# Patent

## Title
Safe mutant viral vaccines

## Abstract

The present invention provides safe vaccines and methods of preparing such vaccines. The vaccines of the present invention contain at least two live mutant viruses of the same family or nucleic acid molecules encoding such viruses, wherein each of the two viruses or the encoding nucleic adds contains a mutation that confers a desirable phenotype and the mutations in the viruses reside in the same genomic site such that the mutant viruses cannot recombine with each other to eliminate the mutations.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 60/490,834, filed Jul. 29, 2003.


FIELD OF THE INVENTION
The present invention relates generally to vaccines suitable for administration to animals against viral infections. More specifically, the present invention relates to safe vaccines and methods of preparing such vaccines. The vaccines of the present invention contain at least two live mutant viruses of the same family or nucleic add molecules encoding such viruses, wherein each of the viruses or the encoding nucleic adds contains a mutation that confers a desirable phenotype and the mutations in the viruses reside in the same genomic site such that the mutant viruses cannot recombine with each other to eliminate the mutations.
BACKGROUND OF THE INVENTION
The virus family Flaviviridae consists of the genera Pestivirus, Flavivirus and Hepacivirus. The genus Pestivirus is represented by the species Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, classical swine fever virus, and Border disease virus. The virions of the family members encapsulate positive-strand RNA genomes of about 9.5 to 12.3 kb. The genomic RNAs contain contiguous long open reading frames (ORFs), which are translated into polyproteins that are processed by cellular and viral proteases to give rise to the mature viral proteins. For members of Pestivirus, the ORF encodes a polyprotein of about 3900 amino acids, which is cotranslationally and posttranslationally processed to the following mature viral proteins (from 5′ to 3′): Npro, C, Ems, E1, E2, NS2-3, NS4A, NS4B, NS5A, and NS5B.
Two biotypes are found among some members of Pestivirus based on their effect on tissue culture cells, namely cytopathogenic (cytopathic or cp) and noncytopathogenic (noncytopathic or ncp). Genome analyses revealed insertions of cellular sequences, sometimes accompanied by duplication of viral sequences, genomic rearrangements, and/or deletions of viral sequences in the genomes of cp pestiviruses, but not in the RNAs of the corresponding ncp pestiviruses. This suggests that cp pestviruses are evolved from ncp pestiviruses by RNA recombination.
BVDV is a widely distributed pathogen of cattle. BVDV-1 usually produces only mild diarrhea in immunocompetent animals, whereas BVDV-2 can produce thrombocytopenia, hemorrhages and acute fatal disease. BVDV is capable of crossing the placenta of pregnant cattle and may result in the birth of persistently infected (PI) calves (Malmquist, J. Am. Vet. Med. Assoc. 152:763-768 (1968); Ross, et al., J. Am. Vet Med. Assoc. 188:618-619 (1986)). Viremic calves are immunotolerant to the virus and persistently viremic for the rest of their lives. They provide a source for outbreaks of mucosal disease (Liess, et al., Dtsch. Tieraerztl. Wschr. 81:481 -487 (1974)) and are highly predisposed to infection with microorganisms causing diseases such as pneumonia or enteric disease (Barber, et al., Vet. Rec. 117:459-464 (1985)). Viruses of either genotype may exist as one of the two biotypes, cp or ncp. The cp phenotype correlates with the expression of NS3, since cells infected with either cp or ncp BVDV both express NS2-3, whereas NS3 is detected only after infection with cp BVDV. NS3 is colinear to the C-terminal part of NS2-3. The expression of NS3 appears to be a result of genomic alterations observed for cp BVDV.
Presently available viral vaccines include killed or attenuated live viral vaccines, live-vectored vaccines, subunit vaccines, and DNA or RNA vaccines. See Roth et al., “New Technology For Improved Vaccine Safety And Efficacy”, Veterinary Clinics North America: Food Animal Practice 17(3): 585-597 (2001). Attenuation of viruses can be achieved by UV irradiation, chemical treatment, or high serial passage in vitro. The number, position and nature of mutations induced by these methods are unknown absent genomic sequence analyses. Attenuation can also be achieved by making defined genetic alterations, for example, specific deletion of viral sequences known to confer virulence, or insertion of sequences into the viral genome. One concern with respect to the use of attenuated live viral vaccines is that attenuated mutant viruses have the potential to recombine in vivo to eliminate the attenuating mutation(s) thereby restoring virulence. For example, in the presence of a virulent (wild type) field strain, attenuated viruses having deletions in the viral genome have the potential to recombine with the virulent strain to restore the deleted sequence. See, e.g., Roth et al., supra. Cytopathic pestviruses having cellular insertions have also been observed to give rise to noncytopathic viruses in cell culture by deletion of the cellular sequences, possibly through RNA recombination. See, e.g., Baroth et al., “Insertion of cellular NEDD8 coding sequences in a pestivirus”, Virology. 278(2): 456-66, (2000), and Becher et al., “RNA recombination between persisting pestvirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease”, Journal of Virology 75(14): 6256-64 (2001). Where it is desired to include two attenuated mutant viruses from the same species, genus or family in a vaccine composition, there is a concern that the two viruses may recombine in the vaccinated animal thereby eliminating the attenuating mutations. See, e.g., Glazenburg et al., “Genetic recombination of pseudorabies virus: evidence that homologous recombination between insert sequences is less frequent than between autologous sequences”, Archives of Virology, 140(4): 671-85 (1995).
There remains a need to develop safe and effective vaccines that protect animals against viral infections.
SUMMARY OF THE INVENTION
The present invention provides safe vaccines which contain at least two live mutant viruses of the same family or nucleic acid molecules encoding such viruses, wherein each virus or the encoding nucleic acid contains a mutation that confers a desirable phenotype, and the mutations in the viruses reside in the same genomic site such that the mutant viruses cannot recombine with each other to eliminate the mutations.
The present invention also provides a method of preparing a safe viral vaccine by selecting or constructing two or more live mutant viruses of the same family, genus or species, wherein each virus contains a mutation that confers a desirable phenotype, and the mutations in the viruses reside in the same genomic site such that the mutant viruses can not undergo homologous recombination to eliminate the mutations.
The present invention further provides a method of protecting an animal against viral infections by administering to the animal a vaccine composition of the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Alignment of the cellular insertions and flanking viral sequences from the NS2-3 regions of BVDV-1 strain NADL and BVDV-2 strain 53637. The sequences shown in this figure are presented as SEQ ID NOs 11-18 in the sequence listinq.



