WO2012163263A1

WO2012163263A1 - Reagents and methods for prrsv detection

Info

Publication number: WO2012163263A1
Application number: PCT/CN2012/076181
Authority: WO
Inventors: Hua Wu; Zhun LIU
Original assignee: SINOVET (BEIJING) BIOTECHNOLOGY CO Ltd
Current assignee: SINOVET (BEIJING) BIOTECHNOLOGY CO Ltd
Priority date: 2011-05-27
Filing date: 2012-05-28
Publication date: 2012-12-06
Anticipated expiration: 2013-11-27
Also published as: CN102731615A; CN102731615B; TW201300421A

Abstract

The present application relates to the detecting reagents and detection methods of porcine reproductive and respiratory virus. In one aspect, the present disclosure provides immunogenic fragments that encoding non-structural protein 2 (Nsp2). Particularly, the immunogenic fragments as provided herein are absent in an Nsp2 protein of an attenuated PRRSV but are present in an Nsp2 protein of a wild type PRRSV. The present disclosure also provides oligonucleotide primers that are useful in distinguishing an attenuated PRRSV from a wild type PRRSV. The present application further provides detection methods and detecting reagents for PRRSV, and also methods of distinguishing pigs immunized by an attenuated PRRSV from pigs infected with a wild type PRRSV.

Description

REAGENTS AND METHODS FOR PRRSV DETECTION

CORSS-REFERENCE TO THE RELATED APPLICATION

[0001] The present application claims the priority to Chinese Patent Application Number 201110143363.7, filed on May 27, 2011, entitled "Reagents and Methods for PRRSV Detection," which is incorporated herein by reference to its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to the field of veterinarian, in particular to reagents and methods for detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). BACKGROUND OF THE INVENTION

[0003] Porcine Reproductive and Respiratory Syndrome (PRRS) is an infectious disease, characterized in high infection rate, acute disease onset and high fatality rate. A swine fever epidemic occurred in China in 2006, during which a large number of pregnant sows were affected by severe symptoms such as miscarriage, fetal death, mummy fetus, hyperpyrexia, anorexia, and even death. The epidemic caused serious economic losses to the pork industry in China. After isolation of the causative agent and subsequent sequencing of its genome, it was found that the swine fever epidemic was caused by the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

[0004] During a PRRSV epidemic, it is desirable to identify animals infected with a pathogenic virus and isolate them from the herds at an early stage, thereby avoid extensive virus transmission and reduce economic loss. However, the existing methods for PRRSV detection have many limitations. For example, it is usually required to isolate and to identify the virus, but the use of a live pathogenic virus can pose a threat on further virus transmission. In addition, the detection methods are complex and usually require high-level experimental conditions. Moreover, it is difficult to distinguish naturally infected animals from vaccinated animals.

[0005] In veterinary practice, it usually requires efficient detection of a large number of serum samples, so as to prevent potential disease outbreaks. Therefore, there exist great needs for new detecting agents and methods that can provide for rapid reaction, convenient operation, accurate results and yet high bio-safety.

SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides isolated polypeptides, comprising one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1. In certain embodiments, the immunogenic fragments comprise at least 6, at least 7, at least 8, or at least 9 continuous amino acids.

[0007] In certain embodiments, the polypeptides are useful in producing an antibody that specifically binds to the immunogenic fragment. In certain embodiments, the polypeptides are useful in detecting in samples for presence of antibodies that specifically bind to the immunogenic fragments.

[0008] In certain embodiments, the polypeptides comprise one or more immunogenic fragments of SEQ ID NO: 1. In certain embodiments, the immunogenic fragments are selected from the group consisting of SVKITRPKYSAQAI (SEQ ID NO: 2), GHLQKEKEA (SEQ ID NO: 3) and PRTPAPSVSAESDLT (SEQ ID NO: 4).

[0009] In certain embodiments, the polypeptides are conjugated to carrier molecules. In certain embodiments, the carrier molecules are carrier proteins or polymers.

[00010] In another aspect, the present disclosure provides detecting reagents, comprising an isolated polypeptide provided herein. In another aspect, the present disclosure also provides detecting devices, which comprise the detecting reagent comprising the polypeptide, in which the detecting reagent is attached to a solid support.

