Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/18—Togaviridae; Flaviviridae
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/18—Togaviridae; Flaviviridae
  • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

• the present invention relates to improved diagnostic methods and kits for differentiating between (a) animals administered a chimeric pestivirus, and (b) animals infected with a wild-type bovine viral diarrhea virus (BVDV) or immunized with a conventional BVDV vaccine.
• BVDV bovine viral diarrhea virus
• Pestiviruses including bovine viral diarrhea virus (BVD virus, or BVDV), have been isolated from several species of animals, both domestic and wild. Identified hosts for BVDV include buffalo, antelope, reindeer and various deer species, while unique pestivirus species have been identified in giraffes and pronghorn antelope. BVDV is a small RNA virus of the family Flaviviridae. It is closely related to other pestiviruses which are the causative agents of border disease in sheep and classical swine fever in pigs.
• BVDV infection in cattle can result in breeding problems, and can cause abortions or premature births.
• BVDV is capable of crossing the placenta of pregnant cattle, and may result in the birth of persistently infected (PI) calves that are immunotolerant to the virus and persistently viremic for the rest of their lives.
• Infected cattle can also exhibit "mucosal disease", characterized by elevated temperature, diarrhea, coughing and ulcerations of the alimentary mucosa.
• microorganisms responsible for causing enteric diseases or pneumonia are responsible for causing enteric diseases or pneumonia.
• BVDV vaccines currently available are those which contain
• BVDV can be attenuated by repeated passage in bovine or porcine cells, or by chemically-induced mutations that confer a temperature-sensitive phenotype on the virus.
• existing inactivated and ML vaccines do not allow for the differentiation between vaccinated and naturally-infected animals.
• a "marked” vaccine that could either contain an additional antigenic determinant which is not present in wild-type virus, or lack an antigenic determinant which is present in wild-type virus could be an effective tool for controlling BVDV infection in the field.
• US Patent Application 2010/0360055 (Luo et al., herein incorporated by reference in its entirety) describes the latter, a vaccine based upon a chimeric pestivirus vaccine in which the E rns protein of the BVDV is replaced with the E rns protein from a pronghorn pestivirus.
• This chimeric pestivirus was deposited as UC 25548 with ATCC ® , and given the ATCC ® deposit designation of PTA-9939. Accompanied by an appropriate diagnostic assay, use of this chimeric pestivirus would allow for the differentiation between animals to which it was administered, versus animals infected with wild-type BVDV or immunized with a conventional BVDV vaccine.
• a method for determining whether an animal was exposed to a BVDV or immunized with a conventional BVDV vaccine, wherein the animal infected with BVDV or immunized with a conventional BVDV vaccine possesses an antibody that specifically binds to at least one E rns epitope which is present in BVDV, but which is not present in a chimeric pestivirus that no longer expresses an E rns protein from a BVDV, but expresses an E rns protein from a pronghorn pestivirus in a BVDV.
• a method for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein comprising the steps of:
• a diagnostic kit for determining whether an animal was exposed to a BVDV or immunized with a conventional BVDV vaccine, said kit comprising reagents capable of detecting antibodies to at least one E rns epitope that is present in BVDV, but which is not present in a chimeric pestivirus that no longer expresses an E rns protein from a BVDV, but expresses an E rns protein from a pronghorn pestivirus in a BVDV.
• a use is provided for an antibody which binds to an epitope present in BVDV or a conventional BVDV vaccine, but which epitope is not present in a chimeric pestivirus that no longer expresses an E rns protein from a BVDV, but expresses an E rns protein from a pronghorn pestivirus in a BVDV.
• a method for determining whether an animal possesses an antibody that specifically binds to a BVDV E rns protein comprising the steps of: a) obtaining a serum sample from the animal;
• a method for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein the step of incubating said sample with pronghorn pestivirus E rns protein or a fragment thereof occurs prior to the step of detecting in said sample the presence or absence of said antibody.
