MX2008005426A - Marked bovine viral diarrhea virus vaccines - Google Patents

Abstract

The present invention is directed to a bovine viral diarrhea virus comprising at least one helicase domain amino acid mutation wherein the mutation in the NS3 domain results in a loss of recognition by a monoclonal antibody raised against wild-type NS3 but wherein viral RNA replication and the generation of infectious virus is retained. The present invention is useful, for example, to produce a marked bovine viral diarrhea virus vaccine or to differentiate between vaccinated and infected or unvaccinated animals.

Description

VACCINES MARKED AGAINST VIRUS OF BOVINE VIRAL DIARRHEA FIELD OF THE INVENTION The present invention relates to a virus of bovine viral diarrhea comprising at least one amino acid mutation in the heiicase domain where the mutation in the NS3 domain causes a loss of recognition by a monoclonal antibody raised against wild-type NS3 but where replication of viral RNA and generation of infectious virus is maintained.

BACKGROUND OF THE INVENTION The virus of bovine viral diarrhea (BVD virus or BVDV) is a small RNA virus of the genus Pestivirus, and the Flaviviridae family. It is closely related to viruses that are the causative agents of border disease in sheep and classical swine fever in pigs. The disease caused by BVDV is widespread, and can be economically devastating. A BVDV infection can cause breeding problems in cattle and can cause miscarriages or premature births. BVDV is capable of crossing the placenta of pregnant cattle, and can cause the birth of persistently infected calves (Pl) that are immunotolerant to the virus and persistently viremic for the rest of their lives (Malmquist, J. Am. Vet. Med. Assoc. 152: 763-768 (1968), Ross, et al., J. Am. Vet. Med. Assoc. 188: 618-619 (1986)). Infected cattle may also show "mucosal disease," characterized by high temperature, diarrhea, catarrh, and ulcerations of the food mucosa (Olafson, et al., Cometí Vet. 36: 205-213 (1946); Ramsey, et al. ., North Am. Vet. 34: 629-633 (1953)). These persistently infected animals provide a source of virus spread in the herd for additional outbreaks of mucosal disease (Liess et al., Dtsch.Tieraerztil.Wschr. 81: 481-487 (1974)) and are highly predisposed to infection with microorganisms. responsible for causing enteric diseases or pneumonia (Barber, et al., Vet. Rec. 17: 459-464 (1985)). BVD viruses are classified into one of two biotypes. Those of "cp" biotype induce a cytopathic effect in cultured cells, while "ncp" biotype viruses do not (Gillespie, et al., Cornell Vet. 50: 73-79 (1960)). In addition, two main genotypes (type 1 and 2) are recognized, which have been shown to cause a variety of clinical syndromes (Pellerin et al., Virology 203: 260-268 (1994); Ridpath, et al., Virology 205: 66- 74 (1994)). The BVD virions are 40 to 60 nm in diameter. The nucleocapsid of BVDV consists of a single molecule of RNA and protein C of the capsid. The nucleocapsid is surrounded by a lipid membrane with two glycoproteins anchored in it, E1 and E2. A third Erns glycoprotein is little associated with the envelope. The BVDV genome is approximately 12.5 kb in length, and contains a single open reading frame located between the 5 'and 3' untranslated regions (NTR) (Collett, et al., Virology 165: 191-199 ( 1988)). An approximately 438 kD polyprotein is translated from this open reading frame, and processed by cellular and viral proteases in at least eleven viral structural and non-structural (NS) proteins (Tautz, et al., J. Virol. 5415-5422 (1997), Xu, et al., J. Virol. 71: 5312-5322 (1997), Elbers, et al., J. Virol. 70: 4131-4135 (1996), and Wiskerchen, et al. ., Virology 184: 341-350 (1991)). The genomic order of BVDV is p20 / Npro, p14 / C, gp48 / Ems, gp25 / E1, gp53 / E2, p54 / NS2, p80 / NS3, p10 / NS4A, p32 / NS4B, p58 / NS5A and p75 / NS5B. P20 / Npro (Stark et al., J. Virol. 67: 7088-7093 (1993); Wiskerchen et al., Virol. 65: 4508-4514 (1991)) is a protease of the papain type that functions in cis which is it clears itself of the rest of the synthesized polyprotein. The capsid protein (C), also referred to as p14, is a basic protein and functions to package genomic RNA and enveloped virion formation. P14 / C is conserved through different pestiviruses. The three envelope proteins, pg48 / Erns, gp25 / E1 and gp53 / E2, are highly glycosylated. Ems forms homodimers, covalently linked by disulfide bridges. The absence of an anchorage region to the hydrophobic membrane suggests that Erns is poorly associated with the envelope. Erns induces high antibody titers in infected cattle, but antisera have limited virus neutralizing activity. E1 is found in virions covalently linked to gp53 / E2 by disulfide bonds. E1 contains two hydrophobic regions that serve to anchor the protein in the membrane, and as a signal peptide to initiate the translocation. E1 does not induce a significant antibody response in infected cattle, suggesting that it may not be exposed on the surface-of the virion. Like E1, E2 also has a membrane anchoring region at its C-terminal end. Unlike E1, however, E2 is very antigenic, being one of the most immunodominant proteins of BVDV. Antibodies that bind to E2 can effectively neutralize a viral infection, suggesting that it may be involved in the entry of the virus. The region of the polyprotein downstream of the structural proteins encodes the non-structural proteins, and is processed by two viral proteolytic enzymes. The NS2-NS3 junction is cleaved by a zinc-dependent protease encoded in NS2. The C-terminal part of the BVDV polyprotein encoding NS3, NS4A, NS4B, NS5A and NS5B is processed by a serine protease encoded by the N-terminal domain of NS3. NS3 is another BVDV main immunogen, since infected cattle develop a strong humoral response against it. In contrast, no serum antibodies are found for the other non-structural proteins in cattle infected with BVDV, and only a slight humoral immune response against NS4A can be detected. NS3 is found exclusively in cytopathic BVDV isolates, and the region encoding the protein is one of the most conserved in the BVDV genome, based on comparisons between BVDV subtypes and other pestiviruses. The C-terminal part of NS3 encodes an RNA-dependent NTPase / helicase, and based on comparisons of highly conserved helicase amino acid sequence sequences, the BVDV helicase has been classified in the helicase 2 superfamily (SF2). In this superfamily there are similar proteins of the NS3 helicase of potyvirus, flavivirus and pestivirus, including porcine cholera virus (classical swine fever), and RNA helicases of other flaviviruses, such as West Nile virus, fever virus yellow fever, the hepatitis C virus (HCV) and the Japanese encephalitis virus. The molecular structure of the protease and helicase domains of HCV NS3 have been resolved (Yao, et al, Nat Struct Biol. 4: 463-7 (1997); Jin and Peterson; Arch Bioxchem Biophys 323: 47-53 (1995)). ). The protease domain contains the double-barrel β fold that is commonly observed among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related β-a-β subdomains, and a third subdomain of seven helices and three short β chains, usually referred to as the a-helical subdomain of helicase. The nucleoside triphosphate (NTP) binding sites and RNA, as well as the helicase active site, are exposed on the surface, whereas the protease active site is not, and is directed towards the helicase domain. The protease and helicase domains are covalently connected by a short chain exposed on the surface, and interact over a large surface area (~ 900 A2). The active helicase site, however, is oriented away from this area of interaction. Among the currently available BVDV vaccines are those containing chemically inactivated wild-type virus (McClurkin, et al., Arch Virol. 58: 1 19 (1978); Fernelius et al., Am. J. Vet. Res. 33: 1421-1431 (1972); and Kolar, et al., Am. J. Vet. Res. 33: 1415-1420 (1972)). These vaccines typically require the administration of multiple doses, and produce a short-lived immune response; they also do not protect against fetal transmission of the virus (Bolín, Vet Clin North Am Food Food Pract 11: 615-625 (1995)). In sheep, a subunit vaccine based on a purified E2 protein has been reported (Bruschke, et al., Vaccine 15: 1940-1945 (1997)). Although this vaccine appears to protect fetuses from becoming infected, protection is limited only to the homologous strain of the virus, and there is no correlation between antibody titers and protection.

