FR2966461A1 - Use of VP2 capsid protein of Bluetongue virus of serotype 8, a composition or compound having the protein, for the preparation of a reagent or a kit for in vitro diagnosis of an infection by BTV virus in an animal e.g. bovine - Google Patents

Abstract

Use of VP2 capsid protein of Bluetongue virus of serotype 8 (BTV-8 virus), or its an antigenic fragment, a composition or compound comprising the protein, for the preparation of a reagent or a kit for in vitro diagnosis of an infection by BTV virus in an animal, is claimed. Use of VP2 capsid protein of Bluetongue virus of serotype 8 (BTV-8 virus), or its an antigenic fragment, a composition or compound comprising the protein, for the preparation of a reagent or a kit for in vitro diagnosis of an infection by BTV virus in an animal, where the antigenic fragment of the VP2 protein is: a fragment capable of specifically binding on monoclonal antibody secreted by the hybridoma referred to as 6C3 Id Vet deposited in the National Collection of Microorganism Cultures (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the 1-4314 number, or on monoclonal antibody secreted by the hybridoma referred to as 10E9 Id Vet deposited at CNCM on June 21, 2010 under number 1-4315; and monoclonal antibody secreted by the hybridoma referred as 6C3 Id Vet deposited under the number I-4314, or antibody secreted by the hybridoma 10E9 Id Vet deposited under number 1-4315 is specifically directed against the antigenic fragment of VP2 protein, is claimed. Independent claims are included for: (1) an in vitro method of detection or diagnosis of an infection of an animal by virus BTV from a biological sample from the animal, comprising (a) contacting the biological sample with the VP2 capsid protein of the BTV-8 virus, or its fragment of the protein VP2, the compound or the composition comprising the protein; and (b) revealing the antigen-antibody complex formed in the step (a) between the VP2 protein, or one of its fragments, and anti-VP2 antibody to be present in the sample; (2) a kit or reagent kit for the detection or diagnosis of infection of an animal by BTV virus, comprising: the compound or the composition comprising the VP2 capsid protein of the BTV-8 virus or its fragment of the protein VP2; and an antibody, or one of its functional fragments, directed against the protein of VP2 capsid of BTV-8 or against an antigenic fragment of the VP2 protein, where the antibody is polyclonal, monoclonal or chimeric; (3) a process of selecting an antibody capable of revealing the presence of anti-VP2 antibody used in a method of competitive ELISA for the detection or diagnosis of an infection of the animal by BTV virus, comprising contacting the antibody to be tested with VP2 capsid protein of BTV-8 virus, or its fragment of the protein VP2, the compound or the composition comprising the protein or its fragment, and revealing the antigen-antibody complex formed in (a) between the protein VP2 and the tested antibody, and in which the revealing step of antigen-antibody complexes formed in (a) is carried out by a competitive ELISA method implementing an antibody, or one of its functional fragments; (4) a hybridoma named 6C3 Id Vet deposited at CNCM, 21 June 2010 under the number 1-4314 or called 10E9 Id Vet deposited at the CNCM on June 21. 2010 under the number 1-4315; and (5) a monoclonal antibody secreted by the hybridoma or one of its functional fragments optionally marked.

Description

Bluetongue (BTV) or Blue Tongue Disease (BT for "Blue Tongue") is an infectious but non-contagious viral disease transmitted by some species of biting midges (genus Culicoides). It is caused by a virus belonging to the genus Orbivirus (Reoviridae) and commonly called "Bluetongue virus" or BTV. 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% [1, 2]. 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. Present throughout the world on a broad band covering North and South America, Africa, South Asia and North Australia, the disease has been tracing for decades in southern Europe remained until now unharmed. 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 affecting sheep and cattle herds ever recorded in Europe. 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. Strictly animal disease, BCF does not affect humans and has no impact on the quality of food but causes significant economic losses for farmers and the community [3]. Directly, CBF causes a reduction in production by triggering abortion in infected females or stunting or even death of animals. These economic consequences are all the more important in the case of the recent epizootic that serotype 8 of the disease is highly pathogenic for European cattle and sheep breeds, especially for cattle. Thus, on 1 July 2008 in France, the Directorate-General for Food (DGLA) assessed mortalities for 65000 cattle and 32000 small ruminants with proportions of up to 30% of a farm.

But it is indirectly that the economic impact of the FCO is the most important with the implementation of embargoes and a restriction of the export of cattle as well as the associated control measures. As an example, the price of the "gossip", which usually represents an exchange volume of 900 million euros per year, fell by 10% in 2007. Similarly, France's expenditure on the compensation of the breeders and the surveillance of the FCO in France amounted to more than 35 million euros in 2008 [4]. Therefore, a vaccination strategy has been put in place by the member countries of the European Union and Switzerland with the aim of reducing the losses in the breeding of domestic animals, to contain any propagation of the disease and to facilitate the market of the livestock of rent. In the crisis situation of the time, vaccines based on attenuation of the live virus, also called attenuated vaccines or MLV vaccines (Modified Live Virus) were imported and used against serotype 2, 4, 9 and 16 BTV. in southern Europe (Corsica, Portugal, Balearic Islands and Italy). However, the risks associated with the use of these last remain important (symptoms of infection, reversion of virulence, etc ...) and a preferable alternative is the use of vaccines based on a total inactivation of the virus, so-called inactivated vaccines [5]. Currently, various inactivated anti-BTV8 vaccines are available in Europe (BLUEVAC® 8, CZ Veterinaria SA, Zulvac® 8, Fort Dodge Animal Health and BTVPUR® AlSap 8, Merial) and others are under development in India and Australia. 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. The detection of BTV genomic 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 mainly used for the detection of anti-BTV group-specific antibodies [6]. It 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 antibodies can be detected in ruminants as early as 7 days after infection and 10 to 40 days after vaccination depending on the animals tested and the vaccines used.

Unfortunately, the VP7 protein being immunologically common between the 24 serotypes, the detection of antibodies directed against this protein does not make it possible to determine the serotype of the BTV virus at the origin of the infection. However, different serotypes can circulate concomitantly in the same regions of the world. The only way to be sure of the health status of an animal and if necessary to determine the serotype of the BTV at the origin of the infection is then to perform a serum neutralization test or molecular biology typing, expensive and long to implement. New serological tests, which are more economical and easy to implement, are therefore necessary for epidemiological studies and the coordination of future vaccination campaigns.

This is precisely the object of the present invention. In order to meet this need, the present Applicant has sought to construct a serological test specific for serotype 8 of the bluetongue virus (BTV), specific in particular with respect to infections with BTV serotype 1 or other serotypes such as such as 2, 4, 9, 10 or 16, and having DIVA properties.

The Applicant has surprisingly demonstrated that the detection of antibodies directed against the VP2 capsid protein of BTV-8 alone made it possible to identify in a BTV infected animal the specific presence of serotype 8, but also to differentiate a infected animal from a vaccinated animal. This VP2 protein is encoded by segment 2 of the viral genome (Seg-2) and forms triskelion-type trimer (triple interwoven spiral). By its association with vimentin, the VP2 protein is involved in intracellular trafficking and the secretion of mature viral particles [7]. It has also been shown that its interaction with a membrane glycoprotein plays an essential role in the cellular attachment phase [8]. At the diagnostic level, the VP2 protein in combination with VP5 is mainly known as a major serotype determinant of the FCO virus [9]. Indeed, it is the target of neutralizing antibodies specific serotype of bluetongue virus. On this basis, a classification was established allowing the discrimination of 24 reference strains (BTV-1 to -24). This serotypic classification has proved to be in perfect adequacy with the phylogenetic classification based on the analysis of the variations of the Seg-2 sequence coding for the VP2 protein [10].

The present Applicant has generated monoclonal antibodies directed against the VP2 protein alone of BTV-8. The epitopes recognized by these monoclonal antibodies have the following characteristics: - to be specific for serotype 8 of BTV, especially with respect to BTV infections of serotype 1 and, where appropriate, serotypes 2, 4, 9, 10 or 16 and, where appropriate, other known BTV serotypes; - not to be involved in the cellular attachment phase of the virus (non-neutralizing monoclonal antibodies); to be recognized by the antibodies of animals naturally infected with BTV-8; - Not to be recognized by the antibodies of animals vaccinated with a mono- or polyvalent inactivated vaccine containing BTV-8.

