US20200096509A1

US20200096509A1 - Method for the detection and classification of prrsv-infections in swine herds and diagnostic antigen compositions for such methods

Info

Publication number: US20200096509A1
Application number: US16/706,371
Authority: US
Inventors: Alexa RAEBER; Franziska Kuhn; Bjorn SCHROEDER; Maria FRIAS BLANCO
Original assignee: Prionics AG
Current assignee: Prionics AG
Priority date: 2013-05-15
Filing date: 2019-12-06
Publication date: 2020-03-26
Also published as: EP3534160A1; US20180364232A1; US10520506B2; WO2014183870A1; EP2997375B1; US20160116471A1; EP2997375A1; US9977022B2; EP2804000A1

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.

Description

CROSS REFERENCE TO PRIORITY APPLICATIONS

This Application is a divisional application under 35 U.S.C. § 120 of pending U.S. application Ser. No. 15/969,773 filed May 3, 2018, which is a divisional of U.S. application Ser. No. 14/890,643 filed Nov. 12, 2015 (now U.S. Pat. No. 9,977,022), which is a U.S. National Phase Stage under § 371 of PCT/EP2014/001301 filed May 14, 2014, which claims priority to EP13002540.6 filed May 15, 2013. The entire contents of the aforementioned applications are incorporated by reference herein.

SEQUENCE LISTINGS

This application contains a nucleotide and/or amino acid Sequence Listing which has been submitted as LT00998US_ST25.txt, via EFS-Web and is hereby incorporated by reference in its entirety.
Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating viral diseases causing significant economic losses to the swine industry. The agent responsible for this endemic disease is the PRRS virus (PRRSV) which is easily transmitted via infected pigs through urine, semen and fomites, infecting almost exclusively pig monocytes and macrophages.
Porcine reproductive and respiratory syndrome (PRRS) emerged first in the United States of America in 1987 and subsequently spread worldwide. The syndrome is characterized mainly by reproductive failure in pregnant sows, high mortality in piglets and respiratory problems such as dyspnoea. The cause of these symptoms is an enveloped positive single-stranded RNA virus. The PRRSV belongs to the Arteriviridae family and it infects almost exclusively swine monocytes and macrophages.
The genome is approximately 15 kb and has an UTR region on 5′ followed by nine open reading frames (ORF): ORF1a, ORF1b, ORFs2a and 2b, 3 to 7. ORF1a and ORF1b constitute about 75% of the virus genome encoding for proteins with replicase and polymerase activities, whereas ORF 2 to 5 appear to encode for membrane-associated proteins or glycoproteins (GP). ORF6 encodes for a viral unglycosylated membrane protein M and ORF7 encodes for a highly conserved basic protein, N, that constitutes the nucleocapsid. Mutations take place predominantly in the nsp2 region of ORF 1a and the envelope proteins GP3, GP4 and GP5.
Both Type I and Type II strains have a number of neutralizing and immunodominant epitopes.
Sensitivity to antibody-mediated neutralization was tested for a selected number of antigen regions (AR) by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, one in GP3 turned out to be targets for virus neutralizing antibodies. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than Lelystad Virus (PRRSV type 1) also induces virus neutralizing antibodies. This provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus (vanhee et al; 2011).
Since ARs GP3 and GP4 were both immunogenic and target for neutralizing antibodies, the serum antibody response against these ARs was further studied (vanhee et al; 2011). While antibodies against GP4 appeared relatively fast upon infection in a majority of the animals, it clearly lasted longer for most animals to develop antibodies against AR GP3. Moreover, antibody titers in sera and alveolar fluids against GP3 were invariably lower than against GP4.
Based on the genetic differences of the virus, it is possible to encounter two main types: European genotype (Lelystad virus or Type I) and American genotype (VR-2332 virus or Type II). These PRRSV genotypes have a nucleotide identity between 55% and 70% when both genomes are compared.
Protection against PRRSV infection is not possible and prevention of virus replication is not mediated by the humoral response alone. Cellular immunity is critical to prevent clinical signs of PRRS.
Nowadays, there are mainly two commercially available modified live vaccines (MLV) on the market; one is composed of an EU type (Porcilis® PRRS) and the other one of a US type PRRSV (Ingelvac® PRRS). Both vaccines demonstrate an overall efficiency and are widely used for the prevention of PRRSV induced clinical symptoms.