[00011] In another aspect, the present disclosure also provides methods for detecting an antibody in a sample obtained from a pig suspected of PR SV infection, comprising contacting the sample to an isolated polypeptide provided herein and detecting the specific binding of the polypeptide and the antibody in the sample.

[00012] In another aspect, the present disclosure also provides methods of distinguishing a pig immunized by an attenuated PRRSV from a pig infected with a wild type PRRSV, using a polypeptide provided herein. In certain embodiments, the attenuated PRRSV is an attenuated live vaccine. In certain embodiments, the attenuated PRRSV comprises a PRRSV having a Deposit Number of CGMCC No. 3121.

[00013] In another aspect, the present disclosure also provides isolated antibodies capable of specifically binding to an immunogenic fragment of the polypeptides provided herein. In certain embodiments, the antibodies provided herein further comprise labels. In certain embodiments, the labels are fluorescent labels, luminescent labels, radioactive labels, enzyme labels or colored substances.

[00014] In another aspect, the present disclosure also provides detecting reagents comprising the isolated antibodies provided herein. In another aspect, the present disclosure also provides detecting devices comprising the detecting reagents comprising the polypeptides, in which the detecting reagents are attached to solid supports.

[00015] In another aspect, the present disclosure also provides methods for detecting the presence of a PRRSV in a sample, comprising contacting the sample to an isolated antibody provided herein and detecting the specific binding of the antibody and the antigen of the PRRSV in the sample.

[00016] In another aspect, the present disclosure also provides methods for distinguishing a pig immunized by an attenuated PRRSV from a pig infected with a wild type PRRSV, using an antibody provided herein. In certain embodiments, the attenuated PRRSV is an attenuated live vaccine. In certain embodiments, the attenuated PRRSV contains a PRRSV having a Deposit Number of CGMCC No. 3121.

[00017] In another aspect, the present disclosure also provides kits, comprising an isolated polypeptide provided herein, wherein the polypeptide is attached to a solid support. In certain embodiments, the kits further comprise a detecting antibody. In certain embodiments, the detecting antibody is conjugated to a label. In certain embodiments, the detecting antibody is an anti-pig antibody.

[00018] In another aspect, the present disclosure also provides oligonucleotide primers, which are useful in detecting the presence of PRRSVs in biological samples.

[00019] In another aspect, the present disclosure also provides methods for detecting a PRRSV in a biological sample, comprising amplifying a reverse transcribed product of RNA in a test sample using a pair of the oligonucleotide primers provided herein, and detecting the presence and/or molecular weights of the amplified product.

BRIEF DESCRIPTION OF THE FIGURES [00020] Figure 1 (a) is a schematic drawing which shows the gene deletions in the Nsp2 sequence of the porcine reproductive and respiratory syndrome virus vaccine strain TJM (PRRSV TJM) and of the highly-pathogenic PRRSV TJ strain when compared with that of the PRRSV standard strain VR-2332.

[00021] Figure 1 (b) shows the regions to which the oligonucleotide primers hybridize on the Nsp2 coding sequence of a PRRSV.

[00022] Figure 2 shows the discontinuous 90 nucleotides which are deleted in the Nsp2 coding sequence of PRRSV TJ strain and TJM strain, but present in that of PRRSV VR-2332 strain.

[00023] Figure 3 shows the 360 nucleotide sequence which is deleted in the Nsp2 coding sequence of PRRSV TJM strain.

[00024] Figure 4 shows the 120 amino acid sequence which is deleted in the Nsp2 protein sequence of PRRSV TJM strain. [00025] Figure 5 shows synthetic B cell epitopes identified in the 120-amino acid sequence which is absent in the Nsp2 protein of PRRSV TJM strain.

[00026] Figure 6 shows the antibody detection results determined in pigs after virus challenge by PRRSV TJ virulent strain.

[00027] Figure 7 shows the antibody detection results determined in pigs after immunization with PRRSV TJM vaccine, and optionally challenged by PRRSV TJ strain.

