METHOD FOR THE DETECTION AND CLASSIFICATION OF PRRSV-INFECTIONS IN SWINE HERDS AND DIAGNOSTIC ANTIGEN COMPOSITIONS FOR SUCH METHODS

Abstract

Method for the detection and classification of PRRSV-infections in swine herds, comprising a) the incubation of tissue samples taken from the animals with at least one antigen capable to bind a neutralizing antibody against the Type I-virus possibly present in the animal and with at least one antigen capable to bind a neutralizing antibody against the Type II-virus possibly present in the animal, b) testing whether a binding of antibodies against the Type I-virus and/or the Type II-virus has taken place and c) determining from the presence of possible epitope-antibody complexes whether an infection of the PRRSV I-Type and/or PRRSV II-Type is present in the herd and diagnostic compositions for such a method.

Claims

1. Method for the detection and classification of PRRSV-infections in swine herds, comprising a) the incubation of tissue samples taken from the animals with at least one antigen capable to bind a neutralizing antibody against the Type I-virus possibly present in the animal and/or with at least one antigen capable to bind a neutralizing antibody against the Type II-virus possibly present in the animal, b) testing whether a binding of antibodies against the Type I-virus and/or the Type II-virus has taken place and c) determining from the presence of possible epitope-antibody complexes whether an infection of the PRRSV I-Type and/or PRRSV II-Type is present in the herd.

2. Method according to claim 1, wherein the antigens used are selected from PRRSV Type I and Type II peptide sequences containing a neutralizing epitope.

3. Method according to claim 1, wherein the peptide sequences are part of the GP4 and/or GP3 protein of the Type I and Type II serotype.

4. Method according to claim 3, wherein the peptide sequences comprise sequences according to SEQ ID NO. 6, ID NO. 7, ID No 8 and SEQ ID No: 10.

5. Method according to claim 4, wherein the peptide sequences are not longer than 50 amino acids.

6. Method according to one of the preceding claims, wherein additionally the titer of antibodies possibly present in the samples is determined.

7. Method according to claim 6, wherein on basis of the antibody titer it is determined whether a possible infection is fresh, chronic or vaccine induced.

8. Method according to claim 7, wherein on basis on the absence of neutralization antibody titers in infected animals, it is determined whether a humoral immune response is limited to non neutralizing antibodies.

9. Diagnostic composition for use in the method according to claims 1-8, including at least one antigen capable to bind a neutralizing antibody against the Type I-virus possibly present in the animal and at least one antigen capable to bind a neutralizing antibody against the Type II-virus possibly present in the animal and the necessary buffers and solutions typically present in an ELISA-assay.

Description

DESCRIPTION OF THE DRAWING FIGURES

[0037] In the following the invention shall be described further in detail by means of figures and examples:

[0038] FIG. 1 Illustration of OD 450-620 nm values for different PRRSV sera measured using EU-specific antigen,

[0039] FIG. 2 Illustration of OD 450-620 nm values for different PRRSV sera measured using US-specific antigen,

[0040] FIG. 3 Illustration of OD 450-620 nm values for different PRRSV sera measured using a combination of further EU-specific antigenic peptides,

[0041] FIG. 4 Illustration of OD 450-620 nm values for different PRRSV sera measured using a further combination of EU-specific antigenic peptides,

[0042] FIG. 5 Illustration of OD 450-620 nm values for different PRRSV sera measured using a combination of further US-specific antigenic peptides,

[0043] FIG. 6 Illustration of OD 450-620 nm values for different PRRSV sera measured using a further combination of US-specific antigenic peptides.

[0044] FIG. 1 illustrates OD 450-620 nm values for PRRS negative (CH Herds 1 to 4) and PRRSV positive sera samples (Porcilis PRRS and InIgelvac PRRS vaccinated animals) using a high binding plate coated with 1 g/ml of GP4_LV4. Stabilcoat had been used for blocking and 0.2% casein in Stabilcoat and PBS 13 1+0.1% Tween-80 had been used as sample diluent and conjugate buffers, respectively.