• a method for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein the step of incubating said sample with pronghorn pestivirus E rns protein or a fragment thereof occurs simultaneously with the step of detecting in said sample the presence or absence of said antibody.
• a method for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein said antibody specifically binds to at least one epitope present in a BVDV E rns protein but not present in a pronghorn pestivirus E rns protein.
• a method for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein the presence of said antibody indicates that the animal has either been infected with BVDV or immunized with a conventional BVDV vaccine.
• a method for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein the absence of said antibody indicates that: a) the animal has not been 1 ) infected with BVDV, or 2) immunized with a conventional BVDV vaccine; or b) has been immunized with a chimeric pestivirus expressing a pronghorn pestivirus E rns protein.
• a method for determining in a sample the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein a serum sample is obtained from an animal that is susceptible to BVDV infections.
• a method for determining in a sample the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein a serum sample is obtained from an animal that is a bovine, ovine, caprine, or porcine species.
• a method for determining in a sample the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein a serum sample is obtained from an animal that is a bovine.
• a diagnostic kit for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein said kit comprises reagents that facilitate the detection of the presence or absence of an antibody to at least one BVDV E rns epitope not present in a pronghorn pestivirus E rns protein, wherein one of said reagents is pronghorn pestivirus E rns protein or a fragment thereof.
• a diagnostic kit for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein said kit comprises reagents that facilitate the detection of the presence or absence of an antibody to at least one BVDV E rns epitope not present in a pronghorn pestivirus E rns protein, wherein one of said reagents is an antibody that specifically binds to an epitope present in BVDV or a conventional BVDV vaccine, but which epitope is not present in a pronghorn pestivirus E rns protein.
• a diagnostic kit for determining the presence or absence of an antibody that specifically binds to a BVDV E rns protein, wherein said kit comprises reagents for carrying out an assay selected from the group consisting of: an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, Western blotting, PCR, radioimmunoassay, solid phase radioimmunoassay, electrochemiluminescent assay, immunoblotting, immunoprecipitation, and immunostaining.
• an enzyme-linked immunosorbent assay ELISA
• a lateral flow assay Western blotting
• PCR radioimmunoassay
• solid phase radioimmunoassay solid phase radioimmunoassay
• electrochemiluminescent assay immunoblotting
• immunoprecipitation immunostaining.
• the present invention provides a method of determining whether an animal possesses an antibody that specifically binds to a BVDV E rns protein, wherein said antibody specifically binds to an epitope present in a BVDV E rns protein, but which epitope is not present in a pronghorn pestivirus E rns protein.
• animal as used herein, is meant to include any animal that is susceptible to BVDV infections, including but not limited to bovine, ovine, caprine, and porcine species, both domesticated and wild.
• antibody refers to an immunoglobulin molecule able to bind to an antigen by means of recognition of an epitope.
• Immunoglobulins are serum proteins composed of "light” and “heavy” polypeptide chains having “constant” and “variable” regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
• An antibody that is "specific" for a given antigen indicates that the variable regions of the antibody recognize and bind a specific antigen exclusively.
• Antibodies can be a polyclonal mixture or monoclonal.
• Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, or can be immunoreactive portions of intact immunoglobulins.
• Antibodies can exist in a variety of forms including, for example, as, Fv, Fab', F(ab') 2 , as well as in single chains.
• An antibody can be converted to an antigen-binding protein, which includes but is not limited to antibody fragments.
• antigen refers to a molecule that contains one or more epitopes (linear, conformational or both) that upon exposure to a subject will induce an immune response that is specific for that antigen.
• the term “antigen” can refer to attenuated, inactivated or modified live bacteria, viruses, fungi, parasites or other microbes.
• the term “antigen” as used herein can also refer to a subunit antigen, which is separate and discrete from a whole organism with which the antigen is associated in nature.
• the term “antigen” can also refer to antibodies, such as antiidiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).
