The present invention relates to an in vitro diagnostic method for distinguishing an animal infected with African swine fever virus (ASFV) from an animal that has been vaccinated against ASFV using an immunogenic composition comprising an attenuated ASFV in which the EP153R gene and the EP402R gene have been inactivated and in which a region of the ASFV genome comprising the EP402R and EP153R genes is replaced with a heterologous gene.
CLAIMS An in vitro diagnostic method to differentiate African Swine Fever Virus (ASFV) infected animals from animals which have been vaccinated against ASFV using an immunogenic composition comprising an attenuated ASFV in which the EP153R gene and the EP402R gene have been inactivated and wherein the region of the ASFV genome comprising the EP402R and the EP153R genes is replaced by a heterologous gene, the method comprising: (i) testing a sample from said animal for the presence of a first marker and of second marker, wherein the first marker is selected from the group consisting: an antibody against an ASFV-specific antigen which is not the EP402R gene product or the EP153R gene product and an ASFV gene or fragment thereof which is not the EP402R gene or the EP153R gene, and wherein the second marker is a “first ASFV vaccine marker” and/or a “second ASFV vaccine marker” wherein the first ASFV vaccine marker is selected from the group consisting of an antibody against the EP402R gene product or against the EP153R gene product and the EP402R gene or a fragment thereof or the EP153R gene or a fragment thereof and wherein the second ASFV vaccine marker is selected from the group consisting of: an antibody specific for the heterologous gene product or the heterologous gene or a fragment thereof and and (ii) identifying the animal as a) having been vaccinated if the at least one first marker and the second ASFV vaccine marker are detected or b) having been infected if the at least one first marker is detected and, optionally, the first ASFV vaccine marker is detected or the second ASFV vaccine marker is not detected. The method according to claim 1 wherein the identification of the animal as vaccinated is further confirmed by testing the first ASFV vaccine marker, wherein the animal is confirmed as vaccinated if said marker is not detected. . The method according to claim 1 wherein the identification of the animal as infected is further confirmed by testing the first ASFV vaccine marker, wherein said first ASFV vaccine marker is a genotype II specific marker, wherein the animal is confirmed as infected by an ASFV strain of genotype II if said marker is detected or as infected by an ASFV strain of a genotype other than genotype II if said marker is not detected. 4. The method according to claim 1 wherein the animal is identified as having been vaccinated and infected with an ASFV strain if the first marker is detected, the first ASFV vaccine marker is detected and the second ASFV vaccine marker are detected. 5. The method according to any one of claims 1 to 4 wherein the antibody against an ASFV-specific antigen which is not the EP402R gene product or the EP153R gene product is an antibody against the p72, an antibody against the CP312 or an antibody against the p30 gene product or wherein the ASFV gene which is not the EP402R gene or the EP153R gene is the p72 gene, the CP312 gene or the p30 gene. 6. The method according to any one of claims 1 to 5 wherein the first ASFV vaccine marker is an antibody against EP153R, an antibody against EP402R, the EP153R gene or a fragment thereof or the EP402R gene or fragment thereof. 7. The method according to claim 6 wherein the antibody against EP153R is specific for the EP153R as defined in SEQ ID NO:1 , wherein the EP153R gene is as defined in SEQ ID NO:2, wherein the antibody against EP402R is specific for the EP402R as defined in SEQ ID NO:3 , wherein the EP402R gene is as defined in SEQ ID NO:4. 8. The method according to any one of claims 1 to 7 wherein the heterologous gene is the eGFP gene. 9. The method according to any of claims 1 to 8 wherein the method does not comprise testing the sample for antibodies against the DP143R, the 9GL/B119L, the MGF_360- 12L, the MGF-13L and the MGF_360-14L gene products and/or wherein the method does not comprise testing the sample for the presence of the DP143R, the 9GL/B119L, the MGF_360-12L, the MGF-13L and the MGF_360-14L genes or fragments thereof. 10. The method according to any one of claims 1 to 9 wherein the first and the second marker are genes or gene fragments. 