[0045] FIG. 2 illustrates OD 450-620 nm values for PRRS negative (CH Herds 1 to 3) and PRRS positive sera samples (Porcilis PRRS and MLV or Ingelvac PRRS vaccinated Animals) using a high binding plate coated with 10 g/ml of GP4_VR_2332_4. Stabilcoat had been used for blocking and 0.2% casein in Stabilcoat and 0.1% Tween-80+0.2% Casein in PBS 13 1 had been used as sample diluent and conjugate buffers, respectively.

[0046] FIGS. 3-6 illustrate the results of tests with combinations of further antigenic peptides on different PRRSV sera. For details see example 11.

Example 1: Development of an Indirect ELISA

[0047] a) Antibodies

[0048] A secondary antibody was used as conjugate for the detection of neutralizing antibodies (Goat anti Pig IgG (Fc) HRP; stock concentration 1 mg/ml [AbD Serotec, AAI41P]) present in the PRRS samples.

[0049] b) Antigen

[0050] The antigen used for the development of this indirect ELISA was GP4 peptide from the European PRRSV strain and the American PRRSV strain according to SEQ ID NOs: 1 and 2. The Type I peptide (EU) used shall also be designated as GP4_LV4. The stock concentration of this peptide was 3 mg/ml in H.sub.2O. Peptide GP4_VR_2332_4 corresponds to the Type II or US strain and had a stock concentration of 2 mg/ml in H.sub.2O. The optimal coating concentration of GP4_LV4 was 1 g/ml whereas for GP4_VR_2332_4 was 10 g/ml.

[0051] c) Sera Samples

[0052] Samples of known status have been used for the development of this indirect ELISA. For Type I PRRSV, a panel of serum samples from 34 pigs that had been previously vaccinated with Porcilis PRRS. For Type II PRRSV, serial sera samples were obtained from 20 pigs that had been vaccinated with Ingelvac PRRS MLV. In addition to PRRSV positive samples, 127 PRRSV negative samples from negative control animals were used for validation of the indirect ELISA. These PRRS negative samples were collected from different locations over Switzerland (Bazenheid, Basel, Zurich [ShZh, Zh]) in 2012. All of these samples were also assayed in the Idexx PRRS ELISA.

[0053] d) Details of Indirect ELISA

[0054] (i) Coating of ELISA plates: A high binding plate (Greiner Bio-One 762071-CED) was coated with 100 l of antigen solution per well. Depending of the antigen used for the coating, the final concentration was 1 g/ml for GP4_LV or 10 g/ml for GP4_VR_2332_4. The coating buffer in which the antigen was diluted in was PBS 13 1 with a pH 9.6. The incubation of the antigen took place at 4 C. without shaking overnight.

[0055] (ii) Blocking: After antigen incubation overnight, the plate was washed four times with 300 l of Washing Buffer (0.5% Tween-80 [Sigma P8074-500 ml] in PBS 13 1) using Tecan Hydroflex washer. Next, 200 l of Blocking Buffer (StabilCoat Immunoassay Stabilizer [SurModics Prod.Nr. SC01-100]) were added per well. The plate was incubated for 2 hours at room temperature without shaking. After incubation period, the Blocking Buffer was drained and the plate was dried in a 37 C. incubator for 2 hours.

[0056] (iii) Dilution of sera samples: Before testing, samples were diluted 1:50. The dilution took place in Sample diluent buffer (0.2% casein in StabilCoat).

[0057] (iv) Capture: To each well on the plate, 100 l of the 1:50 dilution of the serum sample was added. The incubation of the samples took place at room temperature for one hour without shaking. After the incubation period, the plate was washed four times with 300 l of Washing Buffer (0.05% Tween-80 [Sigma P8074-500 ml] in PBS 13 1) using Tecan Hydroflex washer.

