FR2966459A1 - USE OF ANTI-NS1 ANTIBODIES OF BTV IN A DIVA METHOD APPLIED TO BTV VIRUSES - Google Patents

Abstract

The present invention relates to the use of the non-structural NS1 protein of BTV-1 or BTV-8 virus, or an antigenic fragment of said NS1 protein, or a composition or compound comprising such a protein or fragment, for the preparation of a "DIVA-BTV" reagent or kit for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus. It also relates to an in vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus as well as a method for selecting antibodies as a reagent intended to be used in such a DIVA-BTV method. . Hybridomas as well as antibodies secreted by such hybridomas also form part of the present invention.

Description

Bluetongue (BT) is an infectious but non-contagious viral disease that affects ruminants and is transmitted by some species of biting midges (Culicoides genus). It is caused by a virus belonging to the genus Orbivirus (Reoviridae) and commonly called "Bluetongue virus" or BTV. This virus is present worldwide on a broad band covering North and South America, Africa, South Asia and North Australia, leading to bluetongue being one of the most common diseases. affecting domestic livestock. Historically, this virus also occasionally arrived on the shores of southern Europe. BT's recent incursions into this sector have seen a dramatic change in the epizootic situation with the emergence of new serotypes (serotypes 1, 2, 4, 8, 9, 16) in endemic areas and the emergence of the virus in areas previously unaffected. The starting point was observed in 1998 with the arrival of BTV-9 in Greece. Then incursions of BTV-8 were reported almost simultaneously in Belgium, France, Germany and Holland in August 2006, leading to the largest epizootic ever recorded affecting flocks of sheep and cattle. The reasons for this radical change in the epidemiology of bluetongue are complex but seem to be related to the extension of the colonized territory by its main vector, Culicoides imicola, the appearance of new vectors of Culicoides types as well as to global warming.

The clinical signs of a BTV infection are extremely variable. In sheep, they are generally acute and characterized by fever, oral lesions, weight loss, lameness, with a mortality rate of infected sheep of about 20 to 50% (MacLachlan and Pearson, 2004, Verwoerd and Erasmus). , 2004). The symptoms are less apparent in cattle and more than 90% of animals infected with BTV show no or few clinical signs. However, bovine infection is an important factor in the epizoology of BTV due to their role as a viral reservoir. Significant economic losses are therefore associated with BTV infection, directly through the reduction of production or even the decimation of livestock, but above all indirectly through the implementation of embargoes and a restriction of export. cattle and costly control measures. The members of the European Community and Switzerland have therefore adopted a vaccination plan to reduce losses in domestic animal farms, contain any spread of the disease and facilitate the market for livestock. Live virus attenuation vaccines, also known as attenuated vaccines or Modified Live Virus (MLV) vaccines, were the only vaccines available for bluetongue during the last epizootic. This type of vaccine was therefore imported and used as an emergency against BTV Serotype 2, 4, 9 and 16 in southern Europe (Corsica, Portugal, Balearic Islands and Italy). However, the risks associated with the use of these remain important. It has been reported that insufficient attenuation of the virus can lead to clinical manifestations of the disease and teratogenicity. In addition, virulence reversion by gene rearrangement with coexisting wild strains may occur. In order to avoid this kind of problem, a preferable alternative is the use of vaccines based on a total inactivation of the virus, so-called inactivated vaccines. Anti-BTV-2, -4 and -16 vaccines of this type, combining efficacy and risk reduction, have thus been developed. Currently, different inactivated anti-BTV8 vaccines are available in Europe (BLUEVAC 8, CZ Veterinaria SA, Porriuo, Spain, Zulvac® 8, Fort Dodge Animal Health, Naarden, Holland and BTVPUR® AlSap 8, Merial SAS, Lyon, France) and others are under development in India and China against other serotypes.

The diagnosis of bluetongue can be made by virus isolation and identification, by detection of specific sequences of viral RNA or by detection in the serum of antibodies specific for viral antigens. However, the need for a virus amplification phase makes the virus isolation technique complex and time consuming. Detection of BTV RNA by RT-PCR, although faster, remains an expensive method. Serological tests, easier and less expensive, are therefore the most commonly used. This family of tests includes complement fixation, immunodiffusion, immunofluorescence, gel hemolysis, and enzyme-linked immunosorbent assay (ELISA). The ELISA test is essentially used for the detection of anti-BTV antibodies group-specific ("The use of a monoclonal antibody in a blocking ELISA to detect group-specific antibodies to bluetongue virus, Anderson J. et al., J.Immunol Methodes Vo 1.7, pp 139-149, (1984)) is based on the detection of antibodies directed against a particular region of the highly immunogenic and highly conserved VP7 capsid protein between the 24 serotypes of BTV. anti-VP7 can be detected in ruminants from the 7th day after infection and 10 to 40 days after vaccination depending on the animals tested and vaccines used.Unfortunately, it has been shown that the antibodies thus detected are not necessarily neutralizing Thus, a serological status anti-VP7 positive for naturally infected animals can co-exist with a contagious state.In addition, an anti-VP7 seroconversion is also observed after vaccination cattle using commercial vaccines. In this context, the current ELISA tests based on VP7 can not differentiate naturally infected animals from vaccinated animals (DIVA for "Differentiating Infected Vaccinated Animals") and the only way to be sure of the health status of an animal remains of check the absence of a viral genome in the blood by RT-PCR. New serological tests, which are more economical and easier to perform, are therefore necessary for epidemiological studies of BTV and to coordinate future vaccination campaigns.

For this purpose, the non-structural proteins (NS) of bluetongue virus have been identified as a family of interesting antigens. Indeed, the recommended vaccines being inactivated type, this implies that the virus can multiply only in naturally infected animals and not in vaccinated animals. Therefore, a test based on a nonstructural protein that is expressed only during viral replication is theoretically a good candidate for the development of a DIVA test. Similar strategies have been implemented in the past, particularly for avian influenza vaccines ("Overview of avian influenza DIVA test strategies", Biologicals, Vo1.33, pp221-226 (2005)). J.Anderson, P.Mertens and K.Herniman were the first to describe this DIVA strategy for the diagnosis of bluetongue. They developed a competitive ELISA (c-ELISA) using a monoclonal antibody directed against the non-structural protein NS 1 of BTV and called "F58" ("A competitive ELISA for the detection of anti-tubule antibodies"). Using a monoclonal antibody against Bluetongue non-structural protein NSI virus ", Journal of Virological Methods, Vo1.43, pp167- 176 (1993). In this test, the binding capacity of antibody F58 to NS1 is abolished by sera. naturally infected animals, or vaccinated with attenuated vaccines, but not with the sera of animals vaccinated with the inactivated vaccine they produced. inhibition by the sera of animals infected with the epizootic haemorrhagic disease virus or EHDV (epizootic hemorrhagic disease virus), another orbivirus transmitted by culicoids and may interfere with the diagnosis of bluetongue by a standard ELISA test. The test Thus, described combines DIVA characteristics and serogroup specificity. Unfortunately, this approach has its limits. Indeed, nonstructural proteins are partially co-purified with the virus and therefore in small amounts in the formulation of inactivated vaccines. An immunological response directed against these proteins can then be induced. In addition, different serotypes of bluetongue are present in Europe, leading to several cycles of vaccination campaigns (BTV 1 and BTV8 or BTV 1 and BTV 16, etc ...). The association of a lack of traceability with intense commercial exchanges can then lead to situations of multiple vaccinations against the same serotype and for a single animal. In this context, very small amounts of NS proteins contaminating inactivated vaccines can induce seroconversion and thus the appearance of "problem sera" in DIVA tests.

The Applicant has demonstrated that it is possible to use the non-structural protein NS 1 of the BTV virus, or an antigenic fragment of said NS1 protein, for the preparation, selection and production of monoclonal antibodies, as well as associated tests, to differentiate an animal specifically infected with BTV from a vaccinated animal against such a virus. This test is suitable for the analysis of biological samples of animals whose health status with respect to this infection is unknown. Thus, the present invention relates to the use of an antibody, or a functional fragment thereof, directed against the native or recombinant NS 1 protein of the blue tongue virus (referred to as "BTV" for Blue Tongue Virus), for the preparation of a reagent or a kit for its in vitro use and intended to differentiate an animal vaccinated against a BTV virus from an animal infected with a BTV virus, characterized in that said antibody is a monoclonal antibody obtained from a hybridoma culture supernatant, said hybridoma having been obtained by fusion between a myeloma cell and an animal mammalian lymphoid cell, said mammal having been previously immunized with a composition or an immunogenic compound comprising a NS1 protein, native or recombinant, of BTV, or one of its antigenic fragments, and characterized in that said antibody is selected by a process comprising the following steps: in contact with said test antibodies thus obtained from culture supernatant of said hybridomas with a native or recombinant NS1 protein of BTV; and b) - i) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a native or recombinant NS1 protein of epizootic haemorrhagic disease virus ("EHDV" for epizootic hemorrhagic disease virus); and c) selecting said tested antibody if the latter: - 1) specifically recognizes said BTV NS1 protein, or its antigenic fragment, and - ii) does not recognize said EHDV NS1 protein.

In a particularly preferred embodiment, said antibody is selected by a process further comprising in step b): b) - ii) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a serum , or plasma, of an animal infected with EHDV, said serum or plasma comprising polyclonal antibodies directed against the EHDV, and in that said antibody is selected if furthermore in step c): c) - iii) it There is a cross-reaction for the recognition of the BTV NS1 protein between said test antibody and said serum, or plasma, of an animal infected with EHDV.