• the term "antigen” can also refer to an oligonucleotide or polynucleotide that expresses an antigen or antigenic determinant in vivo, such as in DNA immunization applications.
• "Buffer” means a chemical system that prevents change in the concentration of another chemical substance, e.g., proton donor and acceptor systems serve as buffers preventing marked changes in hydrogen ion concentration (pH).
• a further example of a buffer is a solution containing a mixture of a weak acid and its salt (conjugate base) or a weak base and its salt (conjugate acid).
• BVDV bovine viral diarrhea viruses
• type I and type II bovine viral diarrhea viruses include but not limited to type I and type II, that consist of the viral genome, associated proteins, and other chemical constituents (such as lipids).
• a number of type I and type II bovine viral diarrhea viruses are known to those skilled in the art and are available through, e.g., the American Type Culture Collection (ATCC®; Manassas, VA 20108 USA).
• the bovine viral diarrhea virus has a genome in the form of RNA. RNA can be reverse transcribed into DNA for use in cloning.
• references made herein to nucleic acid and bovine viral diarrhea virus sequences encompass both viral RNA sequences and DNA sequences derived from the viral RNA sequences.
• the term "NADL” as used herein refers to a reference strain of BVDV, deposited in the ATCC as VR-534.
• cell line or "host cell”, as used herein, means a prokaryotic or eukaryotic cell in which a virus can replicate or be maintained.
• Cellular immune response or “cell mediated immune response” is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by activated T-cells, white blood cells, or both.
• chimeric or “chimera”, as used herein, means a microorganism, for example a virus, containing genetic or physical components derived from more than one progenitor.
• inventions means a vaccine based on a wild-type BVDV.
• the virus can be attenuated or inactivated.
• the virus is not genetically-modified, however.
• culture means a population of cells or
• microorganisms growing in the absence of other species or types growing in the absence of other species or types.
• IVA means to differentiate infected from vaccinated animals.
• Dose refers to a vaccine or immunogenic composition given to a subject.
• a “first dose” or “priming vaccine” refers to the dose given on Day 0.
• a “second dose” or a “third dose” or an “annual dose” refers to an amount of such composition given subsequent to the first dose, which may or may not be the same vaccine or
• immunogenic composition as the first dose.
• epitopope means the specific site of the antigen which binds to a T-cell receptor or specific antibody, and typically comprises from about 3 amino acid residues to about 20 amino acid residues, and can be continuous or discontinuous.
• “Fragment” refers to a truncated portion of a protein or gene.
• “Functional fragment” and “biologically active fragment” refer to a fragment that retains the biological properties of the full length protein or gene.
• An “immunogenically active fragment” refers to a fragment that elicits an immune response.
• heterologous means derived from a different species or strain.
• homologous means derived from the same species or strain.
• Human immune response refers to one that is at least in part mediated by antibodies.
• Immunogenic response in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen.
• the immunogenic response may be sufficient for diagnostic purposes or other testing, or may be adequate to prevent signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a disease agent.
• Immunogenic refers to the capability to elicit an immune response directed specifically against an antigen.
• immunogenic composition means a composition that capable of being recognized by the immune system, resulting in the generation of a specific immune response (i.e., has immunogenic activity) when administered alone or with a pharmaceutically acceptable carrier, to an animal.
• immunologically effective amount refers to the amount of an antigen effective to induce an immunogenic or immunological response in an animal.
• the immune response can comprise, without limitation, induction of cellular and/or humoral immunity.
• isolated means removed from its naturally occurring environment. When referring to a microorganism, it can be either alone or in a heterologous host cell, or chromosome or vector (e.g., plasmid, phage, etc.).
• isolated bacteria refers to a composition in which the bacteria or virus are substantial free of other microorganisms, e.g., in a culture, such as when separated from it naturally occurring environment.