PRRS MLV vaccine confers effective genotype/strain-specific protection, but provides only partial protection against genetically heterologous PRRSV. Before the emergence of atypical PRRS, it was generally assumed that the reproductive consequences of infection with PRRSV, including abortion, were due to the direct effect of the virus on the conceptus following transplacental infection. While this is certainly true for many cases of PRRSV-induced reproductive failure, it apparently does not explain all cases, because many litters aborted during epidemics of atypical PRRS were found free of infection. The latter observation indicates that abortion can also be the result of a systemic reaction. The additional observation that abortions were common in herds that had previously been vaccinated raised the question of whether an acute—anamnestic immune response contributed to the clinical picture. If so, there is a possibility that exposure to a virulent field strain of PRRSV other than that used in the vaccine elicits a response different than what would have followed exposure of a naive gilt or sow to the same strain. That is not to say vaccination is not beneficial overall, but it is possible that prevalence of one aspect of reproductive failure, namely abortion, may be slightly higher in vaccinated herds than in non-vaccinated herds. (W. L. Mengeling, K. M. Lager, A. C. Vorwald The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance. Animal Reproduction Science 60-61_2000. 199-210)
In such cases, sows often lack neutralizing antibodies against one type of PRRSV. This might be why it is imperative to continuously vaccinate herds with one type of PRRS MLV vaccine since it may displace the other PRRSV type in the herd. In case of a field infection with the counter PRRSV strain, these animals may not be properly protected; cross protection of vaccines against PRRSV of the other type is not fully convincingly shown.
WO 2010/062395 discloses an assay which allows the differentiation of serum antibodies against Type I and/or Type II PRRSV utilizing PRRSV nsp7 from both strains as antigen.
In order to improve vaccine and sanitary management in farms it might be of interest to be aware of a more detailed infectious status of a herd, e.g. to distinguish chronic infections or vaccination from fresh infections.
Such information can be crucial in the further treatment of the animals.
The object of the invention is to provide an improved test method for PRSSV and diagnostic compositions for such method.
According to the invention a method is provided for the detection and classification of PRRSV-infections in swine herds, comprising 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. After incubation it is tested, whether a binding of antibodies against the Type I-virus and/or the Type II-virus with the antigens has taken place and it is determined from the presence of possible antigen-antibody complexes whether an infection of the PRRSV I-Type and/or PRRSV II-Type is present in the herd.
The term tissue is used in a broad sense. Apart from its literal meaning it shall also encompass body fluids like blood, plasma, serum or urine.
The antigens used are selected from PRRSV Type I and Type II peptide sequences containing a neutralizing epitope.
Peptides of GP3 Type I (EU) which can be used with the invention include peptides containing a sequence according to SEQ ID NO. 6. Examples for such peptides are apart from a peptide consisting of SEQ ID NO. 6 peptides with sequences according to SEQ IDs NO. 1 and 5.
Peptides of GP3 Type II (US) which can be used with the invention include peptides containing a sequence according to SEQ ID NO. 8. Examples for such peptides are apart from a peptide consisting of SEQ ID NO. 8 peptides with sequences according to SEQ IDs NO. 3 and 9.
Peptides of GP4 Type I (EU) which can be used with the invention include peptides containing a sequence according to SEQ ID NO. 7. A further example for such a peptide apart from a peptide consisting of SEQ ID NO 7 is a peptide with a sequence according to SEQ ID NO. 2.
Peptides of GP4 Type I (US) which can be used with the invention include peptides containing a sequence according to SEQ ID NO. 10. A further example for such a peptide apart from a peptide consisting of SEQ ID NO 10 is a peptide with a sequence according to SEQ ID NO. 4.
A list of the peptides mentioned above is given in the following:

	Type I (EU) PRRSV
	SEQ ID NO: 1 (GP3 LV 30AA54):
	ICMPCSTSQA ARGRLEPGRS NMCRKGHDRC

	SEQ ID NO: 2 (GP4 LV4)
	DINCFRPHGV SAAQEKISFG KSSQCREA VGTP

	SEQ ID NO. 5 (GP3):
	STSQAARQRL EPGRNMWCKI GHDRCEER

	SEQ ID NO. 6 (GP3)::
	STSQAARQRL EPGRNMW

	SEQ ID NO. 7 (GP4)::
	FRPHGVSAAQ EKISFGKSS

	Type II (US) PRRSV
	SEQ ID NO: 3 (GP3 VR):
	VCPPCLTRQA ATEIYEPGRS LWCRIGYDRC EEDHDELGFM

	SEQ ID NO: 4 (GP4 VR):
	DISCLRHRDS ASEAIRKIPQ CRTAIGTP

	SEQ ID NO. 8 (GP3)::
	YEPGRSLWCR IGYDRCGEDD

	SEQ ID NO. 9 (GP3)::
	IYEPGRSLWC RIGYDRCGED DHDEL

	SEQ ID NO. 10 (GP4)::
	HRDSASEAIR KIPQCRTAI

The above mentioned peptides are examples. It is clear for a person skilled in the art that further peptides containing sequences according to SEQ IDs NO. 6, 7, 8 or 10, respectively, can have similar antigenic properties which allow the diagnostic testing of animals and thus are also encompassed by the invention. The length of such peptides is limited by their ability to differentiate between PRRSV Type I and II in diagnostic tests. Such ability should be given for peptides not longer than 50 amino acids including the above mentioned sequences.
GP4 and GP3 protein is very well conserved between genotypes and it can be used as a target for extremely specific serological detection by e.g. indirect Enzyme-Linked Inmunosorbent Assay (ELISA), due to its capacity to produce neutralizing antibodies. The above mentioned epitopes have been proven to induce the generation of neutralizing antibodies first by vanhee et al; 2011, but previously described by Oleksiewicz M B, Botner A, Toft P, Grubbe T, Nielsen J, Kamstrup S, et al. Emergence of porcine reproductive and respiratory syndrome virus deletion mutants: correlation with the porcine antibody response to a hypervariable site in the ORF 3 structural glycoprotein. Virology 2000; 267: 135-40. Oleksiewicz MB, Botner A, Toft P, Normann P, Storgaard T. Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes. J Virol 2001; 75: 3277-90.
Especially preferred is to use the sequences according to SEQ ID NO: 2, 4 7 and 10 of GP4 which as stated above contains a neutralizing and immunodominant epitope in its N-terminal for both Type I and Type II strains. Although most pigs are able to develop antibodies against both ARs, GP4 clearly shows the highest immunogenicity in the context of infection. There is evidence from recent studies that AR GP4 is susceptible to antibody-mediated selective pressure in vivo, and its high variability confirms that aminoacid changes in this region are generally well tolerated by the virus.
When the GP4 sequences of aminoacids of both Type I and Type II strains are compared ([GenBank: AAA46277.1]; [GenBank: ABB18277.1]), it is observed that the neutralizing epitope is situated within the most variable region of the GP4 protein. Since GP4 shows such great variability in both genotypes, it can be used as a very specific target for the detection of the virus in infected pigs.
As further specified below it is possible to e.g. develop indirect ELISA detection method using a peptide containing a neutralizing epitope in order to discriminate Type 1 and Type 2 PRSSV.
In a further preferred embodiment it is provided that to use a cocktail of peptides, e.g. the above mentioned SEQ ID Nos: 1-4. The use of such a cocktail leads at least in some cases to a broader immune response.
As stated above the main feature of the invention is that method uses antigenic sequences which include neutralizing epitopes. By using such antigens it is possible to detect the presence of neutralizing antibodies possibly present in the animals. However, neutralizing antibodies are only present in animals for a certain period post infection or vaccination. In case that PPRSV is permanent present in a given herd situation (boars) or repeated vaccination (in the following such cases will be referred to as repeated infections) it may happen that in infected animals no neutralizing antibodies are detectable.
For such cases it may be preferable if an additional standard assay for PRRSV is performed, which detects antibodies normally directed against non neutralizing eptopes (e.g. GP7). In an infectious or vaccinated situation animals with repeated infection turned positive in such a screening test.
With this embodiment of the method a very detailed classification of the status of possible infections in the herd is possible especially if further information on the herd normally available, like e.g. date of vaccination etc., is taken into account. If e.g. the standard test is positive and the titre of neutralizing antibodies is negative or low this means that there is a chronic infection in the herd. If on the other hand the standard assay is positive and the test for neutralizing antibodies provides a high titre this means that there is a fresh infection in the herd.
Summing up the method according to the invention allows a much more detailed evaluation of the infection status in a herd compared to known assay formats since it considers the humoral immune response against neutralizing epitopes in addition to the information available on the herd. On the basis of such evaluation pork producers can e.g. assess the risk of facing clinical signs of a PRRSV infection despite vaccination and can adapt their vaccination strategy, e.g. consider treating animals with either a commercially available Type I or Type II modified live vaccine. Additionally, the measurement of the neutralizing antibody titre against PRRSV Type I or Type II may also give an indication of the fitness of a herd against potential PRRSV infection if neutralisation AB titre of only one type can be detected but infected with the other one. This is the reason why a high specificity test as described below capable of distinguishing between Type I and Type II in one single run may benefit the PRRSV diagnostic.
The invention is also directed to diagnostic compositions which can be used in the method according to the invention. Such compositions include at least the GP4 sequences indicated above, but can also include further antigenic sequences (GP3). As a rule the sequences are immobilized, e.g. by coating on a plate or coupling to beads. The compositions furthermore include all buffers and controls which normally are present in such compositions. A detailed composition is given in Example 1.