DETAILED DESCRIPTION OF THE INVENTION
It has been uniquely recognized in accordance with the present invention that live mutant viruses of the same family, which contain mutations at the same genomic site of the viruses, cannot recombine with one another to eliminate the mutations.
Accordingly, in one embodiment, the present invention provides safe vaccine compositions containing at least two, i.e., two or more, live mutant viruses of the same family, or nucleic acid molecules encoding such viruses, wherein the mutations in the viruses reside in the same genomic site such that the mutant viruses cannot recombine with each other to eliminate the mutations.
In another embodiment, the present invention provides a method of preparing a safe viral vaccine, as described hereinabove. Specifically, a safe vaccine is prepared by selecting or constructing two or more live mutant viruses of the same family, genus or species, wherein each virus contains a mutation that confers a desirable phenotype (for example attenuation of virulence, alteration of cellular tropism or biotype, alteration of species tropism, or expression of a foreign gene cassette), and the mutations in the viruses reside in the same genomic site such that the mutant viruses can not undergo homologous recombination with each other to eliminate the mutations.
The term “vaccine” or “vaccine composition” refers to a composition containing live mutant viruses which, upon inoculation into an animal, induces a complete or partial immunity to the pathogenic version of the viruses, or alleviates the symptoms of diseases caused by the pathogenic versions of the viruses. The protective effects of a vaccine composition against a virus are normally achieved by inducing in the subject an immune response, either a cell-mediated or a humoral immune response, or a combination of both. Generally speaking, abolished or reduced incidences of viral infection, amelioration of the symptoms, or accelerated elimination of the viruses from the infected subjects, are indicative of the protective effects of the vaccine composition.
By “animal” is meant to include birds, for example, chickens, turkeys, domestic waterfowl, and any mammal, for example, cattle, sheep, swine, goats, dogs, cats, and horses.
The term “viruses”, “viral isolates” or “viral strains” as used herein refer to viral particles or virions that contain viral genomic DNA or RNA, associated proteins, and other chemical constituents (such as lipids).
By “nucleic acid molecule encoding a virus” or “nucleic acid molecule of a virus” is meant the genomic nucleic acid molecule of the virus, either in the form of RNA or DNA.
By “mutation” is meant to include deletion, insertion or substitution of one or more nucleotides, or a combination thereof. In accordance with the present invention, the mutation preferably confers a desirable phenotype, for example attenuation of virulence, alteration of cellular tropism or biotype, alteration of species tropism, or expression of a foreign gene cassette. Especially preferred mutations are mutations that confer attenuated virulence.
By “attenuation” is meant that the virus has lost some or all of its ability to proliferate and/or cause disease in an animal infected with the virus. For example, an attenuated virus can be a virus that is unable to replicate at all or is limited to one or a few rounds of replication, or restricted in cell or tissue tropism, when present in an animal in which a wild type pathogenic version of the attenuated virus can replicate.
An attenuated virus may have one or more mutations in a gene or genes that are involved in pathogenicity of the virus. Such mutations are also referred to herein as “attenuating mutation(s)”. An attenuated virus can be produced from the wild type, pathogenic virus by UV irradiation, chemical treatment, or high serial passage of the wild type, pathogenic virus in vitro. Alternatively, an attenuated virus can be produced from the wild type, pathogenic virus by making specific deletion of viral sequences known to confer virulence, insertion of sequences into the viral genome, or making one or more point mutations in the viral genome. An attenuated virus can be a viral isolate obtained from an animal, which isolate is derived from the wild type, pathogenic version of the virus through events other than artificial means, e.g., events that have occurred in a host animal such as recombination.
The two or more live mutant viruses present in the vaccine compositions of the present invention contain mutations that reside in the same genomic site. By “same genomic site” is meant that when the genomic nucleotide sequences of the viruses are aligned, the mutations in the viral genomes overlap with one another such that there is no opportunity for homologous recombination between and among the viral genomes to eliminate the mutations. In other words, when the genomic nucleotide sequences of the viruses are aligned, there is at least one contiguous portion of the aligned sequences where the sequences in the aligned viral genomes are mutant sequences. There are a number of computer programs that compare and align nucleic acid sequences which one skilled in the art may use. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a nucleic acid sequence for optimal alignment with a second nucleic acid sequence). For example, the NBLAST and XBLAST programs as described in Altschul, et al., 1990, J Mol. Biol. 215:403-410, the Gapped BLAST program as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, and the PSI-Blast program as described in Altschul et al., 1997, supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see—the United States government web site from the National Center for Biotechnology Information, US National Library of Medicine, National Institutes of Health).
Generally speaking, the concept of the present invention, i.e., including in the same vaccine composition two or more live mutant viruses of the same family having mutations at the same genomic site, applies to mutant viruses from any family where the viral genomes have sufficient sequence identity to permit homologous recombination. It has been shown that a nucleotide identity as short as 15 nucleotides can lead to efficient homologous recombination (Nagy and Bujarski, J. Virol. 69:131-140, 1995).
The present invention applies especially to viruses of the Flaviviridae family. The Flaviviridae family consists of the genera Pestivirus, Flavivirus and Hepacivirus. The virions of the Flaviviridae family members encapsulate positive-strand RNA genomes of about 9.5 to 12.3 kb. The genomic RNAs containing contiguous long open reading frames, which are translated into polyproteins that are processed by cellular and viral proteases to give rise to the mature viral proteins.
Preferably, the mutant viruses of the vaccine composition of the present invention are from the same genus, either the same or different species.
In a preferred embodiment, the vaccine composition of the present invention contains two or more live mutant viruses from the Pestivirus genus. The genus Pestivirus is represented by the species Bovine Viral Diarrhea Virus Type 1 (BVDV-1), Bovine Viral Diarrhea Virus Type 2 (BVDV-2), classical swine fever virus, and Border disease virus. The ORF encodes a polyprotein of about 3900 amino acids, which is co-translationally and post-translationally processed to the following mature viral proteins (from 5′ to 3′): Npro, C, Ems, E1, E2, NS2-3, NS4A, NS4B, NS5A, and NS5B.
Ordinarily, BVDV has a genome in the form of RNA. RNA can be reverse-transcribed into DNA for use in cloning. Thus, references made herein to nucleic acid and BVD viral sequences encompass both viral RNA sequences and DNA sequences derived from the viral RNA sequences. For convenience, genomic sequences of BVDV as depicted in the SEQUENCE LISTING hereinbelow only refer to the DNA sequences. The corresponding RNA sequence for each is readily apparent to those of skill in the art.
In a more preferred embodiment, the vaccine composition of the present invention contains a cytopathic BVDV-1 and a cytopathic BVDV-2, wherein the mutations in both viruses associated with the cytopathic biotype reside in the same genomic site such that the two mutant viruses cannot recombine to eliminate the mutations.
BVDV-1 and BVDV-2 represent two closely related genotypes of BVDV. The nucleotide sequences of the two viruses share about 70% identity over the entire genome, and slightly higher percent identity within the NS2-3 region. It is believed that the percent identity between the viral genomes of BVDV-1 and BVDV-2, at least in the NS2-3 region, is sufficient to permit homologous recombination.
BVDV-1 usually produce only mild diarrhea in animals, whereas BVDV-2 are viruses with high virulence which can produce thrombocytopenia, hemorrhages and acute fatal disease (Corapi et al., J. Virol. 63: 3934-3943; Bolin et al., Am. J. Vet. Res. 53: 2157-2163; Pellerin et al., Virology 203: 260-268, 1994; Ridpath et al., Virology 205: 66-74, 1994; Carman et al., J. Vet. Diagn. Invest. 10: 27-35, 1998). The two types of viruses have distinct antigenicity determined by a panel of MAbs and by cross-neutralization using virus-specific antisera raised in animals (Corapi et al., Am. J. Vet. Res. 51: 1388-1394, 1990). Viruses of either genotype may exist as one of the two biotypes, cytopathogenic (cytopathic or cp) or noncytopathogenic (noncytopathic or ncp). Cp viruses induce cytopathic effects (e.g., cell lysis) on cultured cells, while noncytopathic viruses do not.
It is desirable to prepare vaccines that provide protection against both BVDV-1 and BVDV-2. However, because of the high degree of sequence identity between the two viruses, there is a possibility that a live cytopathic BVDV-1 and a live cytopathic BVDV-2 included in the same vaccine composition, could recombine with each other in the vaccinated animal to yield noncytopathic viruses. Recombinabon between BVDV-1 and BVDV-2 has been documented. See, e.g., Ridpath et al., Virology 212: 259-262 (1995). Infection of the fetus in pregnant cattle with ncp viruses before immunocompetence develops can result in the fetus remaining viremic through the period of gestation and the subsequent birth of a calf that remains persistently viremic. Such a calf can die of mucosal disease upon superinfecton with a cp BVDV. Accordingly, the vaccine compositions provided by the present invention, which contain live cp BVDV-1 and live cp BVDV-2 having mutations at the same genomic site, are especially desirable for protecting animals against both BVDV-1 and BVDV-2.
In one embodiment, BVDV cp isolates obtained from animals can be used in the vaccine composition of the present invention. Cp isolates of both BVDV-1 and BVDV-2 have been reported and are available to those skilled in the art, e.g., BVDV-1 NADL (ATCC# VR1422 or VR-534), BVDV-2 53637 strain (deposited with the ATCC as PTA4859), and type 2 field isolates such as those described by Ridpath and Neill, J. Virol 74:8771-8774, (2000). Cp isolates reported so far typically contain an insertion of a heterologous sequence, e.g., an ubiquitin coding sequence (Genbank accession number M96687 or De Moerlooze et al., J. Gen. Virol. 74:1433-1438, (1993)), a bovine NEDD8 coding sequence (Baroth et al., supra), or a Bos taurus DnaJ1 coding sequence (as described in the Examples hereinbelow), among others.
In another embodiment, a cp BVDV is generated by making defined alterations in the BVDV genome, e.g., by deleting specific viral sequences, inserting sequences into a specific viral genomic site, or making one or more substitutions, or combinations thereof.
Where a cp BVDV is generated by inserting a heterologous (i.e., foreign to the virus) sequence into a specific genomic site, the nature of the sequence to be inserted is generally not critical to the present invention. In addition, the insertion is not limited to any particular site so long as the insertion results in an attenuated phenotype. As heterologous sequences in cp isolates are often found in the NS2-3 region, the NS2-3 region, especially the part surrounding the putative NS2-3 cleavage site which corresponds to, e.g., amino acid residues # 1679 to #1680 of the BVDV-1 NADL strain (the numbering is based on the published genomic sequence Genbank accession No. M31182, SEQ ID NO: 4), is a preferred location for insertions.
An cp BVDV-1 can be generated by making a defined genomic alteration that mimics the mutation identified in a cp BVDV-2 isolate obtained from an animal, such that these viruses have mutations associated with the cp biotype in the same genomic site. Similarly, a cp BVDV-2 can be generated by way of making a defined genomic alteration that mimics the mutation identified in a cp BVDV-1 isolate obtained from an animal.
In a preferred embodiment, the vaccine composition of the present invention contains NADL (a cp BVDV-1 isolate), and BVDV-2 53637 (a cp BVDV-2 isolate), where the two cp isolates each contain a mutation at the same genomic site which results in the cytopathic biotype. The genomic sequence of the BVDV-1 NADL strain is set forth in SEQ ID NO: 4, and the BVDV-2 53637 strain was deposited with the ATCC as PTA-4859. Both isolates contain an insertion in the NS2-3 region. The attenuated cp BVDV-1 contains an insertion of a Bos taurus DnaJ1 coding sequence 3′ of the thymidine at nucleotide position # 4993 (NADL sequence numbering), which is the third nudeotide of the codon encoding the glycine residue at amino acid position 1536. The attenuated cp BVDV-2 contains an insertion of a Bos taurus DnaJ1 coding sequence at the same genomic site.
According to the present invention, the cp BVDV isolates employed in the present vaccine composition have been attenuated and are therefore nonpathogenic. Methods of attenuation are known to those skilled in the art and are also described hereinbelow.
In another embodiment, the vaccine composition of the present invention contains an attenuated BVDV-1 and an attenuated BVDV-2, wherein the attenuating mutations in both viruses reside in the same genomic site such that the two mutant viruses cannot recombine to eliminate the attenuating mutations.
An attenuated BVDV is generated by UV irradiation, chemical treatment, or high serial passage of the pathogenic version of the viruse in vitro. Sequence analysis can be conducted in order to determine the nature and genomic location of mutations generated by these methods. The mutation can be in the form of a deletion, insertion or substitution of one or more nucleotides, or a combination thereof. Alternatively, an attenuated BVDV is generated by making defined alterations in the BVDV genome, e.g., by deleting specific viral sequences, inserting sequences into a specific viral genomic site, or making one or more substitutions, or combinations thereof.
