgtat ggggtttact acaaaggttg ggaggccaag gaactatctg agaggacagc 8160

aggcagaaac ttattcacat tgataatgtt tgaagccttc gagttattag ggatggactc 8220

acaagggaaa ataaggaacc tgtccggaaa ttacattttg gatttgatat acggcctaca 8280

caagcaaatc aacagagggc tgaagaaaat ggtactgggg tgggcccctg caccctttag 8340

ttgtgactgg acccctagtg acgagaggat cagattgcca acagacaact atttgagggt 8400

agaaaccagg tgcccatgtg gctatgagat gaaagctttc aaaaatgtag gtggcaaact 8460

taccaaagtg gaggagagcg ggcctttcct atgtagaaac agacctggta ggggaccagt 8520

caactacaga gtcaccaagt attacgatga caacctcaga gagataaaac cagtagcaaa 8580

gttggaagga caggtagagc actactacaa aggggtcaca gcaaaaattg actacagtaa 8640

aggaaaaatg ctcttggcca ctgacaagtg ggaggtggaa catggtgtca taaccaggtt 8700

agctaagaga tatactgggg tcgggttcaa tggtgcatac ttaggtgacg agcccaatca 8760

ccgtgctcta gtggagaggg actgtgcaac tataaccaaa aacacagtac agtttctaaa 8820

aatgaagaag gggtgtgcgt tcacctatga cctgaccatc tccaatctga ccaggctcat 8880

cgaactagta cacaggaaca atcttgaaga gaaggaaata cccaccgcta cggtcaccac 8940

atggctagct tacaccttcg tgaatgaaga cgtagggact ataaaaccag tactaggaga 9000

gagagtaatc cccgaccctg tagttgatat caatttacaa ccagaggtgc aagtggacac 9060

gtcagaggtt gggatcacaa taattggaag ggaaaccctg atgacaacgg gagtgacacc 9120

tgtcttggaa aaagtagagc ctgacgccag cgacaaccaa aactcggtga agatcgggtt 9180

ggatgagggt aattacccag ggcctggaat acagacacat acactaacag aagaaataca 9240

caacagggat gcgaggccct tcatcatgat cctgggctca aggaattcca tatcaaatag 9300

ggcaaagact gctagaaata taaatctgta cacaggaaat gaccccaggg aaatacgaga 5360

cttgatggct gcagggcgca tgttagtagt agcactgagg gatgtcgacc ctgagctgtc 9420

tgaaatggtc gatttcaagg ggactttttt agatagggag gccctggagg ctctaagtct 9480

cgggcaacct aaaccgaagc aggttaccaa ggaagctgtt aggaatttga tagaacagaa 9540

aaaagatgtg gagatcccta actggtttgc atcagatgac ccagtatttc tggaagtggc 9600

cttaaaaaat gataagtact acttagtagg agatgttgga gagctaaaag atcaagctaa 9660

agcacttggg gccacggatc agacaagaat tataaaggag gtaggctcaa ggacgtatgc 9720

catgaagcta tctagctggt tcctcaaggc atcaaacaaa cagatgagtt taactccact 9780

gtttgaggaa ttgttgctac ggtgcccacc tgcaactaag agcaataagg ggcacatggc 9840

atcagcttac caattggcac agggtaactg ggagcccctc ggttgcgggg tgcacctagg 9900

tacaatacca gccagaaggg tgaagataca cccatatgaa gcttacctga agttgaaaga 9960

tttcatagaa gaagaagaga agaaacctag ggttaaggat acagtaataa gagagcacaa 10020

caaatggata cttaaaaaaa taaggtttca aggaaacctc aacaccaaga aaatgctcaa 10080

cccagggaaa ctatctgaac agttggacag ggaggggcgc aagaggaaca tctacaacca 10140

ccagattggt actataatgt caagtgcagg cataaggctg gagaaattgc caatagtgag 10200

ggcccaaacc gacaccaaaa cctttcatga ggcaataaga gataagatag acaagagtga 10260

aaaccggcaa aatccagaat tgcacaacaa attgttggag attttccaca cgatagccca 10320

acccaccctg aaacacacct acggtgaggt gacgtgggag caacttgagg cgggggtaaa 10380

tagaaagggg gcagcaggct tcctggagaa gaagaacatc ggagaagtat tggattcaga 10440

aaagcacctg gtagaacaat tggtcaggga tctgaaggcc gggagaaaga taaaatatta 10500

tgaaactgca ataccaaaaa atgagaagag agatgtcagt gatgactggc aggcagggga 10560

cctggtggtt gagaagaggc caagagttat ccaataccct gaagccaaga caaggctagc 10620

catcactaag gtcatgtata actgggtgaa acagcagccc gttgtgattc caggatatga 10680

aggaaagacc cccttgttca acatctttga taaagtgaga aaggaatggg actcgttcaa 10740

tgagccagtg gccgtaagtt ttgacaccaa agcctgggac actcaagtga ctagtaagga 10800

tctgcaactt attggagaaa tccagaaata ttactataag aaggagtggc acaagttcat 10860

tgacaccatc accgaccaca tgacagaagt accagttata acagcagatg gtgaagtata 10920

tataagaaat gggcagagag ggagcggcca gccagacaca agtgctggca acagcatgtt 10980

aaatgtcctg acaatgatgt acggcttctg cgaaagcaca ggggtaccgt acaagagttt 11040