11 . The method according to claim 10 wherein the detection of the first and second markers is carried out by a polymerase chain reaction (PCR), preferably a real-time PCR. 12. The method according to claim 11 wherein each PCR fragments is detected using a specific TaqMan probe. 13. The method according to claims 11 or 12 wherein the PCR is a multiplex PCR wherein the first and second markers are detected in the same reaction. 14. The method according to claims 11 to 13 wherein: the first marker is the ASFV p72 gene and the PCR is carried out with the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6. The first ASFV vaccine marker is the EP153R gene and the PCR is carried out with the forward primer SEQ ID NO: 8 and the reverse primer of SEQ ID NO: 9 and/or The second ASFV vaccine marker is the eGFP gene and the PCR is carried out with the forward primer SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12. 15. The method according to claim 14 wherein the p72 gene PCR fragment is detected with a TaqMan probe comprising a sequence as defined in SEQ ID NO: 7, the EP153R gene PCR fragment is detected with a TaqMan probe comprising a sequence as defined in SEQ ID NO: 10 and/or the eGFP gene PCR fragment is detected with a TaqMan probe comprising a sequence as defined in SEQ ID NO: 13. 16. The method according to any of claims 14 or 15 wherein the TaqMan probe specific for p72 gene PCR fragment is labelled with 6-FAM, the TaqMan probe specific for the EP153R gene PCR fragment is labelled with JOE and/or the TaqMan probe specific for the eGFP gene PCR fragment is labelled with Cy5. 17. The method according to claim 16 wherein the TaqMan probe specific for p72 gene PCR fragment contains BHQ1 as quencher, the TaqMan probe specific for the EP153R gene PCR fragment contains BHQ1 as quencher and/or the TaqMan probe specific for the eGFP gene PCR fragment contains BBQ as quencher. 8. The method according to claim 17 wherein the reagents used for the detection of the first marker comprise a primer pair having the sequences of SEQ ID NO:5 and 6 and a TaqMan probe having the sequence 6FAM-TCCTGGCCRACCAAGTGCTT-BHQ1 (SEQ ID NO: 7), the reagents used for the detection of first ASFV vaccine marker comprise a primer pair having the sequences of SEQ ID NO:8 and 9 and a TaqMan probe having the sequence JOE*-AGGAG+AGATTAATAAA+C+CAATA+T+GTTACC- BHQ1 (SEQ ID NO: 10) and the reagents used for the detection of second ASFV vaccine marker comprise a primer pair having the sequences of SEQ ID NO:11 and 12 and a TaqMan probe having the sequence Cy5-TGTAGTTGTACTCCAGCTTGTGCC-BBQ (SEQ ID NO: 13), wherein R indicates A or G and + indicates LNA nucleotides. 19. The method according to any one of claims 1 to 9 wherein the first and second markers are antibodies. 20. The method according to any of claim 18 wherein the detection of the first and second markers is carried out by an immunoassay. 21. The method according to claim 19 wherein the immunoassay involves the capture of the antibodies using immobilized antigens which can be specifically bound by said antibodies. 22. The method according to claim 20 wherein the immunoassay involves the capture of the antibodies against the EP153R gene product and the capture is carried out using the EP153R gene product or a fragment thereof. 23. The method according to any of claims 20 to 22 wherein the immunoassay is a sandwich immunoassay wherein the antibodies are captured using the EP153R gene product or a fragment thereof and the captured antibodies are detected using antibodies specific for swine antibodies. 24. The method according to any of claims 1 to 23 wherein if the first and second markers are antibodies, the sample is a serum sample or wherein if the first and second markers are genes or gene fragments, then the sample is a blood sample. 25. A kit comprising (i) reagents suitable for the detection of a first marker, wherein the first marker is selected from the group consisting: an antibody against an ASFV-specific antigen which is not the EP402R gene product or the EP153R gene product and an ASFV gene or fragment thereof which is not the EP402R gene or the EP153R gene and (ii) reagents suitable for the detection of a second marker, wherein the second marker is a “first ASFV vaccine marker” and/or a “second ASFV vaccine marker” wherein the first ASFV vaccine marker is selected from the group consisting of an antibody against the EP402R gene product or against the EP153R gene product and the EP402R gene or a fragment thereof or the EP153R gene or a fragment thereof and wherein the second ASFV vaccine marker is selected from the group consisting of: an antibody specific for an heterologous gene product or an heterologous gene or a fragment thereof and 26. The kit according to claim 25 wherein the first marker is an antibody against an ASFV- specific antigen which is not the EP402R gene product or the EP153R gene product, in which case the reagent is the ASFV-specific antigen. 