In a still more particularly preferred embodiment, said antibody is selected by a method further comprising in step b): b) iii) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a serum, or plasma, of an animal infected with or vaccinated with BTV, said serum or plasma comprising polyclonal antibodies directed against BTV, and in that said antibody is selected if furthermore at step c) - iiii) a) There is a competition observed and resulting in a significant decrease in the signal obtained for the tested sample compared to the signal obtained for a negative control is significant of the presence of a sample emanating from an animal infected with BTV, preferably a signal representing 0 to 20%, more preferably 0 to 15% of the signal obtained for said negative control; and (3) There is no or little competition observed for the tested sample compared to the signal obtained for a negative control is significant for the presence of a sample emanating from an animal vaccinated against BTV, preferably a signal representing more than 50%, more preferably more than 60% of the signal obtained for said negative control.

In a preferred embodiment, said NS1 protein of the BTV virus, or composition or compound comprising it, or one of its antigenic fragments is chosen from: a) a native NS1 protein isolated and, where appropriate, purified, or recombinant of BTV virus, preferably preferably of amino acid sequence SEQ ID NO: 1 (NS1 of BTV-1, GenBank: CAO85724.1), or SEQ ID NO: 2 (NS1 of BTV-8, GenBank: CAM57246.2 ), or of sequence having at least 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or b) a BTV virus, or a fragment thereof containing the NS1 protein, or one of its antigenic fragments, as defined in a).

According to a also preferred embodiment, said EHDV is a serotype 1 or 2, preferably type 2, EHDV, and said NS1 protein of the EHDV virus is the serotype 1 or 2 NS1 EHDV protein of SEQ ID NO sequence respectively. : 3 (NCBI reference sequence: YP_003240112.1) or SEQ ID NO: 4, (GenBank: CAN89169.1) or sequence with at least 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 3 or SEQ D NO: 4.

According to a particularly preferred embodiment, said antibody is the antibody secreted by said antibody is the antibody secreted by the hybridoma called 9B4 Id Vet deposited at the CNCM July 7, 2009 under the number I-4198.

Functional fragment of said antibody is preferably intended to denote the fragments chosen from the Fab, Fab '), Fab', Fv, scFv and scFv-Fc fragments of said antibody, these fragments having the functional characteristics of the antibody for which they are present. are derived and as defined in particular in step c). The monoclonal antibody fragments according to the invention comprise any fragment of said monoclonal antibody capable of binding to an epitope of the NS1 protein of BTV 1 or 8 and on which the monoclonal antibody of which said fragment is derived is fixed. Examples of such fragments include in particular single-chain monoclonal antibodies or Fab or Fab 'monovalent fragments and divalent fragments such as F (ab') 2, which have the same binding specificity as the monoclonal antibody from which they arise. . A fragment according to the invention may also be a single chain Fv fragment produced by methods well known to those skilled in the art. According to the present invention, monoclonal antibody fragments of the invention can be obtained from the monoclonal antibodies as previously described by methods such as digestion with enzymes, such as pepsin or papain and / or by cleavage. disulfide bridges by chemical reduction. In another way, the monoclonal antibody fragments included in the present invention can be synthesized by automatic peptide synthesizers such as those provided by Applied Biosystems, etc.

In general, for the preparation of monoclonal antibodies or their fragments, reference may be made to the techniques which are in particular described in the "Antibodies" manual (Harlow et al., 1988) or to the preparation technique using hybridomas described by Kohler and Milstein in 1975. The monoclonal antibodies used according to the present invention can be obtained for example from cells of an animal immunized against NS1 protein of BTV 1 or 8 and, or a fragment thereof, comprising epitope specifically recognized by said monoclonal antibody called 9B4 Id Vet according to the invention. Said NS1 protein of BTV 1 or 8 and, or one of its antigenic fragments, may in particular be produced, according to the usual procedures, by genetic recombination from a nucleic acid sequence contained in the sequence of the cDNA coding for the NS1 protein of BTV 1 or 8 and or by peptide synthesis from an amino acid sequence included in the peptide sequence of the NS1 protein of BTV 1 or 8 and, preferably the NS1 protein of BTV 1 or 8 and of sequence SEQ ID NO: 1 or 2. The monoclonal antibodies that can be used according to the invention may, for example, be purified on an affinity column on which the NS1 protein of BTV 1 or 8 has previously been immobilized and or a fragment thereof. comprising the epitope specifically recognized by said monoclonal antibody called 9B4 Id Vet according to the invention. The following use or methods according to the present invention may employ an anti-NS 1 antibody of BTV, preferably of serotype 1 or 8 and or a fragment thereof, characterized in that it is chosen from the chimeric antibodies having the same CDRs ("CDR" for Regions Determining Complementarity as the anti-NS1 monoclonal antibody of BTV and as defined above.) The monoclonal antibodies or their chimeric-type fragments according to the invention can be made in For example, the chimeric monoclonal antibody can be made by cloning a recombinant DNA comprising a promoter and a sequence coding for the variable region of a monoclonal antibody according to the invention and a sequence coding for the region. human antibody constant A chimeric antibody of the invention encoded by such a recombinant gene will be for example a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from murine DNA and its isotype determined by the constant region derived from human DNA. Also included in the invention are differentiation uses or methods DIVA in which the monoclonal antibodies or fragments thereof, or the NS1 proteins of BTV 1 or 8, or their homologs at least 85% identical, or their epitopes / fragments antigenic, are obtained by genetic recombination (recombinant proteins) or by chemical synthesis. The invention also includes uses or methods of detection or diagnosis in which the monoclonal antibodies or fragments thereof, or the BTV 1 or 8 NS1 proteins, their at least 85% identity homologues, or their epitopes / fragments antigenic, are labeled. The monoclonal antibodies or fragments thereof, or the BTV-1 or BTV-8 NS1 proteins or their epitopes / antigenic fragments, may also be labeled in order to obtain a detectable and / or quantifiable signal.

The monoclonal antibodies or fragments thereof, or the NS1 proteins of BTV-1 or -8, their at least 85% identical homologues, or their epitopes / antigenic fragments, include, for example, so-called immunoconjugated antibodies or antigens which can be conjugated by example with enzymes such as peroxidase, alkaline phosphatase, PD-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetyl-cholinesterase, lysozyme, malate dehydrogenase or glucose-6 phosphate dehydrogenase or by a molecule such as biotin, digoxigenin or 5-bromo-deoxyuridine. Fluorescent markers may also be conjugated to the monoclonal antibodies or their fragments of the invention and include, in particular, fluorescein and its derivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone etc. In such conjugates, the monoclonal antibodies of the invention or fragments thereof can be prepared by methods known to those skilled in the art. They can be coupled to fluorescent enzymes or markers directly or via a spacer group or a linker group such as polyaldehyde, such as glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or in the presence of coupling agents such as periodate, etc. Conjugates containing fluoroscein-type labels may be prepared by reaction with an isothiocyanate.

Other conjugates may also include chemiluminescent markers such as luminol and dioxetanes or bioluminescent markers such as luciferase and luciferin.

Among the markers that can be attached to the monoclonal antibody or NS1 antigen of BTV 1 or 8, their homologues, or a fragment thereof, radioactive labels such as 14C, 36C1, 57Co, 58Co, 58Cr can also be mentioned, 59Fe 1'H 125I 121I, 32P or 35S that can be detected by known means such as the gamma or scintillation counter etc. .... According to a also preferred embodiment, - said reagent or kit further comprises a NS1 protein BTV virus, or composition or compound comprising it, or an antigenic fragment thereof.

Preferably, said NS1 protein of the BTV virus, or composition or compound comprising it, or an antigenic fragment thereof is as defined in claim 4 a) and 4 b). According to other features of the invention: said antigenic fragment of the NS1 protein is a fragment capable of binding specifically to the antibody secreted by the hybridoma called 9B4 Id Vet deposited at the CNCM on July 7, 2009 under the number I - 4198. - said BTV NS1 protein or one of its antigenic fragments, or the composition or the compound comprising it, is fixed or adsorbed on a solid support, preferably on a microplate cup.

According to a also preferred embodiment, said animal from which said biological sample originates is a ruminant, preferably chosen from cattle, sheep or goats.

In another aspect, the present invention relates to an in vitro method for differentiating an animal vaccinated against BTV virus from an animal infected with a BTV virus from a biological sample of said animal comprising the following steps: A) the contacting said biological sample with the NS1 protein of a BTV virus, or an antigenic fragment of said NS1 protein, or a composition or compound comprising such a protein or fragment, and B) revealing the antigen complexes -antibody formed in (a) between said NS1 protein, or one of its antigenic fragments, and an anti-NS1 antibody likely to be present in said sample, this revelation being carried out by a competitive immunoassay method implementing an anti-NS1 antibody, or a functional fragment thereof, capable of specifically recognizing the NS1 protein of the BTV virus, or one of its antigenic fragments, said antico rps-anti-NS 1 being a monoclonal antibody obtained from a culture supernatant of a hybridoma, said hybridoma having been obtained by fusion between a myeloma cell and an animal mammalian lymphoid cell, said mammal having been previously immunized with an immunogenic composition or compound comprising a native or recombinant BTV NS1 protein, or an antigenic fragment thereof, and characterized in that - said anti-NS 1 antibody is selected by a method comprising the following steps: ) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a native or recombinant NS1 protein of BTV; and b) - i) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a native or recombinant NS1 protein of epizootic haemorrhagic disease virus ("EHDV" for epizootic hemorrhagic disease virus); and c) selecting said tested antibody if the latter: i) specifically recognizes said BTV NS1 protein, or antigenic fragment thereof, and ii) does not recognize said EHDV NS1 protein. According to a particularly preferred embodiment of the method according to the invention, - said test antibody is selected by a process further comprising in step b): b) - ii) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a serum, or plasma, of an animal infected with EHDV, said serum or plasma comprising polyclonal antibodies directed against EHDV, and in that said antibody is selected if further in step c iii) there is a cross-reaction for the recognition of the BTV NS1 protein between said test antibody and said serum, or plasma, of an animal infected with EHDV.