[0058] (v) Detection: After washing the plate, 100 l of anti Pig IgG (Fc) HRP (20 ng/ml Conjugate Diluent) were added per well. Incubation took place at room temperature for 1 hour without shaking. Next, the plate was washed four times with 300 l of Washing Buffer (0.05% Tween-80 in PBS 13 1) using Tecan Hydroflex washer. After washing, 100 l of TMB substrate (SurModics TTMB-1000-01) were added per well. The plate was then incubated at room temperature for 15 minutes without shaking. For stopping the reaction, 100 l of Stop buffer solution (0.5M H.sub.2SO.sub.4 [Fluka 38294]) were added per well. The absorbance of the coloured reaction was measured using Tecan Sunrise (Measurement wavelength: 450 nm; Reference wavelengths: 620 nm; Read mode: normal).

[0059] e) Determining Cut-Off Values

[0060] To set negative/positive cut off values, both the mean and the standard deviation of the negative samples only (Bazenheid, Basel, Zurich [ShZh, Zh]) were calculated. Once both values were obtained, the standard deviation was multiplied by a factor of 5; the product of this calculation was added to the negative mean previously calculated.

TABLE-US-00002 TABLE 1 Calculation of Cut-Off values Mean Cut-Off Negative Standard Deviation (Neg. Mean + Samples of Negative Samples Std. Dev * 5) GP4_LV4 0.07 0.02 0.17 GP4_VR_2332_4 0.06 0.05 0.32

[0061] f) Calculation of Sensitivity and Specificity

[0062] The sensitivity is calculated to know the probability that a test will give a positive result when the disease is present and it can be calculated using the following formula and following the scheme above (TABLE 5):

Sensitivity=(A/A+C)*100

[0063] On the other hand, the specificity of an assay will provide information about the probability that a test will give a negative result when the disease is not present. It can be calculated using the formula below and following the scheme above (TABLE 2):

Specificity=(D/B+D)*100

TABLE-US-00003 TABLE 2 Scheme for calculating sensitivity and specificity Sample Status Positive Negative Test Positive A B A + B Negative C D C + D A + C B + D Total

[0064] g) Results

[0065] As indicated above animals that were vaccinated with EU or US type as well as PRRSV negative animals were tested in order to calculate the specificity and sensitivity of the assay (TABLES 3 and 4 and FIGS. 1 and 2) to determine the specificity and sensitivity of the indirect ELISA, a cut-off of 0.17 and 0.32 at herd level was calculated for GP4_LV4 and GP4_VR_2332_4, respectively. With the gathering of all the data, we determined that the specificity and sensitivity of GP4_LV4 was 99.41% and 55.80%, respectively, whereas for GP4_VR_2332_4, the specificity was 98.91% and the sensitivity 45%. For the calculation of the diagnostic specificity, the PRRS positive samples that were negative depending on the GP4 peptide used were considered as negative.

[0066] For the ELISA, although the sensitivity is important, it is of higher interest that the assay is specific, since the main objective of this detection method is to be able to correctly classify individuals as disease-free and avoid false positives.

TABLE-US-00004 TABLE 3 Results of optimum ELISA conditions for GP4_LV4 (PRRSV Type I) TEST GP4_LV4 (EU) Total Positive Negative Type 1 - Sera 34 19 15 Type 2 - Sera 20 0* 20 Negative PRRSV 152 1** 151 antibody serum samples

[0067] As one can take from TABLE 3, no cross detection between EU and US could be observed and only one false positive sample was observed by testing PRRSV negative antibody serum.

TABLE-US-00005 TABLE 4 Results of optimum ELISA conditions for GP4_VR_2332_(PRRSV Type II) TEST GP4_VR_2332_4 (US) Total Positive Negative Type 1 - Sera 34 0* 34 Type 2 - Sera 20 9 11 Negative PRRSV 151 2** 149 antibody serum samples

[0068] TABLE 4 shows no cross detection between EU and US and only two false positive were observed by testing PRRSV negative serum.