Vaccines against BVDV of modified live virus (MLV) have been produced using viruses that have been attenuated by repeated passage in bovine or porcine cells (Coggins, et al., Cornell Vet. 51: 539 (1961); and Phillips, et al. , Am. J. Vet. Res. 36: 135 (1975)), or by chemically induced mutations that give a virus-sensitive phenotype (Lobmann, et al., Am. J. Vet. Res. 45: 2498 (1984) and Lobmann, et al., Am. J. Vet. Res. 47: 557-561 (1986)). A single dose of an MLV vaccine against BVDV has been shown to be sufficient to provide protection against infection, and the duration of immunity may be prolonged for years in vaccinated cattle (Coria et al., Can. J. Con. Med. 42 : 239 (1978)). In addition, cross protection has been reported using MLV vaccines (Martin, et al., In "Proceedings of the Conference of Research Workers in Animal Diseases", 75: 183 (1994)). However, safety considerations including fetal transmission of the vaccine strain with respect to the use of these live modified virus vaccines are of primary concern (Bolin, Vet.Clin.NorthAm.Food Anim.Pract. 11: 615-625). (nineteen ninety five)). Based on the above, it is clear that there is a need for new and more effective vaccines to control the spread of BVDV. Such a vaccine could be invaluable in future national or regional BVDV eradication programs, and could also be combined with other vaccines labeled for cattle, representing a substantial advance in livestock. One of these vaccines is a "marked" vaccine. Said vaccine lacks an antigenic determinant present in the wild type virus. Animals infected with the wild type virus mount an immune response against. the determinant immunogenic "marker" while vaccinated animals not infected, as a result of which the determinant is not present in the labeled vaccine. Through the use of an immunological assay directed against the marker determinant, the infected animals could be differentiated from the vaccinated non-infected animals. By selectively rejecting infected animals, the herd could, over time, become BVDV-free. In addition to the benefit of removing the threat of a BVD disease, the certification of a herd as free of BVD has direct freedom from economic benefits of commercialization.

SUMMARY OF THE INVENTION The present invention also relates to a novel bovine viral diarrhea virus-labeled vaccine comprising a bovine viral diarrhea virus having at least one amino acid mutation in the helicase domain, where NS3 is not recognized for an antibody conventional monoclonal against NS3, such as, for example, 20.10.6; 1.11.3; 21.5.8; and 24.8, but where the replication of viral RNA and the generation of infectious virus is maintained. The present invention also relates to an assay for determining whether an animal has been vaccinated, or is not vaccinated or infected with BVDV. In one embodiment of the present invention, a bovine viral diarrhea virus is provided which comprises at least one amino acid mutation in the helicase domain where the mutation in the helicase domain of NS3 results in a loss of recognition by a monoclonal antibody raised against NS3 of wild-type bovine viral diarrhea virus but where replication of viral RNA and generation of infectious virus is maintained. In another embodiment of the present invention, there is provided a bovine viral diarrhea virus comprising at least one amino acid mutation in the helicase domain where NS3 is not recognized by a monoclonal antibody against NS3, and where the antibody against NS3 is selected from the group constituted by 20.10.6; 1.11.3; 21.5.8; and 24.8, but where the replication of viral RNA and the generation of infectious virus is maintained. In another embodiment of the invention, the vaccine against the virus comprises a single amino acid mutation in the helicase domain. In another embodiment of the present invention, the vaccine against the virus comprises a mutation in the helicase domain in the IGR loop. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation in the helicase domain in the IGR loop in the amino acid residue 1841. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation. in the helicase domain in the IGR loop in the amino acid residue 1843. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation in the helicase domain in the IGR loop in the amino acid residue 1845. In another embodiment of The present invention, the bovine viral diarrhea virus, comprises a mutation in the helicase domain in the KHP loop. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation in the helicase domain in the KHP loop in the amino acid residue 1867. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation in the helicase domain in the KHP loop in the amino acid residue 1868. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation in the helicase domain in the KHP loop in the amino acid residue 1869.

In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation in the helicase domain in the SES loop. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation in the helicase domain in the SES loop in the amino acid residue 1939. In another embodiment of the present invention, the bovine viral diarrhea virus comprises a mutation. in the helicase domain in the SES loop in the amino acid residue 1942. In another embodiment of the present invention, the bovine viral diarrhea virus comprises two, three or four amino acid mutations in the helicase domain. In another embodiment of the present invention, the bovine viral diarrhea virus comprises two mutations in the helicase domain. In another embodiment of the present invention, the bovine viral diarrhea virus comprises two mutations in the helicase domain in the IGR loop. In another embodiment of the present invention, bovine viral diarrhea virus comprises two mutations in the helicase domain in the IGR loop in the amino acid residues 1843 and 1845.

In another embodiment of the present invention, the bovine viral diarrhea virus comprises two mutations in the helicase domain in the SES loop. In another embodiment of the present invention, the bovine viral diarrhea virus comprises two mutations in the helicase domain in the SES loop in the amino acid residues 1939 and 1942.

In another embodiment of the present invention, the bovine viral diarrhea virus comprises three mutations in the helicase domain. In another embodiment of the present invention, the bovine viral diarrhea virus comprises three mutations in the helicase domain in the IGR loop. In another embodiment of the present invention, the bovine viral diarrhea virus comprises three mutations in the helicase domain in the IGR loop in the amino acid residues 1867, 1868 and 1869. In another embodiment of the present invention, the viral diarrhea virus bovine comprises three mutations in the helicase domain in the IGR loop and the SES loop in the amino acid residues 1845, 1868 and 1939. In a particularly preferred embodiment of the present invention, a vaccine labeled against bovine viral diarrhea virus is provided, which comprises a virus of bovine viral diarrhea comprising at least one amino acid mutation in the helicase domain where the mutation in the helicase domain of NS3 causes a loss of recognition by a monoclonal antibody produced against NS3 of wild-type bovine viral diarrhea virus but where replication of viral RNA and generation of infectious virus is maintained. In another embodiment of the present invention, there is provided a method for differentiating an animal infected with bovine diarrhea virus from a vaccinated animal with a vaccine against the bovine diarrhea virus. In this embodiment, the vaccine against the bovine diarrhea virus is a labeled vaccine comprising at least one amino acid mutation in the helicase domain, and the method comprises; obtain a test sample from a test animal; detect the bovine diarrhea virus in the test sample; and determine if the bovine diarrhea virus contains the mutation. In another embodiment of the present invention, the method for detecting the bovine diarrhea virus employs the use of at least one monoclonal antibody. A preferred method comprises an amino acid mutation in the helicase domain of the labeled vaccine in the helicase domain of NS3. For example, one embodiment of this differential assay may include the steps of: adding a labeled antibody capable of detecting wild-type bovine diarrhea virus or capable of detecting the virus of the mutated bovine diarrhea to a test sample, where the test sample contains body fluid from a test animal; and measuring the binding affinity of the labeled antibody to the wild-type bovine diarrhea virus or to the virus of the mutated bovine diarrhea by contacting at least one monoclonal antibody against the wild-type bovine diarrhea virus or against the bovine virus. mutated bovine diarrhea; and determining the vaccination status of the test animal by comparing the results of binding affinity using a monoclonal antibody directed against wild-type BVDV versus BVDV with mutated NS3. A preferred method comprises adding a first labeled antibody directed against a non-mutated NS3 domain; and adding a second labeled antibody directed against a mutated part of NS3. In one embodiment of this method, the first antibody is directed against a wild-type virus. In another embodiment of this method, the second antibody is directed against the mutated part of NS3.