According to a more particularly preferred embodiment of the method according to the invention, said antibody is selected by a process further comprising in step b): b) iii) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with serum, or plasma, of an animal infected with or vaccinated against BTV, said serum or plasma comprising polyclonal antibodies directed against BTV, and in that said antibody is selected if in addition to step c): c) - iiii) a) There is a competition observed and resulting in a significant decrease in the signal obtained for the tested sample compared to the signal obtained for a negative control is significant of the presence of a sample from an animal infected with BTV, preferably a signal representing 0 to 20% of the signal obtained for said negative control; and (3) There is no or little competition observed for the tested sample compared to the signal obtained for a negative control is significant for the presence of a sample emanating from an animal vaccinated against BTV, preferably a signal representing more than 50% of the signal obtained for said negative control. According to a mode also preferred of the method according to the invention, said antibody implemented in step b) in the competitive immunoassay method is a labeled antibody, preferably the immunoassay method in competition is a competing ELISA method, preferably said antibody is labeled with peroxidase. According to a more particularly preferred embodiment of the method according to the invention, said antibody used in step b) in the competitive immunoassay method is the antibody secreted by the hybridoma called 9B4 Id V and deposited at the CNCM. July 7, 2009 under number I-4198.

According to a mode also preferred of the method according to the invention, said method comprises the following steps: a) bringing said biological sample into contact with the NS1 protein of a BTV virus, or an antigenic fragment of said NS1 protein, or of a composition or compound comprising such a protein or fragment, said NS1 protein being previously captured by immunocapture on a solid phase coated with a polyclonal anti-NS1-BTV antibody, or, monoclonal type and such as defined in claim 1; b) revealing the antigen-antibody complexes formed in (a) between said NS1 protein, or one of its antigenic fragments, and an anti-NS1 antibody likely to be present in said sample, this revelation being carried out by a method Immunoassay in competition employing an anti-NS1 antibody, or a functional fragment thereof, preferably labeled as defined in claim 1, 2 or 3, preferably 2 and 3.

According to a mode also preferred of the method according to the invention, said method comprises the following steps: in step a): a) the anti-NS1 antibody coated on the solid phase and as defined in claim 1 ; and b) the anti-NS1 antibody, preferably labeled, as defined in claim 2 or 3 is the antibody secreted by the hybridoma designated 9B4 Id V and deposited at the CNCM on July 7, 2009 under the number I- 4198. More preferably, said NS1 protein of the BTV virus used in step a), or a composition or compound comprising it, or one of its antigenic fragments is chosen from: a) a native NS1 protein isolated and, the optionally purified, or recombinant BTV virus, preferably preferably amino acid sequence SEQ ID NO: 1 (NS1 BTV-1) or SEQ ID NO: 2 (NS1 BTV-8), or sequence having at least 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or b) a BTV virus, preferably BTV-1 or BTV-8, or a fragment thereof containing the NS1 protein, or an antigenic fragment thereof.

According to a preferred embodiment: - either said BTV virus NS1 protein, preferably BTV-1 or BTV-8, or one of its antigenic fragments, implemented in step a), or - either said antibody capable of specifically recognizing the NS1 protein of BTV virus, preferably BTV-1 or BTV-8-8, carried out in step b), is labeled, preferably with peroxidase.

According to a preferred embodiment, said biological sample is a sample of biological fluid, preferably selected from plasma, serum, meat juice or whole blood sample of the animal, serum being most preferred. According to a also preferred embodiment, in step b), an observed and resulting competition reaction by a significant decrease in the signal obtained for the tested sample compared to the signal obtained for a negative control is significant of the presence of a sample from an animal infected with BTV, preferably a signal representing 0 to 20%, more preferably 0 to 15% of the signal obtained for said negative control. Any conventional procedure or test may be implemented to perform such detection and / or assay. Said assay may be a two-step or one-step competitive assay, or any competitive antibody immunoassay known to those skilled in the art depending on the formation of an antibody-antigen immune complex. According to the methods of competitive immunoassays implemented in the methods according to the invention, the non-structural NS1 protein of BTV, preferably serotype 1 or 8, or an antigenic fragment of said protein NS1, or a composition or a compound comprising such a protein or a fragment, the monoclonal antibody or a fragment thereof can be immobilized or labeled, and the same is true for the anti-NS1 antibody of BTV, preferably serotype 1 or 8. This immobilization can be performed on many supports known to those skilled in the art. These supports may include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, or natural or modified celluloses. These supports can be either soluble or insoluble. Examples that may be mentioned include microplate cups. In a one-step immunoassay method, a BTV anti-NS1 antibody microplate well, preferably serotype 1 or 8 as described above, is coated and unlabeled. The sample to be tested is then successively added to the sample and the BTV antigen NSV 1, preferably serotype 1 or 8, previously labeled. This step is followed by a step of incubation and washing of the cup before the next step of detection and / or quantification of the marker. Such a one-step competitive ELISA method (as opposed to the two-step competitive ELISA method, as described in particular in the examples below) is also part of the present invention. By way of example, a preferred method involves immunoenzymatic processes according to the ELISA technique, by immunofluorescence, or radioimmunoassay (RIA) or equivalent.

According to a also preferred embodiment: said biological sample previously taken from the animal or obtained from the animal is a sample of biological fluid, preferably selected from plasma, serum, meat juice or else whole blood sample of the animal, serum being the most preferred.

In another aspect, the present invention provides a kit or kit of reagents for differentiating an animal vaccinated against BTV virus from an animal infected with a BTV virus, said kit comprising: A) a compound or composition comprising NS1 protein BTV virus, or an antigenic fragment of said NS 1 protein; B) an antibody, or a functional fragment thereof, directed against the NS1 protein of the BTV virus, or against an antigenic fragment of said NS1 protein, where appropriate labeled, preferably said antibody is polyclonal, monoclonal or chimeric; and, C) where appropriate, the reagents for constitution of the environment conducive to the immunological reaction; and D) where appropriate, reagents for detecting antigen-antibody complexes produced by the immunological reaction.

According to other characteristics of the invention: A) said compound or said composition comprising the NS1 protein of the BTV-8 virus, or said antigenic fragment of said NS1, is chosen from: a) an isolated native NS1 protein and, where appropriate purified, or recombinant, BTV virus, preferably preferably amino acid sequence SEQ ID NO: 1 (NS1 BTV-1) or SEQ ID NO: 2 (NS1 BTV-8), or sequence having at least at least 85%, 90%, 95%, 98 or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or b) a BTV virus, preferably BTV-1 or BTV-8, or a fragment thereof containing the NS1 protein, or an antigenic fragment thereof as defined above in claim 10, and B) said antibody, or one of its functional fragments, directed against the NS1 protein of the BTV virus, or against an antigenic fragment of said NS1 protein, where appropriate labeled, is chosen from the antibody secreted by the hybridoma called 9B4 Id Vet filed at the CNCM on July 7, 2009 under the number I-4198. According to other characteristics, said kit or kit of reagents comprises: a reagent A) comprising a first antibody called anti-NS1-capture; BTV of polyclonal type, or the antibody secreted by the hybridoma as defined in claim 1, optionally coated on a microplate cup; a reagent B) comprising a native NS protein isolated and, where appropriate, purified or recombinant of BTV virus, or else a BTV virus, preferably BTV-1 or an entire BTV-8, where appropriate, reagent B ) having been previously fixed on said cup of reagent A); and a reagent C) comprising the antibody secreted by the hybridoma called 9B4I d Vet deposited at the CNCM on 7 July 2009 under the number I-4198, where appropriate labeled.

In another aspect, the subject of the invention is a method for selecting an antibody for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus, which may be used in a competitive immunoassay method. According to other features, said selection method according to the invention comprises the following step: a), where appropriate the selection of a hybridoma obtained after immunization of an animal with an antigen chosen from: - an NS1 protein isolated native and, if appropriate, purified, or recombinant virus BTV, preferably preferably amino acid sequence SEQ ID NO: 1 (NS1 BTV-1) or SEQ ID NO: 2 (NS1 BTV-8 ), or of sequence having at least 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or - a BTV virus, preferably BTV-1 or BTV-8, or one of its fragments containing the NS1 protein, b) bringing said test antibody into contact with the NS1 protein of the BTV virus as defined in a), or an antigenic fragment of said NS1 protein, or a composition or compound comprising such a protein or fragment, and c) revealing the antigen-antibody complexes formed in (b) between said NS1 protein, or one of its antigenic fragments, and said one antibody tested, and in which step the revelation of the antigen-antibody complexes formed in (a) is carried out by a competitive immunoassay method using an antibody, or one functional fragments, said antibody being the antibody secreted by the hybridoma designated 9B4Id Vet deposited at the CNCM on July 30, 2009 under the number I-4198, or one of its functional fragments, where appropriate labeled .