As described above, the live mutant viruses for use in the vaccine composition of the present invention can be from the same family, genus or species, where the viral genomes have sufficient sequence identity to permit homologous recombination. Additional examples of combinations of viruses appropriate for use in the vaccine composition of the present invention include, but are not limited to, combinations of different types of poliovirus, combinations of multiple live mutant strains of infectious bronchitis virus, combinations of multiple live mutant strains of Newcastle disease virus, combinations of Canine adenovirus-1 and canine adenovirus-2, combinations of equine herpesvirus-1 and equine herpesvirus-4, combinations of multiple live mutant strains of influenza virus, combinations of multiple live attenuated strains of Feline calicivirus, combinations of multiple serotypes of Rotavirus, combinations of multiple serotypes of Rhinovirus, combinations of multiple serotypes of Foot and Mouth Disease virus, combinations of the European and North American genotypes of Porcine reproductive and respiratory syndrome virus, combinations of standard and variant strains of infectious bursal disease virus.
In accordance with the present invention, although viral particles are the preferred form for use in the vaccines, nucleic acid molecules encoding mutant viruses of the same family, genus or species, can be used directly in vaccines as well. The DNA or RNA molecule can be present in a “naked” form or it can be combined with an agent which facilitates cellular uptake (e.g., liposomes or cationic lipids). Vaccines and vaccination procedures that utilize nucleic acids (DNA or mRNA) have been well described in the art, e.g., U.S. Pat. No. 5,703,055, U.S. Pat. No. 5,580,859, U.S. Pat. No. 5,589,466, International Patent Publication WO 98/35562, and by Ramsay et al., 1997, Immunol. Cell Biol. 75:360-363; Davis, 1997, Cur. Opinion Biotech. 8: 635-640; Manickan et al., 1997, Critical Rev. Immunol. 17: 139-154; Robinson, 1997, Vaccine 15(8): 785-787; Robinson et al., 1996, AIDS Res. Hum. Retr. 12(5): 455-457; Lai and Bennett, 1998, Critical Rev. Immunol. 18:449-484; and Vogel and Sarver, 1995, Clin. Microbiol. Rev. 8(3): 406-410, all of which are incorporated herein by reference.
In addition to two or more live mutant viruses from the same family, genus or species, the vaccine compositions can include other antigenic component. Other antigenic components appropriate for use in accordance with the present invention include, but are not limited to, antigens prepared from pathogenic bacteria such as Mycoplasma hyopneumonia, Haemophilus somnus, Haemophilus parasuis, Bordetella bronchiseptica, Bacillus anthracis, Actinobacillus pleuropneumonie, Pasteurella multocida, Mannhemia haemolytica, Mycoplasma bovis, Mycoplasma galanacieum, Mycoplasma gallisepticum, Mycobacterium bovis, Mycobacterium paratuberculosis, Clostridial spp., Streptococcus uberis, Streptococcus suis, Staphylococcus aureus, Erysipelothrix rhusopathiae, Campylobacter spp., Fusobacterium necrophorum, Escherichia coli, Lawsonia intracellularis, Listeria monocytogenes, Rickettsia rickettsii, Borrelia spp., Ehrlichia spp., Chlamydia spp., Brucella spp., Vibrio spp., Salmonella enterica serovars, Leptospira spp.; pathogenic fungi such as Candida; protozoa such as Cryptosporidium parvum, Neospora canium, Toxoplasma gondii, Eimeria spp., Babesia spp., Giardia spp.; helminths such as Ostertagia, Cooperia, Haemonchus, Fasciola; either in the form of an inactivated whole or partial cell preparation, or in the form of antigenic molecules obtained by genetic engineering techniques or chemical synthesis. Additional antigens include pathogenic viruses such as Marek's disease virus, infectious bursal disease virus, Newcastle's disease virus, chicken anemia virus, fowlpox virus, avian leukosis virus, infectious laryngotracheitis virus, reticuloendothelial virus, canine parvovirus, canine distemper virus, canine herpesvirus, canine coronavirus, canine parainfluenza-5, feline panleukopenia virus, feline herpes virus, feline calicivirus, feline immunodeficiency virus, feline infectious peritonitis virus, equine herpesvirus, equine arteritis virus, equine infectious anemia virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, West Nile virus, transmissible gastroenteritis virus, bovine coronavirus, Bovine herpesviruses-1,3,6, Bovine parainfluenza virus, Bovine respiratory syncytial virus, bovine leukosis virus, rinderpest virus, foot and mouth disease virus, rabies virus, African swine fever virus, Porcine parvovirus, PRRS virus, Porcine circovirus, influenza virus, swine vesicular disease virus, Techen fever virus, Pseudorabies virus, either in the form of modified live (attenuated) viral preparation, an inactivated whole or partial virus preparation, or in the form of antigenic molecules obtained by genetic engineering techniques or chemical synthesis. When additional attenuated live viruses are used, such additional viruses should preferably be from a family different from that of the two principal attenuated viruses, as described above.
In a preferred embodiment, the present invention provides a vaccine composition which contains an attenuated cp BVDV-1 derived from the BVDV-1 NADL strain, an attenuated cp BVDV-2 derived from the BVDV-2 53637 strain, where the two cp isolates each contain a mutation associated with the cp biotype at the same genomic site, and at least one (i.e., one or more) of the following antigenic component, either in inactivated or modified live form: bovine herpesvirus-1, bovine respiratory syncytial virus, parainfluenza virus-3, Campylobacter fetus, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, Leptospira pomona, or Mannhemia haemolytica. 
In addition, the vaccine compositions of the present invention can 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 delaying agents, and the like. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. The vaccine compositions can further include one or more other immunomodulatory agents such as, e.g., interleukins, interferons, or other cytokines
Adjuvants suitable for use in the vaccine compositions include, but are not limited to, the RIBI adjuvant system (Ribi inc.), alum, aluminum hydroxide gel, oil-in water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block co polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A, cholesterol, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), or other saponin fractions, monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, or muramyl dipeptide, among many others.
Typically, a live mutant virus is present in a vaccine at an amount of about 1×106 and about 1×108 virus particles per dose, with a veterinarily acceptable carrier, in a volume of between about 0.5 and about 5 ml. The precise amount of a virus in a vaccine composition effective to provide a protective effect can be determined by a skilled veterinarian. Where the DNA or RNA molecule of the virus is used in the vaccine, the amount of the nucleic acids should generally be between about 0.1 μg/ml and about 5.0 mg/ml.
The vaccine compositions of the present invention can be made in various forms depending upon the route of administration. For example, the vaccine compositions can be made in the form of sterile aqueous solutions or dispersions suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized compositions are typically maintained at about 4° C., and can be reconstituted in a stabilizing solution, e.g., saline or and HEPES, with or without adjuvant.
The vaccine compositions of the present invention can be administered to an animal for treating or preventing a disease caused by the pathogenic versions of the viruses in the vaccine compositions. Therefore, methods of vaccinating an animal against a disease caused by a virus are also provided by the present invention.
In practicing the present methods, a vaccine composition of the present invention is administered to an animal preferably via parenteral routes, although other routes of administration can be used as well, such as e.g., by oral, intranasal, intramuscular, intra-lymph node, intradermal, intraperitoneal, subcutaneous, rectal or vaginal administration, or by a combination of routes. Boosting regimens may be required and the dosage regimen can be adjusted to provide optimal vaccination.
The present invention is further illustrated by, but by no means limited to, the following examples.
EXAMPLE I
Determination of the Position of the Cellular Insertion in BVDV2 Strain 53637
A portion of the sequence of the NS2-3 region from BVDV2-53637 was determined, in order to identify and map the location of any cellular insertions in the region. A 670 base RT-PCR product was amplified from viral RNA, using forward primer 53637U1 (5′-CGTCCACAGATGGTTTGGT-3′; SEQ ID NO: 1) and reverse primer 53637L (5′-GGCTATGTATTGGACGTAACCC-3′; SEQ ID NO: 2). The RT-PCR product was purified and submitted for sequence analysis (SEQ ID NO: 3). When aligned with BVDV1-NADL (Genbank accession number M31182, SEQ ID NO: 4), striking similarities were observed (FIG. 1). Both viruses contain an in-frame insertion derived from the Bos taurus DnaJ1 gene. In the case of NADL, the insertion is 90 amino acids (270 nucleotides) in length and is located between glycine-1536 and proline-1627 in the NADL polyprotein. These coordinates correspond to glycine-1536 and proline-1537 in non-cytopathic BVDV1 strains such as SD-1 (Genbank accession number AAA42860, SEQ ID NO: 6), indicating that the genome alteration in NADL is a simple insertion with no concomitant deletion or duplication of flanking viral sequences. Like BVDV1-NADL, there is an insertion of a portion of the Bos taurus DnaJ1 gene in BVDV2-53637. The cellular insertion is longer (131 amino acids, 393 nucleotides), being extended in both directions relative to the insertion in BVDV1-NADL. The location of the cellular insertion within the NS2-3 region is identical in the two viruses. Unlike BVDV1-NADL, the BVDV2-53637 insertion is accompanied by a deletion of 5 amino acids (15 nucleotides) of flanking viral sequences. Three amino acid residues are absent flanking the 5′ end of the insertion, while two amino acids residues are absent flanking the 3′ end of the insertion. Because the cellular insertions are at the same genome position in the two vaccine viruses, they cannot undergo homologous recombination to delete the insertion to generate a non-cytopathic chimeric virus.
EXAMPLE II
Attempts to Detect Non-Cytopathic BVDV Viruses in Co-passaged BVDV1-NADL/BVDV2-53637 Cultures
In order to determine whether the two vaccine viruses are capable of recombining to generate detectable levels of non-cytopathic BVDV, the viruses were co-cultivated on susceptible cells and a sensitive hemi-nested RT-PCR assay was used to detect potential non-cytopathic viruses from among an excess of longer cytopathic products that still contain the cellular insert. To increase the probability of intertypic recombination in vitro, each virus was inoculated simultaneously onto confluent BK-6 cells in 6-well plates at a multiplicity of infection of 2-4 (12 replicates per experiment). After 2-3 days of co-cultivabon the cells were frozen and thawed twice, and cell debris was removed by low speed centrifugation. The resulting supernatant fluid was then used as inoculum for the next passage. A total of seven serial passages were conducted in several studies. During the passages BVDV1-NADL grew more rapidly than BVDV2-53637, but the type II virus was still detectable after seven passages using nested RT-PCR. A sensitive hemi-nested RT-PCR assay was employed in an attempt to detect any non-cytopathic virus.
In first round RT-PCR, forward primers 53637U1 (SEQ ID NO: 1) or NADL4744 (5′-CGTGGCTTCTTGGTACGGG-3′,SEQ ID NO: 7) were used in conjuncton with reverse primers 53637L (SEQ ID NO: 2) or NADL5305 (5′-AGCGGTATATTGTACAAAGCCA-3′, SEQ IDNO: 8). All four combinations of forward and reverse primers were used in order to detect BVDV1, BVDV2, and intertypic recombinants. The expected size of RT-PCR product was 562 bp for cytopathic BVDV1-NADL and 670 bp for cytopathic BVDV2-53637. Non-cytopathic viruses, if present at detectible levels, would be expected to yield first round products of 292 bp (BVDV1-NADL) or 277 bp (BVDV2-53637). Intertypic recombinants should be similar in size to one of the parents, or of intermediate length, depending on the location of the recombination site. Non-cytopathic BVDVs were never detected following first round RT-PCR.
To increase the sensitivity of detecting non-cytopathic BVDV in the presence of a large excess of cytopathic BVDV, a restriction enzyme digestion step was included before the nested PCR to destroy the larger NS2-3 templates derived from the cytopathic viruses. A combination of Mspl and Dral was selected based on the observation that they cut within the Bos taurus DnaJ1 insert but do not cut the flanking viral sequences. In second round (hemi-nested) PCR, forward primers 53637U2 (5′-TGCACGATCTGTGAAGGGAAAGAA-3′, SEQ ID NO: 9) or NADL4844 (5′-TGCACTGTATGTGAGGGCCGAGAG-3′, SEQ ID NO: 10) were used in conjunction with the same two reverse primers 53637L or NADL5305. Appropriate primer combinations were used to attempt to detect intertypic recombinants as well as BVDV1 and BVDV2. The expected size of RT-PCR product is 462 bp for cytopathic BVDV1-NADL and 570 bp for cytopathic BVDV2-53637 (present at low levels due to incomplete digestion of the cytopathic BVDV RT-PCR products). Non-cytopathic viruses, if present at detectable levels, would be expected to yield second round products of 192 bp (BVDV1-NADL) or 177 bp (BVDV2-53637). Intertypic recombinants should be similar in size to one of the parents, or of intermediate length, depending on the location of the recombination site. Non-cytopathic BVDVs were never detected following second round PCR. In a few individual reactions, aberrant bands of various sizes were seen. All bands between 100 and 300 bp were considered to be potential non-cytopathic products and were submitted for DNA sequence analysis. In every case the aberrant band was the result of false priming during PCR. There was no evidence of non-cytopathic virus in any of the studies.