[00028] Figure 8 shows the electrophoresis image of the PCR results using the oligonucleotides as primers and the cDNA derived from the respective samples as templates. Lane 1 is negative control group, lane 2 is the PCR product derived from the classical PRRSV strain, lane 3 is DL2000 DNA Marker, lane 4 is the PCR product derived from the PRRSV TJ strain, and lane 5 is the PCR product derived from the PRRSV TJM strain.

DETAILED DESCRIPTION OF THE INVENTION

[00029] The following description is merely intended to illustrate various embodiments of the present disclosure. As such, the specific modifications discussed are not intended to be limiting. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the spirit or scope of the subject matters presented herein, and it is understood that such equivalent embodiments are to be included herein. All publications, patents or patent applications cited herein are incorporated by reference to their entirety.

[00030] In one aspect, the present disclosure provides one or more immunogenic fragments in an Nsp2 protein sequence as encoded by a PRRSV genome.

[00031] PRRSV is a positive-strand RNA virus for which two genotypes have been identified: American type and European type. The PRRSV genome comprises several open reading frames. The first open reading frame (ORF la and ORF lb) contains 80% sequence of the PRRSV genome, and encodes a RNA replicase that is required for replication of PRRSV (Straw et al, Diseases of Swine, 9^th edition, chapter 24(2006)). ORF la and ORF lb are translated into a poly-protein, which is processed by internal protease domains into multiple nonstructural proteins, including Nspl-Nspl2 (see, for example, Vries et al, Seminars in Virology, 8: 33-47 (1997); Allende et al, Journal of General Virology, 80: 307-315(1999)).

[00032] Nsp2 is one of the nonstructural proteins. Nsp2 protein contains a protease domain, and is believed to mediate the splicing and processing of the poly-protein encoded by ORFl . Nsp2 protein is also believed to contribute to the formation of RNA replicase complex, and plays an important role in the replication of PRRSV.

[00033] Nsp2 protein can be essential for the pathogenicity of PRRSV. Deletion of certain sequences in the Nsp2 coding sequence in a PRRSV genome is reported to render the PRRSV non-viable or with significantly reduced infectivity (see, e.g. Han et al, Journal of Virology, 81(18): 9878-9890 (2007); Kim et al, Virus Genes, 38:118-128 (2008)). Previous reports have provided attenuated PRRSVs, whose Nsp2 coding sequences contain certain deletions that are believed to impair the pathogenicity yet retain the viral replication (see, e.g. Chinese Patent Application CN101633909 A). Such attenuated PRRSVs are particularly useful in the preparation of a PRRSV vaccine for protecting pigs against infection of a wild type and pathogenic PRRSV.

[00034] In one aspect, the present disclosure provides isolated polypeptides comprising one or more immunogenic fragments of an Nsp2 protein sequence. The immunogenic fragments are absent in the Nsp2 protein sequence of an attenuated PRRSV, but present in the Nsp2 protein sequence of a wild type or a pathogenic PRRSV.

[00035] An "attenuated PRRSV," as used herein, refers to a PRRSV which contains a deletion of a partial amino acid sequence in its Nsp2 protein, and is capable of infecting a host yet unable to induce porcine reproductive and respiratory syndrome, or induces less and/or milder symptoms. An attenuated PRRSV can be a live attenuated PRRSV as well as its inactivated product.

[00036] "Porcine reproductive and respiratory syndrome", or "PRRS," as used herein, refers to a series of physiological and pathological symptoms caused by a naturally-occurring pathogenic PRRSV after its infection in a pig. The symptoms can include, without limitation, fever, lethargy, anorexia, listlessness, labored respiration, cough, sow reproductive failure, slow growth and death of piglets, etc.

[00037] A "wild type PRRSV" as used herein, refers to a PRRSV that is capable of infecting a host and causing one or more symptoms of PRRS in the host. A wild type PRRSV can be a field isolated strain or a lab strain, as long as it can infect host and cause PRRS. In certain embodiments, the PRRS symptoms caused by a wild type PRRSV are comparable to those caused by a field isolated pathogenic PRRSV strain or a standard PRRSV strain, or can be even more severe. A standard PRRSV strain can be, for example, without limitation, American type standard strain VR-2332 (GenBank Accession No. of the full genome sequence: AY 150564), and European type standard strain Lelystad (see, for example, W092/21375).