In another embodiment of this method, the second antibody is directed against NS3 and is selected from the group consisting of 20.10.6; 1.11.3; 21.5.8; and 24.8. In another embodiment of the method, the second antibody is directed against at least one mutated part of NS3 selected from the group consisting of the IGR loop., the KHP loop and the SES loop. In another embodiment of this method, the bovine viral diarrhea virus comprises at least one amino acid mutation in the helicase domain in the IGR loop in the amino acid residue selected from the group consisting of 1841, 1843 and 1845. In another embodiment of the method , the virus of bovine viral diarrhea comprises at least one amino acid mutation in the helicase domain in the KHP loop in the amino acid residue selected from the group constituted by 1867, 1868 and 1869. In another embodiment of the procedure, the diarrhea virus bovine virus comprises at least one amino acid mutation in the helicase domain in the SES loop in an amino acid residue selected from the group consisting of 1939 and 1942. In another embodiment of the method, the bovine viral diarrhea virus comprises at least one amino acid in the helicase domain in the IGR loop and the SES loop in the amino acid residues 1845, 1868 and 1939.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features, aspects and advantages of the present invention are illustrated with reference to the following description, appended claims, and accompanying drawings in which: Figure 1 depicts the domains of NS3 Figure 2 shows the alignment of sequence of helicase domains of BVDV and HCV. Figure 3 shows an illustration of the molecular model of the BVDV helicase. Figure 4 shows the location of scanning mutants. Figure 5 shows the domain map of the full-length BVDV precursor structure and the structure of the BVVV subviral replicon.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO. 1 is a peptide sequence of an unprocessed, full-length polyprotein of bovine viral diarrhea virus. The numbering of the remains in this sequence corresponds to the mutations described in this document. For example, a mutation described as "K1845A" means that the lysine residue at position 1845 of SEQ ID No. 1 has been replaced by an alanine residue; SEQ ID No. 2 is a sequence of a plasmid DNA fragment flanking the 5 'end of the p15aDI cloning site to generate exemplary mutants; SEQ ID No. 3 is a sequence of a plasmid DNA fragment flanking the 3 'end of the p15aDI cloning site to generate exemplary mutants; SEQ ID No. 4 is a sequence of a 5 'DNA primer to introduce the 11841 A mutation described herein; SEQ ID No. 5 is a sequence of a 3 'DNA primer to introduce the 11841 A mutation described herein; SEQ ID No. 6 is a sequence of a 5 'DNA primer for introducing the R1843A mutation described herein; SEQ ID No. 7 is a sequence of a 3 'DNA primer for introducing the R1843A mutation described herein; SEQ ID No. 8 is a sequence of a 5 'DNA primer to introduce the K1845A mutation described herein; SEQ ID No. 9 is a sequence of a DNA 3 'primer to introduce the K1845A mutation described herein; SEQ ID No. 10 is a sequence of a 5 'DNA primer to introduce the K1867A mutation described herein; SEQ ID No. 11 is a sequence of a 3 'DNA primer to introduce the K1867A mutation described herein; SEQ ID No. 12 is a sequence of a 5 'DNA primer to introduce the H1868A mutation described herein; SEQ ID No. 13 is a sequence of a 3 'DNA primer to introduce the H1868A mutation described herein; SEQ ID No. 14 is a sequence of a 5 'DNA primer to introduce the P1869A mutation described herein; SEQ ID No. 15 is a sequence of a 3 'DNA primer to introduce the P 1869 A mutation described herein; SEQ ID No. 16 is a sequence of a 5 'DNA primer to introduce the E1939A mutation described herein; SEQ ID No. 17 is a sequence of a 3 'DNA primer to introduce the E1939A mutation described herein; SEQ ID No. 18 is a sequence of a 5 'DNA primer for introducing the R1942A mutation described herein; SEQ ID No. 19 is a sequence of a 3 'DNA primer for introducing the R1942A mutation described herein; SEQ ID No. 20 is a peptide sequence of domains 1 (helicase) and 2 (NTPase) of the NS3 region of translated BVDV; SEQ ID No. 21 is a peptide sequence of domains 1 (helicase) and 2 (NTPase) of the NS3 region of the translated Hepatitis C virus (HCV). Definitions The following definitions can be applied to the terms used in the description of embodiments of the invention. The following definitions are above any contradictory definition contained in each individual reference incorporated in this document as a reference. Unless defined otherwise in this document, the scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those skilled in the art. further, unless otherwise required by the context, the singular terms will include pluralities and plural terms will include the singular. The term "amino acid", as used herein, refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. The amino acids of natural origin are those encoded by the genetic code, as well as the amino acids that are subsequently modified, for example, hydroxyproline, carboxyglutamate and O-phosphoserine. Stereoisomers (eg, D-amino acids) of the twenty essential amino acids, non-natural amino acids such as a and disubstituted amino acids, N-alkyl amino acids, lactic acid and other non-conventional amino acids may also be suitable components for polypeptides of the present invention. Examples of non-conventional amino acids include: 4-hydroxyproline,? -carboxyglutamate, e-?,?,? - trimetillysin, e -? - acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, s -? - methylarginine, and other amino acids and similar imino acids. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, ie, a carbon that is attached to a hydrogen, a carboxyl group, an amino group, and a R group. Analogs of Exemplary amino acids include, for example, homoserine, norleucine, methionine sulfoxide, and methylsulfonium methionine. Such analogs have modified R groups (e.g., norleucine) or modified peptide structures, but maintain the same essential chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to an amino acid of natural origin. The amino acids can be mentioned in this document by their three-letter symbols usually known or by their symbols of a letter recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The term "animal subjects", as used herein, is understood to include any animal that is susceptible to BVDV infections, such as cattle, sheep and pigs. By "treatment" or "vaccination" is meant to prevent or reduce the risk of infection by a virulent strain of BVDV, by improving the symptoms of a BVDV infection, or by accelerating the recovery of a BVDV infection. "Viruses", "viral isolates" or "viral strains" BVD as used herein, refer to BVD viruses that consist of a viral genome, associated proteins, and other chemical constituents (such as lipids). Normally, the BVD virus has a genome in the form of RNA. RNA can be reverse transcribed to DNA for use in cloning. Thus, references made in this document to viral sequences of nucleic acid and BVD include both viral RNA sequences and DNA sequences derived from the viral RNA sequences. For convenience, the genomic sequences of BVD, represented in the LIST OF SEQUENCES hereinafter, refer to the polypeptide sequence, and DNA primer sequences used in the preparation of the exemplary mutations. The corresponding RNA sequence for each is apparent to those skilled in the art. Various BVD type I and type II viruses are known to those skilled in the art and are available through, for example, the American Type Culture Collection. The term "conservative amino acid substitutions", as used herein, are those that generally occur in a family of amino acids that are related in their side chains. In particular, as used herein, a conservative amino acid substitution is one that has no effect on the recognition by the antibody of a given peptide as compared to the wild type derived peptide. The genetically encoded amino acids are generally divided into four groups: (1) acids = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are also classified together as aromatic amino acids. Accordingly, the term "non-conservative amino acid substitutions", as used herein, are those that are likely to have different properties, particularly with respect to recognition by an antibody. Thus, a non-conservative amino acid substitution will elicit a differential immune response, such as, for example, loss of recognition "by an antibody raised against a wild-type derived peptide." The term "immunogenic" as used herein, means the capacity of a mutated or wild-type BVD virus to elicit an immune response in an animal against BVD type I or type II viruses, or BVD viruses of both type I and type II. The immune response can be a cellular immune response mediated mainly by cytotoxic T cells, or a humoral immune response mediated mainly by helper T cells, which in turn activate B cells that lead to the production of antibodies. As used herein, the term "naked DNA" refers to a plasmid comprising a nucleotide sequence encoding an agent of the present invention together with a short promoter region to control its production. It is called "naked DNA" because the plasmids do not go in any supply vehicle. When said DNA plasmid enters a host cell, such it takes a eukaryotic cell, the proteins it encodes are transcribed and translated into the cell. The term "plasmid" as used herein, refers to any nucleic acid that encodes an expressible gene and induces linear or circular nucleic acids and double-stranded or single-stranded nucleic acids. The nucleic acid can be DNA or RNA and can comprise modified nucleotides or ribonucleotides, and can be chemically modified by means such as methylation or the inclusion of protective groups or hood or glue-like structures. The term "vaccine", as used herein, refers to a composition that prevents p reduces the risk of infection and improves the symptoms of an infection. The protective effects of a vaccine composition against a pathogen are usually achieved by inducing in the subject an immune response, either a cell-mediated or humoral immune response or a combination of both. Generally speaking, a elimination or reduction of the incidences of infection by BVDV, improvement of symptoms, or accelerated elimination of the viruses of the infected subjects are indicative of the protective effects of a vaccine composition. The vaccine compositions of the present invention provide protective effects against infections caused by BVD virus. The term "vector", as used herein, means a tool that allows or facilitates the transfer of a nucleic acid from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant DNA techniques allow nucleic acids, such as a DNA segment (such as a heterologous DNA segment, eg, a heterologous cDNA segment) , is transferred into a host cell or target for the purpose of replication of the nucleic acids and / or expression of the proteins encoded by the nucleic acids. The examples of the vectors used in techniques of Recombinant DNAs include, but are not limited to, plasmids, chromosomes, artificial chromosomes and viruses.