SEQ



ID NO
DESCRIPTION
















1
forward primer 53637U1


2
reverse primer 53637L


3
670 bp RT-PCR product from the NS2-3 region of BVDV2



strain 53637


4
genomic sequence of BVDV1-NADL (Genbank accession



number M31182)


5
polyprotein sequence of BVDV1-NADL (Genbank accession



number AAA42854)


6
polyprotein sequence of non-cytopathic BVDV1 strain SD-1



(Genbank accession number AAA42860)


7
forward primer NADL4744


8
reverse primer NADL5305


9
forward primer 53637U2


10
forward primer NADL4844

















SEQ NO: 1



cgtccacagatggtttggt


 


SEQ NO: 2


ggctatgtattggacgtaaccc


 


SEQ NO: 3


cgtccacagatggtttggtgaggaggaaatatatggggcacccaaggtgatcaccatcataaaagctagtac


 


cctaagtaaaaacaggcactgcataatctgcacgatctgtgaagggaaagaatggaacggagccaactgccc


 


aaagtgtggaagacaaggaaagcccataacatgtggaatgacactcgcagactttgaggagaaacattacaa


 


aaagatatttataagagaaggacgccaagaagcaatgaatacgatgatgtgcagccgatgccagggaaagca


 


taggaggtttgaaacggaccgggaacctaagagtgccagatactgtgctgagtgtaataggctgcatcctgc


 


tgaggaaggtgacttttgggcagagtcaagcatgttgggcctcaaaatcacctactttgcgctgatggatgg


 


aaaggtgtatgatatcacagagtgggctggatgccagcgtgtgggaatctccccagatacccacagagtccc


 


ttgtcacatctcatttggttcacggatgccaggcaccagtgggcggcagagagctactccagatgcccctcc


 


tgctgaccttcaggatttcttgagccggatctttcaagtacccccaggccagatgtccagggaagagtataa


 


gggttacgtccaatacatagcc


 


SEQ ID: 4


gtatacgaga attagaaaag gcactcgtat acgtattggg caattaaaaa taataattag


 


gcctagggaa caaatccctc tcagcgaagg ccgaaaagag gctagccatg cccttagtag


 


gactagcata atgagggggg tagcaacagt ggtgagttcg ttggatggct taagccctga


 


gtacagggta gtcgtcagtg gttcgacgcc ttggaataaa ggtctcgaga tgccacgtgg


 


acgagggcat gcccaaagca catcttaacc tgagcggggg tcgcccaggt aaaagcagtt


 


ttaaccgact gttacgaata cagcctgata gggtgctgca gaggcccact gtattgctac


 


taaaaatctc tgctgtacat ggcacatgga gttgatcaca aatgaacttt tatacaaaac


 


atacaaacaa aaacccgtcg gggtggagga acctgtttat gatcaggcag gtgatccctt


 


atttggtgaa aggggagcag tccaccctca atcgacgcta aagctcccac acaagagagg


 


ggaacgcgat gttccaacca acttggcatc cttaccaaaa agaggtgact gcaggtcggg


 


taatagcaga ggacctgtga gcgggatcta cctgaagcca gggccactat tttaccagga


 


ctataaaggt cccgtctatc acagggcccc gctggagctc tttgaggagg gatccatgtg


 


tgaaacgact aaacggatag ggagagtaac tggaagtgac ggaaagctgt accacattta


 


tgtgtgtata gatggatgta taataataaa aagtgccacg agaagttacc aaagggtgtt


 


caggtgggtc cataataggc ttgactgccc tctatgggtc acaacttgct cagacacgaa


 


agaagaggga gcaacaaaaa agaaaacaca gaaacccgac agactagaaa gggggaaaat


 


gaaaatagtg cccaaagaat ctgaaaaaga cagcaaaact aaacctccgg atgctacaat


 


agtggtggaa ggagtcaaat accaggtgag gaagaaggga aaaaccaaga gtaaaaacac


 


tcaggacggc ttgtaccata acaaaaacaa acctcaggaa tcacgcaaga aactggaaaa


 


agcattgttg gcgtgggcaa taatagctat agttttgttt caagttacaa tgggagaaaa


 


cataacacag tggaacctac aagataatgg gacggaaggg atacaacggg caatgttcca


 


aaggggtgtg aatagaagtt tacatggaat ctggccagag aaaatctgta ctggcgtccc


 


ttcccatcta gccaccgata tagaactaaa aacaattcat ggtatgatgg atgcaagtga


 


gaagaccaac tacacgtgtt gcagacttca acgccatgag tggaacaagc atggttggtg


 


caactggtac aatattgaac cctggattct agtcatgaat agaacccaag ccaatctcac


 


tgagggacaa ccaccaaggg agtgcgcagt cacttgtagg tatgataggg ctagtgactt


 


aaacgtggta acacaagcta gagatagccc cacaccctta acaggttgca agaaaggaaa


 


gaacttctcc tttgcaggca tattgatgcg gggcccctgc aactttgaaa tagctgcaag


 


tgatgtatta ttcaaagaac atgaacgcat tagtatgttc caggatacca ctctttacct


 


tgttgacggg ttgaccaact ccttagaagg tgccagacaa ggaaccgcta aactgacaac


 