• isolated when used to describe any particularly defined substance, such as a polynucleotide or a polypeptide, refers to the substance that is separate from the original cellular environment in which the
• polynucleotide of the invention makes use of the "isolated" nucleic acid.
• a particular protein or a specific immunogenic fragment is claimed or used as a vaccine or other composition, it would be considered to be isolated because it had been identified, separated and to some extent purified as compared to how it may exist in nature.
• the protein or a specific immunogenic fragment thereof is produced in a recombinant bacterium or eukaryote expression vector that produces the antigen, it is considered to exist as an isolated protein or nucleic acid.
• a recombinant cell line constructed with a polynucleotide makes use of an "isolated" nucleic acid.
• MMI multiplicity of infection
• pathogen or "pathogenic microorganism”, as used herein, means a microorganism - for example a virus, bacterium, fungus, protozoan, or helminth - which is capable of inducing or causing a disease, illness, or abnormal state in its host animal.
• pestivirus means a RNA virus from the genus
• Pestivirus of the family Flaviviridae. Pestiviruses include, but are not limited to, BVDV (type 1 and type 2), Classical Swine Fever Virus (CSFV), and Border Disease Virus (BDV), as well as pestiviruses isolated from species such as wild boar, buffalo, eland, bison, alpaca, pudu, bongo, various deer species, giraffe, reindeer, chamois and pronghorn antelope (Vilcek and Nettleton; Vet Microbiol. 1 16:1 -12 (2006)).
• BVDV type 1 and type 2
• CSFV Classical Swine Fever Virus
• BDV Border Disease Virus
• “Pharmaceutically acceptable” refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio and effective for their intended use.
• prevent means to inhibit the replication of a microorganism, to inhibit transmission of a
• microorganism or to inhibit a microorganism from establishing itself in its host.
• These terms and the like as used herein can also mean to inhibit or block one or more signs or symptoms of infection.
• Protection means that the vaccine or composition prevents or reduces the symptoms of the disease caused by the organism from which the antigen(s) used in the vaccine or composition is derived.
• protection and “protecting” and the like, also mean that the vaccine or composition can be used to therapeutically treat the disease or one of more symptoms of the disease that already exists in a subject.
• telomere binding is defined as two or more molecules that form a complex that is measurable under physiologic or assay conditions, and is selective.
• An antibody or other inhibitor is said to "specifically bind" to a protein if, under appropriately selected conditions, such binding is not substantially inhibited, while at the same time non-specific binding is inhibited.
• Binding in IgG antibodies is generally characterized by an affinity of at least about 10 "7 M or higher, such as at least about 10 "8 M or higher, or at least about 10 "9 M or higher, or at least about 10 "10 or higher, or at least about 10 "11 M or higher, or at least about 10 "12 M or higher.
• the term is also applicable where, e.g., an antigen-binding domain is specific for a particular epitope that is not carried by numerous antigens, in which case the antibody carrying the antigen-binding domain will generally not bind other antigens.
• terapéuticaally effective amount means an amount of a microorganism, or a subunit antigen, or polypeptides, or polynucleotide molecules, and combinations thereof, or a vaccine or a composition, needed to treat a disease in the subject to which it is administered.
• treat means to reduce or eliminate an infection by a microorganism.
• These terms and the like as used herein can also mean to reduce the replication of a microorganism, to reduce the transmission of a microorganism, or to reduce the ability of a microorganism to establish itself in its host.
• These terms and the like as used herein can also mean to reduce, ameliorate, or eliminate one or more signs or symptoms of infection by a
• microorganism or accelerate the recovery from infection by a microorganism.
• vaccinate and “vaccinating” and the like, as used herein, mean to administer to an animal a vaccine or immunogenic composition.
• vaccine and "vaccine composition,” as used herein, mean a composition which prevents or reduces an infection, or which prevents or reduces one or more signs or symptoms of infection.
• the protective effects of a vaccine composition against a pathogen 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.