DETAILED DESCRIPTION OF THE INVENTION The following detailed description is provided to assist those skilled in the art to practice the present invention. However, this detailed description should not be considered as unduly limiting the present invention since those skilled in the art can make modifications and variations of the embodiments discussed herein without departing from the spirit or scope of the present disclosure of the invention. The contents of each of the references cited in this document, including the contents of the references cited in these main references, are incorporated herein by reference. Conventional methods can be used to propagate and purify a plasmid useful in the present invention. The preferred prokaryotic host cell for the propagation of plasmids is the cell line of £. GM2163 coli, but some other cell types can also be used. The RNA transcribed from the plasmid can be introduced by transfection into eukaryotic host cells capable of supporting the production of viruses, such as MDBK cells. The virus can be produced in said host cells and isolated therefrom in highly purified form using known separation techniques such as sucrose gradient centrifugation. In one embodiment, the present invention provides immunogenic compositions in which one or more mutant BVD viruses described above have been included. Another embodiment of the present invention relates to genomic nucleic acid molecules isolated from the mutant BVD viruses as described above. The nucleic acid molecules used in this document include both RNA and DNA. In this embodiment, the genomic nucleic acid molecule isolated from a BVD virus contains a genomic sequence of a type I virus, where at least a portion of the NS3 domain is mutated in the helicase domain. In another embodiment, the present invention provides vectors in which the genomic nucleic acid sequence of a BVD virus has been incorporated as described herein above. Said vectors can be introduced into appropriate host cells, for the production of large amounts of the genomic nucleic acid molecules or for the production of descending mutant BVD viruses. The vectors may contain other sequence elements to facilitate the propagation, isolation and subcloning of the vector, for example, genes selection markers and origins of replication that allow propagation and selection in bacteria and host cells. A particularly preferred vector of the present invention is p15aDI (see Fig. 5). Yet another embodiment of the present invention relates to host cells into which the genomic nucleic acid molecule of a mutated BVD virus of the present invention has been introduced. "Host cells", as used herein, includes any prokaryotic cell transformed with the genomic nucleic acid molecule, preferably provided with an appropriate vector, of a mutated BVD virus. "Host cells", as used herein, also includes any eukaryotic cell infected with a BVD virus mutated or otherwise carrying the genomic nucleic acid molecule of a mutated BVD virus. A preferred prokaryotic host cell for the propagation of plasmids is the cell line of £. GM2163 coli, but other cell types can also be used. Preferred eukaryotic host cells include mammalian cells such as DBK cells (ATCC CCL 22). However, other cultured cells can also be used. The invention further includes descending viruses produced in said host cells. In another embodiment of the present invention, the viruses can be attenuated by chemical inactivation or by serial passages in cell culture before use in an immunogenic composition. The attenuation procedures are well known to those skilled in the art. The immunogenic compositions of the present invention may also include an additional active ingredient such as other immunogenic compositions against BVDV., for example, those described in the co-pending U.S. patent application Serial No. 08 / 107,908, U.S. Patent No. 6,060,457.; U.S. Patent No. 6,015,795; U.S. Patent No. 6,001,613, and U.S. Patent No. 5,593,873, which are all incorporated by reference in their entirety. In addition, the immunogenic compositions of the present invention may include one or more veterinarily acceptable carriers. As used herein, "a veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retarding agents, and Similar. The diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. The stabilizers include albumin, among others. Adjuvants include, but are not limited to, the adjuvant system RIBI (Ribi inc), alum, aluminum hydroxide gel, emulsions of oil in water, water-in-oil emulsions such as, for example, complete and incomplete Freund's adjuvants, block copolymers (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), adjuvant AMPHIGEN®, saponin , Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), Or other fractions of saponin, monophosphoryl lipid A, lipid-amine adjuvant Avridine, heat-unstable enterotoxin of E. coli (recombinant or otherwise) , cholera toxin, or muramyl dipeptide, among many others. Immunogenic compositions may additionally include one or more other immunomodulatory agents such as, for example, interleukins, interferons, or other cytokines. The immunogenic compositions of the present invention can be prepared in various forms depending on the route of administration. For example, immunogenic compositions can be prepared in the form of aqueous solutions or sterile dispersions suitable for use in injection, or prepared in lyophilized forms using freeze drying techniques. The lyophilized immunogenic compositions are typically maintained at about 4 ° C, and can be reconstituted in a stabilizing solution, for example, saline and / or HEPES, with or without adjuvant. The immunogenic compositions of the present invention can be administered to animal subjects to induce an immune response against BVD virus. Accordingly, another embodiment of the present invention provides methods for stimulating an immune response against BVD virus by administering to an animal subject an effective amount of an immunogenic composition of the present invention described above. According to the methods of the present invention, a preferred immunogenic composition for administration to an animal subject includes a mutated BVD virus.An immunogenic composition containing a mutated virus, preferably attenuated by chemical inactivation or serial passages in culture, is It is preferably administered to cattle via the parenteral route, although other routes of administration can also be used, such as, for example, orally, intranasally, intramuscularly, through the lymph nodes, intradermally, intraperitoneally, subcutaneously, rectally or vaginally, or by A combination of routes Immunization protocols can be optimized using procedures well known in the art.A single dose can be administered to animals or, alternatively, two or more inoculations can take place at intervals of two to ten weeks. nature of the immune responses induced in cattle can evaluate yourself using a variety of techniques. For example, serum from the inoculated animals can be collected and assayed for the presence of antibodies specific for BVD virus, for example, in a neutralization assay of conventional virus. The detection of responding CTLs in lymphoid tissues can be achieved by assays such as T cell proliferation, which is indicative of the induction of a cellular immune response. Relevant techniques are well described in the art, for example, Coligan et al. Current Protocols ¡n Immunology, John Wiley & Sons Inc. (1994). Another embodiment of the present invention relates to vaccine compositions. In one embodiment, the vaccine compositions of the present invention include an effective amount of one or more of the mutated BVD viruses described above. The purified mutated viruses can be used directly in a vaccine composition, or the mutated viruses can be further attenuated by chemical inactivation or in vitro serial passage. Typically, a vaccine contains between about 1x106 and about 1x108 viral particles, with a veterinarily acceptable carrier, in a volume between 0.5 and 5 ml. The precise amount of a virus in a vaccine composition effective to provide a protective effect can be determined by a veterinarian skilled in the art. Veterinarily acceptable vehicles suitable for use in vaccine compositions can be any of those described herein. In another embodiment, the vaccine compositions of the present invention include the nucleic acid molecule of a mutated virus. DNA or RNA molecules that encode all or part of the BVD virus genome in vaccines can be used. The DNA or RNA molecule may be present in a "naked" form or may be administered together with an agent that facilitates cellular uptake (eg, liposomes or cationic lipids). The typical route of administration will be intramuscular injection of between about 0.1 and about 5 ml of vaccine. The total polynucleotide in the vaccine should generally be between about 0.1 μg ml and about 5.0 mg / ml. The polynucleotides may be present as part of a suspension, solution or emulsion, but aqueous vehicles are generally preferred. Vaccines and vaccination procedures using nucleic acids (DNA or mRNA) have been well described in the art, for example, U.S. Patent No. 5,703,055, U.S. Patent No. 5,580,859, U.S. Pat. of the United States No. 5,589,466, which are all incorporated herein by reference. The vaccine compositions of the present invention may also include an additional active ingredient such as other BVDV vaccine compositions, for example, those described in U.S. Patent No. 6,060,457, U.S. Patent No. 6,015. 795, U.S. Patent No. 6,001,613 and U.S. Patent No. 5,593,873.

Vaccination can be achieved by a single inoculation or through multiple inoculations. If desired, the serum of the inoculated animals can be collected and assayed for the presence of antibodies against the BVD virus. In another embodiment of the present invention, the above vaccine compositions of the present invention are used for the treatment of BVDV infections. Accordingly, the present invention provides methods for treating infections in animal subjects caused by BVD virus by administering to an animal a therapeutically effective amount of a mutated BVD virus of the present invention. Those skilled in the art can easily determine if a genetically modified virus needs to be attenuated prior to administration. A mutated virus of the present invention can be administered directly to an animal subject without further attenuation. The amount of a virus that is therapeutically effective may vary depending on the particular virus used, the condition of the livestock and / or the degree of infection, and may be determined by a veterinarian. In the practice of the present methods, a vaccine composition of the present invention is preferably administered to cattle parenterally, although other routes of administration may also be used, such as, for example, by oral, intranasal, intramuscular, through of the lymph nodes, intradermal, intraperitoneal, subcutaneous, rectal or vaginal, or by a combination of pathways. Reinforcement regimens may be necessary and the dosage regimen may be adjusted to provide an optimal immunization. A further aspect of the present invention provides methods for determining the attenuated virus from a previous vaccination as the origin of the BVD virus present in an animal subject. The mutant BVD viruses of the present invention are distinguished from the wild type BVD strains both in the genomic composition and in the expressed proteins. This distinction allows discrimination between vaccinated and infected animals, and allows the identification of BVDV in the case of outbreaks associated with a supposed vaccine. For example, a determination can be made to see whether an animal that has tested positive for BVDV in certain laboratory assays carries a virulent or pathogenic BVD virus, or simply carries a mutant BVD virus of the present invention previously inoculated through a virus. vaccination. A variety of assays can be used to make the determination. For example, viruses can be isolated from the animal subject that tested positive for BVDV, and nucleic acid-based assays can be used to determine the presence of a BVD viral genome. mutant as indicative of a BVD virus used in a previous vaccination. Nucleic acid-based assays include Southern or Northern blot analysis, PCR, and sequencing. As an alternative, protein-based assays can be employed. In protein-based assays, cells or tissues suspected of having infection of the animal that has tested positive for BVDV can be isolated. The cell extracts can be prepared from said cells or tissues and can be subjected to, for example, Western blot, using appropriate antibodies against viral proteins that can distinctly identify the presence of the previously inoculated mutant virus, as opposed to the presence of Wild type BVDV. FORMS AND ADMINISTRATION PARENTERAL ADMINISTRATION The compounds of the invention can also be administered directly into the blood stream, into the muscle or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle injectors (including microneedle), needleless injectors and infusion techniques. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH of 3 to 9) but, for some applications, may be more adequately formulated as a sterile non-aqueous solution or as a dried form to be used together with a suitable vehicle such as sterile, pyrogen-free water. The preparation of the parenteral formulations under sterile conditions, for example, by lyophilization, can be easily achieved using conventional pharmaceutical techniques well known to those skilled in the art. The solubility of the compounds of formula I used in the preparation of parenteral solutions can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility enhancing agents. Formulations for parenteral administration can be formulated for immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, directed and programmed release. Thus, the compounds of the invention can be formulated in solid, semi-solid or thixotropic form for administration as an implanted reservoir that provides a modified release of the active compound. The examples of such formulations include drug-coated stents and poly (d / -lactic-co-glycolic acid) (PGLA) microspheres. TOPICAL ADMINISTRATION The compounds of the invention can also be administered topically to the skin or mucosa, ie, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, bandages, foams, films, dermal patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes can also be used. Typical vehicles include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers can be incorporated - see, for example, Transdermal Penetration Enhancers: Applications; Limitations, and Potential J. Pharm Sci, 88 (10), 955-958, by Finnin and Morgan (October 1999). Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis, and microneedle or needle-free injection (e.g., Powderjet ™, Bioject ™, efe). Formulations for topical administration can be formulated for immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, directed and programmed release. INHALED / INTRANASAL ADMINISTRATION The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (alone, as a mixture, for example, in a dry mixture with lactose, or as a one-component particle mixed, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, sprayer, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant such as 1,1,1,2-tetrafluoroethane or 1, 1, 1, 2,3, 3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin. The pressurized container, pump, spray, atomizer or nebulizer contains a solution or suspension of the compound or compounds of the invention comprising, for example, ethanol, aqueous ethanol, or an alternative agent suitable for dispersing, solubilizing, or extending the release of the ingredient. active, a propellant or propellants as a solvent and an optional surfactant such as sorbitan trioleate, oleic acid or an oligolactic acid.