ctggttaggc aagcagctcg ggatactagg aaaaaagttg gaaaacaaga gtaagacgtg


 


gtttggagca tacgctgctt ccccttactg tgatgtcgat cgcaaaattg gctacatatg


 


gtatacaaaa aattgcaccc ctgcctgctt acccaagaac acaaaaattg tcggccctgg


 


gaaatttggc accaatgcag aggacggcaa gatattacat gagatggggg gtcacttgtc


 


ggaggtacta ctactttctt tagtggtgct gtccgacttc gcaccggaaa cagctagtgt


 


aatgtaccta atcctacatt tttccatccc acaaagtcac gttgatgtaa tggattgtga


 


taagacccag ttgaacctca cagtggagct gacaacagct gaagtaatac cagggtcggt


 


ctggaatcta ggcaaatatg tatgtataag accaaattgg tggccttatg agacaactgt


 


agtgttggca tttgaagagg tgagccaggt ggtgaagtta gtgttgaggg cactcagaga


 


tttaacacgc atttggaacg ctgcaacaac tactgctttt ttagtatgcc ttgttaagat


 


agtcaggggc cagatggtac agggcattct gtggctacta ttgataacag gggtacaagg


 


gcacttggat tgcaaacctg aattctcgta tgccatagca aaggacgaaa gaattggtca


 


actgggggct gaaggcctta ccaccacttg gaaggaatac tcacctggaa tgaagctgga


 


agacacaatg gtcattgctt ggtgcgaaga tgggaagtta atgtacctcc aaagatgcac


 


gagagaaacc agatatctcg caatcttgca tacaagagcc ttgccgacca gtgtggtatt


 


caaaaaactc tttgatgggc gaaagcaaga ggatgtagtc gaaatgaacg acaactttga


 


atttggactc tgcccatgtg atgccaaacc catagtaaga gggaagttca atacaacgct


 


gctgaacgga ccggccttcc agatggtatg ccccatagga tggacaggga ctgtaagctg


 


tacgtcattc aatatggaca ccttagccac aactgtggta cggacatata gaaggtctaa


 


accattccct cataggcaag gctgtatcac ccaaaagaat ctgggggagg atctccataa


 


ctgcatcctt ggaggaaatt ggacttgtgt gcctggagac caactactat acaaaggggg


 


ctctattgaa tcttgcaagt ggtgtggcta tcaatttaaa gagagtgagg gactaccaca


 


ctaccccatt ggcaagtgta aattggagaa cgagactggt tacaggctag tagacagtac


 


ctcttgcaat agagaaggtg tggccatagt accacaaggg acattaaagt gcaagatagg


 


aaaaacaact gtacaggtca tagctatgga taccaaactc ggacctatgc cttgcagacc


 


atatgaaatc atatcaagtg aggggcctgt agaaaagaca gcgtgtactt tcaactacac


 


taagacatta aaaaataagt attttgagcc cagagacagc tactttcagc aatacatgct


 


aaaaggagag tatcaatact ggtttgacct ggaggtgact gaccatcacc gggattactt


 


cgctgagtcc atattagtgg tggtagtagc cctcttgggt ggcagatatg tactttggtt


 


actggttaca tacatggtct tatcagaaca gaaggcctta gggattcagt atggatcagg


 


ggaagtggtg atgatgggca acttgctaac ccataacaat attgaagtgg tgacatactt


 


cttgctgctg tacctactgc tgagggagga gagcgtaaag aagtgggtct tactcttata


 


ccacatctta gtggtacacc caatcaaatc tgtaattgtg atcctactga tgattgggga


 


tgtggtaaag gccgattcag ggggccaaga gtacttgggg aaaatagacc tctgttttac


 


aacagtagta ctaatcgtca taggtttaat catagctagg cgtgacccaa ctatagtgcc


 


actggtaaca ataatggcag cactgagggt cactgaactg acccaccagc ctggagttga


 


catcgctgtg gcggtcatga ctataaccct actgatggtt agctatgtga cagattattt


 


tagatataaa aaatggttac agtgcattct cagcctggta tctgcggtgt tcttgataag


 


aagcctaata tacctaggta gaatcgagat gccagaggta actatcccaa actggagacc


 


actaacttta atactattat atttgatctc aacaacaatt gtaacgaggt ggaaggttga


 


cgtggctggc ctattgttgc aatgtgtgcc tatcttattg ctggtcacaa ccttgtgggc


 


cgacttctta accctaatac tgatcctgcc tacctatgaa ttggttaaat tatactatct


 


gaaaactgtt aggactgata cagaaagaag ttggctaggg gggatagact atacaagagt


 


tgactccatc tacgacgttg atgagagtgg agagggcgta tatctttttc catcaaggca


 


gaaagcacag gggaattttt ctatactctt gccccttatc aaagcaacac tgataagttg


 


cgtcagcagt aaatggcagc taatatacat gagttactta actttggact ttatgtacta


 


catgcacagg aaagttatag aagagatctc aggaggtacc aacataatat ccaggttagt


 


ggcagcactc atagagctga actggtccat ggaagaagag gagagcaaag gcttaaagaa


 


gttttatcta ttgtctggaa ggttgagaaa cctaataata aaacataagg taaggaatga


 


gaccgtggct tcttggtacg gggaggagga agtctacggt atgccaaaga tcatgactat


 


aatcaaggcc agtacactga gtaagagcag gcactgcata atatgcactg tatgtgaggg


 


ccgagagtgg aaaggtggca cctgcccaaa atgtggacgc catgggaagc cgataacgtg


 


tgggatgtcg ctagcagatt ttgaagaaag acactataaa agaatcttta taagggaagg


 


caactttgag ggtatgtgca gccgatgcca gggaaagcat aggaggtttg aaatggaccg


 


ggaacctaag agtgccagat actgtgctga gtgtaatagg ctgcatcctg ctgaggaagg


 


tgacttttgg gcagagtcga gcatgttggg cctcaaaatc acctactttg cgctgatgga


 


tggaaaggtg tatgatatca cagagtgggc tggatgccag cgtgtgggaa tctccccaga


 


tacccacaga gtcccttgtc acatctcatt tggttcacgg atgcctttca ggcaggaata


 


caatggcttt gtacaatata ccgctagggg gcaactattt ctgagaaact tgcccgtact


 


ggcaactaaa gtaaaaatgc tcatggtagg caaccttgga gaagaaattg gtaatctgga


 


acatcttggg tggatcctaa gggggcctgc cgtgtgtaag aagatcacag agcacgaaaa


 


atgccacatt aatatactgg ataaactaac cgcatttttc gggatcatgc caagggggac


 


tacacccaga gccccggtga ggttccctac gagcttacta aaagtgagga ggggtctgga


 


gactgcctgg gcttacacac accaaggcgg gataagttca gtcgaccatg taaccgccgg


 


aaaagatcta ctggtctgtg acagcatggg acgaactaga gtggtttgcc aaagcaacaa


 


caggttgacc gatgagacag agtatggcgt caagactgac tcagggtgcc cagacggtgc


 


cagatgttat gtgttaaatc cagaggccgt taacatatca ggatccaaag gggcagtcgt


 


tcacctccaa aagacaggtg gagaattcac gtgtgtcacc gcatcaggca caccggcttt


 


cttcgaccta aaaaacttga aaggatggtc aggcttgcct atatttgaag cctccagcgg


 


gagggtggtt ggcagagtca aagtagggaa gaatgaagag tctaaaccta caaaaataat


 


gagtggaatc cagaccgtct caaaaaacag agcagacctg accgagatgg tcaagaagat


 


aaccagcatg aacaggggag acttcaagca gattactttg gcaacagggg caggcaaaac


 


cacagaactc ccaaaagcag ttatagagga gataggaaga cacaagagag tattagttct


 


tataccatta agggcagcgg cagagtcagt ctaccagtat atgagattga aacacccaag


 


catctctttt aacctaagga taggggacat gaaagagggg gacatggcaa ccgggataac


 


ctatgcatca tacgggtact tctgccaaat gcctcaacca aagctcagag ctgctatggt


 


agaatactca tacatattct tagatgaata ccattgtgcc actcctgaac aactggcaat


 


tatcgggaag atccacagat tttcagagag tataagggtt gtcgccatga ctgccacgcc


 


agcagggtcg gtgaccacaa caggtcaaaa gcacccaata gaggaattca tagcccccga


 


ggtaatgaaa ggggaggatc ttggtagtca gttccttgat atagcagggt taaaaatacc


 


agtggatgag atgaaaggca atatgttggt ttttgtacca acgagaaaca tggcagtaga


 


ggtagcaaag aagctaaaag ctaagggcta taactctgga tactattaca gtggagagga


 


tccagccaat ctgagagttg tgacatcaca atccccctat gtaatcgtgg ctacaaatgc


 


tattgaatca ggagtgacac taccagattt ggacacggtt atagacacgg ggttgaaatg


 


tgaaaagagg gtgagggtat catcaaagat acccttcatc gtaacaggcc ttaagaggat


 


ggccgtgact gtgggtgagc aggcgcagcg taggggcaga gtaggtagag tgaaacccgg


 


gaggtattat aggagccagg aaacagcaac agggtcaaag gactaccact atgacctctt


 


gcaggcacaa agatacggga ttgaggatgg aatcaacgtg acgaaatcct ttagggagat


 


gaattacgat tggagcctat acgaggagga cagcctacta ataacccagc tggaaatact


 