[00038] A "PRRSV" as used herein includes a PRRSV virus and a genetic material that can be used to produce a PRRSV virus, for example, without limitation, an RNA molecule containing the complete genome of a PRRSV, multiple RNA molecules containing materials that can be assembled into a complete genome of a PRRSV, a DNA molecule encoding the complete genome of a PRRSV, and multiple DNA molecules encoding PRRSV genome fragments which can be assembled into the complete genome of a PRRSV.

[00039] In certain embodiments, the attenuated PRRSV differs from the wild type PRRSV in the Nsp2 protein sequence, wherein the Nsp2 protein of the attenuated PRRSV lacks a polypeptide fragment when compared with the Nsp2 protein of the wild type PRRSV. [00040] The sequence of the Nsp2 protein for a PRRSV may be determined by methods known in the art. For example, the genome sequence of the PRRSV can be aligned with that of a known PRRSV, and the Nsp2 coding sequence may be determined based on the reported Nsp2 coding sequence in the known PRRSV genome. Coding sequences of Nsp2 proteins have been identified in genomes of different PRRSV strains (see, for example, Allende et al, Journal of General Virology, 80: 307-315(1999); US Patent Application US20100215694). Alternatively, the coding sequence of the Nsp2 protein in a PRRSV can be determined by identifying the N-terminal and the C-terminal cleavage sites of Nsp2 in the ORFla of the PRRSV genome. The N-terminal cleavage site and C-terminal cleavage site of Nsp2 protein have both been reported (see, for example, Nielsen et al, Journal of General Virology, 82:1263-1272 (2001); Ziebuhr et al, Journal of General Virology, 81 :853-879 (2000)). The Nsp2 protein sequence can be readily translated from the coding sequence, in accordance to the genetic codons. [00041] In certain embodiments, the polypeptide fragment, which is contained in the wild type PRRSV but not in the attenuated PRRSV, comprises one or more immunogenic fragments. An "immunogenic fragment" as used herein refers to a polypeptide fragment that can be specifically recognized by an antibody. The immunogenic fragments of a polypeptide can be identified using various methods known in the art, for example, without limitation, by using suitable sequence predicting software, or by using analytical methods.

[00042] In certain embodiments, the immunogenic fragments can be predicted using software known in the art, for example, without limitation, DNAStar's Lasergene software (see, for example, T. Burland et al, DNASTAR's Lasergene Sequence Analysis Software, Methods in Molecular Biology, Vol 132, 71-91(1999)), PEOPLE software (Alix et al, Vaccine, 18(324):311-314, 1999), BEPITOPE software (Odorico M et al, J. Mol. Recognit, 16:20-22, 2003), Bcepred, ABCpred software (Saha S et al, Proteins, 65(1): 40-48, 2006), CEP software (Kulkarni-Kale U et al, Nucleic Acids Res, 33:W168-W171 (2005)), DiscTope software (Haste A P et al, Protein Science, 15:2558-2567 (2006)), MEPS software (Castrignano T et al, BMC Bioinformatics, 8 (Suppl 1): S6-S10, 2007), etc. A person skilled in the art can choose the appropriate software according to the polypeptide sequence, and provide the sequence information and parameters with the guidance of the software developer's directions or instructions, and thereby obtain the predicted immunogenic fragments.

[00043] In certain embodiments, the immunogenic fragments of the polypeptide fragment can also be predicted by suitable analytical methods with suitable devices and techniques, for example, without limitation, X ray crystal diffraction technique, nuclear magnetic resonance, mass spectrum, etc. (for detailed review, please see, e.g. Methods in Molecular Biology, Vol 66: Epitope Mapping Protocols, Humana Press, Edited by: Glenn E. Morris, 1996).

[00044] The immunogenic fragments as predicted can be prepared or synthesized, for subsequent determination of its specific binding to an antibody or its capability in eliciting antibody production, so as to confirm that the predicted immunogenic fragment indeed has immunogenicity.