In a context of mass vaccination of European flocks, a serological test based on this VP2 protein and using the associated monoclonal antibodies constitutes a major advance in the diagnosis of FCO. It will differentiate the two main serotypes (BTV-1 and BTV-8) that affect France, Spain and Portugal (and more weakly Greece and Italy) and assess the spread of the virus under vaccine cover. by the quick method to implement and adapted to a large number of samples. The applicant has demonstrated that it is possible to use BTV-8 VP2 capsid protein, or an antigenic fragment of said VP2 protein, for the preparation, selection and production of monoclonal antibodies, as well as that associated tests, making it possible to establish a specific serological diagnosis of BTV-8, in particular vis-à-vis infections with BTV serotype 1 and, where appropriate with respect to other known BTV serotypes such as in particular the serotype 2, 4, 9, 10 or 16, these antibodies having the further property of being able to differentiate between naturally infected animals and animals vaccinated against BTV-8. This test is suitable for the analysis of biological samples of animals whose health status with respect to this infection is unknown.

In the detailed description below, the terms used are defined as follows: a) The term "animal" from which the biological sample originates is defined as a ruminant, preferably selected from cattle, sheep or goats. b) The term "biological sample" is a biological fluid sample containing polyclonal antibodies, preferably selected from milk, whey, plasma, serum, meat juice or whole blood sample of the animal. , the serum being the most preferred. c) The term "naive" refers to the absence of exposure of an animal to the disease, in this case BCF, giving it a healthy health status with respect to this disease. d) The term "infection" refers to the exposure of the animal to the disease, ie, BAF, regardless of the presence of clinical signs in this animal. An infection is described as "natural" if this exposure was made by one of the natural transmission modes of the disease, for example a transmission by culicoides. The term infection does not include exposure to FCO through vaccine treatment. E) The term "vaccinated" refers to the administration of a vaccine composition preventing or reducing the effects of infection caused by a pathogen by enabling the establishment or improvement of immunity to of this pathogen. The vaccine composition comprising inactivated pathogens, classifies inactivated vaccines, and its administration is performed, unless otherwise indicated, in accordance with the manufacturer's recommendations. f) The term "negative control" refers to a set of biological samples from naïve animals.

The present invention relates to the use of the BTV-8 VP2 capsid protein, or an antigenic fragment of said VP2 protein, or a composition or compound comprising such a protein or fragment, for the preparation of a reagent or a kit for the in vitro diagnosis of BTV infection in animals. According to other features and preferably: said reagent is further intended to differentiate a BTV-8 infection from an infection with BTV-1 in said animal; said reagent is intended to differentiate a BTV-8 infection from a serotype BTV infection selected from serotypes 1, 2, 4, 9, 10 or 16; said reagent is intended to differentiate a BTV-8 infection from a BTV infection of another serotype, preferably chosen from known serotypes 1 to 7 or 9 to 24.

In a preferred embodiment, said VP2 capsid protein of BTV-8, or a composition or compound comprising it, is chosen from: an isolated and, where appropriate purified VP2 protein, BTV-8, or one of its antigenic fragments; a recombinant VP2 protein, preferably of amino acid sequence SEQ ID NO: 1 or one of its antigenic fragments; or a BTV-8 virus, or one of its fragments containing the VP2 protein or one of its antigenic fragments, characterized in that: said antigenic fragment of the VP2 protein is a fragment capable of binding specifically on the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited at the National Collection of Cultures of Microorganisms (CNCM), Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number I- 4314 or on the antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I-4315; or the monoclonal antibody secreted by the hybridoma designated 6C3 Id Vet deposited under the number I-4314 or the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number I-4315 is specifically directed against said fragment antigenic protein VP2.

In another equally preferred embodiment, said reagent or kit comprises, preferably separately from said reagent containing said VP2 protein, an antibody, or a functional fragment thereof, directed against the VP2 capsid protein of BTV-8, or against an antigenic fragment of said VP2 protein, for the preparation of a reagent for the in vitro diagnosis of an infection with BTV-8 in animals. said antibody or antibody fragment is chosen from monoclonal antibodies, polyclonal antibodies, chimeric antibodies, or their functional fragments, in particular the Fab, (Fab ') z, Fab', Fv, scFv, scFv-Fc fragments previous antibodies. The monoclonal antibody fragments according to the invention comprise any fragment of said monoclonal antibody capable of binding to an epitope of the VP2 protein of BTV-8 and on which the monoclonal antibody from 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. Alternatively, 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 hybridoma preparation technique 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 the VP2 protein of BTV-8, or a fragment thereof, comprising the epitope specifically recognized by said monoclonal antibodies called 6C3 Id. Vet and 10E9 Id Vet according to the invention.

Said VP2 protein of BTV-8, 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 encoding the VP2 protein of BTV-8 or by peptide synthesis from an amino acid sequence included in the peptide sequence of the VP2 protein of BTV-8, preferably the BTV-8 VP2 protein of sequence SEQ ID NO: 1 The monoclonal antibodies that can be used according to the invention may, for example, be purified on an affinity column on which the BTV-8 VP2 protein has been previously immobilized or one of its fragments containing the epitope specifically recognized by said monoclonal antibodies called 6C3. Id Vet and 10E9 Id Vet according to the invention. The following use or methods according to the present invention may employ an anti-VP2-BTV-8 antibody or a fragment thereof, characterized in that it is chosen from chimeric antibodies having the same CDRs (" CDR "for Regions Determining the Complementarity That the Anti-VP2-BTV-8 Monoclonal Antibody As Defined Above The monoclonal antibodies or their chimeric-type fragments according to the invention can be made using genetic recombination techniques. For example, the chimeric monoclonal antibody can be made by cloning a recombinant DNA comprising a promoter and a coding sequence for the variable region of a monoclonal antibody according to the invention and a sequence coding for the constant region of human antibody. 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 the uses or methods of detection or diagnosis in which the monoclonal antibodies or their fragments, or the VP2-BTV-8 proteins or their epitopes / antigenic fragments 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 VP2-BTV-8 proteins or epitopes / antigenic fragments thereof are labeled. The monoclonal antibodies or their fragments or the VP2-BTV-8 proteins or their epitopes / antigenic fragments may also be labeled in order to obtain a detectable and / or quantifiable signal. The monoclonal antibodies or their fragments, or the VP2-BTV-8 proteins or their epitopes / antigenic fragments, include, for example, so-called immunoconjugated antibodies or antigens which can be conjugated for example with enzymes such as peroxidase or alkaline phosphatase. PD-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose-6 phosphate dehydrogenase or by a molecule such as biotin, digoxigenin or 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, rhodamine and its derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone 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 the 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 having fluorescein-type markers can 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 the VP2 antigen or a fragment thereof, it is also possible to mention the radioactive labels such as 14C "Cl," Co, "Co," Cr, 1 "Eu" Fe 13H 1 "I 121I, 32P or" s which can be detected by known means such as gamma or scintillation counter etc. In a particularly preferred embodiment, the anti-VP2-BTV-8 monoclonal antibodies used according to the present invention. invention are chosen from: - the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited at the National Collection of Cultures of Microorganisms (CNCM), Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number I-4314 or by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I-4315 or - said monoclonal antibody 6C3 Id Vet or 10E9 Id Vet, or one of its functional fragments, coupled to a marker.

More preferably, the reagent or kit is for the in vitro diagnosis of BTV virus infection in a ruminant, preferably a bovine, ovine or goat. The present invention also relates to an in vitro method for detecting or diagnosing an infection of an animal with a BTV virus from a biological sample of said animal, characterized in that it comprises the following steps: a) contacting said biological sample with the BTV-8 virus VP2 capsid protein, or an antigenic fragment of said VP2 protein, or a composition or compound comprising such a protein or fragment, and b) implementing evidence or revealing antigen-antibody complexes formed or likely to be formed in (a) between said VP2 protein, or one of its antigenic fragments, and an anti-VP2 antibody likely to be present in said biological sample of the animal.

According to other characteristics and preferably - in step b), the revelation of the antigen-antibody complexes formed in (a) is carried out by a competitive immunoassay method, preferably by a competing ELISA using A monoclonal antibody, or one of the functional fragments, capable of specifically recognizing the VP2 protein of BTV-8, or one of its antigenic fragments, is used. said monoclonal antibody used in step b) in the competitive immunoassay method, preferably by a competing ELISA, is a monoclonal antibody labeled, preferably with peroxidase - even more preferably, said monoclonal antibody implemented in step b) in the competitive immunoassay method, preferably by a competitive ELISA is the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number I-4314 or by the hybridoma called 10E9 Id Vet filed at the CNCM June 21, 2010 under the number I-4315. According to a preferred embodiment, the subject of the invention is an in vitro method of detection or diagnosis according to the present invention, characterized in that it comprises the following steps: a) bringing said biological sample into contact with the VP2 protein of a BTV-8 virus, or an antigenic fragment of said VP2 protein, or a composition or compound comprising such a protein or fragment, said VP2 protein being previously captured by immunocapture on a solid phase coated with an anti-antibody -VP2-BTV-8 polyclonal type, or, monoclonal type and as defined in the present invention; b) revealing the antigen-antibody complexes formed in (a) between said VP2 protein, or one of its antigenic fragments, and an anti-VP2 antibody likely to be present in said sample, this revelation being carried out by a method immunoassay in competition using an anti-VP2 antibody, or one of its functional fragments, preferably labeled, as defined in the present invention. More preferably, said method is intended to differentiate a BTV-8 infection from a serotype 1 BTV infection, preferably to differentiate a BTV-8 infection from a serotype BTV infection. selected from serotypes 1, 2, 4, 9, 10 or 16 or for differentiating a BTV-8 infection from a serotype BTV infection selected from serotypes 1 to 7 or 9 to 24.