DESCRIPTION OF THE DRAWING FIGURES

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

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

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

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

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

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

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

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 μg/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.
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 μg/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.
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

a) Antibodies
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.
b) Antigen
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 H₂O. Peptide GP4_VR_2332_4 corresponds to the Type II or US strain and had a stock concentration of 2 mg/ml in H₂O. The optimal coating concentration of GP4_LV4 was 1 μg/ml whereas for GP4_VR_2332_4 was 10 μg/ml.
c) Sera Samples
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.
d) Details of Indirect ELISA
(i) Coating of ELISA plates: A high binding plate (Greiner Bio-One 762071-CED) was coated with 100 μl of antigen solution per well. Depending of the antigen used for the coating, the final concentration was 1 μg/ml for GP4_LV or 10 μg/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.
(ii) Blocking: After antigen incubation overnight, the plate was washed four times with 300 μl of Washing Buffer (0.5% Tween-80 [Sigma P8074-500 ml] in PBS 13 1×) using Tecan Hydroflex washer. Next, 200 μl 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.
(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).
(iv) Capture: To each well on the plate, 100 μl 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 μl of Washing Buffer (0.05% Tween-80 [Sigma P8074-500 ml] in PBS 13 1×) using Tecan Hydroflex washer.
(v) Detection: After washing the plate, 100 μl 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 μl of Washing Buffer (0.05% Tween-80 in PBS 13 1×) using Tecan Hydroflex washer. After washing, 100 μl 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 μl of Stop buffer solution (0.5M H₂SO₄[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).
e) Determining Cut-Off Values
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 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

f) Calculation of Sensitivity and Specificity
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
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 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

g) Results
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.
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 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

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 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

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

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.
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.
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:
Peptides Used:

	Type I (EU) PRRSV
	GP3 LV 30AA54 (SEQ ID NO. 1):
	ICMPCSTSQA ARGRLEPGRS NMCRKGHDRC

	GP4 LV4 (SEQ ID NO. 2):
	DINCFRPHGV SAAQEKISFG KSSQCREA VGTP

	Type II (US)
	GP3 VR (SEQ ID NO. 3):
	VCPPCLTRQA ATEIYEPGRS LWCRIGYDRC EEDHDELGFM

	GP4 VR (SEQ ID NO. 4):
	DISCLRHRDS ASEAIRKIPQ CRTAIGTP

	The additional peptide sequences used in
	example 11 are indicated in the following:
	Type I (EU) PRRSV
	GP3 (SEQ ID NO. 5):
	STSQAARQRL EPGRNMWCKI GHDRCEER

	GP3 (SEQ ID NO. 6):
	STSQAARQRL EPGRNMW

	GP4 (SEQ ID NO. 7):
	FRPHGVSAAQ EKISFGKSS

	Type II (US)
	GP3 (SEQ ID NO. 8):
	YEPGRSLWCR IGYDRCGEDD

	GP3 (SEQ ID NO. 9):
	IYEPGRSLWC RIGYDRCGED DHDEL

	GP4 (SEQ ID NO. 10):
	HRDSASEAIR KIPQCRTAI

Example 2: PRRS Negative Herd (Nucleus Herd)

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.
Animal Status: Animals are predicted to be negative for PRRSV directed antibodies.
Screening ELISA is negative as well as the discrimination ELISA.
Test interpretation: Animals are negative for PRRSV.
In the following the results are summarized in TABLES 5 and 6.

	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 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

Herd Status: Unknown. However, boars will never be vaccinated against PRRSV and are closed meshed measured for the presence of PRRSV.
Animal Status: Unknown
Screening ELISA is negative as well as the discrimination ELISA.
Test interpretation: Animals are negative for PRRSV.
In the following the results are summarized in TABLES 7 and 8.

	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 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

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.
Animal Status: Unknown
Screening ELISA is positive but not the discrimination ELISA.
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.
→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).
In the following the results are summarized in TABLES 9 and 10.

	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 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

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.
Animal Status: Unknown
Screening ELISA is positive and discrimination ELISA too.
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.
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.
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.
In the following the results are summarized in TABLES 11 and 12.

	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 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

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).
Animal Status: Unknown
Screening ELISA is positive and discrimination ELISA, too.
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.
In the following the results are summarized in TABLES 13 and 14.

	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 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)

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.
Animal Status: Unknown
Screening ELISA is positive and discrimination ELISA too.
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).
In the following the results are summarized in TABLES 15 and 16.

	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 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

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.
Animal Status: Unknown
Screening ELISA is positive and discrimination ELISA for EU and US, too.
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.
Titre for EU PRRSV are low (<1 OD in average) indicating that an older infection with EU PRRSV occurred in the herd.
→Old infection with EU PRRSV and fresh infection with US PRRSV
In the following the results are summarized in TABLES 17 and 18.

	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 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

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).
The results are depicted in TABLE 19.
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 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)

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

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

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.
a) Cocktail of Type I (EU)-Specific GP3 and GP4 Peptides
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.
b) Cocktail of Type I (EU)-Specific GP3 and GP4 Peptides
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.
c) Cocktail of Type II (US)-Specific GP3 and GP4 Peptides
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.
d) Cocktail of Type II (US)-Specific GP3 and GP4 Peptides
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.

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.