[00045] The immunogenic fragments as provided herein are absent in the Nsp2 protein sequence of an attenuated PRRSV, but are present in that of a wild type PRRSV. Such immunogenic fragments can be identified through sequence alignment. For example, the Nsp2 protein sequence or nucleotide sequence of an attenuated PRRSV can be aligned with that of a wild type PRRSV, and therefore the polypeptide fragment that is present in the Nsp2 protein of the wild type PRRSV but is absent in that of the attenuated PRRSV can be identified, and the immunogenic fragments contained in the polypeptide fragment can be predicted by methods known in the art. Optionally, the immunogenicity of the immunogenic fragments can be further validated.

[00046] In certain embodiments, the immunogenic fragments as provided herein are absent in the Nsp2 protein sequence of PRRSV T JM strain but is present in that of a wild type PRRSV.

[00047] A "PRRSV TJM strain" refers to the PRRSV with a Deposit Accession No. of CGMCC No. 3121, and with the following deposit information: Microorganism Deposit Accession No.: CGMCC No. 3121; Taxonomic Name: porcine reproductive and respiratory syndrome virus; Deposit Address: Institute of Microbiology, Chinese Academy of Sciences, No. l West Beichen Road, Chaoyang District, Beijing, China; Deposit Center: China General Microbiological Culture Collection Center; and Deposit Date: June 15, 2009. The complete genome sequence of the PRRSV TJM strain has been sequenced, and the genome sequence is shown in SEQ ID NO: 6. The amino acid sequence of Nsp2 protein encoded in the genome sequence is shown in SEQ ID NO: 7.

[00048] In certain embodiments, the present disclosure provides isolated polypeptides comprising one or more immunogenic fragments of a polypeptide fragment which is absent in the Nsp2 protein sequence (e.g. SEQ ID NO: 7) of PRRSV TJM strain but is present in that of a wild type PRRSV.

[00049] In certain embodiments, the wild type PRRSV is a highly-pathogenic PRRSV. The term "highly-pathogenic PRRSV" refers to a PRRSV comprising an Nsp2 nucleotide encoded by a DNA sequence which, when compared with SEQ ID NO: 20, lacks discontinuous 90 nucleotides within the portion of SEQ ID NO: 21 (i.e. the fragment from the 1440th to the 1680th nucleotide of SEQ ID NO: 20). PRRSV isolates lacking such 90 discontinuous nucleotides (see Figure 1 (a)) are found to have higher pathogenicity than PRRSV VR-2332 strain (see, e.g. Tian et al, PLoS ONE 2(6): e526, (2007) doi: 10.1371). In certain embodiments, the discontinuous 90 nucleotides include the "TTT" from the 1440 to the 1442 nucleotide of SEQ ID NO: 20 and the sequence as shown in SEQ ID NO: 22 (see, for example, Figure 2).

[00050] In certain embodiments, the highly-pathogenic PRRSV comprises an Nsp2 protein sequence as shown in SEQ ID NO: 23, or a homologous sequence thereof. In certain embodiments, the highly-pathogenic PRRSV is PRRSV TJ strain, whose genome is encoded by a sequence having a GenBank Accession number of EU860248 (provided herein as SEQ ID NO: 5). The PRRSV TJ strain is an American type PRRSV strain, which can cause porcine reproductive and respiratory syndrome after the infection of porcine host (see, Chinese Patent ZL200710121202.1).

[00051] In certain embodiments, the isolated polypeptides provided herein comprises one or more immunogenic fragments of a polypeptide fragment having at least 75% homology to SEQ ID NO: 1. SEQ ID NO: 1 is a partial sequence of the Nsp2 protein of PRRSV TJ strain, a wild type highly-pathogenic strain, and is absent in the Nsp2 protein of PRRSV TJM strain, an attenuated PRRSV strain. The Nsp2 protein sequence of PRRSV TJ strain is homologous to that of many American type PRRSV strains, for example, it has 77.9% homology to the Nsp2 protein sequence of VR-2332, the standard strain for American type PRRSV.