aaataatcta ctcatctcag aagacttgcc agccgctgtt aagaacataa tggccaggac


 


tgatcaccca gagccaatcc aacttgcata caacagctat gaagtccagg tcccggtcct


 


attcccaaaa ataaggaatg gagaagtcac agacacctac gaaaattact cgtttctaaa


 


tgccagaaag ttaggggagg atgtgcccgt gtatatctac gctactgaag atgaggatct


 


ggcagttgac ctcttagggc tagactggcc tgatcctggg aaccagcagg tagtggagac


 


tggtaaagca ctgaagcaag tgaccgggtt gtcctcggct gaaaatgccc tactagtggc


 


tttatttggg tatgtgggtt accaggctct ctcaaagagg catgtcccaa tgataacaga


 


catatatacc atcgaggacc agagactaga agacaccacc cacctccagt atgcacccaa


 


cgccataaaa accgatggga cagagactga actgaaagaa ctggcgtcgg gtgacgtgga


 


aaaaatcatg ggagccattt cagattatgc agctggggga ctggagtttg ttaaatccca


 


agcagaaaag ataaaaacag ctcctttgtt taaagaaaac gcagaagccg caaaagggta


 


tgtccaaaaa ttcattgact cattaattga aaataaagaa gaaataatca gatatggttt


 


gtggggaaca cacacagcac tatacaaaag catagctgca agactggggc atgaaacagc


 


gtttgccaca ctagtgttaa agtggctagc ttttggaggg gaatcagtgt cagaccacgt


 


caagcaggcg gcagttgatt tagtggtcta ttatgtgatg aataagcctt ccttcccagg


 


tgactccgag acacagcaag aagggaggcg attcgtcgca agcctgttca tctccgcact


 


ggcaacctac acatacaaaa cttggaatta ccacaatctc tctaaagtgg tggaaccagc


 


cctggcttac ctcccctatg ctaccagcgc attaaaaatg ttcaccccaa cgcggctgga


 


gagcgtggtg atactgagca ccacgatata taaaacatac ctctctataa ggaaggggaa


 


gagtgatgga ttgctgggta cggggataag tgcagccatg gaaatcctgt cacaaaaccc


 


agtatcggta ggtatatctg tgatgttggg ggtaggggca atcgctgcgc acaacgctat


 


tgagtccagt gaacagaaaa ggaccctact tatgaaggtg tttgtaaaga acttcttgga


 


tcaggctgca acagatgagc tggtaaaaga aaacccagaa aaaattataa tggccttatt


 


tgaagcagtc cagacaattg gtaaccccct gagactaata taccacctgt atggggttta


 


ctacaaaggt tgggaggcca aggaactatc tgagaggaca gcaggcagaa acttattcac


 


attgataatg tttgaagcct tcgagttatt agggatggac tcacaaggga aaataaggaa


 


cctgtccgga aattacattt tggatttgat atacggccta cacaagcaaa tcaacagagg


 


gctgaagaaa atggtactgg ggtgggcccc tgcacccttt agttgtgact ggacccctag


 


tgacgagagg atcagattgc caacagacaa ctatttgagg gtagaaacca ggtgcccatg


 


tggctatgag atgaaagctt tcaaaaatgt aggtggcaaa cttaccaaag tggaggagag


 


cgggcctttc ctatgtagaa acagacctgg taggggacca gtcaactaca gagtcaccaa


 


gtattacgat gacaacctca gagagataaa accagtagca aagttggaag gacaggtaga


 


gcactactac aaaggggtca cagcaaaaat tgactacagt aaaggaaaaa tgctcttggc


 


cactgacaag tgggaggtgg aacatggtgt cataaccagg ttagctaaga gatatactgg


 


ggtcgggttc aatggtgcat acttaggtga cgagcccaat caccgtgctc tagtggagag


 


ggactgtgca actataacca aaaacacagt acagtttcta aaaatgaaga aggggtgtgc


 


gttcacctat gacctgacca tctccaatct gaccaggctc atcgaactag tacacaggaa


 


caatcttgaa gagaaggaaa tacccaccgc tacggtcacc acatggctag cttacacctt


 


cgtgaatgaa gacgtaggga ctataaaacc agtactagga gagagagtaa tccccgaccc


 


tgtagttgat atcaatttac aaccagaggt gcaagtggac acgtcagagg ttgggatcac


 


aataattgga agggaaaccc tgatgacaac gggagtgaca cctgtcttgg aaaaagtaga


 


gcctgacgcc agcgacaacc aaaactcggt gaagatcggg ttggatgagg gtaattaccc


 


agggcctgga atacagacac atacactaac agaagaaata cacaacaggg atgcgaggcc


 


cttcatcatg atcctgggct caaggaattc catatcaaat agggcaaaga ctgctagaaa


 


tataaatctg tacacaggaa atgaccccag ggaaatacga gacttgatgg ctgcagggcg


 


catgttagta gtagcactga gggatgtcga ccctgagctg tctgaaatgg tcgatttcaa


 


ggggactttt ttagataggg aggccctgga ggctctaagt ctcgggcaac ctaaaccgaa


 


gcaggttacc aaggaagctg ttaggaattt gatagaacag aaaaaagatg tggagatccc


 


taactggttt gcatcagatg acccagtatt tctggaagtg gccttaaaaa atgataagta


 


ctacttagta ggagatgttg gagagctaaa agatcaagct aaagcacttg gggccacgga


 


tcagacaaga attataaagg aggtaggctc aaggacgtat gccatgaagc tatctagctg


 


gttcctcaag gcatcaaaca aacagatgag tttaactcca ctgtttgagg aattgttgct


 


acggtgccca cctgcaacta agagcaataa ggggcacatg gcatcagctt accaattggc


 


acagggtaac tgggagcccc tcggttgcgg ggtgcaccta ggtacaatac cagccagaag


 


ggtgaagata cacccatatg aagcttacct gaagttgaaa gatttcatag aagaagaaga


 


gaagaaacct agggttaagg atacagtaat aagagagcac aacaaatgga tacttaaaaa


 


aataaggttt caaggaaacc tcaacaccaa gaaaatgctc aacccaggga aactatctga


 


acagttggac agggaggggc gcaagaggaa catctacaac caccagattg gtactataat


 


gtcaagtgca ggcataaggc tggagaaatt gccaatagtg agggcccaaa ccgacaccaa


 


aacctttcat gaggcaataa gagataagat agacaagagt gaaaaccggc aaaatccaga


 


attgcacaac aaattgttgg agattttcca cacgatagcc caacccaccc tgaaacacac


 


ctacggtgag gtgacgtggg agcaacttga ggcgggggta aatagaaagg gggcagcagg


 


cttcctggag aagaagaaca tcggagaagt attggattca gaaaagcacc tggtagaaca


 


attggtcagg gatctgaagg ccgggagaaa gataaaatat tatgaaactg caataccaaa


 


aaatgagaag agagatgtca gtgatgactg gcaggcaggg gacctggtgg ttgagaagag


 


gccaagagtt atccaatacc ctgaagccaa gacaaggcta gccatcacta aggtcatgta


 


taactgggtg aaacagcagc ccgttgtgat tccaggatat gaaggaaaga cccccttgtt


 


caacatcttt gataaagtga gaaaggaatg ggactcgttc aatgagccag tggccgtaag


 


ttttgacacc aaagcctggg acactcaagt gactagtaag gatctgcaac ttattggaga


 


aatccagaaa tattactata agaaggagtg gcacaagttc attgacacca tcaccgacca


 


catgacagaa gtaccagtta taacagcaga tggtgaagta tatataagaa atgggcagag


 


agggagcggc cagccagaca caagtgctgg caacagcatg ttaaatgtcc tgacaatgat


 


gtacggcttc tgcgaaagca caggggtacc gtacaagagt ttcaacaggg tggcaaggat


 


ccacgtctgt ggggatgatg gcttcttaat aactgaaaaa gggttagggc tgaaatttgc


 


taacaaaggg atgcagattc ttcatgaagc aggcaaacct cagaagataa cggaagggga


 


aaagatgaaa gttgcctata gatttgagga tatagagttc tgttctcata ccccagtccc


 


tgttaggtgg tccgacaaca ccagtagtca catggccggg agagacaccg ctgtgatact


 


atcaaagatg gcaacaagat tggattcaag tggagagagg ggtaccacag catatgaaaa


 


agcggtagcc ttcagtttct tgctgatgta ttcctggaac ccgcttgtta ggaggatttg


 


cctgttggtc ctttcgcaac agccagagac agacccatca aaacatgcca cttattatta


 


caaaggtgat ccaatagggg cctataaaga tgtaataggt cggaatctaa gtgaactgaa


 


gagaacaggc tttgagaaat tggcaaatct aaacctaagc ctgtccacgt tgggggtctg


 


gactaagcac acaagcaaaa gaataattca ggactgtgtt gccattggga aagaagaggg


 


caactggcta gttaagcccg acaggctgat atccagcaaa actggccact tatacatacc


 


tgataaaggc tttacattac aaggaaagca ttatgagcaa ctgcagctaa gaacagagac


 


aaacccggtc atgggggttg ggactgagag atacaagtta ggtcccatag tcaatctgct


 


gctgagaagg ttgaaaattc tgctcatgac ggccgtcggc gtcagcagct gagacaaaat


 


gtatatattg taaataaatt aatccatgta catagtgtat ataaatatag ttgggaccgt


 


ccacctcaag aagacgacac gcccaacacg cacagctaaa cagtagtcaa gattatctac


 


ctcaagataa cactacattt aatgcacaca gcactttagc tgtatgagga tacgcccgac


 


gtctatagtt ggactaggga agacctctaa cag


 