Examples 2-8 and 11

[0069] Examples 2-8 show the results of ELISA analysing blinded samples derived from different pig herds or sera with known status (example 11). The herein described ELISA can also be used for the assessment about the immunological status of a herd related to the presence or absence of neutralization antibodies directed against special epitopes of GP3 and GP4. Further on statements about a fresh or continuous infection situation in a given herd situation can be drawn by consideration the titer level in animals.

[0070] Result interpretation for examples 2-8 need additional information alongside the discriminating ELISA e.g. information about the single animal, herd status, and the result of a screening ELISA is needed.

[0071] For examples 2-8 and 11 the experiments were done in the identical manner as described for Example 1 with the exceptions that the antigens represented combinations of GP3 and GP4 peptides. The used peptide sequences in examples 2-8 are indicated in the following:

[0072] Peptides Used:

TABLE-US-00006 TypeI(EU)PRRSV GP3LV30AA54(SEQIDNO.1): ICMPCSTSQAARGRLEPGRSNMCRKGHDRC GP4LV4(SEQIDNO.2): DINCFRPHGVSAAQEKISFGKSSQCREAVGTP TypeII(US) GP3VR(SEQIDNO.3): VCPPCLTRQAATEIYEPGRSLWCRIGYDRCEEDHDELGFM GP4VR(SEQIDNO.4): DISCLRHRDSASEAIRKIPQCRTAIGTP Theadditionalpeptidesequencesusedin example11areindicatedinthefollowing: TypeI(EU)PRRSV GP3(SEQIDNO.5): STSQAARQRLEPGRNMWCKIGHDRCEER GP3(SEQIDNO.6): STSQAARQRLEPGRNMW GP4(SEQIDNO.7): FRPHGVSAAQEKISFGKSS TypeII(US) GP3(SEQIDNO.8): YEPGRSLWCRIGYDRCGEDD GP3(SEQIDNO.9): IYEPGRSLWCRIGYDRCGEDDHDEL GP4(SEQIDNO.10): HRDSASEAIRKIPQCRTAI

Example 2: PRRS Negative Herd (Nucleus Herd)

[0073] Herd Status: Animal tested derived from a boar nucleus herd. Boars will never be vaccinated against PRRSV and are closed meshed measured for the presence of PRRSV.

[0074] Animal Status: Animals are predicted to be negative for PRRSV directed antibodies.

[0075] Screening ELISA is negative as well as the discrimination ELISA.

[0076] Test interpretation: Animals are negative for PRRSV.

[0077] In the following the results are summarized in TABLES 5 and 6.

TABLE-US-00007 TABLE 5 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU) (US) Info Animal Site Info Neg X X X Boars PRRSV negative Pos farm Titre* <0.3 <0.3 Nucleus herd

TABLE-US-00008 TABLE 6 Actual test results Information Discriminatory ELISA to animals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status (EU) (US) Idexx X3 ELISA 12-2414 BOARS 0.026 0.068 Neg 12-2415 PRRS 0.060 0.032 Neg 12-2416 negativer 0.051 0.069 Neg 12-2417 animals 0.036 0.034 Neg 12-2418 0.065 0.092 Neg

Example 3: PRRS Negative Herd

[0078] Herd Status: Unknown. However, boars will never be vaccinated against PRRSV and are closed meshed measured for the presence of PRRSV.

[0079] Animal Status: Unknown

[0080] Screening ELISA is negative as well as the discrimination ELISA.

[0081] Test interpretation: Animals are negative for PRRSV.

[0082] In the following the results are summarized in TABLES 7 and 8.