Any conventional procedure or test may be implemented to perform such detection and / or assay. Said test 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 competitive immunoassay methods used in the methods according to the invention, the VP2 capsid protein of BTV-8, or an antigenic fragment of said VP2 protein, or of a composition or compound comprising such a protein or such a fragment can be immobilized or labeled, and so is the anti-VP2-BTV-8 antibody. 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, an anti-VP2-BTV-8 monoclonal antibody microplate well as described above, unlabeled, is coated. Then, the sample to be tested and the previously labeled VP2-BTV-8 antigen are successively added to the well. 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.

The present invention also relates to a kit or kit of reagents for detecting or diagnosing an infection of an animal with a BTV virus, said kit comprising: a) a compound or composition comprising the BTV virus VP2 capsid protein -8, or an antigenic fragment of said VP2 protein; b) an antibody, or one of its functional fragments, directed against the VP2 capsid protein of BTV-8, or against an antigenic fragment of said VP2 protein, where appropriate labeled, preferably said antibody is polyclonal, monoclonal or chimerical. c) where appropriate, the reagents for the constitution of the environment conducive to the immunological reaction; and d) reagents for detecting antigen-antibody complexes produced by the immunological reaction.

Preferably, in the kit or the kit of reagents according to the invention: a) said compound or said composition comprising the VP2 capsid protein of BTV-8, or said antigenic fragment of said VP2 protein, is chosen from: a protein VP2 isolated and, if appropriate purified, BTV-8, or one of its antigenic fragments; a recombinant VP2 protein, preferably of amino acid sequence SEQ ID NO: 1 or one of its antigenic fragments; or a BTV-8 virus, preferably inactivated, or one of its fragments containing the VP2 protein or one of its antigenic fragments, and characterized in that: said antigenic fragment of the VP2 protein is a fragment capable of to specifically bind to the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited at the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number I-4314 or on the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I-4315; or the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited under the number I-4314 or the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number I-4315 is directed specifically against said antigenic fragment VP2 protein; and b) said monoclonal antibody, or one of its functional fragments, directed against the VP2 capsid protein of the BTV-8 virus, or against an antigenic fragment of said VP2 protein, where appropriate labeled is chosen from the secreted antibody by the hybridoma called 6C3 Id Vet deposited at the CNCM, on June 21, 2010 under the number I-4314 or by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I-4315, or the one of its functional fragments.

The present invention also relates to a method for selecting a monoclonal antibody capable of revealing the presence of an anti-VP2 antibody for use in a competitive immunoassay method, in particular by ELISA in competition for the detection or diagnosis of an infection of an animal with BTV, said method is intended to differentiate a BTV-8 infection from a serotype 1 BTV infection, preferably to differentiate a BTV-8 infection from an infection. by the serotype BTV selected from serotypes 1, 2, 4, 9, 10 or 16 or intended to differentiate a BTV-8 infection from a BTV serotype infection selected from other serotypes than serotype 8, especially serotypes 1 to 7 or 9 to 24, characterized in that it comprises the following step: a) contacting said monoclonal antibody to be tested with the VP2 capsid protein of BTV-8, or an antigenic fragment of said it e VP2 protein, or a composition or compound comprising such a protein or fragment, and b) revealing the antigen-antibody complexes formed in (a) between said VP2 protein, or one of its antigenic fragments, and said monoclonal antibody tested, is carried out by a competitive immunoassay method, in particular by competitive ELISA using a monoclonal antibody, or one of the functional fragments, said monoclonal antibody being chosen from the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited at the CNCM, June 21, 2010 under the number I-4314 or the hybridoma called 10E9 Id Vet filed at the CNCM June 21, 2010 under the number I-4315, or one of its functional fragments, if necessary marked. The subject of the present invention is also the monoclonal antibody, or a functional fragment thereof, characterized in that it is produced by the hybridoma called 6C3 Id Vet deposited at the CNCM on June 21, 2010 under the number I-4314. or produced by the hybridoma called 10E9 Id Vet deposited at the CNCM on June 21, 2010 under the number I-4315, where appropriate labeled.

The present invention also relates to the hybridoma called 6C3 Id Vet deposited at the CNCM, June 21, 2010 under the number I-4314 and the hybridoma called 10E9 Id Vet filed at the CNCM June 21, 2010 under the number I-4315.

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.

LEGENDS OF THE FIGURES - Figure 1 is an evaluation of the anti-VP2 and anti-VP7 immune response of immunized mice. Reactivity test of murine immune sera post-immunization on the VP2 protein BTV-8 or recombinant VP7. FIG. 2 represents a reactivity test of the anti-VP2 "6C3" and "10E9" monoclonal antibodies. Peroxidase-coupled and constant concentration antibody (6c3 and 10E9) reagent test (51ag / mL) on recombinant VP2 BTV-8 protein at different concentrations. FIG. 3 represents a competition of anti-VP2 "6C3" and "10E9" monoclonal antibodies. Competition test for the recognition of the recombinant VP2 BTV-8 protein between the uncoupled 6C3 and 10E9 monoclonal antibodies and the peroxidase-coupled monoclonal antibody 6C3 (A) or 10E9 (B). Figure 4 is an evaluation of the sensitivity and specificity of the cELISA VP2-BTV8 assay. Detection of the presence of antibodies directed against the BTV-8 VP2 protein in a panel of naive (n = 66) or BTV-8 infected (n = 67) bovine sera by the cELISA test ELISA technique VP2-BTV8. 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). FIG. 5 represents the validation of the serotype-differentiating character of the cELISA DIVA-BTV8 test. Evaluation of serum of animals infected with BTV serotype 1, 8 or 10, or vaccinated with a monovalent attenuated vaccine BTV-2, -4, -9 or -16 by a standard competition ELISA test ( ID Screen® Bluetongue Competition, IDVET) based on the recognition of the VP7 protein of BTV (A.) and the cELISA VP2-BTV8 test based on the recognition of the VP2 protein of BTV-8 (B.) The values are represented in percentage of the value obtained for the sample on the average of the negative controls (S / N) according to the type of BTV infection. FIG. 6 represents the validation of the serotype-differentiating character of the cELISA DIVA-BTV8 test. Evaluation of sera from infected or vaccinated animals (OJ and D + 45) against serotype 8 BTV by a classical competitive ELISA test (ID Screen® Bluetongue Competition, IDVET) based on the recognition of the BTV VP7 protein (A .) and the cELISA VP2-BTV8 assay based on the recognition of the BTV-8 VP2 protein (B.) The values are represented as a percentage of the value obtained for the sample on the average of the negative controls (S / N). depending on the type of BTV infection.

EXAMPLES Example 1: Materials and reagents 1.1 Preparation of the native VP2 antigen A monolayer of Vero cells at 80% confluence in a flask of 75 cm 2 (MEM, 0.5% FCS, 50 μg / mL of gentamycin + 100 U of penicillin + 100 μg / mL of streptomycin) was infected with strain BTV-8 with a moi of 0.5 (10106 TCID50 / mL). 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 VP2 antigen was recovered and stored at -20 ° C. 1.2 Preparation of the recombinant VP2 antigen. 1.2.1 Construction of Expression Vectors for the Recombinant VP2-BTV8 Protein i. Coding sequence for the VP2-BTV8 protein (see SEQ ID NO: 2) The sequence corresponding to the complete Segment Seg-2 of the viral genome and coding for the VP2 protein of BTV-8 (NET2006 / 04 isolate) was used for the construction of the expression vectors (Mann S., et al., Virology, 2008; 377 (2): 308-18). Nucleotide sequence (GenBank AM498052.2, GL-195972621) (SEQ ID NO: 2) Corresponding amino acid sequence (GenBank CAM57243.2) (SEQ ID NO: 1) ü. Cloning vector of the recombinant VP2-BTV8 protein The complete DNA sequence corresponding to the Seg-2 region of BTV-8 was amplified and the PacI and AscI restriction sites were added at the 5 'and 3' ends. , respectively. The synthetic gene obtained was cloned into the plasmid vector pGA15 (kanR) after PacI / AscI digestion. The plasmid was purified (Pure Yie1dTM Plasmid Midiprep, Promega) from transformed bacteria (E. coli K12 XL10 gold) and the concentration was determined by UV spectroscopy at 260 nm. iii. Expression Vector of the Recombinant VP2-BTV8 Protein The plasmid encoding the VP2 protein of BTV-8 was used to construct a baculovirus-type viral expression vector. Briefly, the plasmid was digested with BamHI / EcoRI restriction enzymes. The ends were made blunt by the action of the Klenow fragment of the DNA polymerase and then digested with the restriction enzyme SalI. The fragments thus obtained were inserted into the expression vector pFastBacHTb (Invitrogen) previously opened with the restriction enzymes SalI then XhoI and the ends made blunt by the action of the Klenow fragment of the DNA polymerase. A sequence of the TEV protease recognition site (underlined, cleavage between Q and G) was thus positioned between the fusion partner 6His (in green) and the beginning of the gene encoding the VP2 BTV-8 (in red). The resulting fusion protein is 997 aa and has a theoretical size of 115.5 kDa. His theoretical pI is 6.47. The sequence of the 6His-VP2 gene cloned into pFastBacHTb is represented by the sequence SEQ ID NO: 3 (nucleic sequence) and SEQ ID NO: 4 (amino acid sequence encoded by SEQ ID NO: 3).