[00052] The term "percent (%) homology to" as used herein refers to the percentage of identity between two amino acid sequences or two polynucleotide sequences, after aligning the candidate and the reference sequences, and if necessary introducing gaps, to achieve the maximum number of identical amino acids or nucleotides. The comparison of amino acid (or nucleotide) sequences and calculation of homology can be achieved by well-known software in the art, for example, but without limitation, BLAST software (available from the website of National Center for Biotechnology Information (NCBI) h tt ://blast.ncbi ,nlm..nih. gov/B 1 ast.cg also see, for example, Altschul S.F. et al, J. Mol. Biol, 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 software (available from the website of European Bioinformatics Institute http://www.ebi.ac.uk/Tools/msa clustalw2/, see also, for example, Higgins D.G. et al, Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)) and TCoffee software (available from the website of Sweden Bioinformatics Institute http://tcoffeeA^tal-it h/cgi-bin/Tcoffee/tcoffee c i index. cei. see also, for example, Poirot O. et al, Nucleic Acids Res., 31(13): 3503-6 (2003); Notredame C. et al, J. Mol. Boil, 302(1): 205-17 (2000)). If the alignment of the sequences is performed using software, the default parameters available in the software may be used, or otherwise the parameters may be customized to suit the alignment purpose. All of these are within the scope of knowledge of a person of ordinary skill in the art.

[00053] In certain embodiments, the isolated polypeptides as provided herein comprise one or more immunogenic fragments of a polypeptide fragment having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the isolated polypeptides comprise one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1. In certain embodiments, the isolated polypeptides do not encompass the full length of a Nsp2 protein of a wild type PRRSV.

[00054] In certain embodiments, the isolated polypeptides comprise one, two, three, four, five, six or seven immunogenic fragments of a polypeptide fragment having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the immunogenic fragment comprises at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen or at least fourteen continuous amino acids.

[00055] In certain embodiments, the isolated polypeptides comprise one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1, wherein the immunogenic fragments comprise at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen or at least fourteen continuous amino acids.

[00056] In certain embodiments, the isolated polypeptides as provided herein can be used to produce an antibody that specifically binds to the immunogenic fragment. For example, the isolated polypeptides can be recognized by immune system, and thereby induces the immune system to produce one or more antibodies that can specifically bind to the one or more immunogenic fragments of the polypeptides.

[00057] In certain embodiments, the polypeptides as provided herein can specifically bind to an antibody that recognizes the polypeptide on the immunogenic fragments. In certain embodiments, the polypeptides as provided herein can be used to detect in the sample for the absence or presence of an antibody that specifically binds to the immunogenic fragment. For example, the sample may contain one or more antibodies that can specifically bind to one or more of the immunogenic fragments of the polypeptide, and when the polypeptide is contacted with the sample, the polypeptide can specifically bind to the antibody and provide for the detection of the antibody in the sample.

[00058] In certain embodiments, the isolated polypeptides as provided herein comprise one or more immunogenic fragments of SEQ ID NO: 1. In certain embodiments, the immunogenic fragments are selected from the group consisting of SVKITRPKYSAQAI (SEQ ID NO: 2), GHLQKEKEA (SEQ ID NO: 3) and PRTPAPSVSAESDLT (SEQ ID NO: 4). In certain embodiments, the immunogenic fragment is SVKITRPKYSAQAI (SEQ ID NO: 2).

[00059] The isolated polypeptides as provided herein can be prepared by any suitable methods well-known in the art, for example, without limitation, by chemical synthesis or biological recombinant expression, and etc.

[00060] Any suitable chemical methods well-known in the art can be used, for example, without limitation, liquid phase-based polypeptide synthesis and solid phase-based polypeptide synthesis, etc. (see, for example, Michael W. Pennington, Peptide synthesis protocols, Methods in molecular biology, volume 35, published by Humana Press, 1994; John M. Waler et al., Molecular biomethods handbook, Chapter 32, published by Springer, 2008).

[00061] The polypeptide as provided herein can also be prepared by biological recombinant expression. For example, a polynucleotide sequence that encodes the polypeptide can be introduced into a suitable expression host (for example, prokaryotic cells such as Escherichia coli, or eukaryotic cells such as yeast or mammalian cells, etc.), which are cultured under the condition that allows for expression of the polypeptide. In certain embodiments, an expression vector containing a coding sequence for the polypeptide can be introduced into a suitable expression host to allow the expression. The recombinantly expressed polypeptide can be appropriately recovered, for example, without limitation, by recovering from the cell lysate of the recombinant host cells or from the supernatant of the cell culture. Optionally, the polypeptide can be further separated and/or purified by any suitable methods known in the art, for example, without limitation, by molecular-exclusion chromatography, affinity column chromatography, or ion-exchange chromatography, etc. (see, for example, Deutscher, Methods in enzymology, 182 (1990); Scopes, Protein purification: Principles and Practices, Springer- Verlag, New York (1982)).