Before use in a dry powder or suspension formulation, the drug product is micronized to a suitable size for delivery by inhalation (typically less than 5 μ ??). This can be achieved by any suitable grinding process, such as spiral jet grinding, fluid bed jet grinding, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying. Capsules (prepared, for example, from gelatin or hydroxypropylmethylcellulose), blister packs and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound of the invention, a suitable powder base such as lactose or starch. and a performance modifier such as / -leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose. A solution formulation suitable for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from 1 μg to 20 mg of the compound of the invention per actuation and the actuation volume may vary from 1 μ? at 100 μ ?. A typical formulation may comprise a compound of formula I, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents that can be used in place of propylene glycol include glycerol and polyethylene glycol. Suitable flavors, such as menthol and levomenthol, or sweeteners such as saccharin, or sodium saccharin may be added to these formulations of the invention intended for inhaled / intranasal administration. Formulations for inhaled / intranasal administration can be formulated for immediate and / or modified release using, for example, PGLA. Modified release formulations include delayed, sustained, pulsed, controlled, directed and programmed release.

In the case of inhalers and dry powder aerosols, the dosing unit is determined by means of a valve that supplies a measured quantity. The units according to the invention are typically arranged to administer a measured dose or "puff" containing from 10 ng to 100? of the compound of formula I. The overall daily dose will typically be in the range of 1 μ? to 100 mg that can be administered in a single dose or, more usually, as divided doses throughout the day.

RECTAL / INTRAVAGINAL ADMINISTRATION The compounds of the invention can be administered rectally or vaginally, for example, in the form of a suppository, pessary or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate. Formulations for rectal / vaginal administration can be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, directed and programmed release. EYE / AURAL ADMINISTRATION The compounds of the invention can also be administered directly to the eye or ear, typically in the form of drops of a suspension or micronized solution in isotonic, pH adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems such as niosomes or liposomes. A polymer such as crosslinked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose or methylcellulose, or a heteropolysaccharide polymer, for example, geiano gum, can be incorporated together with a preservative, such as benzalkonium chloride. Said formulations can also be supplied by iontophoresis. Formulations for ocular / aural administration can be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, or programmed release. PARTS KIT Since it may be desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a vaccine according to the invention, can be suitably combined in the form of a kit suitable for the co-administration of the compositions. Thus, the kit of the invention comprises two or more different pharmaceutical compositions, at least one of which contains a vaccine according to the invention, and a means for retaining said compositions from forming, such as a container, divided bottle, or divided sheet container. An example of such a kit is a syringe and needle, and the like.

The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering compositions other than different dosage intervals, or to assess the different compositions against each other. To assist the veterinarian, the kit typically comprises administration instructions. The present invention is further illustrated, but without limitation, by the following examples. EXAMPLES Example 1. Mapping of Epitopes of NS3 Domains An epitope mapping method was applied to identify the specific epitopes recognized in the NS3 protein by a panel of mAb monoclonal antibodies. The procedure includes the PCR amplification of each test fragment, followed by the translation of the truncated protein in vitro, and finally the assay of its reactivity with various mAbs. To preliminarily identify the antigenic regions in NS3, a set of seven DNA fragments representing the region was amplified (Figure 1). Each fragment contained at its 5 'end a T7 promoter followed by a start codon and a stop codon at the 3' end. These DNA fragments were used as template for the generation of S35-labeled protein fragments by in vitro transcription / translation using the rabbit reticulocyte lysate system TnT® Rabbit Reticulocyte Lysate System (Promega; Madison, Wl) and radiolabeled methionine and cysteine. The resulting translated protein fragments included the full-length NS3 protein, helicase and protease, as well as individual subdomains of the helicase. (The limits of protease, helicase and helicase subdomains were identified based on the sequence alignment of the NS3 proteins of BVDV and HCV.) A panel of 12 mAb recognizing BVDV NS3 was used, including several used by laboratories of diagnosis for the detection of BVDV infection in cattle, to immunoprecipitate the translated proteins. These monoclonal antibodies are known in the art, and are described as "previously prepared" in Deregt et al., Mappinq of two antioenic domains on the NS3 protein of the pestivirus bovine viral diarrhea virus. Veterinarv Microbioloqy (2005), 108 (1-2), 13-22. The immunoprecipitates were then analyzed by SDS-PAGE and fluorography. The results of the immunoprecipitation are summarized in Table 1. The 12 mAb and the polyclonal serum (POLY) recognized full length NS3 and one or more helicase subdomains, although they did not recognize the protease fragment. Three mAb (1.1 1.3, 21.5.8 and 24.8) immunoprecipitated both the full-length helicase and the domain 1-domain 2 fragment (d1-d2) but not. the d2-d3 fragment, suggesting that these three antibodies recognize domain 1 of the helicase protein. The two mAbs 21.5.8 and 24.8 were bound to the d1 fragment but the mAb 1.1 1.3 did not, suggesting that the 1.1 1.3 antibody was more sensitive to the conformation of epitope than mAbs 21.5.8 and 24.8. MAb 2.32.5 recognized the full-length helicase and to some extent the d1-d2 fragment, but not the d2-d3 fragment, suggesting that it may also recognize domain 1. The weak binding of the d1-d2 fragment may indicate that the epitope recognized by 2.32.5 differs between the d1-d2 fragment and the full-length helicase. Mabs 4.11.4 and 16.1.5 bound both full-length NS3 and helicase, but only weakly to the d1-d2 and d2-d3 fragments, suggesting that they may be specific for an epitope in the second domain of the helicase. Four mAb, 5.2.1, 9.10.4, 12.7.3, and 15.14.6 recognize full length NS3 and helicase. They also bound weakly to the d2-d3 fragment but not to the d1-d2 fragment, suggesting that they recognize epitopes located in domain 3. None of them bound to the single d3 fragment suggesting that proper folding of d3 may not occur in absence of the other subdomains. MAb 19.7.6 bound NS3 and the full-length helicase, but not any of the other fragments. The recognition of this antibody may require the presence of the intact helicase protein. The mAb 20.10.6 bound to NS3, the full-length helicase, and to the d1-d2 and d2-d3 fragments very well. It also recognized the unique d2 fragment, suggesting that the epitope of domain 2, recognized by this antibody, was unaffected by the absence of domains 1 and 3. It was surprising that none of the 12 mAb bound to the full-length protease, as the polyserum (POLY) of a cow infected with BVDV did not recognize the protease in these experiments, strongly suggesting that the protease is not very antigenic. This is consistent with the molecular orientation of the protease, helicase, and NS4A (protease cofactor) proteins in HCV, because the orientation of the protease between the helicase and NS4A proteins leaves very little of its surface structure accessible to antibody binding. Based on these results, domain 1 is an exemplary target for the introduction of a mutation or mutations producing a labeled virus. Table 1. Immunoprecipitation of Subdomains NS3 Example 2. Sequence Alignment of BVDV and HCV Helicases To generate a labeled virus based on a mutation in domain 1 of the BVDV helicase, further refinement of the epitopes in this domain is desirable. It is desirable to delete an immunodominant epitope without significantly altering the function of the helicase. To facilitate the identification of candidate epitopes to mutate, a molecular model of the BVDV helicase would be extremely useful. As the crystal structure of the HCV helicase is known, it can be used as a template for modeling. To begin the procedure for generating a molecular model of domain 1, the amino acid sequences of domain 1 of the helicases of BVDV and HCV were aligned. The primary sequence identity between them is about 34%. To elucidate the secondary structure of domain 1 of the BVDV helicase, 47 NS3 sequences derived from various BVDV isolates and other pestiviruses were aligned using the Pileup program from the Genetics Computer Group software package (University of Wisconsin; adison, Wl), and the NADL BVDV strain as a prototype sequence. From the aligned sequences, a multiple sequence file (MSF) was generated, and presented to the JPred server (Cuff, et al., Bioinformatics, 14: 892-893 (1998)) for the prediction of secondary structure using the PHD prediction procedure (Rost and Sander, J. Mol. Biol. 235: 13-26 (1993)). A Silicon Graphics lndigo2 Impact 1000Ó workstation (Silicon Graphics, Mountain View, CA) was used for all molecular modeling studies. The Molecular Operating Environment (MOE) version 2001.01 (Chemical Computing) was used Group, Inc .; Montreal, Quebec) and the SYBYL 6.7 software (Tripos Associates Inc.; St. Louis, MO) for molecular modeling and visualizations. The amino acid sequences of domain 1 and 2 of HCV NS3 proteins (SEQ ID No. 21) and BVDV (SEQ ID No. 20) were aligned (Figure 2) based on primary sequence homology and structure predictions high school. A preliminary molecular model of domain 1 and 2 of BVDV NS3 was then generated, using the corresponding region of the HCV protein as a template. As shown in Figure 3, the presence of several loops and turns between alpha helices and beta chains, including a1-β2 (IGR loop), a2-β3 (KHP), β4-β5 (DMA) and a3-β7 ( SES), leads to the formation of an exposed surface away from the helicase catalytic center and the helicase-protease interaction surface. This area has the potential to be a highly antigenic region. Three of the identified loops, KHP loop, IGR loop and SES loop were used as targets for a mutagenesis study. Example 3. Location of mAb Binding Sites by Exploration Mutagenesis To further define the epitopes of domain 1 linked by various mAbs, a scanning mutagenesis procedure was used. Briefly, short segments of the BVDV helicase domain 1 sequence (SEQ ID NO: 20) were replaced with the corresponding HCV sequence (SEQ ID NO: 21) using PCR amplification, followed by digestion with restriction enzymes and ligation of the resulting fragments, generating the "exploration mutants" indicated in Figure -4. In vitro transcription and translation were performed, as well as immunoprecipitation, as described in Example 1. A summary of the reactivity of the various mAbs with the mutants is shown in Table 2.