• the vaccine compositions of the present invention provide protective effects against infections caused by BVDV.
• vehicle-acceptable carrier refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.
• the present invention provides methods of determining whether an animal has had exposure to specific pestiviruses, either through infection or vaccination.
• Vaccination which utilizes a DIVA vaccine - one which allows differentiation between infected and vaccinated animals - provides a means for assessing the exposure history of the animal subject. This differentiation can be accomplished via any of various diagnostic methods, including but not limited to an enzyme-linked
• ELISA immunosorbent assay
• SPRIA radioimmunoassay
• ECL electrochemiluminescent assay
• the chimeric pestiviruses described herein can be distinguished from wild-type BVDV strains in both their genomic composition and proteins expressed. Such distinction can allow for discrimination between vaccinated and infected animals. For example, a determination can be made as to whether an animal testing positive for BVDV in certain laboratory tests carries a wild-type BVDV strain or has been immunized with a conventional BVDV vaccine, or whether it has been administered a chimeric pestivirus or is uninfected.
• virus can be isolated from the animal testing positive for infection.
• Nucleic acid-based assays can include, but are not limited to, Southern or Northern blot analysis, PCR, and sequencing.
• protein-based assays can be employed.
• cells or tissues suspected of an infection can be isolated from the animal testing positive for BVDV.
• Cellular extracts can be made from such cells or tissues and can be subjected to, e.g., Western Blot, using appropriate antibodies against viral proteins that can distinctively identify the presence of either the chimeric pestivirus previously administered in a vaccine, or wild-type BVDV.
• the extent and nature of the immune responses induced in the animal can be assessed by using a variety of techniques. For example, sera can be collected from the inoculated animals and tested for the presence or absence of antibodies specific for the chimeric virus.
• This can be accomplished by incubating the sera from animals in the presence of pronghorn E rns protein.
• the protein can be native - that is, purified from pronghorn pestivirus - or recombinantly-expressed.
• the addition of the pronghorn E rns protein can be done prior to addition of sera to the assay plate(s), or at the same time sera is added to the assay plate(s). This is effective in removing E rns cross- reactive antibodies, and results in a more accurate and reliable assay.
• kits of the present invention can comprise one or more reagents useful for the detection of and differentiation between (1 ) a BVDV-infected animal or one immunized with a conventional BVDV vaccine, and (2) an animal administered a chimeric pestivirus.
• the kit can include reagents for analyzing a sample for the presence of whole BVDV, or BVDV polypeptides, epitopes or polynucleotide sequences which are not present in the chimeric pestivirus.
• kits of the present invention can include reagents for analyzing a sample for the presence of a chimeric pestivirus, or polypeptides, epitopes or polynucleotide sequences which are not present in wild-type BVDV.
• the presence of virus, polypeptides, or polynucleotide sequences can be determined using antibodies, PCR, hybridization, and other detection methods known to those of skill in the art.
• kits of the present invention can provide reagents for the detection of antibodies against particular epitopes.
• the epitopes are either present in the chimeric pestivirus and not present in wild type BVDV, or alternatively, are present in wild-type BVDV and not present in the chimeric pestivirus.
• Such reagents are useful for analyzing a sample for the presence of antibodies, and are readily known and available to one of ordinary skill in the art.
• the presence of antibodies can be determined using standard detection methods known to those of skill in the art.
• kits can include a set of printed instructions or a label indicating that the kit is useful for the detection and differentiation of BVDV- infected or BVDV-vaccinated animals from animals administered a chimeric pestivirus.
• Antibodies can either be monoclonal, polyclonal, or recombinant.
• the antibodies can be prepared against the immunogen or a portion thereof. For example, a synthetic peptide based on the amino acid sequence of the immunogen, or prepared
• Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art, such as described generally in Harlow and Lane, "Antibodies: A Laboratory
• Antibody fragments can also be prepared from the antibodies, and include Fab, F(ab') 2 , and Fv, by methods known to those skilled in the art.