In another aspect, the invention comprises the hybridoma designated 9B4Id Vet deposited at the CNCM on July 7, 2009 under the number I-4198, or one of its functional fragments, where appropriate labeled. The invention also comprises the antibody secreted by the hybridoma called 9B4Id Vet deposited at the CNCM, on 7 July 2009 under the number I-4198, or one of its functional fragments, where appropriate labeled. Other features and advantages of the invention appear in the following description with examples and figures. The figures whose legends are hereinafter, as well as the examples which follow, are intended to illustrate the invention without limiting its scope. FIGURE LEGENDS FIG. 1 shows a competition test between monoclonal antibodies 11G1 and 9B4. The monoclonal antibodies 9B4 or 11G1 were brought into contact with the monoclonal antibody 11G1 coupled to the peroxidase (11G1-HRP) at different concentrations (Mab / 11G1-HRP ratio) on plates sensitized with the native NS1 protein (A). or recombinant (B). FIG. 2 represents an evaluation of the monoclonal antibodies 11G1 and 9B4 in Western blot. A native NS1 antigen preparation sample was analyzed by SDS-PAGE electrophoresis and then transferred to PVDF membrane before being contacted with anti-NS1 monoclonal antibodies (10B1, 9B4 and 11G1). The revelation was performed using an anti-mouse antibody coupled to peroxidase. FIG. 3 represents the DIVA BT potential of the monclonal antibodies 11G1 and 9B4 on sera of multivaccinated animals. A sheep flock (n = 6) was vaccinated against BTV8 with a commercial vaccine (Merial). The animals received 2 injections of one dose at 36 days intervals (OJ and D + 36) then a double dose at the 74th day (D + 74). Sera were collected regularly for 108 days. The presence of competitive anti-NS1 antibodies of monoclonal antibodies 11G1 (A., white triangle) or 9B4 (B., white squares) was compared with the presence of anti-VP7 antibodies (black diamonds) by ELISA technique. FIG. 4 represents an evaluation of anti-VP7 and anti-NS1 seroconversion after infection with BTV. A panel of naive (n = 71) or BTV-8 infected (n = 87) bovine sera were tested for the presence of antibodies against VP7 (A.) and NS1 (B) proteins by the technique of Competitive ELISA using a standard test (ID Screen® Bluetongue Competition) or the cELISA DIVA-BT NS test 1. The results are represented as a distribution of the samples according to the percentage of the value obtained for each sample. on the average of the negative controls (S / N). Figure 5 represents an evaluation of anti-VP7 and anti-NS 1 seroconversion after vaccination against BTV in cattle: a bovine herd (n = 30) was vaccinated against BTV8 with a commercial vaccine . The sera were collected on the day of the first vaccination injection (OJ) and day 64 (1 + 64) and the presence of antibodies directed against the proteins VP7 (A.) and NS 1 (B) was highlighted. by competition ELISA technique. The results are represented as a sample distribution based on the percentage of the value obtained for each sample on the average of the negative controls (S / N). - Figure 6 represents an evaluation of anti-VP7 and anti-NS 1 seroconversion after vaccination against BTV in sheep: a sheep flock (n = 75) was vaccinated against BTV8 with a commercial vaccine (Merial). The sera were collected on the day of the first vaccination injection (JO) and on the 33rd day (1 + 33) and the presence of antibodies directed against the proteins VP7 (A.) and NS 1 (B. evidence by competition ELISA technique. The results are represented as a sample distribution based on the percentage of the value obtained for each sample on the average of the negative controls (S / N).

EXAMPLES Example 1 Materials and Reagents 1.1 Preparation of Native NS1 Antigen A monolayer of Vero cells at 80% confluence in a flask of 75 cm 2 (MEM, 0.5% FCS, gentamycin 40 μg / mL, amphotericin B 1 μg / mL) was infected with BTV-1. The cells were then unhooked and separated from the culture medium by centrifugation for 10 min at 1000 rpm when the percentage of lysis observed is greater than 70%. The cell pellet is taken up to 1 / 100th of the initial volume using lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 0.5% Tx100, protease inhibitors). The suspension was incubated for 1 h at +4 ° C., sonicated for 5 min. on ice then centrifuged 20 min. at 2200 x g ..

The supernatant containing the native NS1 antigen was recovered and stored at -20 ° C.

1.2 Preparation of the recombinant NS1 antigen The complete sequence of the DNA corresponding to the M6 region of BTV-1 was obtained by RT-PCR. The construction of the recombinant baculovirus containing the sequence of interest was carried out by the company GenScript (Piscataway, USA). Briefly, the gene coding for the BTV-1 NS1 protein was subcloned into the plasmid pGSF1 using the EcoR I and Sal I restriction sites to graft a 6xHIS N-terminal tag of the recombinant NS1 protein. . This fragment encoding 6xHIS-NS1 was transferred into the DH10Bac vector to allow protein expression in insect cells. The Sf9 cells were thus transfected with the recombinant baculovirus DH1OBac-6xHIS-NS1 using Cellfectin (Invitrogen, USA) and cultured for 5 to 7 days in SF-9000 medium (GIBCO, USA). Sf9 cells expressing NS1 were lysed using lysis buffer (50 mM Tris, 500 mM NaCl, 20 mM Imidazole, 0.1% NP40, PMSF and Protease Inhibitors) and sonicated for 5 min. After centrifugation, the supernatant was deposited on a nickel column and the NS1 protein eluted by adding 250 mM imidazole. The collected fraction containing the recombinant NS1 antigen was verified on SDSPAGE gel and by Western-blot using an anti-6xHIS antibody. The concentration was determined by measuring the optical density at 280 nm. 1.3 Banks of serums

1.3.1 Naïve animal serums The serums of the naïve animals were recovered during 2006 from the slaughterhouse in Alès (France).

1.3.2 Serums naturally infected animals BTV sera from animals infected with BTV come from several sampling campaign areas. BTV-8 positive cattle sera were provided by the Arsia laboratory (Belgium) and relate to the 2007 epidemic episode in the Liège region (Belgium). 1.3.3 Serums of infected animals EHDV

The EHDV sera were provided by Veterinary Diagnostic Technology (Wheat Ridge, USA) and correspond to the serological monitoring of an experimental infection of 6 cattle inoculated with serotype 1 or 2 EHDV, as well as positive reference sera. qualified by agarose gel immunodiffusion (AGID technique).

1.3.4 Serums of BTV vaccinated animals 1.3.4.1 Sheep serum: Sheep sera free from natural infection and vaccinated against BTV-8 were provided by Merial and correspond to a merial / Fort Dodge / CZ Veterinaria SA round robin test. in which the animals were vaccinated with inactivated BTV-8 vaccines (BLUEVACe 8, CZ Veterinaria SA, Zulvac® 8, n = 5, Fort Dodge Animal Health, n = 3 and BTVPUR® AlSap 8, Merial, n = 3) according to the manufacturers' protocol, as well as a Mérial anti-BTV8 vaccine trial (BTVPUR® AlSap 8). The protocol followed in the latter case consists of a triple vaccination injection 14 days apart between each dose, followed by a sampling 21 days after the last injection. The animals which received two additional doses per standard protocol establish for this vaccine, the sera obtained are considered as "hyperimmune sera" The sera of sheep free of natural infection and vaccinated against BTV-1 and -8 were provided by the National Veterinary School of Toulouse (ENVT, Toulouse, France). The animals received one dose and two doses of inactivated vaccine BTV-8 (BTVPUR® AlSap 8, Merial) and BTV-1 (Zulvac®1 sheep, Fort Dodge Animal Health), respectively, according to the manufacturers' protocol.

Sheep sera free of natural infection and vaccinated against BTV-2 and -4 were obtained by IDVET and correspond to immunization using the bivalent inactivated vaccine BTVPUR® AlSap 2 - 4 (Merial).

1.3.4.2 Bovine serums:

Bovine serum free of natural infection and vaccinated against BTV-8 were provided by Merial and correspond to a Merial / Fort Dodge / CZ Veterinaria SA interlaboratory trial in which the animals were vaccinated with inactivated BTV vaccines. -8 (BLUEVACe 8, CZ Veterinaria SA, Zulvac® 8, n = 6, Fort Dodge Animal Health, n = 7 and BTVPUR® AlSap 8, Merial, n = 4) according to the manufacturers protocol.

1.4 Reagents for ELISA tests

Unless otherwise indicated, all the reagents used in the context of the ELISA technique are input elements into the composition of the IDVET commercial kits. In order to define the terms used, the various solutions used are identified below: Wash Solution: Solution SL (from the IDScreen® test or PBST-type washing solution (0.01 M PBS containing 0.05% Tween-20, pH 7.2)

Saturation Solution: ST solution (PBST saturation solution containing 3% BSA). Revelation Solution: SR solution ("Revelation Solution" from IDScreen® test or 3.3 'revealing solution). , 5'-tetramethylbenzidine (TMB)) Stop Solution: Solution SA (IDScreen® Test Stop Solution or 0.5 M Sulfuric Acid Stop Solution). Serum dilution solution: DL18 or SL solution containing 2% skimmed milk.

Dilution solution of monoclonal antibody conjugated with peroxidase: DL19 or SL solution.

Such solutions are well known to those skilled in the art and are described in particular in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2009, OIE) or in standard manuals such as for example "Antibodies A". Laboratory Manual "(Ed Harlow and David Lane, 1998, Cold Spring Harbor Laboratory).