SEQ ID: 5


melitnelly ktykqkpvgv eepvydqagd plfgergavh pqstlklphk rgerdvptnl


 


aslpkrgdcr sgnsrgpvsg iylkpgplfy qdykgpvyhr aplelfeegs mcettkrigr


 


vtgsdgklyh iyvcidgcii iksatrsyqr vfrwvhnrld cplwvttcsd tkeegatkkk


 


tqkpdrlerg kmkivpkese kdsktkppda tivvegvkyq vrkkgktksk ntqdglyhnk


 


nkpqesrkkl ekallawaii aivlfqvtmg enitqwnlqd ngtegiqram fqrgvnrslh


 


giwpekictg vpshlatdie lktihgmmda sektnytccr lqrhewnkhg wcnwyniepw


 


ilvmnrtqan ltegqpprec avtcrydras dlnvvtqard sptpltgckk gknfsfagil


 


mrgpcnfeia asdvlfkehe rismfqdttl ylvdgltnsl egarqgtakl ttwlgkqlgi


 


lgkklenksk twfgayaasp ycdvdrkigy iwytknctpa clpkntkivg pgkfgtnaed


 


gkilhemggh lsevlllslv vlsdfapeta svmylilhfs ipqshvdvmd cdktqlnltv


 


elttaevipg svwnlgkyvc irpnwwpyet tvvlafeevs qvvklvlral rdltriwnaa


 


tttaflvclv kivrgqmvqg ilwlllitgv qghldckpef syaiakderi gqlgaegltt


 


twkeyspgmk ledtmviawc edgklmylqr ctretrylai lhtralptsv vfkklfdgrk


 


qedvvemndn fefglcpcda kpivrgkfnt tllngpafqm vcpigwtgtv sctsfnmdtl


 


attvvrtyrr skpfphrqgc itqknlgedl hncilggnwt cvpgdqllyk ggsiesckwc


 


gyqfkesegl phypigkckl enetgyrlvd stscnregva ivpqgtlkck igkttvqvia


 


mdtklgpmpc rpyeiisseg pvektactfn ytktlknkyf eprdsyfqqy mlkgeyqywf


 


dlevtdhhrd yfaesilvvv vallggryvl wllvtymvls eqkalgiqyg sgevvmmgnl


 


lthnnievvt yflllylllr eesvkkwvll lyhilvvhpi ksvivillmi gdvvkadsgg


 


qeylgkidlc fttvvlivig liiarrdpti vplvtimaal rvtelthqpg vdiavavmti


 


tllmvsyvtd yfrykkwlqc ilslvsavfl irsliylgri empevtipnw rpltlillyl


 


isttivtrwk vdvaglllqc vpilllvttl wadfltlili lptyelvkly ylktvrtdte


 


rswlggidyt rvdsiydvde sgegvylfps rqkaqgnfsi llplikatli scvsskwqli


 


ymsyltldfm yymhrkviee isggtniisr lvaalielnw smeeeeskgl kkfyllsgrl


 


rnliikhkvr netvaswyge eevygmpkim tiikastlsk srhciictvc egrewkggtc


 


pkcgrhgkpi tcgmsladfe erhykrifir egnfegmcsr cqgkhrrfem drepksaryc


 


aecnrlhpae egdfwaessm lglkityfal mdgkvydite wagcqrvgis pdthrvpchi


 


sfgsrmpfrq eyngfvqyta rgqlflrnlp vlatkvkmlm vgnlgeeign lehlgwilrg


 


pavckkiteh ekchinildk ltaffgimpr gttprapvrf ptsllkvrrg letawaythq


 


ggissvdhvt agkdllvcds mgrtrvvcqs nnrltdetey gvktdsgcpd garcyvlnpe


 


avnisgskga vvhlqktgge ftcvtasgtp affdlknlkg wsglpifeas sgrvvgrvkv


 


gkneeskptk imsgiqtvsk nradltemvk kitsmnrgdf kqitlatgag kttelpkavi


 


eeigrhkrvl vliplraaae svyqymrlkh psisfnlrig dmkegdmatg ityasygyfc


 


qmpqpklraa mveysyifld eyhcatpeql aiigkihrfs esirvvamta tpagsvtttg


 


qkhpieefia pevmkgedlg sqfldiaglk ipvdemkgnm lvfvptrnma vevakklkak


 


gynsgyyysg edpanlrvvt sqspyvivat naiesgvtlp dldtvidtgl kcekrvrvss


 


kipfivtglk rmavtvgeqa qrrgrvgrvk pgryyrsqet atgskdyhyd llqaqrygie


 


dginvtksfr emnydwslye edsllitqle ilnnllised lpaavknima rtdhpepiql


 


aynsyevqvp vlfpkirnge vtdtyenysf lnarklgedv pvyiyatede dlavdllgld


 


wpdpgnqqvv etgkalkqvt glssaenall valfgyvgyq alskrhvpmi tdiytiedqr


 


ledtthlqya pnaiktdgte telkelasgd vekimgaisd yaagglefvk sqaekiktap


 


lfkenaeaak gyvqkfidsl ienkeeiiry glwgthtaly ksiaarlghe tafatlvlkw


 


lafggesvsd hvkqaavdlv vyyvmnkpsf pgdsetqqeg rrfvaslfis alatytyktw


 


nyhnlskvve palaylpyat salkmftptr lesvvilstt iyktylsirk gksdgllgtg


 


isaameilsq npvsvgisvm lgvgaiaahn aiesseqkrt llmkvfvknf ldqaatdelv


 


kenpekiima lfeavqtign plrliyhlyg vyykgweake lsertagrnl ftlimfeafe


 


llgmdsqgki rnlsgnyild liyglhkqin rglkkmvlgw apapfscdwt psderirlpt


 


dnylrvetrc pcgyemkafk nvggkltkve esgpflcrnr pgrgpvnyrv tkyyddnlre


 


ikpvaklegq vehyykgvta kidyskgkml latdkweveh gvitrlakry tgvgfngayl


 


gdepnhralv erdcatitkn tvqflkmkkg caftydltis nltrlielvh rnnleekeip


 


tatvttwlay tfvnedvgti kpvlgervip dpvvdinlqp evqvdtsevg itiigretlm


 


ttgvtpvlek vepdasdnqn svkigldegn ypgpgiqtht lteeihnrda rpfimilgsr


 


nsisnrakta rninlytgnd preirdlmaa grmlvvalrd vdpelsemvd fkgtfldrea


 


lealslgqpk pkqvtkeavr nlieqkkdve ipnwfasddp vflevalknd kyylvgdvge


 


lkdqakalga tdqtriikev gsrtyamkls swflkasnkq msltplfeel llrcppatks


 


nkghmasayq laqgnweplg cgvhlgtipa rrvkihpyea ylklkdfiee eekkprvkdt


 


virebnkwil kkirfqgnln tkkmlnpgkl seqldregrk rniynhqigt imssagirle


 


klpivraqtd tktfheaird kidksenrqn pelhnkllei fhtiaqptlk htygevtweq


 


leagvnrkga agflekknig evldsekhlv eqlvrdlkag rkikyyetai pknekrdvsd


 


dwqagdlvve krprviqype aktrlaitkv mynwvkqqpv vipgyegktp lfnifdkvrk


 


ewdsfnepva vsfdtkawdt qvtskdlqli geiqkyyykk ewhkfidtit dhmtevpvit


 


adgevyirng qrgsgqpdts agnsmlnvlt mmygfcestg vpyksfnrva rihvcgddgf


 


litekglglk fankgmqilh eagkpqkite gekmkvayrf ediefcshtp vpvrwsdnts


 


shmagrdtav ilskmatrld ssgergttay ekavafsfll myswnplvrr icllvlsqqp


 


etdpskhaty yykgdpigay kdvigrnlse lkrtgfekla nlnlslstlg vwtkhtskri


 


iqdcvaigke egnwlvkpdr lissktghly ipdkgftlqg khyeqlqlrt etnpvmgvgt


 


eryklgpivn lllrrlkill mtavgvss


 