Table 2. Reactivity of the Scan Mutants with mAb Example 4. Detailed Resolution of mAb Binding Sites - by Alanine Replacement Mutagenesis. To further define the epitopes of domain 1 bound by various mAbs, and to identify the critical residues in these regions for antibody binding, a total of sixteen single amino acid (alanine) replacement mutants were generated in three regions, I1841- R1846, K1867-S1872 and S1938-I1941 and were tested for antibody binding. The coordinates of the amino acid residue are according to SEQ ID No. 1. Thus, "1841?" Represents a replacement of isoleucine with alanine at the coordinate 1841 listed in SEQ ID No. 1. Of course, in other isolates of BVDV can present different specific amino acids at the particular coordinates of the exemplary sequence.Therefore, a mutation at the same locus of the helicase domain of a variant BVD virus, or plasmid constructed to express a variant BVD virus, will produce an equivalent loss of recognition by antibodies produced against the unmodified virus peptide, variant. Replacement mutants were constructed using a PCR overlap extension technique known in the art (see, for example, Ho et al., Gene, 77 (1): 51-9 (1989)). Briefly, PCR was used to generate the alanine replacement fragments, which each encode domain 1 and domain 2 of the helicase. Each fragment encoded a T7 promoter sequence and a start codon of translation at its 5 'end, and a stop codon at the 3' end. Initially, two different reactions were performed to generate overlapping fragments encoding the 5 'and 3' halves of the replacement region. In the overlapping region, a single mutation with alanine was introduced into the sequence of both fragments thanks to the mutagenic oligonucleotide primers used in the PCR. The products of each PCR were separated by electrophoresis in an agarose gel, and a single band of the correct size of each reaction was purified. The purified DNA fragments were mixed and used as templates for a second PCR to generate a single replacement fragment. This complete procedure was repeated to generate each of the desired replacement fragments. The sequence of each fragment was verified by DNA sequencing. S35-labeled protein fragments were generated, using these fragments as a template by in vitro transcription / translation as described above. Immunoprecipitation was used using mAb, followed by analysis by SDS-PAGE to determine if the mutated epitopes were still recognized by the antibodies.

E1939A and R1942A, completely altered the binding by mAb 1.1 1.3, suggesting that these two residues are crucial for the binding of antibodies. That these two amino acids are in the same 3-ß7 loop (SES) (Figure 3) suggests that the epitope recognized by this antibody is formed by this loop. Two different mutants, I1841A and K1867A, which are located in two different regions of the helicase molecule (loops α1-β2 (IGR) and α2-β3 (β)), had a significantly reduced binding by mAb 21.5.8, but not by the other antibodies. One conclusion that could be drawn from these results would be that the epitope recognized by this mAb could encompass two different loops that are located in close proximity to the native molecule. This is consistent with the molecular model shown in Figure 3. The mutant R1843A destroyed the binding by mAb 24.8 but had no effect on the binding of the other antibodies. Again, this would suggest that this remainder is part of a key epitope located in the a1-β2 loop (IGR). The partial effect of the mutant R1942A on the binding of mAb 24.8 suggests that the a3-p7 loop (SES) together with the loop a1-p2 (IGR), constitutes the epitope recognized by this antibody. In conclusion, the epitopes recognized by three mAbs were mapped accurately in domain 1 of the BVDV helicase. The key residues in these epitopes were identified, being located in three different regions of the primary sequence, but in close proximity in the tertiary conformation. The function of these epitopes was further examined in the context of a subviral replicon of BVDV. Table 3. Immunoprecipitation of Alanine Replacement Mutants Example 5. Construction of Mutations in Domain 1 of Helicasa in the Context of a Subviral Replicon of BVDV Construction of the Subviral Replicon A desirable quality for the production of a successful vaccine against a virus is the ability to obtain high-virus yields. Therefore, a marker mutation should not significantly impede the replication of the virus. As the helicase activity is essential for the replication of the BVDV RNA, we wanted to evaluate all the point mutants of domain 1 prepared, not only with respect to the loss of recognition by antibody, but also with respect to the preservation of the catalytic activity helicase . The amplification and genetic manipulation of a proviral molecular clone of full length BVDV in Esc erichia coli (E. coli) is difficult because the plasmid is unstable during propagation. Therefore, p15aDI was created, which contains a truncated subviral replicon that expresses NS3 and supports the replication of viral RNA, although lacking the viral structural genes, to facilitate the exploration of the mutants. P15aDI of the infectious proviral parental plasmid (pNADL.p15a) containing the full-length BVDV genome was obtained. More manipulable because it lacks most of the structural genes and of the coding region of NS2, the only sequence located upstream of NS3 consists of a fusion between a part of the N protein to the bovine ubiquitin (Figure 5). The NS3 protein expressed from this replicon is detectable by immunohistochemistry only when efficient RNA replication leads to the amplification of the transcripts, producing an increase in the expression of viral proteins. Therefore, the detection of NS3 serves as an indirect confirmation of the efficient replication of RNA and the catalytic activity of helicase. Generation of Untans in Domain 1 of Helicase of BVDV A set of twelve mutants was generated in domain 1 of different helicase in the context of the subviral replicon, and analyzed for viral RNA replication and loss of epitope recognition. Eight of these mutants contained only a single amino acid change and included: in the IGR loop, l > A (amino acid residue 1841), R > A (1843), and K > A (1845); in the KHP loop, K > A (1867), H > A (1868), and P > A (1869); in the SES loop, E > A (1939), and R > A (1942). Two mutants had changes in two amino acids: in the IGR loop, R > A (1843) and K > A (1845), and in the SES loop, E > A (1939), and R > A (1942). Two contained three changes: K > A (1867), H > A (1868), and P > A (1869), all in the IGR loop, and K > A (1845), H > A (1868), and E > A (1939), which affects multiple loops. Although alanine was used in exemplary mutations for convenience, non-conservative amino acid substitutions may be used as appropriate mutations. Each mutant was generated using the overlapping PCR strategy described above. A specific set of overlapping primers was designed for each desired mutation (Table 4). For screening purposes, each set of primers also contained additional silent nucleotide changes, which would result in the creation of a single cleavage site by new restriction enzymes near the site of the mutation. The overlapping PCR fragments served as templates in the second round of amplification, performed using only the two external primers. To generate fragments containing multiple amino acid changes, the amplification reaction was repeated, using the previous mutant fragment as a template. The completely mutated fragment was then cloned into the structure of the subviral replicon by means of two unique restriction enzyme sites (Bsm B I and Sma I) created during the PCR procedure. The mutant PCR fragment and the subviral replicon structure were both digested with Bsm BI and Sma I, treated with alkaline phosphatase (NEB, Inc.), purified by agarose gel electrophoresis and ligated overnight at 16 °. C using T4 DNA ligase (New England Biolabs, Inc., Beverly, MA). E.coli STBL2 cells were transformed (Invitrog'en; Carlsbad, CA) with an aliquot of the bound reaction and plated on selective media plates. Colonies were screened by purification of plasmid DNA, followed by digestion with restriction enzymes. Plasmids of the expected size were further confirmed by sequence analysis. Table 4 Example 6. Characterization of Mutant Subviral Replicons Transcription in vitro and RNA Transfection RNA transcripts were synthesized in vitro using T7 RNA polymerase and MEGAscript ™ (Ambion, Austin, TX). The DNA templates were linearized with Ksp I and treated with T4 DNA polymerase to remove the 3 'overhang. The products of the transcription reaction were analyzed by agarose gel electrophoresis before transfection. 1-5 μg of RNA was added to 200 μ? of Opti-MEM (Invitrogen) containing 6 μg of Lipofectin (Invitrogen) and incubated for 10 to 15 minutes at room temperature. Simultaneously, monolayers (50 to 60% confluent) of Madin Darby Bovine Kidney Cells (MDBK) that had grown in six-well plates (35 mm diameter) with RNase-free PBS, and once with Opti-MEM. After the final wash, the transfection mixtures were added to each well, followed by incubation for 10 minutes at room temperature with gentle break. Then 1 ml of Opti-MEM was added to each well, and the plates were incubated for an additional 3 hours at 37 ° C. Then, three ml of Opti-MEM containing bovine serum from the 2-3% bovine donor were added to each well. RNA Replication Analysis and Antibody Recognition After incubation for 24-48 hours at 37 ° C , the transfected cells were fixed with 80% acetone, and subjected to an immunohistochemistry (IHC) assay, using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Monoclonal antibody 20.10.6, which recognizes helicase domain 2, was used to visualize NS3-positive cells as an indicator of efficient RNA replication. Cells transfected with wild-type BVDV RNA, as well as many of the replicons mutants showed strong staining with mAb 20.10.6, indicating that these individual mutant viral helices supported efficient replication of vRNA. Only the mutant K1867A / H1868A / P1869A failed to produce detectable NS3 protein, suggesting that this set of mutations significantly impeded replication of viral RNA. All cells transfected with wild-type or mutant replicons were also tested with mAbs 1.11.3, 21.5.8, and 24.8. (Table 5). It appeared that each loop was recognized by one of these three antibodies, since mutations in each loop produced loss of recognition by one of the three antibodies. In particular, the mutation of residues R1843A and K1845A in the IGR loop, individually and together, produced the complete loss of recognition by mAb 24.8. At the same time, recognition by mAb 20.10.6, 1.11.3 and 21.5.8 was not affected. In the KHP loop, the K1867A mutation suppressed recognition by mAb 21.5.8, without affecting recognition by the other three antibodies. In addition, both point mutations in the SES loop lead to a loss of recognition by mAb 1.11.3, as did the double mutant. In addition, the triple mutant (K1845A / H1868A / E1939A) produced a loss of recognition by mAbs 1.11.3 and 24.8, although recognition by antibody by mAbs 20.10.6 and 21.5.8 was not affected. In summary, vain mutations were identified in the three helicase loops that produced a deletion of mAb recognition and binding. In addition, it was discovered that it is feasible to simultaneously alter recognition sites for two antibodies, while still maintaining the helicase function. Therefore, each of these individual mutations, or a combination of the same, could serve as a vaccine labeled against BVDV, which contains a mutation or mutations in the helicase region.