TABLE-US-00009 TABLE 7 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU) (US) Info Animal Site Info Neg X X X Boars PRRS Pos negative herd Titre* <0.3 <0.3

TABLE-US-00010 TABLE 8 Actual test results Information Discriminatory ELISA to animals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status (EU) (US) Idexx X3 ELISA 13-1107 PRRS 0.109 0.119 Neg 13-1108 negative 0.079 0.082 Neg 13-1109 herd 0.029 0.049 Neg 13-1110 0.037 0.057 Neg 13-1111 0.057 0.058 Neg

Example 4: Herd with Repeated PRRS Infection

[0083] Herd Status: Herd was positive tested in the past for the presence of PRRSV EU directed antibodies over years. Boars are continuously infected (repeated infections) with PRRSV. Boars will never be vaccinated against PRRSV and are closed meshed measured for the presence of PRRSV.

[0084] Animal Status: Unknown

[0085] Screening ELISA is positive but not the discrimination ELISA.

[0086] Test interpretation: Animals losing their capability to establish a neutralising humoral immune response in a continuous, repeated PRRSV infection situation. However, these animals turned positive in a screening ELISA not measuring neutralising antibodies. These animals, even tested positive in the screening ELISA do not have anymore neutralising antibodies, indicating that a considerable proportion of antibodies are not present anymore.

[0087] .fwdarw.Even aware that boars demonstrate only minor clinical signs of a PRRSV infection (e.g. fever) those animals may have a minor resistance capability against a heterologous PRRSV challenge (infection with PRRSV Type II in this assumed case, as animals are infected with EU type PRRSV).

[0088] In the following the results are summarized in TABLES 9 and 10.

TABLE-US-00011 TABLE 9 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU) (US) Info Animal Site Info Neg X X Boars Farm with Pos X seroreactors, Titre* <0.3 <0.3 Continuous EU infected herd

TABLE-US-00012 TABLE 10 Corresponding ELISA Results Information Discriminatory ELISA to animals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status (EU) (US) Idexx X3 ELISA 13-19 Farm with 0.082 0.131 Pos! 13-22 seroreactors, 0.070 0.061 Pos! 13-24 Continuous 0.076 0.100 Pos! 13-25 EU infected 0.043 0.072 Pos! 13-26 herd 0.038 0.087 Pos!

Example 5: Repeated EU Infected Boar Herd, Freshly Infected with US

[0089] Herd Status: Herd was positive tested in the past for the presence of PRRSV EU directed antibodies over years and turned recently positive for US PRRSV as proved by positive US PRRSV PCR results. Boars are continuously infected (repeated infections) with PRRSV. Boars will never be vaccinated against PRRSV and are closed meshed measured for the presence of PRRSV.

[0090] Animal Status: Unknown

[0091] Screening ELISA is positive and discrimination ELISA too.

[0092] Test interpretation: Animals losing their capability to establish a neutralising humoral immune response in a continuous, repeated PRRSV infection situation against EU PRRSV. However, these animals turned positive in the discrimination ELISA for PRRSV Type II (US). These animals tested positive in the screening ELISA.

[0093] Titre for US PRRSV are very high (>2 OD in average) indicating that a fresh infection with US PRRSV occurred in the herd. This test interpretation could be confirmed by PCR test results, demonstrating that the infection turned out to be in the viraemic phase.

[0094] Animals may have a minor resistance capability against a heterologous PRRSV challenge (infection with PRRSV Type II in this assumed case, as animals are infected with EU type PRRSV). Sows at gestation day 90, vaccinated with one of the MLVs but infected with a counter field PRRSV isolate would be the more profound sub species to prove the limited protection of the animals.

[0095] In the following the results are summarized in TABLES 11 and 12.