SEQ ID NO: 6


melitnelly ktykqkpvgv eepvydqagn plfgergaih pqstlklphk rgernvptsl


 


aslpkrgdcr sgnskgpvsg iylkpgplfy qdykgpvyhr aplelfeegs mcettkrigr


 


vtgsdgklyh iyicidgcit vksatrshqr vlrwvhnrld cplwvtscsd tkeegatkkk


 


qqkpdrlekg rmkivpkese kdsktkppda tivvdgvkyq vkkkgkvksk ntqdglyhnk


 


nkppesrkkl ekallawail avvlievtmg enitqwnlqd ngtegiqram fqrgvnrslh


 


giwpekictg vpshlatdve lktihgmmda sektnytccr lqrhewnkhg wcnwyniepw


 


ilimnrtqan ltegqpprec avtcrydrds dlnvvtqard sptpltgckk gknfsfagvl


 


trgpcnfeia asdvlfkehe ctgvfqdtah ylvdgvtnsl esarqgtakl ttwlgkqlgi


 


lgkklenksk twfgayaasp ycdvdrkigy iwftknctpa clpkntkiig pgkfdtnaed


 


gkilhemggh lsevlllslv vlsdfapeta samylilhfa ipqshvditd cdktqlnlti


 


elttadvipg svwnlgkyvc irpdwwpyet aavlafeevg qvvkivlral rdltriwnaa


 


tttaflvcli kmvrgqvvqg ilwlllitgv qghldckpey syaiakndrv gplgaegltt


 


vwkdyshemk ledtmviawc kggkftylsr ctretrylai lhsralptsv vfkklfegqk


 


qedtvemddd fefglcpcda kpivrgkfnt tllngpafqm vcpigwtgtv scmlanrdtl


 


dtavvrtyrr svpfpyrqgc itqktlgedl ydcalggnwt cvtgdqsryt ggliesckwc


 


gykfqksegl phypigkcrl nnetgyrlvd dtscdregva ivphglvkck igdttvqvia


 


tdtklgpmpc kpheiisseg piektactfn ytrtlknkyf eprdsyfqqy mlkgdyqywf


 


dlevtdhhrd yfaesilvvv vallggryvl wllvtymvls eqkasgaqyg agevvmmgnl


 


lthdnvevvt yffllylllr eesvkkwvll lyhilvahpl ksvivillmi gdvvkadpgg


 


qgylgqidvc ftmvviiiig liiarrdpti vplitivasl rvtgltyspg vdaamaviti


 


tllmvsyvtd yfrykrwlqc ilslvsgvfl irclihlgri etpevtipnw rpltlilfyl


 


isttvvtmwk idlaglllqg vpilllittl wadfltlili lptyelvkly ylktiktdie


 


kswlggldyk rvdsiydvde sgegvylfps rqkaqknfsm llplvratli scvsskwqli


 


ymaylsvdfm yymhrkviee isggtnmisr ivaalielnw smeeeeskgl kkfyllsgrl


 


rnliikhkvr netvagwyge eevygmpkim tiikastlnk nkhciictvc egrkwkggtc


 


pkcgrhgkpi tcgmsladfe erhykrifir egnfegpfrq eyngfiqyta rgqlflrnlp


 


ilatkvkmlm vgnlgeevgd lehlgwilrg pavckkiteh erchinildk ltaffgimpr


 


gttprapvrf ptsllkvrrg letgwaythq ggissvdhvt agkdllvcds mgrtrvvcqs


 


nnkltdetey gvktdsgcpd garcyvlnpe avnisgskga vvhlqktgge ftcvtasgtp


 


affdlknlkg wsglpifeas sgrvvgrvkv gkneeskptk imsgiqtvsk ntadltemvk


 


kitsmnrgdf kqitlatgag kttelpkavi eeigrhkrvl vliplraaae svyqymrlkh


 


psiafnlrig dmkegdmatg ityasygyfc qmpqpklraa mveysyifld eyhcatpeql


 


aiigkihrfs esirvvamta tpagsvtttg qkhpieefia pevmegedlg sqfldiaglk


 


ipvdemkgnm lvfvptrnma vevakklkak gynsgyyysg edpanlrvvt sqspyvivat


 


naiesgvtlp dldtvvdtgl kcekrvrvss kipfivtglk rmavtvgeqa qrrgrvgrvk


 


pgryyrsqet atgskdyhyd llqaqrygie dginvtksfr emnydwslye edsllitqle


 


ilnnllised lpaavknima rtdhpepiql aynsyevqvp vlfpkirnge vtdtyenysf


 


lnarklgedv pvyiyatede dlavdllgld wpdpgnqqvv etgkalkqva glssaenall


 


valfgyvgyq alskrhvpmi tdiytiedqr ledtthlqya pnaiktegte telkelasgd


 


vekimgaisd yaaggldfvk sqaekiktap lfkenveaar gyvqklidsl iedkdviiry


 


glwgthtaly ksiaarlghe tafatlvlkw lafggetvsd hirqaavdlv vyyvmnkpsf


 


pgdtetqqeg rrfvaslfis alatytyktw nynnlskvve palaylpyat salkmftptr


 


lesvvilstt iyktylsirk gksdgllgtg isaameilsq npvsvgisvm lgvgaiaakm


 


aiesseqkrt llmkvfvknf ldqaatdelv kenpekiima lfeavqtign plrliyhlyg


 


vyykgweake lsertagrnl ftlimfeafe llgmdsegki rnlsgnyild lihglhkqin


 


rglkkivlgw apapfscdwt psderirlpt dsylrvetkc pcgyemkalk nvsgkltkve


 


esgpflcrnr pgrgpvnyrv tkyyddnlre irpvaklegq vehyykgvta ridyskgktl


 


latdkweveh gtltrltkry tgvgfrgayl gdepnhrdlv erdcatitkn tvqflkmkkg


 


caftydltis nltrlielvh rnnleekeip tatvttwlay tfvnedvgti kpvlgervip


 


dpvvdinlqp evqvdtsevg itiigkeavm ttgvtpvmek vepdtdnnqs svkigldegn


 


ypgpgvqtht lveeihnkda rpfimvlgsk ssmsnrakta rninlytgnd preirdlmae


 


grilvvalrd idpdlselvd fkgtfldrea lealslgqpk pkqvtkaair dllkeerqve


 


ipdwftsddp vfldiamkkd kyhligdvve vkdqakalga tdqtrivkev gsrtytmkls


 


swflqasskq msltplfeel llrcppatks nkghmasayq laqgnweplg cgvhlgtvpa


 


rrvkmhpyea ylklkdlvee eekkprirdt virehnkwil kkikfggnln tkkmlnpgkl


 


seqldreghk rniynnqist vmssagirle klpivraqtd tksfheaird kidknenrqn


 


pelhnkllei fhtiadpslk htygevtweq leaginrkga agflekknig evldsekhlv


 


eqlvrdlkag rkiryyetai pknekrdvsd dwqagdlvde kkprviqype aktrlaitkv


 


mynwvkqqpv vipgyegktp lfnifnkvrk ewdlfnepva vsfdtkawdt qvtsrdlhli


 


geiqkyyyrk ewhkfidtit dhmvevpvit adgevyirng qrgsgqpdts agnsmlnvlt


 


miyafcestg vpyksfnrva kihvcgddgf litekglglk fsnkgmqilh eagkpqklte


 


gekmkvaykf ediefcshtp vpvrwsdnts symagrdtav ilskmatrld ssgergttay


 


ekavafsfll myswnplvrr icllvlsqrp etapstqtty yykgdpigay kdvigrnlse


 


lkrtgfekla nlnlslstlg iwtkhtskri iqdcvaigke egnwlvnadr lissktghly


 


ipdkgftlqg khyeqlqlga etnpvmgvgt eryklgpivn lllrrlkvll maavgass


 


SEQ ID NO: 7


cgtggcttcttggtacggg


 


SEQ ID NO: 8


agcggtatattgtacaaagcca


 


SEQ ID NO: 9


tgcacgatctgtgaagggaaagaa


 


SEQ ID NO: 10


tgcactgtatgtgagggccgagag

## Claims

1. A vaccine comprising at least two live mutant viruses, of the same family, wherein each virus contains a mutation in the viral genome, and the mutations in the viruses reside in the same genomic site such that the mutant viruses cannot recombine with each other to eliminate the mutations, and wherein two of the live mutant viruses consist of a mutant Bovine Viral Diarrhea Virus Type 1 (BVDV-1) and a mutant Bovine Viral Diarrhea Virus Type 2 (BVDV-2), wherein the BVDV-1 and the BVDV-2 comprise a mutation comprising an insertion of a heterologous sequence in the NS2-3 region that results in a cytopathic biotype, wherein said heterologous sequence is inserted at 3′ of nucleotide #4993 of the BVDV-1 genome, and at 3′ of nucleotide #4993 of the BVDV-2 genome.

2. A vaccine comprising at least two live mutant viruses of the same family, wherein each virus contains a mutation in the viral genome, and the mutations in the viruses reside in the same genomic site such that the mutant viruses cannot recombine with each other to eliminate the mutations, and wherein two of the live mutant viruses consist of a mutant Bovine Viral Diarrhea Virus Type 1 (BVDV-1) and a mutant Bovine Viral Diarrhea Virus Type 2 (BVDV-2), wherein the BVDV-1 and the BVDV-2 comprise a mutation comprising an insertion of a heterologous sequence in the NS2-3 region that results in a cytopathic biotype, wherein said heterologous sequence is a DnaJ coding sequence or a portion thereof.

3. The vaccine of claim 2 wherein said DnaJ coding sequence or a portion thereof is inserted at 3′ of nucleotide #4993 of the BVDV-1 genome, and at 3′ of nucleotide #4993 of the BVDV-2 genome.

4. The vaccine of claim 3 wherein the cp BVDV-1 is BVDV-1 NADL, and the cp BVDV-2 is BVDV-2 53637.

5. A vaccine comprising at least two live mutant viruses of the same family, wherein each virus contains a mutation in the viral genome, and the mutations in the viruses reside in the same genomic site such that the mutant viruses cannot recombine with each other to eliminate the mutations, and wherein two of the live mutant viruses consist of a mutant cytopathic (cp) Bovine Viral Diarrhea Virus Type 1 (BVDV-1) and a mutant cp Bovine Viral Diarrhea Virus Type 2 (BVDV-2), and wherein the vaccine further comprises at least one of bovine herpesvirus-1, bovine respiratory syncytial virus, parainfluenza virus-3, Campylobacter fetus, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, Leptospira pomona, or Mannhemia haemolytica.