[00062] In certain embodiments, the isolated polypeptides as provided herein can be conjugated to a carrier molecule. A person skilled in the art can conjugate the polypeptides with a suitable carrier molecule, for example, by conjugating, coupling or fusing the carrier molecule to the polypeptide. In certain embodiments, the carrier molecules can be used to enhance the immunogenicity of the polypeptide. For example, the polypeptide conjugated with a carrier molecule can have an enhanced stimulation on the immune system, and thereby facilitates with the production of an antibody specific for the polypeptide. In certain embodiments, the carrier molecule can be used to increase the molecular weight of the polypeptide, which can improve recognization by the immune system, or provide for better attachment to a solid support, or be desired for sample detection. In certain embodiments, the carrier molecules can facilitate with the separation and purification of the polypeptides. For example, the carrier molecule can be a purification tag that can be pulled down using commercially available antibodies or kits. In certain embodiments, the carrier molecules can be desirable for recombination expression and/or secretion of the polypeptide herein. For example, the carrier molecules can be a secretion tag that allows the polypeptide to be secreted into the extracellular or periplasm of recombinant host cells, and thereby can be desirable for expression and recovery of the polypeptides. In certain embodiments, the carrier molecules can be used to increase or decrease the solubility of the polypeptides. Any carrier molecules suitable for the above-mentioned purposes are within the scope of this present disclosure.

[00063] In certain embodiments, the carrier molecule is a carrier protein. Exemplary carrier proteins include, without limitation, albumin, keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, avidin, streptavidin, thyroglobulin, glutathione- S- transferase, maltose-binding protein, and chitin-binding protein, etc.

[00064] In certain embodiments, the carrier molecule is a polymer. Exemplary carrier polymers include, without limitation, polyamino acids (such as polyhistidine, polylysine, polyglutamates), polyethylene glycol, Ficoll, glucosan, and dendrimer, etc.

[00065] A person skilled in the art can conjugate a polypeptide provided herein with a suitable carrier molecule using any suitable methods known in the art, for example, without limitation, by chemical methods (for example, using a di-functional coupling agent, see, for example, Susan Hockfield, Selected methods for antibody and nucleic acid probes, published by CSHL Press, 1993, page 72-75; John D. Pound, Immunochemical Protocols, published by Humana Press, 1998, page 74-78; J. Mark Carter, The protein protocols handbook, Part VII, 1996, page 679-687; Francesco M. Veronese, Biomaterials, 22: 405-417 (2001); Greg T. Hermanson, Bioconjugate techniques, published by Academic Press, 2008, page 754-757) or by biological recombinant expression (for example, expressing the polypeptide and the carrier molecule as a fusion protein, see, for example, S. J. Higgins et al. Protein expression: a practical approach, published by Oxford University Press, 1999, page 174-199).

[00066] In certain embodiments, the polypeptides provided herein can be combined with an adjuvant to provide an immunogenic composition. An adjuvant can protect the antigen from in vivo degradation, and/or can non-specifically stimulate immune system, which is desirable for enhancing the immune response against the polypeptide. Exemplary adjuvants include, without limitation, mineral salts (such as aluminum hydroxide, aluminum phosphate, calcium hydroxide), water-in-oil emulsion (such as Freund's complete adjuvant, Freund's incomplete adjuvant), saponin adjuvant (such as Stimulon™ etc.), bacteria or microorganism derivatives (such as lipopolysaccharide, lipid A derivatives, etc.), and microparticles (such as poly-a hydroxy-acid, etc.).