• ELISAs Western blotting are types of immunoassays that can be used, and both are well known to those skilled in the art.
• polyclonal and monoclonal antibodies can be used in the assays.
• the antibody used to bind BVDV E rns protein can be bound to a solid support substrate.
• Antibody can be conjugated with a detectable moiety or label.
• the detectable moieties contemplated for use in the present invention can include, but are not limited to, fluorescent, metallic, enzymatic, and radioactive markers including but not limited to biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C, and iodination.
• the antibody conjugated with a detectable moiety can bind to BVDV E rns protein, as in a competitive ELISA assay, or it can bind to antibodies from an animal which bind to BVDV E rns protein, as in an indirect ELISA assay.
• compositions include a label conjugate comprising a binding component incorporated with a label.
• the binding component in the conjugate participates with other
• a binding reaction system producing two species or forms of the conjugate, e.g., a bound-species (conjugate complex) and a free-species.
• a bound-species conjugate complex
• a free-species e.g., the binding component of the conjugate is bound by a corresponding binding partner whereas in the free species, the binding component is not so bound.
• the amount of analyte is proportional to the amount of bound versus unbound conjugate.
• Cattle to which have been administered a chimeric pestivirus, in which the E rns protein of the BVDV is replaced with the E rns protein from a pronghorn pestivirus can be distinguished from cattle naturally infected with a wild-type BVDV or immunized with a conventional BVDV vaccine through the use of the invention described herein.
• Cattle of various ages are either not treated or are administered three doses of either a live or inactivated chimeric pestivirus, or a live or inactivated BVDV, with about three weeks between each dose. Serum samples are collected 2-3 weeks or later following each administration, but prior to the next administration.
• wild-type BVDV or chimeric pestivirus E rns protein can be used as an antigen source in the assay. If E rns protein present on wild-type BVDV is used as the test antigen, a lack of binding by the labeled wild type BVDV E rns -specific mAb indicates the presence of antibodies in the cattle serum that bind to the wild-type BVDV-specific epitope, indicative of a natural (wild-type) infection or immunization with a conventional BVDV vaccine.
• serum from cattle given the chimeric pestivirus will not contain antibodies which bind to the wild-type BVDV E rns protein coating the plate. Therefore, the labeled wild type BVDV E rns -specific mAb will bind to the bound protein, and result in subsequent color development.
• a recombinant baculovirus expressing BVDV-NADL E rns was constructed.
• a DNA molecule encoding a genetic fusion of a 3' portion of the C gene of BVDV and the full length E rns gene was amplified by PCR from a plasmid containing full length of BVDV- NADL cDNA with primers Oligo 250 (SEQ ID NO: 1 ;
• Competent E. coli Competent E. coli (Invitrogen) according to the manufacturer's instructions.
• the recombinant plasmid was extracted and the insert was confirmed by sequencing. This plasmid was designated pENTR-E rns .
• pENTR-E rns and BaculoDirectTM Baculovirus Expression System were used to construct recombinant baculovirus expressing BVDV-NADL E rns protein according to the manufacturer's instructions.
• the recombinant baculovirus expressing BVDV-NADL E rns protein was generated, plaque purified, expanded, and stored at both 4°C and -80°C.
• BVDV-NADL E rns protein in the recombinant baculovirus was confirmed by immunofluorescent staining and Western blot using BVDV E rns specific MAb 15C5 (Idexx Laboratories Inc. ; Westbrook, ME).
• pronghorn pestivirus E rns protein was expressed in baculovirus using a similar strategy. Expression of pronghorn E rns protein was confirmed by
• Sf21 or Sf9 cells in 100 ml suspension culture were infected with recombinant baculovirus stock at MOI's of 0.2 to 5.
• the cells were harvested after 2 to 4 days incubation at 27°C.
• the cells were centrifuged at low speed (about 800g) for 10 min to collect the cells.