TABLE-US-00013 TABLE 11 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU) (US) Info Animal Site Info Neg X Boar Continuous EU Pos X X infected Titre* <0.3 >1 herd (minimal 3 years), US positive since January 2013

TABLE-US-00014 TABLE 12 Corresponding ELISA Results Information Discriminatory ELISA to animals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status (EU) (US) Idexx X3 ELISA 12-2448 Continuous 0.140 0.381 Pos! 12-2449 EU infected 0.040 0.052 Neg 12-2450 herd 0.040 0.061 Neg 12-2451 (minimal 3 0.045 0.057 Neg 12-2452 years), US 0.044 0.051 Neg 12-2453 positive 0.043 0.055 Neg 12-2454 since 0.062 2.291 Pos! 12-2455 January 0.095 2.055 Pos! 12-2456 2013 0.044 2.341 Pos! 12-2457 0.177 2.478 Pos!

Example 6: Maternal Interference or Building of First Neutralizing Antibodies

[0096] Herd Status: Piglets four weeks of age. Normally the age one week after vaccination against PRRSV. It is unknown if the sows have been vaccinated or not (based on the discrimination ELISA): It is predicted that the sows have been vaccinated with Porcilis PRRSV (EU PRRSV).

[0097] Animal Status: Unknown

[0098] Screening ELISA is positive and discrimination ELISA, too.

[0099] Test interpretation: Animals are actually too young to establish a humoral immune response against PRRSV. It is likely to assume that maternal interference (transfer of neutralising antibodies from sow to pig via suckling). It might be also possible that some mAB derived from the piglet immune system. Infection with wild PRRSV is unlikely due to the age of the piglets and the low titre measured in the discrimination ELISA.

[0100] In the following the results are summarized in TABLES 13 and 14.

TABLE-US-00015 TABLE 13 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU) (US) Info Animal Site Info Neg X piglets of 4 weeks, unknown Pos X X fattening Titre* <1 <0.3

TABLE-US-00016 TABLE 14 Corresponding ELISA Results Discriminatory Information ELISA to animals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status (EU) (US) Idexx X3 ELISA 13-2721 unknown 0.682 0.098 Neg 13-2722 0.596 0.250 Neg 13-2724 0.175 0.035 Pos! 13-2725 0.923 0.206 Pos! 13-2727 0.079 0.059 Neg

Example 7: Ingelvac Vaccinated Herd (US PRRSV Positive)

[0101] Herd Status: Piglets are ten weeks of age. Normally, this is the time six weeks after the vaccination against PRRSV normally took place. Pigs should demonstrate an intensive humoral immune response and high titre level are expected.

[0102] Animal Status: Unknown

[0103] Screening ELISA is positive and discrimination ELISA too.

[0104] Test interpretation: Animals are six weeks after vaccination. Based on the fact that only US PRRSV could be detected, with very high titres (average >1.0 OD), it is likely that these animals have been vaccinated with Ingelvac PRRSV (US PRRSV).

[0105] In the following the results are summarized in TABLES 15 and 16.

TABLE-US-00017 TABLE 15 Discriminatory Reference ELISA Test Type I Type II Idexx X3 (EU) (US) Info Animal Site Info Neg X fattening, 30 kg, unknown Pos X X ca. 10 weeks of age Titre* <0.3 >1

TABLE-US-00018 TABLE 16 Corresponding ELISA Results Information to animals Discriminatory ELISA and OD 450 nm-620 nm Sample PRRS Type I Type II Reference Test No Status (EU) (US) Idexx X3 ELISA 13-2738 unknown, 0.024 0.126 Neg 13-2743 30 kg 0.051 0.498 Pos! 13-2744 0.038 0.205 Pos! 13-2748 0.031 0.756 Pos! 13-2749 0.033 0.691 Pos! 13-2753 0.055 2.894 Pos! 13-2754 0.028 0.757 Neg 13-2755 0.030 0.177 Neg 13-2756 0.042 0.297 Pos! 13-2757 0.085 0.405 Neg 13-2761 0.033 2.703 Pos! 13-2762 0.068 2.545 Pos! 13-2763 0.023 0.395 Pos! 13-2764 0.039 0.193 Neg 13-2765 0.063 2.748 Pos! 13-2767 0.047 2.379 Neg 13-2768 0.113 2.788 Pos! 13-2772 0.066 1.031 Pos! 13-2777 0.107 2.769 Pos! 13-2778 0.070 2.783 Pos!