The data was obtained by reading the optical density at 450nm using a multichannel spectrophotometer (Sunrise, Tecan).

Example 2 Hybridoma preparation method 2.1 Preparation of BTV for inoculation Vero cell lines (ATCC, CCL-81) were cultured and brought to 80% confluency in DMEM medium containing 10% fetal calf serum (SVF), 50 μg / mL gentamycin + 100 U penicillin + 100 μg / mL streptomycin (Gibco). The culture medium was replaced by infection medium (MEM + 0.5% FCS + 50 μg / mL gentamycin + 100 U penicillin + 100 μg / mL streptomycin) and the cells were infected with strain BTV-1. Three days after infection, the infection medium was removed, the cells were washed with PBS and 10 mL of PBS containing 5% sucrose was added. The culture dish underwent a rapid freeze / thaw cycle -70 ° C / + 37 ° C. The native BTV-1 virus contained in the cell lysate thus recovered was determined by limit dilution and detection of PF0 by indirect immunofluorescence on Vero cells and using a mouse anti-VP7 BTV (IDVET) monoclonal antibody and an anti-mouse IgG antibody coupled to fluorescein (Sigma), then stored at -20 ° C before being used for immunization of mice. 35

24 2.2 Immunization of mice

BALB / c adult female mice were immunized intraperitoneally with the previously obtained viral stock BTV-1 (106 PFU BTV-1 / injection) at 0.5 mL containing 50% (vol / ml). vol) of Freund's complete adjuvant. The mice were resensitized at 14 and 35 days using an identical dose of BTV-1 containing 50% (vol / vol) incomplete Freund's adjuvant. Finally, an inoculation of BTV-1 (106 PFU of BTV-1 / injection) was carried out at the 3 rd month post-infection before the cell fusion step and the search for anti-BTV antibodies in the supernatants. hybridomas.

2.3 Test of immunized mouse sera

After 3 months, the presence of murine anti-VP7 and anti-NS1 antibodies of BTV1 was verified by indirect ELISA method. The NS1 native protein and recombinant BTV-1 NS1 and VP7 proteins were plated in 96-well plates at predetermined PBS dilutions and incubated at room temperature overnight. After washing to remove excess unbound antigen, saturation of the plates was performed. The ST solution was then removed and 50 μL / well of immune mouse serum was removed. After incubation for 5 h at 37 ° C., the plates were washed three times with the SL solution and 50 μl / well with anti-mouse IgG antibody coupled to peroxidase (Sigma-Aldrich, A4416 at 1 ° C.). / 1000e) were added and incubated for 30 min. at room temperature. 50 μL / well of SR solution was added after the last washes. The reaction was stopped after 10 min. by adding an identical volume of SA solution.

2.4 Preparation of splenocytes at fusion The mice were sacrificed then the spleens of each animal were removed under aseptic conditions and placed in a petri dish containing 10 ml of medium without serum (DMEM + 50 μg / ml of gentamycin + 100 U penicillin + 100 μg / mL streptomycin). After scalpel dilation, the residual splenic aggregates were dissociated by suction-discharge with the pipette and the suspension obtained was passed through a cell strainer. The cell suspension thus obtained was centrifuged for 5 min. at 500 x g. and the supernatant was removed. Lysis of red blood cells was performed by 5 min. incubation at room temperature after taking up the cell pellet in 5 ml of ammonium chloride solution. The reaction was stopped by adding 40 mL of serum-free medium and 5 min. at 500 x g. The supernatant was removed and the pellet containing the splenocytes was washed twice more (40 mL serum-free medium, centrifuged for 5 min at 500 xg) and then taken up in 10 mL of serum-free DMEM and the concentration determined by cell counting. Malassez.

2.5 Fusion Preparation of Myeloma Cells SP2 / O-Ag14 myeloma cells (ATCC # CRL 1581) were used for fusion with splenocytes of immunized mice. These cells have the particularity of being resistant to 8-azaguanine at a concentration of 20 μg / ml and sensitive to hypoxanthine-aminopterin-thymidine (HAT) because they lack the enzyme hypoxanthine phosphoribosyltransferase. These myeloma cells were maintained in culture in a complete medium DMEM / HEPES / pyruvate (DMEM + 10% FCS + 10 mM HEPES + 1 mM sodium pyruvate + 100 U penicillin + 100 μg / ml streptomycin) and passed every 3 days before use for the merger. On the day of fusion, 20 mL of suspension was washed once with serum-free DMEM medium preheated to 37 ° C and centrifuged for 5 min. at 400 x g. The cell pellet was taken up in 10 mL and the concentration determined by cell counting of Malassez.

2.6 Preparation of Antibody Secreting Hybridomas

The obtaining of the hybridomas was carried out on the basis of the protocols described by Galfre et al. (Nature, 1977, 266: 550-2) and Yokoyama W.M. et al. (Current Protocols in Immunology, 1995; 2.5.1-2.5.17) and is described below. Briefly, 0.5 g of polyethylene glycol 4000 (PEG 4000) was melted at 60 ° C in a water bath and then placed at 37 ° C after addition of 0.5 ml of serum-free DMEM. Cell suspensions of splenocytes and myeloma cells were returned to the same final cell concentration for a final volume of 10 ml of serum-free medium. The two suspensions were then pooled in a single tube (cell ratio "SP2 / O-Ag14" / "splenocytes" of 1: 1). The mixture was centrifuged for 5 minutes. at 800 x g for 5 min. then the supernatant was carefully removed. The PEG 50% was gently added using a Pasteur pipette on the cell pellet with slow stirring in a water bath at 37 ° C and for a total duration of one minute. The mixture was stirred for one more minute and then 10 mL of serum free culture medium (preheated to 37 ° C) was added dropwise over 2 min. additional. The cells were then centrifuged for 5 min. at 400 xg, the supernatant removed then the pellet resuspended in OmL then 200mL of complete medium (DMEM + 20% of SVF + 10 mM HEPES + 1 mM sodium pyruvate + 100 U / ml penicillin + 100 μg / ml streptomycin ). The cell suspension (2.5 × 10 6 cells / ml) was distributed in 96-well culture plates at a rate of 100 μl / well and incubated at 37 ° C. (5% CO 2). The following day, 50 μL / well of hybridoma selection medium (complete medium + 1X HAT) was added and half of the culture medium was replaced by this same selection medium every 2 days for 2 weeks. The supernatant of each hybridoma obtained was evaluated as described below and the selected antibody secreting hybridomas were amplified and then cloned by limiting dilution technique. The corresponding monoclonal antibodies could thus be produced by affinity purification of the culture supernatants and according to the technique described by Eaton et al. (1988).

Example 3 Identification of anti-BTV NS1 monoclonal antibody secreting hybridomas The native or recombinant NS1 protein of BTV-1 was plated in 96-well plates at predetermined PBS dilutions and then incubated at room temperature overnight. After washing to remove excess unbound antigen, saturation of the plates was performed. The ST solution was then removed and 50 μl / well of hybridoma culture supernatant was removed. After incubation for 3 h at 37 ° C., the plates were washed three times with SL solution and 50 μl / well of anti-mouse IgG antibody coupled to peroxidase (Sigma-Aldrich, A4416 at 1 / 1000e) were added and incubated for 30 min. at room temperature. 50 μL / well of SR solution was added after the last washes. The reaction was stopped after 10 min. by adding an identical volume of SA solution. Non-sensitized plates were used as negative controls in this experiment. Hybridomas whose supernatants gave a positive signal on the non-sensitized plates were discarded. Those responding to both types of sensitized plaques (native or recombinant NS1) were selected. The hybridomas thus retained are secretors of anti-BTV monoclonal antibodies whose epitopes are carried by the viral protein NS1.

Example 4 Identification of hybridoma secretion hybridomas of anti-BTV NS1 monoclonal antibodies with DIVA potential

The native or recombinant NS1 protein of BTV-1 was plated in 96-well plates at predetermined PBS dilutions and then incubated at room temperature overnight. After washing to remove the excess of unfixed antigen, saturation of the plates was performed. The ST solution was then removed and 50 μl / well of sera from naive, infected or vaccinated animals diluted with A in SL solution were deposited. After incubation for 2 h at 37 ° C. and 50 μl / well of hybridoma supernatant were added for an additional 1 h of incubation at 37 ° C. The plates were washed three times and 50 μl / well of peroxidase-coupled anti-mouse IgG antibody (Sigma-Aldrich, A4416 1/1000 "was added and incubated for 30 min at room temperature 50 μL / well). SR solution was added after the last washings, the reaction was stopped after 10 min by the addition of an identical volume of solution SA and the absorbance values at 450 nm were recorded. supernatants gave a positive signal in the presence of serum of naive or vaccinated animals and negative in the presence of serum of naturally infected animals were selected.The hybridomas thus retained are secretors of anti-NS1 monoclonal antibodies of BTV and able to discriminate naturally infected animals naive or vaccinated animals using commercial inactivated vaccine (DIVA character).

Example 5 Characterization of monoclonal antibodies 11G1 and 9B4

28 5.1 Isotype Determination The 11G1 and 9B4 antibodies were subjected to an ELISA isotyping test. The ascites fluid induced by each of the hybridomas was diluted 1/1000 in SL solution and 100 μL was deposited on a previously sensitized plate (SIGMA antibody kit, 1/1000 dilution.) After 90 min incubation at 37 ° C, the plate was washed and 100 μl of anti-mouse Ig antibodies were deposited (SIGMA) The plate was revealed using 100 μl of peroxidase substrate after an additional incubation of 30 min. at 37 ° C and washes.