The recombinant expression vectors were transformed into E. coli strain DH10Bac. Bacmidic DNA was extracted from positive colonies and then transfected into Sf9 insect cells. Cell supernatants were harvested seven days after transfection. The presence of 6His-VP2 proteins was tested on SDSPAGE gel by Western-blot using an anti-Histidines monoclonal antibody. Positive supernatants were amplified (50 mL), assayed by the "plate assay" method, and stored at 4 ° C and -80 ° C. 1.2.2 Production and purification of the VP2 fusion protein BTV-8. A viral stock of recombinant baculovirus was prepared in order to produce 4 liters of Sf9 cells at 1.5 × 10 6 cells / ml. At the end of production, the cells were centrifuged, washed with PBS and stored at -80 ° C. Sf9 cell pellets were taken up in lysis buffer (50 mM Tris pH 7.9, 300 mM NaCl, 20 mM imidazole supplemented with a cocktail of anti-proteases). The cells were then lysed under the action of a mechanical cell grinder (2 passes at 1000 bar). After centrifugation of the cell lysate, the supernatant was recovered, filtered and passed through a 1 mL nickel column (HisTrap HP - GE Healthcare) using an AKTA range purification pilot. After a step of washing the column in the same buffer containing 60 mM imidazole, the proteins were eluted at a concentration of imidazole of 250 mM. The proteins harvested during the elution were analyzed on SDS-PAGE gel and then quantified by the BCA method using a standard range of BSA of known concentration. The concentration of the purified VP2 protein BTV-8 was determined by measuring the optical density at 280 nm, and then the protein was stored at -20 ° C after addition of glycerol (1: 1 vol / vol). 1.3 Serum banks 1.3.1 Naïve animal serums The serums of naive animals were collected during the year 2006 from the abattoir of Alès (France), before any incursion of FCO in France. 1.3.2 sera from BTV-infected animals sera from BTV-infected animals originate from several sampling campaign areas. - Sheep serums: BTV-8 positive sheep sera were collected in Belgium in December 30, 2007. BTV-1 positive sheep sera were provided by the National Veterinary School of Toulouse (ENVT, Toulouse, France) in December 2008.

BTV-2, -4, -9 and -16 positive sheep sera were provided by the Center for Agricultural Research for the Development of Southern Countries (CIRAD, UMR15 "Control of Exotic and Emerging Animal Diseases", Montpellier, France) and correspond to animals vaccinated with an attenuated, therefore replicative, monovalent vaccine (Onderstepoort Biological Products). - Cattle serums: The BTV-8 positive serum sera were provided by the Regional Association for Animal Health and Identification (ARSIA, Belgium) and relate to the 2007 epidemic episode in the Liège region (Belgium).

The BTV-1 positive cattle sera were provided by the National Veterinary School of Toulouse (ENVT, Toulouse, France) in August 2008. The BTV-10 positive bovine serum was provided by Veterinary Diagnostic Technology, Inc. (Wheat Ridge, Colorado 80033 USA) and corresponds to a control positive serum characterized by agarose gel immunodiffusion technique (AGID) from the "BT antibody test kit" test. 1.3.3 Serums of BTV vaccinated animals using an inactivated vaccine Bovine serum free from natural infection and vaccinated against BTV-8 (BTVPUR® AlSap 8, Merial) corresponds to a vaccination and sampling campaign carried out by the veterinary services (Veterinary Clinic Dr. Pierrard and Dr. Pekus, (Sainte-Menehould, 51800, FRANCE) between April and June 2008. The sera are taken from samples taken before (OJ) and after 0 + 45) field vaccination using inactivated vaccine BTV-8 (BTVPUR® AlSap 8, Merial) according to the manufacturer's protocol. 1.4 Reagents for ELISA Assays Unless otherwise specified, the reagents used in the ELISA technique are input into the composition of IDVET's commercial kits. In order to define the terms used, the various solutions used are identified below: Wash Solution: Solution SL ("Washing Solution" of the IDScreen® test or PBST-type washing solution (0.01 M PBS containing 0.05% Tween- 20, pH 7.2) Saturation solution: Solution ST (PBST-based saturation solution containing 3% BSA) Revelation solution: SR solution ("Revelation Solution" of the IDScreen® test or 3-based revelation solution, 3 ', 5,5'-tetramethylbenzidine (TMB)) Stop Solution: Solution SA (IDScreen® Test Stop Solution or 0.5M Sulfuric Acid Stop Solution). dilution of the sera: DL18 or SL solution containing 2% of skim milk Dilution solution of the monoclonal antibody conjugated to 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 s 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 Description of Monoclonal Antibodies 2.1 Preparation of BTV-8 Inoculum: Vero line cells (ATCC, CCL-81) were cultured and brought to 80% confluency in MEM medium containing 10% calf serum fetal (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-8. Three days after infection, the infection medium was removed, the cells were washed with PBS and mL of PBS containing 10% sucrose was added. The culture dish underwent a rapid freeze / thaw cycle -70 ° C / + 37 ° C. The native BTV-8 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). 2.2 Immunization of mice BALB / c adult female mice were immunized intraperitoneally with the previously obtained viral stock BTV-8 (106 PFU of BTV-8 / injection) at a rate of 0.5 mL containing 50 % (vol / vol) of Freund's complete adjuvant. Mice were resensitized at 14th and 35th day using an identical dose of BTV-8 containing 50% (vol / vol) incomplete Freund's adjuvant. Finally, an inoculation of BTV-8 (106 PFU of BTV-8 / injection) was carried out at the 3rd month post-infection before the cell fusion step and the search for anti-BTV antibodies in the hybridoma supernatants. . 2.3 Establishment of Hybridomas and Cloning 2.3.1 Testing of Immunized Mouse sera The presence of murine anti-VP7 and anti-VP2 BTV8 antibodies was verified by indirect ELISA. 96-well plates were sensitized with recombinant VP7 and VP2 proteins of BTV-8 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 titrated. After incubation for 5 h at 37 ° C., the plates were washed three times with the SL solution and 50 μl / well of anti-mouse IgG antibody coupled to peroxidase (SIGMA, A4416 at 1 / 1200th ) were added and incubated for 30 min. at room temperature. After washing, 100 μL / well of SR solution was added. The reaction was stopped after 10 min. by adding an identical volume of solution SA and the absorbance values at 450nm were measured.

The results obtained show that the mice immunized with the BTV-8 virus triggered a humoral response against the latter with the presence of a strong anti-VP7 response. Although weaker, a response against the VP2 protein of BTV-8 is also present and justifies the transition to establishment and selection of hybridoma secreting monoclonal antibodies from these animals. 2.3.2 Splenocyte preparation 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. 2.3.3 Preparation of myeloma cells for fusion 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 phosphoribosyl transferase. 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. 2.3.4 Preparation of Antibody-secreting Hybridomas Hybridomas were obtained 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.