Example 8: Pig Herd Infected with Type I and II (EU and US) of PRRSV

[0106] Herd Status: Herd was positive tested in the past for the presence of PRRSV EU directed antibodies over years and turned recently positive for US PRRSV. Boars are continuously infected (repeated infections) with PRRSV. Boars will never be vaccinated against PRRSV and are closed meshed measured for the presence of PRRSV.

[0107] Animal Status: Unknown

[0108] Screening ELISA is positive and discrimination ELISA for EU and US, too.

[0109] Test interpretation: Animals tested positive in the past for EU PRRSV and very recently for US PRRSV. These animals tested positive in the screening ELISA. Titre for US PRRSV are very high (>1 OD in average) indicating that a fresh infection with US PRRSV occurred in the herd.

[0110] Titre for EU PRRSV are low (<1 OD in average) indicating that an older infection with EU PRRSV occurred in the herd.

[0111] .fwdarw.Old infection with EU PRRSV and fresh infection with US PRRSV

[0112] In the following the results are summarized in TABLES 17 and 18.

TABLE-US-00019 TABLE 17 Reference Discriminatory ELISA Test Type I Type II Idexx X3 (EU) (US) Info Animal Site Info Neg Boars Pos X X X Titre* <1 >1

TABLE-US-00020 TABLE 18 Corresponding ELISA Results Information Discriminatory ELISA to animals OD 450 nm-620 nm Sample and PRRS Type I Type II Reference Test No Status (EU) (US) Idexx X3 ELISA Boar 1 Boars, 0.037 0.044 Neg Boar 2 0.031 0.029 Neg Boar 3 0.052 0.063 Neg Boar 4 0.039 0.038 Neg Boar 5 0.115 2.408 Pos! Boar 6 0.130 0.958 Pos! Boar 7 0.030 0.038 Neg Boar 8 0.035 0.063 Pos! Boar 9 0.071 1.544 Pos! Boar 10 0.267 2.584 Pos! Boar 11 0.084 0.095 Neg Boar 12 0.125 2.614 Pos! Boar 13 0.035 0.423 Pos! Boar 14 2.310 0.964 Pos! Boar 15 0.440 1.539 Pos! Boar 16 0.455 1.156 Pos! Boar 17 0.035 0.044 Pos! Boar 18 0.041 0.057 Pos! Boar 19 0.029 0.031 Pos! Boar 20 0.037 0.036 Neg Boar 21 0.037 0.079 Pos! Boar 22 0.092 0.257 Pos! Boar 23 0.057 0.092 Pos! Boar 24 0.103 1.192 Pos! Boar 25 0.040 0.053 Pos!

Example 9: Results of ELISA Assays Analyzing Serum Samples of Known PRRS Status

[0113] Discriminatory ELISA assays were performed on pig serum samples with known PRRS Status. 32 sera derived of vaccinated pigs (Type I (EU)), 20 sera derived of vaccinated pigs (Type II (US)) and 46 sera of PRRSV negative pigs were analyzed on EU specific peptides (left column) and US specific peptides (right column).

[0114] The results are depicted in TABLE 19.

[0115] The first number indicates amount of sample values over cutoff, right number indicates the total number of sample analyzed per cohort. In the 2 last rows the respective specificities and sensitivity is calculated.