The reaction was stopped after 10 min. by adding an identical volume of solution SA and the absorbance values at 450 nm were recorded. Serotype IgG1 gG2a gG2b gG3 IgM 0.536 0.364 1.798 0.194 0.008 0.354 1.915 0.215 0.043 0.050 0.121 0.206 Monoclonal Antibody 11G1 9B4 Table 1: Isotypic Determination of 11 GI and 9B4 Monoclonal Antibodies. The results obtained show that the monoclonal antibodies 11G1 and 9B4 are respectively of IgG2b and IgG1 isotype.

5.2 Competition 11G1 / 9B4 The native or recombinant NS1 protein of BTV-1 was plated in 96-well plates at predetermined PBS dilutions and then incubated at room temperature overnight. After washing to remove excess unbound antigen, saturation of the plates was performed. The ST saturation solution was then removed and 50 μL / well of unlabelled monoclonal antibody 9B4 or 11G1 was added at different concentrations and incubated overnight at +4 ° C, the plates washed three times at room temperature. SL solution aid and 50 μl / well of 11G1 monoclonal antibody coupled to peroxidase (11G1-HRP) were added and incubated for 30 min. at room temperature. After the last washings, 50 μl / well of SR solution was added and the reaction was stopped after 10 min. by the addition of an identical volume of solution SA.15 The results obtained with the native protein (Fig.1A) or recombinant NS1 (Fig. 1B) are similar. There is a drop followed by a signal extinction for the 11G1 / 11G1-HRP control torque at ratios 1/140 and 8/1, respectively. This characteristic profile of a competition effect between two antibodies confirms that the monoclonal antibodies 11G1 and 11G1 coupled to peroxidase recognize the same epitope on the NS1 protein. On the contrary, no competitive effect is observable between monoclonal antibodies 9B4 and 11G1-HRP. These results were confirmed in the reciprocal system (9B4 / 9B4-HRP and 11G1 / 9B4-HRP pair).

The monoclonal antibodies 9B4 and 11G1 therefore recognize distinct epitopes of the NS1 protein

5.3 Functionality of the 9B4 and 11G1 monoclonal antibodies in Western blot technique

Define whether monoclonal antibodies 9B4 and 11G1 recognize conformational or linear epitopes of NS1 protein.

In order to determine whether the NS1 epitopes recognized by the monoclonal antibodies 11G1 and 9B4 are of conformational or linear type, a western-blot experiment was conducted. Five microliters of native antigen preparation previously evaluated by ELISA were deposited on SDS-PAGE gel (NuPAGE® Bis-Tris 10%, Invitrogen) and then transferred to vinylidene polyfluoride membrane (Immobilon PSQ, Millipore). The corresponding migration tracks were then brought into contact with the antibody 9B4 or 11G1 (3 μg / ml in TBS-milk 3%) overnight at 4 ° C. The membranes were then incubated for one hour at room temperature in the presence of anti-mouse IgG antibody coupled to peroxidase (A4416 at 1 / 5000th, Sigma). After washing, the revelation was carried out by addition of 3,3 ', 5,5'-tetramethylbenzidine (TMB small particles, BioFX) and then stopped by rinsing with bidistilled water. Monoclonal antibody 10BI directed against a linear epitope of the C-terminal portion of NS1 of BTV-10 (P.Roy et al., J. Virol., 1995) was also used as a positive control.

The results obtained showed that the monoclonal antibody 1OB1 allowed the detection of the NS1 protein of BTV 1 at 62 kDa. However, no signal was observed with the use of monoclonal antibodies 11G1 and 9B4.

The epitopes of the NS1 protein recognized by the monoclonal antibodies 11G1 and 9B4 therefore appear to be of conformational type.

5.4 Reactivity of the 11G1 and 9B4 Monoclonal Antibodies on the EHDV The search for a possible cross-reaction of the monoclonal antibodies 11G1 and 9B4 with a protein of EHDV related to the NS1 protein of BTV-1 was conducted. A native antigen preparation of the EHDV was performed according to the protocol established for the BTV. Fifty microliters of solution diluted 1/100 in PBS were deposited in 96-well plates and incubated at room temperature overnight. After washing to remove excess unbound antigen, saturation of the plates was performed. The ST solution was then removed and 50 μl / well of peroxidase-coupled monoclonal antibody 9B4 or 11G1 was added and incubated for 1 h at room temperature, the plates washed three times with SL solution and 50 μl. It was added with SR solution and the reaction was stopped after 10 min. by adding an identical volume of SA solution. An anti-EHDV serum (Veterinary Diagnostic Technology Inc., USA) diluted 1: 100 in SL and revealed using a bovine anti-mouse IgG antibody coupled to peroxidase (IDVET) was used as positive control in this experiment.

OD 450nm 11G1 0.110 9B4 0.022 Anti-EHDV Serum 2.280 Table 1: Reactivity test of the anti-NS1 11 GI and 9B4 monoclonal antibodies on the EHDV virus.

These results showed that monoclonal antibodies 9B4 and 11G1 are very little or not reactive on native EHDV proteins.

5.5 Reactivity of 11G1 and 9B4 Monoclonal Antibodies to sera from EHDV35 Infected Animals A competition-type ELISA NS1 experiment was conducted with EHDV positive sera. Fifty microliters of native NS1 antigen preparation were diluted 1: 100 in PBS were plated in 96-well plates and incubated at room temperature overnight. After washing to remove excess non-fixed antigen saturation of the plates was performed using ST solution. The excess solution was then removed and 50 μl / well of sera from naive or infected animals BTV-1, -8 or EHDV diluted to A in SL solution were deposited. After overnight incubation at room temperature, plates were washed three times and 50 μl / well of peroxidase-coupled monoclonal antibody was added for 30 min. additional incubation at room temperature. 50 ul / well of SR solution was added after the last washings and the reaction was stopped after 10 min. by adding an identical volume of SA solution. Absorbance values at 450 nm were recorded. DO450nm INS (%) 11G1 9B4 11G1 9B4 0.095 0.147 7 11 0.254 0.129 18 9 1.100 0.155 79 11 1.323 0.299 95 21 1.773 0.704 127 49 1.753 1.106 126 77 Table 2: Evaluation of cross-reactivity of positive EHDV sera on the NS1 system -BTV / 11 GI or 9B4.

The results obtained show a reduction of the signal percentage at 7 and 11%, respectively with the antibodies 11G1 and 9B4, in the presence of a serum of an animal infected with BTV-1. Similarly, this signal is only 18 and 9% in the presence of a serum of an animal infected with BTV-8. The BTV serums are therefore in blockage in the NS1-BTV / 11G1 and NS1-BTV / 9B4 systems. Titration to 1 / 40th of a strong EHDV serum induced a 79 to 126% variation of the signal with the 11G1 antibody against 11 to 77% with 9B4 antibody. We can thus conclude that there is little or no cross-reaction for the recognition of the BTV NS1 protein between EHDV sera BTV-1 BTV-8 Pur Control EHDV 1/20 1/40 and the monoclonal antibody 11G1. On the contrary, this cross-reaction is important with the 9B4 antibody. The two monoclonal antibodies therefore have different behaviors with respect to cross reactions with EHDV sera. 5.6 DIVA potential difference between monoclonal antibodies 11G1 and 9B4

In order to obtain problematic serums due to a multiplication of vaccination procedures, 6 animals were vaccinated with a commercial vaccine (BTVPUR Alsap 8, Merial) according to a dose protocol at D0, D + 38. and 2 doses at D + 74, instead of a standard protocol with 2 simple injections. The sera were collected regularly and the detection of anti-VP7 and anti-NS1 antibodies was carried out by competitive ELISA method. Detection of anti-VP7 antibodies was performed using the IDVET BTC commercial kit. The detection of anti-NS1 antibodies was carried out by direct competition ELISA using antibodies 11G1 or 9B4 coupled to peroxidase and according to the protocol described below. Fifty microliters of serum diluted in SL solution were deposited per well on a plate presensitized with native NS1 protein. After incubation overnight at room temperature, the plates were washed three times and 50 μl / well of monoclonal antibody 11G1 or 9B4 coupled with peroxidase were added for 30 min. additional incubation at room temperature. 50 μL / well of SR solution was added after the last washings and the reaction was stopped after 10 min. by adding an identical volume of SA solution. Absorbance values at 450 nm were recorded. The values are represented as a percentage of the value obtained for the sample on the average of the negative controls (S / N).