The cell suspensions of splenocytes and myeloma cells were brought back 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 × g, the supernatant removed and the pellet resuspended in 10 ml and then 200 ml 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). 2.3.5 Evaluation and selection of monoclonal antibody secreting hybridomas An identification test of monoclonal antibody secretion hybridomas directed against the VP7 protein was performed in the first instance. Hybridomas positive for this assay were discarded and the remaining hybridomas were tested in competitive ELISA for their ability to recognize the serotype-specific virus-specific BTV-8 virus VP2 protein and then distinguish naturally infected animals from BTV-8. 8 animals vaccinated with inactivated mono- or multivalent vaccines containing BTV-8. Identification of secretion hybridomas of anti-BTV8 and non-anti-VP7 monoclonal antibodies The supernatant of the hybridomas was deposited at a rate of 50 μl / well on 96-well plates sensitized with recombinant VP7 protein or BTV-virus. 8 native and then incubated for 2 h at 37 ° C. After three washings, 100 μl / well of anti-mouse IgG antibody coupled to peroxidase was added and incubated for 30 min. at room temperature with stirring. After washing, 100 μL / well of SR solution was added and the reaction was stopped after 10 min. by adding an identical volume of SA solution. The absorbance values at 450 nm were recorded. Non-sensitized plates were used as negative controls in this experiment. Hybridomas whose supernatants gave a positive signal on the non-sensitized plates, as well as those which proved negative for the presence of anti-BTV8 antibodies and positive for the presence of anti-VP7 antibodies were discarded. The hybridomas thus retained are secretors of monoclonal antibodies and BTV8 whose epitopes are not carried by the VP7 viral protein. Identification of hybridoma secreting secretions of serotype-specific and DIVA potential-and-BTV8 monoclonal antibodies The anti-BTV-8 antibodies secreted by the previously selected hybridomas were evaluated in a competitive ELISA test involving sera of naturally infected, naive cattle. by BTV-1 or BTV-8, or vaccinated against BTV-8 with a commercial inactivated vaccine. The sera were deposited at a rate of 50 μl / well (dilution in solution SL + 2% skim milk) on 96-well plates sensitized with native BTV-8 virus, then incubated for 2 h at 37 ° C. After three washes, the hybridoma supernatant was added at 50 μL / well and the plate incubated at 37 ° C. After three washings, 100 μl / well of anti-mouse IgG antibody coupled to peroxidase was added and incubated for 30 min. at room temperature with stirring. After washing, 100 μL / well of SR solution was added and the reaction was stopped after 10 min. by adding an identical volume of SA solution. The absorbance values at 450 nm were measured and expressed as a percentage of the average of the values obtained for said negative control.

Hybridomas secreting monoclonal antibodies corresponding to all of the following expectations were retained: Characteristic serotype 1/8 differentiating: Total inhibition when using serums of animals naturally infected with BTV-8, and weak, even null, when using sera from animals naturally infected with BTV-1. Characteristic DIVA:, Partial, low or no inhibition, when using sera of animals vaccinated against BTV-8 using a commercial inactivated vaccine. The hybridomas thus retained are secretors of anti-BTV8 monoclonal antibodies, differentiating serotype 1-8, of DIVA character and whose epitopes are not carried by the VP7 viral protein. 2.4 Characterization of the differentiating and DIVA-differentiating serotype 1-8 monoclonal anti-BTV8 antibodies Following this triple selection of the monoclonal antibody secreting hybridomas, two potentially interesting candidates were selected. These hybridomas as well as the associated monoclonal antibodies were identified as "6C3" and "10E9" and further characterization experiments were performed. These two hybridomas have been deposited with the National Collection of Cultures of Microorganisms (CNCM Pasteur Institute, Paris, France) under the order number 20 CNCM I-4314 and I-4315, respectively. 2.4.1 Reactivity Test of 6C3 and 10E9 Monoclonal Antibodies on Recombinant BTV-8 VP2 Protein Serotype 1-8 differentiating previously identified monoclonal antibodies suggests that their respective epitopes are probably carried by the major serotypic determinant of the virus. of the FCO that is the VP2 protein. In order to verify this hypothesis, a reactivity test of the 6C3 and 10E9 antibodies on the recombinant VP2 protein of BTV-8 was undertaken. The monoclonal antibodies 6C3 and 10E9 coupled to the peroxidase were deposited at a rate of 100 μL / well (5 μg / mL, SL solution + 2% skimmed milk) on a plate sensitized with recombinant VP2 BTV-8 and in increasing concentration from 0 to 56 μg / mL. After 30 min. incubation at room temperature, the plate was washed 3 times, 100 μL / well of SR solution were added, and the reaction was stopped after 15 min. by adding an identical volume of SA solution. Absorbance values at 450 nm were recorded. The results obtained showed that the antibodies 6C3 and 10E9 are well directed against the VP2 protein of BTV-8 and that these two monoclonal antibodies exhibit different behaviors with greater reactivity of the 6C3 antibody at low concentrations of antigen compared to to the 10E9 antibody. All of these results indicate that the epitopes recognized by the monoclonal antibodies 6C3 and 10E9 are carried by the VP2 protein of BTV-8 and that under our conditions of use the 6C3 antibody shows a better affinity than the 10E9 for the recombinant VP2 protein. 2.4.2 Isotype of monoclonal antibodies The 6C3 and 10E9 antibodies were subjected to an ELISA isotyping test using the ISO2 kit "Monoclonal Antibody Isotyping Reagents" (SIGMA) and according to the manufacturer's recommendations. The ascites liquids induced by each of the hybridomas were diluted in SL solution and 100 .mu.l were deposited on a plate previously sensitized. After 90min. incubation at 37 ° C, the plate was washed and 100 μL of anti-IgG1, IgG2a, IgG2b, IgG3, IgA or mouse IgM antibodies were deposited. The plate was revealed using 100 μl of peroxidase substrate after an additional 30 min incubation. 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 450nm were recorded. The results obtained showed that the monoclonal antibodies 6C3 and 10E9 are of IgG2b and IgG1 isotype, respectively. 2.4.3 Competition of BTV-8 anti-VP2 monoclonal antibodies The 6C3 and 10E9 monoclonal antibodies are deposited at increasing concentrations of 0 to 10 μg / mL (SL solution + 2% skim milk) at a rate of 100 μL / well on a plate presensitized with recombinant VP2 BTV-8. After incubation for 2 hours at room temperature, 100 μl of monoclonal antibody 6C3 or 10E9 coupled to peroxidase and at a constant concentration (75 ng / ml or 500 ng / ml in SL solution + 2% skimmed milk, respectively) are added. for 30 min. additional incubation. After 3 washes in SL solution, 100 μL / well of SR solution was added and the reaction was stopped after 15 min. by adding an identical volume of SA solution. Absorbance values at 450 nm were recorded. The results showed a drop, followed by a signal extinction, for the 6C3 / 6C3-HRP control pair (Fig. 3A) and the 10E9 / 10E9-HRP couple (Fig. 3B). This characteristic profile of a competition effect between two antibodies confirms that a monoclonal antibody coupled or not to peroxidase recognizes the same epitope on the VP2 protein. On the other hand, no competition effect is observable between the monoclonal antibodies 10E9 and 6C3-HRP (Fig. 3A) or their reciprocals (Fig. 3B). The monoclonal antibodies 6C3 and 10E9 thus recognize distinct epitopes of the VP2 protein of BTV-8. 2.4.4 Western-blot anti-BTV8 and recombinant anti-VP2 BTV-8 using antibodies 6C3 and 10E9 2.4.5 Evaluation of the seroneutralizing potential of monoclonal antibodies 6C3 and 10E9 No seroneutralizing potential could be demonstrated for monoclonal antibodies 6C3 and 10E9. 2.4.6 Evaluation of Serotype 1-8 Potential Differentiating Monoclonal Antibodies 6C3 and 10E9 The differentiating serotype 1-8 potentials of the 6C3 and 10E9 monoclonal antibodies were compared on the basis of sera from cattle either naive or naturally infected with BTV-1. or BTV-8. To do this, the sera were diluted to SL solution containing 2% skim milk and deposited on a plaque presensitized with recombinant VP2 BTV-8 at 100μL / well. Naïve animal sera were used as negative controls and the BTV-8 serum was further diluted 1: 20 and 1:40 to assess the detectability limit. After 45 min. incubation at 37 ° C, the plate was washed 3 times in SL solution and 100 μl of monoclonal antibody 6C3 or 10E9 at a concentration of 0.2 or 0.04 μg / mL (in solution SL + 2% milk skimmed) added for 45 min. additional incubation at 37 ° C. After 3 washings in SL solution, 100 μl / well of anti-mouse IgG antibody coupled to peroxidase was added. The plate was incubated for 30 min at room temperature, washed three times and 100 μl of SR solution was added. The reaction was stopped after 15 minutes. by adding an identical volume of solution SA and the absorbance values at 450 nm were recorded. The results were represented in units of optical density at 450 nm (Table 1, left) and as a percentage of the value obtained for the sample on the average of the negative controls (Table 1, right). OD 450nm 6C3 10E9 10E9 6C3 0.2 μg / mL 0.2 μg / mL 0.04 μg / mL 0.2 μg / mL 0.04 μg / mL 0.04 μg / mL 0.2 μg / mL 0, 04 μg / mL 0 333 0 637 1 455 18 11 20 57 30 70 76 47 82 0 352 1 221 1 516 0 681 2 443 2 849 0 611 1 678 2 640 14 49 60 BTV-8 BTV-8 (at 1 / 20) BTV-8 (at 1/40) BTV-1 96 77 87 69 1,742 2,378 3,334 Serum type Serum-free 3,479 3,100 3,471 2,508 Table 1: Comparison of potential serotypes BTV-I / -8 differentiating monoclonal antibodies 6C3 and 10E9 . VP2 BTV-8 competition ELISA using sera of cattle naive or naturally infected with BTV-I or BTV-8, and anti-VP2 monoclonal antibodies BTV-8 6C3 and 10 I0E9. The results were shown in units of optical density at 450 nm (left) and percentage of the value obtained for the sample on the average of the negative controls (right). As previously, the results obtained (Table 1) showed a greater detection loss of the recombinant VP2 BTV-8 signal with the 10E9 monoclonal antibody than with the 6C3 monoclonal antibody following a dilution of 0.2 at 0.04 μg / mL of these ("serum-free" point). This indicates a greater affinity of the 6C3 antibody for the VP2 BTV-8 protein. For serum BTV8 positive, values obtained from 18 to 11% and 20 and 14% of the signal with serum BTV-8 for monoclonal antibodies 6C3 and 10E9 at concentrations of 0.2 to 0.04 μg / mL, respectively, demonstrated a better blocking by serum antibodies of the binding of 6C3 on VP2 BTV-8 than that of 10E9. The results obtained with the complementary dilutions of the serum BTV-8 positive confirmed this difference, thus testifying to a better sensitivity with the use of the monoclonal antibody 6C3. Similarly, the values obtained for BTV-1 positive serum showed lower signal inhibition with the use of 6C3 monoclonal antibody than with 10E9. Finally, the results showed a distribution of the control naive sera more homogeneous with the use of the monoclonal antibody 6C3 than with the 10E9.