caacagggtg gcaaggatcc acgtctgtgg ggatgatggc ttcttaataa ctgaaaaagg 11100

gttagggctg aaatttgcta acaaagggat gcagattctt catgaagcag gcaaacctca 11160

gaagataacg gaaggggaaa agatgaaagt tgcctataga tttgaggata tagagttctg 11220

ttctcatacc ccagtccctg ttaggtggtc cgacaacacc agtagtcaca tggccgggag 11280

agacaccgct gtgatactat caaagatggc aacaagattg gattcaagtg gagagagggg 11340

taccacagca tatgaaaaag cggtagcctt cagtttcttg ctgatgtatt cctggaaccc 11400

gcttgttagg aggatttgcc tgttggtcct ttcgcaacag ccagagacag acccatcaaa 11460

acatgccact tattattaca aaggtgatcc aataggggcc tataaagatg taataggtcg 11520

gaatctaagt gaactgaaga gaacaggctt tgagaaattg gcaaatctaa acctaagcct 11580

gtccacgttg ggggtctgga ctaagcacac aagcaaaaga ataattcagg actgtgttgc 11640

cattgggaaa gaagagggca actggctagt taagcccgac aggctgatat ccagcaaaac 11700

tggccactta tacatacctg ataaaggctt tacattacaa ggaaagcatt atgagcaact 11760

gcagctaaga acagagacaa acccggtcat gggggttggg actgagagat acaagttagg 11820

tcccatagtc aatctgctgc tgagaaggtt gaaaattctg ctcatgacgg ccgtcggcgt 11880

cagcagctga gacaaaatgt atatattgta aataaattaa tccatgtaca tagtgtatat 11940

aaatatagtt gggaccgtcc acctcaagaa gacgacacgc ccaacacgca cagctaaaca 12000

gtagtcaaga ttatctacct caagataaca ctacatttaa tgcacacagc actttagctg 12060

tatgaggata cgcccgacgt ctatagttgg actagggaag acctctaaca gcccccgcgg 12120

atctagagga gcatgcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 12180

tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 12240

aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 12300

ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 12360

aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 12420

acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 12480

ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 12540

gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 12600

atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 12660

acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 12720

tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 12780

ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 12840

aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 12900

aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 12960

ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 13020

atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 13080

aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 13140

accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 13200

tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 13260

tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 13320

tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 13380

cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 13440

caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 13500

cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 13560

cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 13620

gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 13680

acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 13740

atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 13800

cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 13860

gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 13920

tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 13980

tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 14040

agcgcagcga gtcagtgagc gaggaagcgg aagagcgc 14078

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<400>2

aaaggtctcg agatgccacg 20

<210>3

<211>39

<212>DNA

<213> Artificial sequence

<400>3

gtctgacatg tgccatgtac agcagagatt tttagtagc 39

<210>4