27. The kit according to claim 25 or 26 wherein the first marker is an ASFV gene or fragment thereof which is not the EP402R gene or the EP153R gene and wherein the reagents is a primer pair and/or probe which is specific for said gene or gene fragment. 28. The kit according to any one of the claims 25 to 27 wherein if the second marker is an antibody against the EP402R gene product, then the reagent is the EP402R gene product or an immunogenic fragment thereof and wherein if the second marker is an antibody against the EP153R gene product, then the reagent is the EP153R gene product or an immunogenic fragment thereof. 29. The kit according to any one of the claims 25 to 28 wherein if the second marker is the EP402R gene or a fragment thereof, then the reagent is a primer or probe specific for the EP402R gene product or the fragment thereof and wherein if the second marker is the EP153R gene or a fragment thereof, then the reagent is a primer or probe specific for the EP153R gene product or the fragment thereof. 30. The kit according to claims 28 or 29 wherein the reagent specific for the antibody against EP153R is specific for the antibody against EP153R of SEQ ID NO:1 and/or wherein the reagent specific for the antibody against EP402R is specific for the antibody against EP402R of SEQ ID NO:2. 31. The kit according to any one of claims 25 to 30 wherein if the second marker is an antibody against the heterologous gene product, then the reagent is the heterologous gene product or an immunogenic fragment thereof. 32. The kit according to any one of claims 25 to 31 wherein if the second marker is the heterologous gene or a fragment thereof, then the reagent is a primer or probe specific for the heterologous gene product or the fragment thereof. 33. The kit according to any of claims 25 to 32 wherein the ASFV-specific antigen which is not the EP402R gene product or the EP153R gene product is the p72, the CP312 and/or the p30 antigen or wherein the ASFV gene which is not the EP402R gene or the EP153R gene is the p72 gene, the CP312 gene or the p30 gene. 34. The kit according to any of claims 25 to 33 wherein the heterologous gene is the eGFP gene or wherein the heterologous gene product is the eGFP protein. 35. The kit according to any of claims 25 to 34 wherein the kit does not comprise reagents for the detection of antibodies against the DP143R, the 9GL/B119L, the MGF_360-12L, the MGF-13L and the MGF_360-14L gene products and/or does not comprise reagents for the detection of the DP143R, the 9GL/B119L, the MGF_360-12L, the MGF-13L and the MGF_360-14L genes or fragments thereof. 36. The kit according to any of claims 25 to 35 wherein the first and second reagents are primers and/or probes. 37. The kit according to claim 36 wherein the primers are the primers according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 12. 38. The kit according to claim 36 or 37 wherein the probes are the probes according to SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13. 39. The kit according to claim 38 wherein the TaqMan probe specific for p72 gene PCR fragment contains BHQ1 as quencher, the TaqMan probe specific for the EP153R gene PCR fragment contains BHQ1 as quencher and/or the TaqMan probe specific for the eGFP gene PCR fragment contains BBQ as quencher. The kit according to any of claims 25 to 35 wherein the first and second reagents are polypeptides. The kit according to claim 40 wherein the polypeptides are immobilized on a support. The kit according to claims 40 or 41 wherein the kit further comprises antibodies specific for swine antibodies. Use of a kit according to any of claims 25 to 42 in an in vitro diagnostic method to differentiate African Swine Fever Virus (ASFV) infected animals from animals which have been vaccinated against ASFV using an immunogenic composition comprising an attenuated ASFV in which the EP153R gene and the EP402R gene have been inactivated and wherein the region of the ASFV genome comprising the EP402R and the EP153R genes is replaced by a heterologous gene.