The BTV-8 anti-VP2 monoclonal antibodies studied here are therefore both usable in a differentiating serotype 1-8 serotype ELISA-type serum test. However, the 6C3 monoclonal antibody has a better affinity for the recombinant VP2 BTV-8 protein produced according to our method of manufacture, and its use relative to that of 10E9 improves the detectability and specificity of the test. 2.4.7 DIVA BTV8 Potential of 6C3 and 10E9 Monoclonal Antibodies The BTV DIVA potentials of the 6C3 and 10E9 monoclonal antibodies were compared on the basis of sera from naive cattle, naturally infected with BTV-1 or BTV-8, or vaccinated against BTV-8 using a commercial inactivated vaccine.

To do this, the sera were diluted with SL solution containing 2% skim milk and deposited on a plaque presensitized with recombinant VP2 BTV-8 at 100 μL / well. After 45 min. incubation at 37 ° C, the plate was washed 3 times in SL solution and 100 μl of monoclonal antibody 6C3 or 10E9 at a predefined and optimal concentration (SL solution + 2% skim milk) were added for 45 min. additional incubation at 37 ° C. After 3 washings in SL solution, 100 μl / well of anti-mouse IgG antibody coupled to peroxidase was added. The plate was incubated for 30 min at room temperature, washed three times and 100 μL of SR solution was added. The reaction was stopped after 15 minutes. by adding an identical volume of solution SA and the absorbance values at 450 nm were recorded. The results were shown in optical density units at 450 nm. The values are represented as a percentage of the value obtained for the sample on the average of the negative controls (S / N).

D.O.450nm 10E9 6C3 6C3 10E9, 923 4. 896 5 264 BTV-8 611 8 566 Table 2: Comparison of differentiating serotypes BTV-I / -8 and DIVA BTV-8 potentials of 6C3 and 10E9 monoclonal antibodies. VP2 BTV-8 competition ELISA using sera of naive cattle, naturally infected with BTV-I or -8, or vaccinated against BTV-8, and anti-VP2 monoclonal antibodies BTV-8 6C3 and 10E9 . The results were shown in units of optical density at 450 nm (left) and as a percentage of the value obtained for the sample on the average of the negative controls (right).

The results obtained showed a fall in the signal percentage between the sera of animals naturally infected with BTV-1 and those infected with BTV-8 by 70.2 points of average (94.5% on BTV-1 and 24). , 3% on BTV-8) for the 6C3 antibody and only 54.4 points (88% on BTV-1 and 33.7% on BTV-8) for the 10E9 antibody. As previously, a better differential BTV-1 / BTV-8 on the sera of naturally infected animals is therefore observable with the use of the monoclonal antibody 6C3 instead of 10E9. For sera from animals vaccinated against BTV-8, mean signal percentages of 91.6% and 96.6% were obtained for antibodies 6C3 and 10E9, respectively. The difference in percentages observed between animals naturally infected with BTV-8 and animals vaccinated against it is therefore 67.3 points on average with the use of the 6C3 antibody and 62.9 points with the of the 10E9 antibody. This demonstrates a strong and similar DIVA BTV-8 character of the two anti-VP2 monoclonal antibodies. All these results made it possible to better characterize the monoclonal antibodies 6C3 and 10E9. These antibodies are respectively of IgG2b and IgG1 isotype, directed against epitopes carried by the VP2 protein alone bluetongue virus serotype 8 and surprisingly are not neutralizing towards it. These epitopes are strongly recognized by the antibodies of animals naturally infected with BTV-8 but not by those infected with the BTV-1 virus. Therefore, the use of monoclonal antibodies 6C3 or 10E9 in a competition ELISA serological test gives it a high differentiating serotype 1-8 potential. Surprisingly, these epitopes are little or not recognized by the antibodies of animals vaccinated against BTV-8 using a commercial inactivated vaccine. The use of the monoclonal antibodies 6C3 or 10E9 in a competitive ELISA serological test thus also confers a high potential DIVA BTV-8. Although both monoclonal antibodies can be used in the development of a competition-type serological test with DIVA BTV-8 and differentiating serotype 1-8 properties, all the data obtained show better results with the use of 6C3 antibody in our conditions of use.

DIVA VP2-BTV8 competition ELISA test differentiating serotype 2.5 Principle of the DIVA VP2-BTV8 competition ELISA test differentiating serotype The serological test described in this document allows the detection of antibodies directed against bluetongue virus serotype 8 (BTV-1). 8). It differentiates, on the one hand, animals naturally infected with serotype 8 from those naturally infected with other serotypes (eg BTV-1, -2, -4, -9, -10, -16), or vaccinated against this serotype with a mono- or multivalent inactivated vaccine. This method is a competition enzyme immunosorbent assay (ELISA) assay (cELISA) and uses monoclonal antibodies coupled to an enzyme (peroxidase) and a synthetic substrate (3,3 ', 5,5'). -tetramethylbenzidine, TMB) to detect antibodies specific for BTV-8 after infection.

If the biological samples contain polyclonal antibodies against the antigen, the antibodies bind to it. This antigen is subsequently placed in the presence of a labeled monoclonal antibody. If it has not previously been complexed by the polyclonal antibodies of the sample at the level of the epitope of the monoclonal antibody then the antigen will bind the labeled monoclonal antibody. On the contrary, if it is already engaged in an immune complex with the polyclonal antibodies of the sample masking the epitope of the monoclonal antibody, the antigen will not bind the labeled monoclonal antibody. The measurement of the resulting enzymatic activity makes it possible to estimate the amount of monoclonal antibodies bound to the non-complexed antigen by the polyclonal antibodies of the sample. The amount of polyclonal antibodies directed against the BTV-8 antigen present in the test sample can therefore be determined by calculating the difference. The results obtained are expressed in units of optical density at 450 nm or percentage of the value obtained for the sample on the average of the negative controls (S / N).

The test was developed on panels of sera from different ruminant species (sheep, cattle, goats). The BTV-8 antigen used in this test is the VP2 capsid protein alone of BTV-8, and more particularly the epitopes carried by the VP2 protein and having the following characteristics: - be specific to serotype 8 of the bluetongue virus (BTV-8), - not to be involved in the viral phases adsorption and penetration (non-neutralizing monoclonal antibodies), - to be recognized by the antibodies of animals naturally infected with BTV-8 - to be little, or not, recognized by antibodies from animals vaccinated with an inactivated mono- or multivalent vaccine containing BTV-8.