Table 5. Immunoreactivity of mAb with Helicase Mutants Example 7. Generation and Analysis of Marked Viruses To evaluate the effect or effects of mutations directed on the NS3 protein on viral replication and infectivity, it was necessary to move the mutations in a proviral piásmido containing the full-length BVDV sequence (pNADLp15A). The three mutated sequences chosen for the additional study were: K1845A-H1868A-E1939A, R1942A, and E 1939 A. A DNA fragment containing each respective mutated sequence of interest was cloned into pNADLp15A, once again using the unique restriction sites Bsm Bl and Sma I. The ligation mixtures were transformed into E. coli GM2163 cells (New England Biolabs, Inc.; Beverly, MA), and then plated on selective media. After incubation overnight, the colonies were screened for the presence of the pyramide that contained the correct sequence. A representative clone of each mutation (R1942A, E1939A, and KHE) was selected, and from these clones, viral RNA was prepared as described in Example 6. MDBK cells were transfected with each RNA preparation, and incubated 37 ° C for 64 hours. Duplicate transfections of RD cells (ATCC, Rockville, D) were established for each mutant. A set of transfected cells was fixed for IHC staining as described in Example 6, and from the second set, the cells were scraped of the seed flasks and stored at -80 ° C as stock solutions for future propagation. To further evaluate the virus produced by the three clones, the culture fluids collected from the transfection experiment were passed over the monolayers of fresh RD cells. After adsorption and incubation overnight, the cells were fixed for the IHC analysis. The results of that analysis are shown in Table 6. Both wild type and mutant viruses were recognized by mAb 20.10.6 (control antibody). The wild-type virus was also recognized by mAb 1.1.3 and 24.8. The E1939A mutant was linked by mAb 24.8, but not by 1.1 1.3. The mutant K-H-E was recognized only by mAb 20.10.6, and not by 1.1 1.3 or 24.8. The mutant R1942A demonstrated reactivity with mAb 24.8, but not with 1.1 1.3. Table 6. IHC Analysis of Cells Infected with Marked Viruses The growth kinetics of each labeled virus was also evaluated. Stock virus titers were predetermined for each using a conventional virus titration protocol. In a time course study, fresh monolayers of RD cells were seeded into tissue culture flasks, incubated overnight and the next day infected with a predetermined amount of each virus. After the adsorption and washing, an initial set of samples was collected (Hour "0"). Samples were collected later at 14, 19, 24, 39, 43, 47, and 65 hours after infection. Virus titers were determined using the Spearman-Karber procedure (Hawkes, RA in EH Lennette (ed.), Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, pp. 33-35; 7th ed., American Public Health Association Publications, Washington, DC) and expressed themselves as TCID5o / ml. Compared to wild-type (parental) BVD virus, all mutants grew at a rate similar to, or in some cases, slightly better than the wild-type (Table 7) Table 7. Comparative Titles of Wild Type BVD Viruses and Mutants (TCID50 / ml) Some of the mutations generated produced the alteration of specific immunologically distinct epitopes, determined by a panel of monoclonal antibodies. Similar results were obtained when recognition by antibodies was analyzed in the context of an infectious viral particle. Clones that contain mutations that did not prevent the generation of infectious viruses, but led to a loss of recognition by mAb, represent novel strains that serve as vaccine strains labeled against effective BVDV. Example 8. Testing of Vaccine Efficacy in a Young Calf Model Healthy calves negative to BVDV are obtained, randomly assigned to study groups, and kept under the supervision of a veterinarian. The test vaccine is combined with a sterile adjuvant and is administered by intramuscular (IM) or subcutaneous (SC) injection. Two doses of vaccine are administered, separated from each other for 21 to 28 days. The animals are subsequently stimulated on days 21 to 28 after the final vaccination with a Type 1 or Type 2 strain of BVDV. The stimulation inoculum is given intranasally in a divided dose of 4 ml, 2 ml per nostril. Control groups consisting of unvaccinated, unstimulated and / or unvaccinated, stimulated animals were also maintained throughout the study. Clinical parameters are controlled daily, including rectal temperature, depression, anorexia and diarrhea. The neutralization titers in serum are determined by a test of constant virus, decreasing serum in bovine cell culture, using serial dilutions of serum combined with a type 1 or type 2 BVDV strain. Isolation after stimulation of BVDV in bovine cell culture is attempted from peripheral blood. A culture of BVDV positive cells is determined by indirect immunofluorescence. To demonstrate protection after stimulation, a reduction in the incidence of infection in vaccinated groups versus control groups must be demonstrated.