• the cells were lysed with native lysis/binding buffer (pH 8.0 50 mM NaH2PO4, 300 mM NaCI, 10 mM Imidazole, and 1 % IGEPAL CA-630).
• native lysis/binding buffer pH 8.0 50 mM NaH2PO4, 300 mM NaCI, 10 mM Imidazole, and 1 % IGEPAL CA-630.
• the mixture was pipetted up and down to break up cell clumps, and then frozen at -80°C for > 1 hour.
• the ELISA plates were coated overnight at 4°C with 100 ⁇ /well of MAb WB210 (Veterinary Laboratory Agency, Surrey, UK), which specifically binds to BVDV Type 1 E rns protein, diluted to 1 ug/ml in carbonate/bicarbonate buffer (pH 9.0). The next day, the plates were washed three times with PBST wash buffer (PBS containing 0.05% Tween 20) and incubated with blocking buffer (PBST plus 1 % casein sodium salt) at 37°C for 1 hour.
• PBST wash buffer PBS containing 0.05% Tween 20
• blocking buffer PBST plus 1 % casein sodium salt
• the plates were subsequently washed three times with PBST, and 100 ⁇ of Baculo-BVDV E rns lysate (diluted 1 :1 600 in PBS) was added to each well, and the plates were incubated at 37°C for 1 hour. During this incubation period, 60 ⁇ of a cattle serum sample was added to 60 ⁇ sample diluent containing blocking buffer and a pre-determined concentration of Baculo-Pronghorn E rns lysate. The serum-diluent mixtures were incubated at room temperature for at least 30 minutes. Following three washes with PBST, serum-diluent mixtures were transferred to the wells of ELISA plates.
• % Inhibition (OD of Sample) ⁇ (Mean OD of 15C5-HRP Controls) x 100%.
• BVDV sero-negative cattle per treatment group were vaccinated with inactivated BVDV (NADL strain), the chimeric pestivirus, or no vaccine (NTX).
• Vaccinations were on three-week intervals. Serum samples were collected at each time point, prior to administering the vaccines. Samples were then tested in the above- described assay.
• Table 1 represents a comparison of serologic responses of cattle administered experimental antigens and untreated controls. Data presented were generated using the original diagnostic assay and the improved diagnostic assay, which is the present invention. Serum samples were collected after administration to cattle of two doses of inactivated BVDV, or two doses of inactivated chimeric pestivirus. Based on several known BVDV positive and negative samples, the cutoff values for positive (“Pos”), negative (“Neg”) or uncertain (“+/-”) were defined as follows:
• Table 2 represents a continuation of the experiment described above for Table 1 .
• serum samples were collected after administration of a third dose of either the inactivated BVDV or inactivated chimeric pestivirus.
• the DIVA assay described in Example 1 is combined in tandem with another BVDV-specific antigen capture assay or antibody assay, to determine if a negative sample detected with the DIVA assay of Example 1 (i.e., one that is from an animal either vaccinated with the chimeric pestivirus, or a na ' ive uninfected animal) is indeed na ' ive, or is instead from an animal vaccinated with the chimeric pestivirus.
• the negative sample from the DIVA assay of Example 1 is tested for the presence of a second antigen or antibody to BVDV that will be present even in an animal that has been vaccinated with the chimeric pestivirus.
• a sample from an animal that is determined "negative” for BVDV with the DIVA assay of Example 1 is further analyzed for the presence or absence of another BVDV-specific antigen or antibody.
• the DIVA assay of Example 1 is combined with an antigen capture test or an antibody detection test for an antibody that specifically binds another BVDV antigen, e.g., BVDV E2 protein.
• the sample from an animal that is determined "negative” for BVDV with the DIVA assay of Example 1 is analyzed for antibodies specific for BVDV p80/125 (i.e. NS 2/3) non-structural protein. Assays for the detection of such proteins are commercially available and described, e.g.,

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