The monoclonal antibodies used in this test are the antibodies labeled "6C3" (CNCM I-4314) and are coupled directly and covalently to horseradish peroxidase. Their preparation, selection and production has been described previously in this document. 2.6 Protocol of the DIVA VP2-BTV8 competition ELISA test differentiating serotype The reagents used in the context of the ELISA technique are input elements into the composition of IDVET's commercial kits. In order to define the terms used, the different solutions used are identified below: Wash Solution: SL Solution Saturation Solution: ST Solution Revelation Solution: SR Solution Stop Solution: SA Solution Serum Dilution Solution: DL18 or SL solution containing 2% skimmed milk. Dilution solution of the monoclonal antibody conjugated to peroxidase: DL19 or SL solution The data were obtained by reading the optical density at 450 nm using a multichannel spectrophotometer (ex: Sunrise, Tecan) The protocol chosen for carrying out the assay is described below: The recombinant VP2 protein of BTV-8 was plated in 96-well plates at a predetermined optimal PBS dilution and then incubated at room temperature overnight. After washing to remove the excess of unfixed antigen, saturation of the plates was performed with 2041 of ST solution. The saturation solution was then removed and 50 μl / well of sera of naive, infected or vaccinated animals were deposited after dilution in DL18 solution. After 1h incubation at 37 ° C, the plates were washed three times with SL solution and 100μL / well of murine anti-VP2 murine monoclonal antibody coupled to peroxidase was added and incubated for 30min. at room temperature. After 3 washes in SL solution, 100 μL / well of SR solution was added and the reaction was stopped after 15 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). 2.7 Evaluation of the specificity and sensitivity of the test: Discrimination of animals naturally infected with BTV8 from naive animals. In order to determine the discriminating power of the cELISA VP2-BTV8 assay between naive or BTV-infected animals, a panel of naive (n = 66) or naturally infected BTV8 (n = 67) bovine serum sera were tested in ELISA. competition to compare their anti-VP7 and anti-NS1 seroconversions. Detection of anti-VP7 antibodies was performed using IDVET's ID Screen® Bluetongue Competition commercial kit according to the associated protocol. The absorbance values at 450 nm were recorded and 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 blockage percentage of between 60 and 130%. On the contrary, the sera of infected animals have a distribution of between 0 and 15% of blocking. The specificity and sensitivity of this test are therefore satisfactory and allow a clear distinction between naive animals and those naturally infected with BTV-8. 2.8 Evaluation of the BTV8-differentiating potential of the test: Discrimination of animals naturally infected with BTV8 from those infected with other BTV serotypes.

The differentiating serotype 1-8 character was a selection criterion for anti-BTV8 monoclonal antibody secreting hybridomas at the base of this serodiagnostic test. IDVET wished to evaluate, and validate if necessary, this differentiating property between serotype 8 and the other main BTV serotypes encountered in Europe and the United States, namely BTV-1, -2, -4, -9 , -16 and BTV-10, respectively. To do this, the anti-VP7 (Fig. 5A.) And anti-VP2 BTV8 (Fig. 5B.) Serological response of animals (cattle and sheep) that were naive or infected with the different serotypes of interest was evaluated at using a BTV standard non-differentiating serotype competition test (ID Screen® Bluetongue Competition, IDVET) and the cELISA VP2-BTV8 test.

The results obtained showed that all the animals tested were positive during an evaluation by conventional competitive ELISA (anti-VP7) test. In this diagnostic approach, no distinction regarding the serotype of the BTV responsible for the infection could thus be highlighted. In contrast, the results obtained in the VP2-BTV8 competition ELISA showed that only animals infected with BTV serotype 8 induced complete inhibition of binding of the monoclonal antibody to the VP2 protein (S / N). %) and therefore a positive status. Other serotypes produced only low or no inhibitions (S / N> 75%). This cELISA VP2-BTV8 test is therefore able to differentiate animals infected with BTV-8 from animals infected with BTV-1 but also from other serotypes such as -2, -4, -9, -16 or -10. 2.9 Evaluation of DIVA BTV-8 potential of the test: Discrimination between animals naturally infected with BTV-8 and those vaccinated against this virus. Studies on the 6C3 antibody showed the DIVA potential of this antibody.

In order to verify the maintenance of this characteristic in the cELISA VP2-BTV8 assay, a panel of cattle sera (n = 46) obtained before (OJ) and after vaccination 0 + 45) against BTV-8 using an inactivated vaccine was tested. The vaccination protocol was performed according to the manufacturer's recommendations and these sera were analyzed using a standard competition ELISA test (ID Screen® Bluetongue Competition, IDVET) and the cELISA VP2-BTV8 assay. In order to evaluate the discriminating potential between vaccinated animals and animals naturally infected with BTV-8, a panel of serums of naturally infected animals was also analyzed using these two tests. The results obtained using the classical competition ELISA test (Fig.6.A.) show that the distribution of vaccinated animal samples increases from 80 to 130% of the average value of the negative control signal, with a maximum of at 90-100%, before vaccination (OJ) at a distribution between 0 and 15%, with a maximum at 5-10%, after vaccination against BTV-8 and this for all the animals studied. These latter values are very close to those obtained with the sera of naturally infected animals which, for their part, have a distribution of between 0 and 10%, with a maximum of 0-5%.

These results highlight a strong anti-VP7 vaccine seroconversion of BTV-8 leading to the transition from a seronegative status before vaccination to a strong seropositive status after vaccination preventing any distinction between vaccinated animals and animals naturally infected with BTV-8. .

With the use of the cELISA VP2-BTV8 assay the distribution of vaccinated animal samples (Fig.6.B.) is different from that described above for a standard cELISA test. Indeed, it goes from 50 to 130% of the average value of the signal of the negative controls, with a maximum at 100-110%, before vaccination (JO) at a distribution between 30 and 130%, with a maximum at 70-80 % after vaccination against BTV-8. These latter values are thus clearly distinct from those obtained with serums of naturally infected animals which have a distribution of between 0 and 15%, with a maximum of 5-10%.

This cELISA VP2-BTV8 test thus demonstrates a more moderate anti-VP2 vaccine seroconversion than that observed during a natural infection and thus makes it possible to distinguish vaccinated animals from animals naturally infected with BTV-8.

REFERENCES

1. MacLachlan, N.J. and B.I. Osburn, Impact of bluetongue virus infection on the international movement and trade of ruminants. J Am Vet Med Assoc, 2006. 5 228 (9): p. 1346-9. 2. Erasmus, B.J., Bluetongue in sheep and goats. Aust Vet J, 1975. 51 (4): p. 165-70. 3. Calistri, P., et al., Bluetongue in Italy: Part L Vet Ital, 2004. 40 (3): p. 243-51. 4. Bricq, N., Management of the bluetongue epizootic (BLD): learning for the future. Senate Information Report 2008. No. 460. 5. EFSA, Scientific Report on the Scientific Panel on Animal Health and Welfare on Request from the Commission (EFSA-Q-2006-311) and EFSA Selfmandate (EFSA-Q-2007-063) on bluetongue. The EFSA Journal, 2007. 480: p. 1-20. 6. Anderson, J., Use of a monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus. J Immunol Methods, 1984. 74 (1): p. 139-49. 7. Grimes, J.M., et al., The atomic structure of the bluetongue virus core. Nature, 1998. 395 (6701): p. 470-8. 8. Hassan, S.S. and P. Roy, Expression and functional characterization of bluetongue VP2 protein: yole in cell entry. J Virol, 1999. 73 (12): p. 9832-42. 9. Mertens, P. P., et al., Analysis of the yole of bluetongue virus outer capsid protein VP2 and VP5 in determination of serotype virus. Virology, 1989. 170 (2): p. 561-5. 10. Maan, S., et al., Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes. J Gen Virol, 2007. 88 (Pt 2): p. 621-30. 37

Claims (4)