<211>29

<212>DNA

<213> Artificial sequence

<400>4

cacatgtcag acacgaaaga agagggagc 29

<210>5

<211>22

<212>DNA

<213> Artificial sequence

<400>5

caggtttgca atccaagtgc cc 22

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<400>6

ggagcatacg ctgcttcccc 20

<210>7

<211>21

<212>DNA

<213> Artificial sequence

<400>7

gccttgccta tgagggaatg g 21

<210>8

<211>19

<212>DNA

<213> Artificial sequence

<400>8

cacttgcatc catcatacc 19

<210>9

<211>27

<212>DNA

<213> attenuated diarrhea virus

<400>9

tgtacatggc acatgtcaga cacgaaa 27

Claims (18)

1. A method of modifying an isolated wild-type bovine viral diarrhea virus genome to make it suitable for use as a vaccine, comprising mutating the nucleic acid of the isolated wild-type bovine viral diarrhea virus genome to result in N^proThe protease gene is inactivated.

2. The method of claim 1, wherein N is substituted^proProtease gene inactivation is achieved by the following method, including:

a) reverse transcribing the wild type bovine viral diarrhea virus genomic RNA to cDNA;

b) cloning the cDNA of step a);

c) n in cDNA cloned in step b)^proA protease gene is mutated such that the gene does not produce a gene product having full activity; and is

d) Cloning the cDNA mutated in step c).

3. The method of claim 2, wherein the viral diarrhea is caused by deletion of all or a portion of the N of the wild-type bovine viral diarrhea virus genome^proThe protease gene sequence inactivates the gene.

4. A bovine viral diarrhea virus genome obtained by the method of claim 1.

5. A vector comprising an independent sequence element consisting of the bovine viral diarrhea virus genome of claim 4.

6. A host cell transfected with the viral genome of claim 4 or the vector of claim 5.

7. Progeny bovine viral diarrhea virus produced by the host cell of claim 6.

8. A vaccine comprising the viral genome of claim 4 and a veterinarily acceptable carrier.

9. A method of attenuating wild-type bovine viral diarrhea virus for use in a vaccine comprising mutating the viral genome nucleic acid such that N is^proThe protease gene is inactivated.

10. The method of claim 9, wherein attenuation is achieved by a method comprising:

a) isolating the wild-type bovine viral diarrhea virus;

b) cloning the genomic nucleic acid of the virus isolated in step a);

c) mutating the genomic nucleic acid cloned in step b) to result in N^proInactivation of protease gene;

and is

d) Transforming or transfecting the nucleic acid mutated in step c) into a host cell to obtain an attenuated virus.

11. The method of claim 10, wherein the viral diarrhea is caused by deletion of all or a portion of N from the wild-type bovine viral diarrhea virus genome^proThe protease gene inactivates the gene.

12. An attenuated bovine viral diarrhea virus obtained by the method of claim 9.

13. A host cell infected with the attenuated bovine viral diarrhea virus of claim 12.

14. The infected host cell of claim 13, wherein the host cell is an MDBK cell.

15. The infected host cell of claim 14, wherein the MDBK cell is ATCC CCL-22.

16. Progeny virus obtained using the host cell of claim 14.

17. A vaccine comprising the attenuated bovine viral diarrhea virus of claim 12 and a veterinarily acceptable carrier.

18. Use of the viral genome of claim 4, the vector of claim 5, or the attenuated bovine viral diarrhea virus of claim 12 for the preparation of a pharmaceutical composition for inducing the production of anti-bovine viral diarrhea virus antibodies in an antibody-producing animal.