All the results obtained show homogeneous responses between animals. The establishment of anti-VP7 seroconversion is observable as of the day after injection with a percentage of about 53% of the signal which reaches an average of 12.5% at 108 days. Anti-NS1 seroconversion only appears after the 2nd vaccination injection with a 40% and 44% decrease in the signal of monoclonal antibody 11G1 and 9B4, respectively, between D + 38 and D + 73. A differential is established between the two types of anti-NS1 monoclonal antibodies after the third vaccination injection. Indeed, the signal decreases from 51% to J + 74 at 16% at D + 108 for the antibody 1 1G1. Under these conditions, the DIVA potential limits of the monoclonal antibody 11G1 therefore seem to be reached. On the contrary, the signal of antibody 9B4 remains stable at around 48%. In view of these results, it appears that the monoclonal antibody 9B4 is more efficient in DIVA BTV than the monoclonal antibody 11G1. Example 6 Development of a test cELISA DIVA-BT NS1

6.1 Evaluation of the specificity and sensitivity of the cELISA DIVA-BT NS1 test: Discrimination of naive or BTV-infected animals 10 In order to determine the discriminating power of the cELISA DIVABT NS1 test between naive or BTV-infected animals, a panel Naïve (n = 71) or infected (BTV8 positive, n = 87) cattle sera were tested in competitive ELISA to compare their anti-VP7 and anti-NS1 seroconversions. Anti-VP7 antibody detection was performed using IDVET's ID Screen® Bluetongue Competition commercial kit according to the associated protocol. The detection of anti-NS1 antibodies was carried out as follows. Fifty microliters of anti-NS1 monoclonal antibodies (8 μg / ml in PBS) were deposited in 96-well plates and incubated at room temperature overnight. After washing to remove excess unbound antigen, saturation of the plates was performed using ST solution. The excess solution was then removed and 50 μL / well NS1 antigen preparation at a predefined concentration was deposited on the sensitized plates and incubated overnight at room temperature. The plates were washed three times and 50 μl / well of serum diluted in SL solution were deposited. After overnight incubation at room temperature, the plates were washed three times and 50 μl / well of peroxidase-coupled monoclonal antibody 9B4 was added for 30 min. additional incubation at room temperature. 100 μL / well of SR solution was added after the last washings and the reaction was stopped after 10 min. by adding an identical volume of SA solution. Absorbance values at 450 nm were recorded. The values are represented as a percentage of the value obtained for the sample on the average of the negative controls (S / N).

The results obtained show that all the sera of naive animals have a signal percentage between 70 and 110% for the standard BT5 test and 60 to 110% for the cELISA DIVA-BT NS1 test. The specificity is therefore substantially similar between these two tests.

Sera from BTV-infected animals show 5-10% blockage with the use of the standard BT test. These sera have a distribution between 5 and 40% of the signal with the use of the cELISA DIVA-BT NS1 test. There is therefore a seroconversion anti-VP7 but also anti-NS 1 in animals naturally infected with BTV. . Although less efficient than the classical sensitivity test, the cELISA DIVA-BT NS 1 test shows a very good discrimination between the sera of naive animals and those of naturally infected animals.

6.2 Evaluation of the DIVA potential of the cELISA DIVA-BT NS 1 test: Discrimination of animals vaccinated or infected by BTV A flock of 30 cattle and 75 sheep was vaccinated with a commercial vaccine according to the protocol defined by the maker. The sera were taken on the day of the first vaccine dose and on day 64 or day for cattle and sheep, respectively. These 2 types of samples were tested in competition ELISA in order to compare their anti-VP7 and anti-NS 1 seroconversions. The detection of anti-VP7 antibodies was carried out using the commercial kit ID Screen® Bluetongue Competition d IDVET according to the associated protocol. The detection of anti-NS1 antibodies was carried out as previously described. The values are represented as a percentage of the value obtained for the sample on the average of the negative controls (S / N).

The results obtained using the classical cELISA test of BT show that the distribution of cattle serum passes values between 90 and 130% of the signal before vaccination, with a maximum between 100 and 110%, at values between 0 and 40% of the signal after vaccination, with a maximum of 5-10%. Regarding sheep sera, they range between 60 and 130% of the signal before vaccination and between 10 and 90% after vaccination with maximums between 100-110% and 30-40%, respectively. One animal that has not seroconverted. As expected, there is therefore globally the acquisition of an anti-VP7 status of BTV seropositive following vaccination against BTV-8.

On the contrary, the use of the BTV DIVA-NS 1 test described in this document demonstrates that these animals have maintained an anti-NS 1 seronegative status. In fact, the bovine serums before vaccination have a distribution between 70 and 110% of the signal, with a maximum at 90-110%, against a distribution between 70 and 120% of the signal, with a maximum at 100-110%, after vaccination. The sheep serums retain a distribution between 60 and more than 130% of the signal before vaccination, with a maximum that goes from values greater than 130% to values of 90-120%.

All these results show an infectious and non-vaccinal seroconversion against the NS1 protein of BTV. Although the cELISA DIVABT NS 1 test does not discriminate between vaccinated animals and naive animals (distribution between 70 and 120% of the signal in both cases), it allows the animals vaccinated with an inactivated vaccine to be distinguished from naturally infected animals. (5 to 40% of the signal).

Claims (29)