REVENDICATIONS1. Use of the VP2 capsid protein of Bluetongue virus serovar 8 called BTV-8 ("BTV" for "Blue Tongue Virus"), or an antigenic fragment of said VP2 protein of BTV-8, or a composition or compound comprising such a protein or fragment for the preparation of a reagent or kit for the in vitro diagnosis of BTV infection in animals, said antigenic fragment of the VP2 protein being characterized in that that: it is a fragment capable of binding specifically on the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris , France, on June 21, 2010 under the number I-4314 or on the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM on June 21, 2010 under the number I-4315; or the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited under the number I-4314 or the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number I-4315 is directed specifically against said antigenic fragment of the VP2 protein. 2. Use according to claim 1, characterized in that said reagent is further intended to differentiate a BTV-8 infection from an infection with BTV-1 in said animal. 3. Use according to claim 1 or 2, characterized in that said reagent is intended to differentiate a BTV-8 infection from a serotype BTV infection selected from serotypes 1, 2, 4, 9, 10 or 16. 4. Use according to one of claims 1 to 3, characterized in that said reagent is intended to differentiate a BTV-8 infection from a serotype BTV infection selected from serotypes 1 to 7 or 9 to 24.. Use according to one of Claims 1 to 4, characterized in that the said VP2 capsid protein of BTV-8 or the said antigenic fragment of the said VP2 protein of BTV-8, or the compound comprising it, is chosen from: a protein Isolated and, if appropriate, purified VP2 of BTV-8, or one of its antigenic fragments as defined in claim 1; a recombinant VP2 protein, preferably of amino acid sequence SEQ ID NO: 1 or one of its antigenic fragments as defined in claim 1; or BTV-8, or one of its fragments containing the VP2 protein or one of its antigenic fragments as defined in claim 1. Use according to one of claims 1 to 5, characterized in that said reagent or kit comprises, preferably separately from said reagent containing said VP2 protein, an antibody, or a functional fragment thereof, directed against the BTV-8 VP2 capsid protein, or against an antigenic fragment of said protein VP2, for the preparation of a reagent for the in vitro diagnosis of an infection with BTV-8 in animals. 7. Use according to claim 6, characterized in that said antibody or antibody fragment is chosen from monoclonal antibodies, polyclonal antibodies, chimeric antibodies, or their functional fragments, in particular Fab fragments, (Fab ') 2, Fab ', Fv, scFv, scFv-Fc of the above antibodies. 8. Use according to claim 6 or 7, characterized in that said antibody 25 is the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France, June 21, 2010 under the number I-4314 or the hybridoma called 10E9 Id Vet filed at the CNCM June 21, 2010 under the number I-4315. 9. Use according to any one of claims 6 to 8, characterized in that said antibody or antibody fragment is coupled to a marker. 10. Use according to one of claims 1 to 9, characterized in that said animal is a ruminant, preferably a bovine, an ovine or a goat. 11. In vitro method for detecting or diagnosing an infection of an animal with a BTV from a biological sample of said animal, characterized in that it comprises the following steps: a) contacting said sample with the BTV-8 VP2 capsid protein, or an antigenic fragment of said VP2 protein, or a composition or compound comprising such a protein or fragment, and b) revealing the antigen-antibody complexes formed in a) between said VP2 protein, or one of its antigenic fragments, and an anti-VP2 antibody likely to be present in said biological sample, said antigenic fragment of the VP2 protein being characterized in that: - it is a fragment capable of binding specifically to the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75 724 Paris, France, on June 21, 2010 under the number I-4314 or on the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM on June 21, 2010 under the number I-4315; or the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited under the number I-4314 or the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number I-4315 is directed specifically against said antigenic fragment of the VP2 protein. 12. In vitro method for detecting or diagnosing an infection of an animal with BTV from a biological sample of said animal according to claim 11, characterized in that in step b), the revelation of antigen-antibody complexes formed in (a) is carried out by a competitive immunoassay method, preferably a competitive ELISA method, employing a monoclonal antibody, or a functional fragment thereof, capable of specifically recognizing the protein VP2 of BTV-8, or one of its antigenic fragments. In vitro detection or diagnostic method according to claim 12, characterized in that: - either said VP2 protein of BTV-8, or one of its antigenic fragments, carried out in step a), or - either said antibody capable of specifically recognizing the VP2 protein of BTV-8 used in step b) is labeled, preferably with peroxidase. 14. In vitro method of detection or diagnosis according to claim 12 or 13, characterized in that said monoclonal antibody used in step b) in the competing ELISA method is the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited at the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number I-4314 or by the hybridoma called 10E9 Id Vet deposited at the CNCM on June 21st, 2010 under the number I-4315. 15. In vitro method of detection or diagnosis according to one of claims 11 to 14, characterized in that it comprises the following steps: a) bringing said biological sample into contact with the VP2 protein of BTV-8, or an antigenic fragment of said VP2 protein, or of a composition or compound comprising such a protein or fragment, said VP2 protein being previously captured by immunocapture on a solid phase coated with an anti-VP2-BTV-8 antibody of the type polyclonal, or, monoclonal type and as defined in claims 6 to 8; b) revealing the antigen-antibody complexes formed in (a) between said VP2 protein, or one of its antigenic fragments, and an anti-VP2 antibody likely to be present in said sample, this revelation being carried out by a method immunoassay in competition employing an anti-VP2 antibody, or one of its functional fragments, preferably labeled, as defined in one of claims 6 to 8, said antigenic fragment of the VP2 protein being characterized in that: - it is a fragment capable of binding specifically to the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number I-4314 or on the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I-4315 or - the ant monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited under the number 1-4314 or the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number 1-4315 is directed specifically against said antigenic fragment of the VP2 protein . 16. In vitro method for detecting or diagnosing an animal infection with BTV according to claim 11, characterized in that said method is intended to differentiate a BTV-8 infection from a BTV infection. serotype 1, preferably to differentiate BTV-8 infection from BTV serotype infection selected from serotypes 1, 2, 4, 9, 10 or 16 or to differentiate BTV-1 infection. 8 of a serotype BTV infection selected from serotypes 1 to 7 or 9 to 24. 17. In vitro method for detecting or diagnosing an infection according to one of claims 11 to 16, characterized in that said biological sample is a biological fluid sample, preferably selected from milk, whey, plasma, serum, meat juice or a whole blood sample of the animal, serum being most preferred. 18. A kit or kit of reagents for detecting or diagnosing an infection of an animal with a BTV, said kit comprising: a) a compound or composition comprising the BTV-8 VP2 capsid protein, or an antigenic fragment said VP2 protein of BTV-8; and b) an antibody, or a functional fragment thereof, directed against the VP2 capsid protein of BTV-8, or against an antigenic fragment of said VP2 protein of BTV-8, if appropriate labeled, preferably said antibody is polyclonal, monoclonal or chimeric, said antigenic fragment of the VP2 protein being characterized in that: - it is a fragment capable of binding specifically to the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM), Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number 1-4314 or on the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM on June 21, 2010 under number I-4315; or - the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited under the number 1-4314 or the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number I-4315 is directed specifically against said antigenic fragment of the VP2 protein. 19. Kit or kit of reagents according to claim 18, characterized in that: a) said compound comprising the VP2 capsid protein of BTV-8, or said antigenic fragment of said VP2 protein of BTV-8, is chosen from: an isolated and, where appropriate, purified VP2 protein, BTV-8, or one of its antigenic fragments; a VP2 protein of recombinant BTV-8, preferably of amino acid sequence SEQ ID NO: 1 or one of its antigenic fragments; or BTV-8, preferably inactivated, or one of its fragments containing the VP2 protein of BTV-8 or one of its antigenic fragments, and characterized in that: said antigenic fragment of the VP2 protein of BTV-8 is a fragment capable of binding specifically to the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France, June 21, 2010 under the number I-4314 or on the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I-4315; or the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited under the number I-4314 or the antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number I-4315 is directed specifically against said antigenic fragment of the VP2 protein and b) said antibody, or one of its functional fragments, directed against the VP2 capsid protein of BTV-8, or against an antigenic fragment of said VP2 protein of BTV-8, if appropriate labeled is chosen among the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited at the CNCM, on June 21, 2010 under the number I-4314 or by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I -4315, or one of its functional fragments. A method of selecting an antibody capable of revealing the presence of an anti-VP2 monoclonal antibody useful in an ELISA method in competition for the detection or diagnosis of an infection of an animal by a BTV, said method is intended to differentiate BTV-8 infection from BTV serotype 1 infection, preferably to differentiate BTV-8 infection from BTV serotype 1 infection selected from serotype 1, 2, 4, 9, 10 or 16 or for differentiating a BTV-8 infection from a serotype BTV infection selected from serotypes 1 to 7 or 9 to 24, characterized in that it comprises the next step: a) contacting said test antibody with the VP2 capsid protein of BTV-8, or an antigenic fragment of said VP2 protein, or a composition or compound comprising such a protein or fragment, and b) the revelation of antigen-antic complexes orps formed in (a) between said VP2 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 an ELISA method. competition using a monoclonal antibody, or one of the functional fragments, said antibody being chosen from the monoclonal antibody secreted by the hybridoma called 6C3 Id V and deposited at the CNCM, June 21, 2010 under the number I-4314 or by the hybridoma called 10E9 Id Vet deposited at the CNCM on June 21, 2010 under the number I-4315, or one of its functional fragments, where appropriate labeled, said antigenic fragment of the VP2 protein being characterized in that it is a fragment capable of binding specifically to the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited in the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 25 rue du Docte ur Roux, 75724 Paris, France, June 21, 2010 under the number 1-4314 or on the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited at the CNCM June 21, 2010 under the number I-4315; or - the monoclonal antibody secreted by the hybridoma called 6C3 Id Vet deposited under the number 1-4314 or the monoclonal antibody secreted by the hybridoma called 10E9 Id Vet deposited under the number I-4315 is directed specifically against said antigenic fragment of the VP2 protein. 21. Hybridome called 6C3 Id Vet filed at the CNCM, June 21, 2010 under the number I-4314 or called 10E9 Id Vet filed at the CNCM June 21, 2010 under the number 1-4315. A monoclonal antibody secreted by a hybridoma according to claim 21, or a functional fragment thereof, if appropriate labeled.