Example 9. Vaccine Efficacy Test in a Cow-Pregnant-Calf Model Cows and heifers of BVDV-negative reproductive age are obtained and randomly assigned to a vaccination test group and a placebo (control) group . Cows are inoculated twice by intramuscular (IM) or subcutaneous (SC) injection with vaccine or placebo, separated 21 to 28 days. After the second vaccination, all the cows receive an IM injection of prostaglandin to synchronize the estrus. Cows that exhibit estrus are pregnant by artificial insemination with semen negative to BVDV certified. At approximately 60 days of gestation, the pregnancy status of the cows is determined by rectal palpation. Approximately 6 weeks later, cows with confirmed pregnancy are randomly selected from each test group. Each of these cows is stimulated by intranasal BVDV inoculation of Type 1 or 2. Blood samples are collected on the day of stimulation and at multiple intervals after stimulation for BVDV isolation purposes. Twenty-eight days after the stimulation, laparotomies of the left flank are performed and amniotic fluid is extracted from each cow. Immediately before surgery, a blood sample is collected from each cow for serum neutralization tests. After delivery by caesarean section, a blood sample is taken from each fetus. The fetuses are then sacrificed with euthanasia, and the tissues are aseptically collected for BVDV isolation purposes. In cases in which a miscarriage occurs, blood samples are taken from the baby when the abortion is detected and two weeks later. Paired blood samples and aborted fetuses are subjected to serological testing and virus isolation. The effectiveness of the vaccine is demonstrated by an absence of fetal infection and late phase abortion. Example 10. Diagnostic Test for Vaccines Marked Against BVDV Cattle of various ages can be vaccinated with a live attenuated BVDV vaccine or inactivated mutated NS3 (labeled) according to the instructions provided. Serum samples can be collected 2-3 weeks or later after vaccination. To differentiate between cows, which received the BVDV vaccine labeled against those infected with a field strain (wild type) of BVDV, serum samples can be assayed by a differential diagnostic assay. The NS3 protein with epitope-specific amino acid mutations can, when presented to cows in the context of a labeled vaccine, elicit the production of specific antibodies that will bind to the mutated epitopes of the NS3 protein, but not to the non-mutated epitopes present in the wild type virus. In the context of wild-type virus, the opposite is true, since specific antibodies can recognize wild-type epitopes in the NS3 protein, but not in the mutated form. Methods for testing the specificity and binding affinity of antibodies are well known in the art and include, but are not limited to, immunoassay formats such as ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays, and the like. A competitive ELISA can be a direct or indirect assay. This document describes an example of a direct competitive trial. Full or partial wild type viral antigens can be used, including the NS3 protein (naturally occurring or synthetically obtained) as a source of antigens. After coating the ELISA plate with antigen under alkaline conditions, the cow serum samples and dilutions are added together with an optimized dilution of the epitope-specific mAb, and incubated for 30-90 minutes. Horseradish horseradish peroxidase or alkaline phosphatase has been conjugated to the mAb to allow colorimetric detection of the binding. After washing the plates, a chromogenic substrate specific for the enzyme is added, and after a final incubation step, the optical density of each well is measured at an appropriate wavelength for the substrate used. Depending on the level of reactivity of the cow serum with the plate coated with the NS3 protein, the binding of the labeled mAb could be inhibited. An absence of binding by the mAb indicates the presence of antibodies in the cow serum that recognizes the wild-type specific epitope, indicative of a natural (wild-type) binding. In contrast, serum from cows immunized with the labeled vaccine having a mutation or epitope-specific mutations will contain antibodies that will bind to the NS3-coated plate. Therefore, the mAb will bind to the NS3 protein, and will produce a further development of color. Numerous variations will occur to those skilled in the art in light of the above description. For example, other cytopathic strains of BVDV in the helicase domain of NS3 can be mutated in a manner analogous to that exemplified herein by the NADL strain. Although exemplary mutations in this document use alanine, other non-conservative amino acid replacements, or other mutations that result in retention of replication, but loss of recognition for antibodies raised against wild-type NS3 are within the scope of the invention. These are merely exemplary.

Claims (39)

1. A virus of bovine viral diarrhea comprising at least one amino acid mutation in the helicase domain in which the mutation in the helicase domain of NS3 causes a loss of recognition by a monoclonal antibody raised against NS3 of the bovine viral diarrhea virus of wild type, but in which the replication of viral RNA and the generation of infectious virus is maintained.

2. A virus of bovine viral diarrhea comprising at least one amino acid mutation in the helicase domain in which NS3 is not recognized by a monoclonal antibody against NS3, wherein the antibody against NS3 is selected from the group consisting of 20.10.6; 1.11.3; 21.5.8; and 24.8, but in which the RNA replication and the generation of infectious virus is maintained.

3. The bovine viral diarrhea virus of claim 1, wherein the vaccine against the virus comprises a single amino acid mutation in the helicase domain.

4. The bovine viral diarrhea virus of claim 1 comprising a mutation in the helicase domain in the IGR loop.

5. The virus of bovine viral diarrhea of claim 4 comprising a mutation in the helicase domain in the IGR loop in the amino acid residue 1841.

6. The virus of bovine viral diarrhea of claim 4 comprising a mutation in the helicase domain in the IGR loop in the amino acid residue 1843.

7. The bovine viral diarrhea virus of claim 4 comprising a mutation in the helicase domain in the IGR loop in the amino acid residue 1845.

8. The bovine viral diarrhea virus of claim 1 comprising a mutation in the helicase domain in the KHP loop.

9. The bovine viral diarrhea virus of claim 8 which comprises a mutation in the helicase domain in the KHP loop in the amino acid residue 1867.

10. The virus of bovine viral diarrhea of claim 8 comprising a mutation in the helicase domain in the KHP loop in the amino acid residue 1868.

11. The virus of bovine viral diarrhea of claim 8 comprising a mutation in the helicase domain in the KHP loop in the amino acid residue 1869.

12. The bovine viral diarrhea virus of claim 1 comprising a mutation in the helicase domain in the SES loop.

13. The virus of bovine viral diarrhea of claim 12 comprising a mutation in the helicase domain in the SES loop in the amino acid residue 1939.

14. The bovine viral diarrhea virus of claim 12 comprising a mutation in the helicase domain in the SES loop in the amino acid residue 1942.

5. The bovine viral diarrhea virus of claim 1, wherein the virus comprises two, three or four amino acid mutations in the helicase domain.

16. The virus of bovine viral diarrhea of claim 15 comprising two mutations in the helicase domain.

17. The bovine viral diarrhea virus of claim 16, wherein the two mutations in the helicase domain are in the IGR loop.

18. The virus of bovine viral diarrhea of claim 17, wherein the two mutations in the helicase domain in the IGR loop are in the amino acid residues 1843 and 1845.

19. The bovine viral diarrhea virus of claim 16, wherein the two mutations in the helicase domain are in the SES loop.

20. The virus of bovine viral diarrhea of claim 19, wherein the two mutations in the helicase domain in the SES loop are in the amino acid residues 1939 and 1942.

21. The virus of bovine viral diarrhea of claim 5, comprising three mutations in the helicase domain.

22. The bovine viral diarrhea virus of claim 21, wherein all three mutations in the helicase domain are in the IGR loop.

23. The virus of bovine viral diarrhea of claim 22, wherein the three mutations in the helicase domain in the IGR loop are in the amino acid residues 1867, 1868, and 869.

24. The virus of bovine viral diarrhea of claim 21, wherein the three mutations in the helicase domain are in the IGR loop and the SES loop are in the amino acid residues 1845, 1868, and 1939.

25. A bovine viral diarrhea virus-labeled vaccine comprising a virus of bovine viral diarrhea comprising at least one amino acid mutation in the helicase domain, wherein the mutation in the helicase domain of NS3 results in a loss of recognition by a monoclonal antibody produced against NS3 of wild-type bovine viral diarrhea virus, but in which the replication of RNA and the generation of infectious virus is maintained.

26. A method for differentiating an animal infected with bovine diarrhea virus from an animal vaccinated with a vaccine with a vaccine against bovine diarrhea virus wherein said bovine diarrhea virus vaccine is a labeled vaccine comprising at least an amino acid mutation in the helicase domain, said method comprising; obtain a test sample from a test animal; detect the virus of bovine diarrhea in said test sample; and determine if the bovine diarrhea virus contains the mutation.

27. The method of claim 26, wherein said method for detecting the bovine diarrhea virus employs the use of at least one monoclonal antibody.

28. The method of claim 26, wherein the amino acid mutation in the helicase domain of the labeled vaccine is in the helicase domain of NS3.

29. The method of claim 27, comprising the steps of: adding labeled antibody capable of detecting the wild-type bovine diarrhea virus or capable of detecting the virus of the bovine diarrhea mutated to a test sample, wherein the sample test contains body fluid from the test animal and; measuring the binding affinity of said labeled antibody to said wild-type bovine diarrhea virus or to said bovine diarrhea virus mutated by contacting at least one monoclonal antibody against said wild-type bovine diarrhea virus or said bovine mutated bovine diarrhea; determining the vaccination status of the test animal by comparing binding affinity results using a monoclonal antibody directed against wild type BVDV versus BVDV with mutated NS3.

30. The method of claim 27 comprising the steps of: adding a first labeled antibody directed against a domain other than the mutated NS3; and adding a second labeled antibody directed against a mutated part of NS3.

31. The method of claim 30, wherein the first antibody is directed against a wild-type virus.

32. The method of claim 30, wherein the second antibody is directed against the mutated part of NS3.

33. The method of claim 32, wherein the second antibody is directed against NS3 and is selected from the group consisting of 20.10.6; 1.11.3; 21.5.8; and 24.8.

34. The method of claim 32, wherein the second antibody is directed against at least one mutated part of NS3 selected from the group consisting of the IGR loop, the KHP loop and the SES loop.

35. The method of claim 34, wherein the bovine viral diarrhea virus it comprises at least one amino acid mutation in the helicase domain in the IGR loop in an amino acid residue selected from the group consisting of 1841, 1843 and 1845.

36. The method of claim 34, wherein the bovine viral diarrhea virus comprises at least one amino acid mutation in the helicase domain in the KHP loop in the amino acid residue selected from the group consisting of 1867, 1868, and 1869.

37. The method of claim 34, wherein the bovine viral diarrhea virus comprises at least one amino acid mutation in the helicase domain in the SES loop in an amino acid residue selected from the group consisting of 1939 and 1942.

38. The method of claim 34, wherein the bovine viral diarrhea virus comprises at least one amino acid mutation in the helicase domain in the IGR loop and the SES loop in the amino acid residues 1845, 1868, and 1939.