REVENDICATIONS1. Use of an antibody, or functional fragment thereof, against the native or recombinant NS1 protein of the blue tongue virus (referred to as "BTV" for Blue Tongue Virus), for the preparation of a reagent or a kit for its in vitro use and intended to differentiate an animal vaccinated against a BTV virus from an animal infected with a BTV virus, characterized in that said antibody is a monoclonal antibody obtained from a culture supernatant of a hybridoma, said hybridoma having been obtained by fusion between a myeloma cell and an animal mammalian lymphoid cell, said mammal having been previously immunized with an immunogenic composition or compound comprising a native or recombinant NS1 protein of BTV, or one of its antigenic fragments, and characterized in that said antibody is selected by a process comprising the following steps: a) bringing said test antibodies into contact with each other; obtained from culture supernatant of said hybridomas with a native or recombinant NS1 protein of BTV; and b) - i) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a native or recombinant NS1 protein of epizootic haemorrhagic disease virus ("EHDV" for epizootic hemorrhagic disease virus); and c) selecting said tested antibody if the latter: - i) specifically recognizes said BTV NS1 protein, or its antigenic fragment, and - ii) does not recognize said EHDV NS1 protein. 25 2. Use of an antibody, or a functional fragment thereof, according to claim 1, characterized in that said antibody is selected by a process further comprising in step b): b) - ii) setting in contact with said test antibodies thus obtained from culture supernatant of said hybridomas with a serum, or plasma, of an animal infected with EHDV, said serum or plasma comprising polyclonal antibodies directed against the EHDV, and in that said antibody is selected if furthermore in step c): c) - iii) there is a cross-reaction for the recognition of the BTV NS1 protein 1 between said test antibody and said serum, or plasma, of an infected animal by the EHDV. 3. Use of an antibody, or a functional fragment thereof, according to claim 1 or 2, characterized in that said antibody is selected by a method further comprising in step b): b) - iii ) bringing said test antibodies thus obtained from culture supernatant of said hybridomas into contact with a serum, or plasma, of an animal infected with or vaccinated with BTV, said serum or plasma comprising polyclonal antibodies directed against BTV, and in that said antibody is selected if further in step c): c) - iiii) a) There is observed competition and resulting in a significant decrease in the signal obtained for the tested sample compared to the signal obtained for a negative control is significant of the presence of a sample from an animal infected with BTV, preferably a signal representing 0 to 20% of the signal obtained for said negative control; and (3) There is no or little competition observed for the tested sample compared to the signal obtained for a negative control is significant for the presence of a sample emanating from an animal vaccinated against BTV, preferably a signal representing more than 50% of the signal obtained for said negative control. 30 4. Use according to one of claims 1 to 3, characterized in that said NS1 protein of the BTV virus, or composition or compound comprising it, or one of its antigenic fragments is chosen from: a) an isolated native NS1 protein and, where appropriate purified, or recombinant BTV virus, preferably preferably amino acid sequence SEQ ID NO: 1 (NS1 BTV-1) or SEQ ID NO: 2 (NS1 BTV-8), or sequence having at least 85% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or b) a BTV virus, or a fragment thereof containing the NS1 protein, or one of its antigenic fragments, as defined in a). 5. Use according to one of claims 1 to 4, characterized in that said EHDV is a serotype 1 or 2 EHDV, preferably of serotype 2 and in that said NS1 protein of the EHDV virus is serotype NS1 EHDV protein 1 or 2 of respective sequence SEQ ID NO: 3 and SEQ ID NO: 4 or sequence having at least 85% identity with SEQ ID NO: 3 or SEQ ID NO: 4. 6. Use according to claim 1 to 5, characterized in that said antibody is the antibody secreted by the hybridoma called 9B4 Id Vet deposited at the CNCM July 7, 2009 under the number I-4198. 7. Use according to one of claims 1 to 6, characterized in that the functional fragment of said antibody is selected from Fab, (Fab ') z, Fab', Fv, scFv, scFv-Fc fragments of said antibody, these fragments having the functional characteristics of the antibody from which they are derived and as defined in step c). 8. Use according to any one of claims 1 to 7, characterized in that said antibody or antibody fragment is coupled to a marker. 30 9. Use according to one of claims 1 to 8, characterized in that said reagent or kit further comprises a NS1 protein of BTV virus, or composition or compound comprising it, or an antigenic fragment thereof. 10. Use according to claim 9, characterized in that said NS1 protein of the BTV virus, or composition or compound comprising it, or an antigenic fragment thereof is as defined in claim 3 a) and 3 b). and characterized in that: said antigenic fragment of the NS1 protein is a fragment capable of binding specifically to the antibody secreted by the hybridoma called 9B4 Id Vet deposited at the CNCM on July 7, 2009 under the number I-4198. 11. Use according to claim 9 or 10, characterized in that said NS1 protein BTV or one of its antigenic fragments, or the composition or the compound comprising it, is fixed or adsorbed on a solid support, preferably on a microplate cup. 12. Use according to one of claims 1 to 11, characterized in that said animal is a ruminant, preferably selected from cattle, sheep or goats. 13. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus from a biological sample of said animal, characterized in that it comprises the following steps: a) the implementation of contacting said biological sample with the NS1 protein of a BTV virus, or an antigenic fragment of said NS1 protein, or a composition or compound comprising such a protein or fragment, and b) revealing the antigen-antibody complexes formed in (a) between said NS1 protein, or one of its antigenic fragments, and an anti-NS1 antibody likely to be present in said sample, this revelation being carried out by a competitive immunoassay method using a anti-NS1 antibody, or a functional fragment thereof, capable of specifically recognizing the NS1 protein of BTV virus, or one of its antigenic fragments, said anti-NS-1 antibody being a monoclonal antibody obtained from a culture supernatant of a hybridoma, said hybridoma having been obtained by fusion between a myeloma cell and an animal mammalian lymphoid cell, said mammalian having been previously immunized with an immunogenic composition or compound comprising a protein NS1 of BTV, native or recombinant, or one of its antigenic fragments, and characterized in that said anti-NS 1 antibody is selected by a method comprising the following steps: a) contacting said test antibodies as well as obtained from culture supernatant of said hybridomas with a native or recombinant NS1 protein of BTV; and b) - i) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with native or recombinant NS1 protein of epizootic haemorrhagic disease virus; and c) selecting said tested antibody if the latter: i) specifically recognizes said BTV NS1 protein, or antigenic fragment thereof, and ii) does not recognize said EHDV NS1 protein. 14. In vitro method for differentiating an animal vaccinated against BTV virus from an animal infected with a BTV virus according to claim 13, characterized in that said test antibody is selected by a process further comprising in step 20 b) b) - ii) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a serum, or plasma, of an animal infected with EHDV, said serum or plasma comprising polyclonal antibodies directed against the EHDV, and in that said antibody is selected if furthermore in step c): c) - iii) there is a cross-reaction for the recognition of the BTV NS1 protein between said test antibody and said serum, or plasma from an animal infected with EHDV. 30 15. In vitro method for differentiating an animal vaccinated against BTV virus from an animal infected with a BTV virus according to claim 13 or 14, characterized in that said antibody is selected by a process further comprising in step b) b) - iii) contacting said test antibodies thus obtained from culture supernatant of said hybridomas with a serum, or plasma, of an animal infected with BTV or vaccinated against it, said serum or plasma comprising polyclonal antibodies directed against BTV, and in that said antibody is selected if furthermore in step c): c) - iiii) a) There is an observed competition and resulting in a significant decrease in the signal obtained for the tested sample compared to the signal obtained for a negative control is significant of the presence of a sample emanating from an animal infected with BTV, preferably a signal representing 0 to 20%, more preferably 0 to 15% d. u signal obtained for said negative control; and (3) There is no or little competition observed for the tested sample compared to the signal obtained for a negative control is significant for the presence of a sample emanating from an animal vaccinated against BTV, preferably a signal representing more than 50%, more preferably more than 60% of the signal obtained for said negative control. 16. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus according to one of claims 13 to 15, characterized in that: - either said NS1 protein of the BTV virus, preferably BTV -1 or BTV-8, or one of its antigenic fragments, implemented in step a), or - either said antibody capable of specifically recognizing the NS1 protein of BTV virus, preferably BTV-1 or BTV -8-8, carried out in step b), is labeled, preferably with peroxidase. 17. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus according to one of claims 13 to 16, characterized in that said antibody used in step b) in the immunoassay method in competition is a labeled antibody, preferably the immunoassay method in competition is a competitive ELISA method, preferably said antibody is labeled with peroxidase 18. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus according to one of claims 13 to 17, characterized in that said antibody used in step b) in the immunoassay method in competition is the antibody secreted by the hybridoma called 9B4 Id Vet deposited at the CNCM July 7, 2009 under the number I-4198. 19. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus according to claim 18, characterized in that it comprises the following steps: a) bringing said biological sample into contact with the protein NS1 of a BTV virus, or an antigenic fragment of said NS1 protein, or of a composition or compound comprising such a protein or fragment, said NS1 protein being previously captured by immunocapture on a solid phase coated with an anti-antibody -NS1-BTV polyclonal type, or, monoclonal type and as defined in claim 1; b) revealing the antigen-antibody complexes formed in (a) between said NS1 protein, or one of its antigenic fragments, and an anti-NS1 antibody likely to be present in said sample, this revelation being carried out by a method competitive immunoassay method employing an anti-NS1 antibody, or one of its functional fragments, preferably labeled, as defined in claim 2. 20. In vitro method for differentiating an animal vaccinated against BTV virus from an animal infected with a BTV virus according to claim 19, characterized in that: - in step a) the anti-NS1 antibody coated on the solid phase is an antibody as defined in claim 1; and in step b) the anti-NS1 antibody, preferably labeled, as defined in claim 2, is the antibody secreted by the hybridoma called 9B4 Id V and deposited at the CNCM on July 7, 2009 under the number I-4198. 21. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus according to one of claims 13 to 20, characterized in that said NS1 protein BTV virus implemented at step a), or a composition or compound comprising it, or an antigenic fragment thereof, is chosen from: a) an isolated, and, where appropriate, purified or recombinant, BTV virus native NS1 protein, preferably preferably amino acid SEQ ID NO: 1 (NS1 of BTV-1) or SEQ ID NO: 2 (NS1 of BTV-8), or of sequence having at least 85% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or b) a BTV virus, preferably BTV-1 or BTV-8, or a fragment thereof containing the NS1 protein, or an antigenic fragment thereof. 22. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus according to one of claims 13 to 21, characterized in that said biological sample is a fluid sample. biological, preferably selected from plasma, serum, meat juice or a whole blood sample of the animal, the serum being most preferred. 23. In vitro method for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus according to one of claims 13 to 22, characterized in that in step b), a competition reaction observed and resulting in a significant decrease in the signal obtained for the tested sample compared to the signal obtained for a negative control is significant of the presence of a sample emanating from an animal infected with BTV, preferably a signal representing 0 to 20% , more preferably 0 to 15% of the signal obtained for said negative control. 25 24. Kit or kit of reagents for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus, said kit comprising: a) a compound or composition comprising the NS1 protein of the BTV virus, or an antigenic fragment of said NS1 protein; B) an antibody, or a functional fragment thereof, directed against the NS1 protein of the BTV virus, or against an antigenic fragment of said NS1 protein, where appropriate labeled, preferably said antibody is polyclonal, monoclonal or chimeric; and, where appropriate, c) the reagents necessary for the reaction. 25. Kit or kit of reagents according to claim 24, characterized in that: A) said compound or said composition comprising the NS1 protein of the BTV-8 virus, or said antigenic fragment of said NS1, is chosen from: a) a protein Isolated and, where appropriate, purified or recombinant BTV virus native NS1, preferably preferably amino acid sequence SEQ ID NO: 1 (BTV-1 NS1) or SEQ ID NO: 2 (BTV-8 NS1) ), or of sequence having at least 85% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or b) a BTV virus, preferably BTV-1 or BTV-8, or a fragment thereof containing the NS1 protein, or an antigenic fragment thereof as defined above in claim 10, and B) said antibody, or one of its functional fragments, directed against the NS1 protein of the BTV virus, or against an antigenic fragment of said NS1 protein, where appropriate labeled, is chosen from the antibody secreted by the hybridoma called 9B4 Id Vet filed at the CNCM on July 7, 2009 under number I-4198. 26. Kit or reagent kit according to claim 25, characterized in that it comprises: a reagent A) comprising a first polyclonal anti-NS1-BTV capture antibody, or the antibody as defined in US Pat. claim 1, optionally coated on a microplate cup; a reagent B) comprising a native NS1 protein and, where appropriate purified, or recombinant virus BTV, or a BTV virus, preferably BTV-1 or BTV-8 whole, where appropriate, the reagent B) having previously fixed on said cup of reagent A); and a reagent C) comprising the antibody secreted by the hybridoma designated 9B4Id V and deposited at the CNCM on 7 July 2009 under the number I-4198, where appropriate labeled. 27. A method of selecting an antibody for differentiating an animal vaccinated against the BTV virus from an animal infected with a BTV virus, where appropriate for use in a competitive immunoassay method, characterized in that it comprises the next step: a), where appropriate the selection of a hybridoma obtained after immunization of an ammal with an antigen selected from: - an isolated native NS1 protein and, where appropriate purified, or recombinant BTV virus, preferably of amino acid sequence preference SEQ ID NO: 1 (NS1 of BTV-1) or SEQ ID NO: 2 (NS1 of BTV-8), or of sequence having at least 85% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or one of its antigenic fragments; or a BTV virus, preferably BTV-1 or BTV-8, or one of its fragments containing the NS1 protein, b) bringing said test antibody into contact with the NS1 protein of the BTV virus as defined in FIG. ), or an antigenic fragment of said NS1 protein, or a composition or compound comprising such a protein or fragment, and c) revealing the antigen-antibody complexes formed in (b) between said NS1 protein, or one of its antigenic fragments, and said one antibody tested, and in which step the revelation of the antigen-antibody complexes formed in (a) is carried out by a competitive immunoassay method using an antibody, or one of the fragments functional, said an antibody being the antibody secreted by the hybridoma called 9B4Id Vet deposited at the CNCM on July 7, 2009 under the number I-4198, or one of its functional fragments, where appropriate labeled. 28. Hybridoma called 9B4Id Vet deposited at the CNCM on July 7, 2009 under number I-4198, or one of its functional fragments, if appropriate labeled. 29. The antibody designated 9B4 Id Vet secreted by the hybridoma according to claim 28, or one of its functional fragments, if appropriate labeled.