TABLE-US-00021 TABLE 19 ELISA for ELISA for PRRSV Type 1 PRRSV Type 2 (EU) Peptides (US) Peptides GP3 and GP4 GP3 and GP4 Number of Number of PPRSV corectly tested corectly tested status of Type of PRRSV animals/Number animals/Number samples strain of tested animals of tested animals Positive PRRSV Type 1 (EU) 20/32 7/32 Positive PRRSV Type 2 (US) 0/46 19/20 Negative 0/46 2/46 Sensitivity 63% 95% Specificity 100% 97%

Example 10: Diagnostic Composition (Kit)

[0116] A typical kit which can be used in the method according to the invention includes the components given in TABLE 20:

TABLE-US-00022 TABLE 20 Component Name Description Component 1 High binding 96-well plate coated with PRRSV Type I/II Test Plate specific peptides as follows: row 1, 3, 5, 7, 9, 11 coated with GP3/GP4 peptides specific for PRRSV Type I (EU) row 2, 4, 6, 8, 10 12 coated with GP3/GP4 peptides specific for PRRS Type II (US) Component 2 Buffer used for diluting serum samples to be analyzed on Sample Diluent the Test Plate (Component 1) Component 3 Buffer used for rinsing unbound assay components after Washing Fluid (20x) the sample incubation step and conjugate incubation step. Component 4 Horse radish peroxidase labelled anti-pig antibody used for Conjugate (30x) detection of antibodies bound to PRRSV Type I and Type II peptides. Component 5 Buffer used for diluting the Conjugate (Component 4) Conjugate Diluent during the conjugate incubation step. Component 6 Serum of pig containing high antibodies titer specific for Positive Control EU PRRSV Type I (EU) Component 7 Serum of pig containing high antibodies titer specific for Positive Control US PRRSV Type II (US) Component 8 Serum of pig containing low antibodies titer specific for Weak Positive Control EU PRRSV Type I (EU) Component 9 Serum of pig containing low antibodies titer specific for Weak Positive Control US PRRSV Type II (US) Component 10 Serum of pig containing no antibody to the PRRS virus Negative Control Component 11 Solution used as Enzymatic Substrate for HRP producing a Substrate color reaction Component 12 Solution used for stopping and stabilizing the color Stop Solution development

Example 11: Performance of Cocktails Including Further GP3 and GP 4 Peptides in Diagnostic Tests

[0117] For this example for each case 4 Type I (EU) positive, 4 Type II (US) positive and 4 PRRS negative samples were analyzed by indirect ELISA (as described under example 1 in detail) using different cocktails of antigens. The results are discussed below referring to FIGS. 3-6.

[0118] a) Cocktail of Type I (EU)-Specific GP3 and GP4 Peptides

[0119] In this assay peptides according to SEQ ID NO. 5 (GP3) and SEQ ID NO. 7 (GP4) were used in combination. The results are shown in FIG. 3. One can see that all 4 Type I positive samples showed a clear signal with high intensity while the remaining further samples were identified as clearly negative.

[0120] b) Cocktail of Type I (EU)-Specific GP3 and GP4 Peptides

[0121] In this assay peptides according to SEQ ID NO. 6 (GP3) and SEQ ID NO. 2 (GP4) were used in combination. The results are shown in FIG. 4. One can see that all 4 Type I positive samples were identified positive with, however, signals of different intensity depending on the sample. The remaining Type II (US) positive and PRRS negative samples showed only weak signals and could still be clearly discriminated as negative.

[0122] c) Cocktail of Type II (US)-Specific GP3 and GP4 Peptides

[0123] In this assay peptides according to SEQ ID NO. 8 (GP3) and SEQ ID NO. 10 (GP4) were used in combination. The results are shown in FIG. 5. One can see that all 4 Type II positive samples were identified positive with, however, signals of different intensity depending on the sample. The remaining Type I (EU) positive and PRRS negative samples showed only weak signals and could still be clearly discriminated as negative.

[0124] d) Cocktail of Type II (US)-Specific GP3 and GP4 Peptides

[0125] In this assay peptides according to SEQ ID NO. 9 (GP3) and SEQ ID NO. 10 (GP4) were used in combination. The results are shown in FIG. 6. One can see that all 4 Type II positive samples were identified positive with signals of higher intensity compared to example c. The remaining Type I (EU) positive and PRRS negative samples again showed only weak or no signals and could still be clearly discriminated as negative.