Detecting Reagents Comprising the Polypeptide and Detection Methods

[00067] In another aspect, the present disclosure provides detecting reagents comprising an isolated polypeptide provided herein. The detecting reagent can comprise a suitable amount of the isolated polypeptide in a suitable purity and/or at a suitable concentration, such that the polypeptide in the detecting reagent is suitable for the detection purpose. In certain embodiments, the polypeptide in the detecting reagent is in a freeze-dried form or is dissolved in a suitable vehicle. In certain embodiments, the detecting reagent can further comprise a suitable excipient, for example, without limitation, suitable buffers, preservatives, proteins, and etc.

[00068] In another aspect, the present disclosure provides detecting devices, comprising a detecting reagent comprising the polypeptide provided herein, wherein the detecting reagent is attached to a solid support. The solid support can be any material that is suitable for detection purpose and attachment of a detecting reagent. Examples include, without limitation, plastic dish, plastic plate, nitrocellulose filter, glass fiber membrane, glass plate, beads, and etc.

[00069] The detecting reagent can be attached to the solid support using any suitable methods, for example, but without limitation, by fixation, coating, adsorption, spray painting, printing, and etc. In certain embodiments, the solid support can be contacted with a solution containing the detecting reagent for a period of time, and then the solution is removed. In certain embodiments, a solution, suspension or emulsion containing the detecting reagent can be sprayed or coated on the solid support, and then the solvent component can be removed appropriately, for example by drying. In certain embodiments, the detecting reagent can be also printed or pressed on the solid support.

[00070] In another aspect, the present disclosure provides methods for detecting an antibody in a sample, wherein the sample is from a pig suspected of PRRSV infection, comprising contacting the sample with an isolated polypeptide as provided herein, and detecting in the sample for the presence of an antibody that specifically binds to the polypeptide.

[00071] The specific binding of the polypeptide with an antibody in the sample can be detected by any suitable detection methods known in the art.

[00072] In certain embodiments, the detection is based on a Sandwich method. Briefly, an un-labeled capture molecule (such as the polypeptide as provided herein) can be immobilized on a solid support, and then contacted with a sample containing a test antibody for a sufficient period of time to allow for sufficient binding of the antibody with the capture molecule, followed by reaction with a detecting antibody which specifically binds to the test antibody, thereby forming a triple-complex of capture molecule - test antibody - detecting antibody. The test antibody in the sample can be indirectly detected by detecting the signals produced by detecting antibody in this triple-complex.

[00073] The detecting antibody can be an anti-pig antibody. The term "anti-pig antibody" used herein refers to any binding partners that can specifically bind to the Fc region of a pig antibody. For example, an anti-pig antibody can be a full antibody which is produced by a species other than pig and specifically binds to the Fc region of a pig antibody, or an antigen-binding fragment thereof (e.g. Fab, Fab', F(ab')₂, Fv fragment, dsFv, scFv, scFv dimer, BsFv, a camelized single domain antibody, a nanobody, or a domain antibody, etc.). Anti-pig antibody useful as a detecting antibody include, for example, without limitation, mouse anti-pig antibody, rat anti-pig antibody, rabbit anti-pig antibody, goat anti-pig antibody, chicken anti-pig antibody, and donkey anti-pig antibody, etc. Anti-pig antibody can be monoclonal antibody or a polyclonal antibody, or any antigen-binding fragment thereof. Anti-pig antibodies are commercially available, or can be obtained by conventional antibody production methods (see, for example, Harlow et al, Antibodies: A laboratory manual, Cold Spring Harbor, 1988).

[00074] The detecting antibody can be conjugated with a label. The term "label" as used herein refers to a molecule that can be directly or indirectly detected. Labels may produce directly detectable signals, such as color, luminescence, fluorescence, radioactivity, and etc.; or may be indirectly detected by reaction with a specific molecule (such as the reaction between an enzyme and a substrate) that produces a detectable signal such as color change, fluorescence, luminescence, and etc. Labels can be an enzyme, a fluorescent molecule, a chemoluminescent substance, a radioactive molecule, or a colored substance, etc.

[00075] In certain embodiments, the detection is based on a direct detection method. The polypeptides provided herein can be labeled. The labeled polypeptide can specifically bind to the test antibody and form a complex, thereby allow for detection of the test antibody in the sample by detecting the label in the complex.

[00076] In certain embodiments, the detection is based on competitive binding. A labeled competitive binding molecule, which can form a complex with the test antibody or the polypeptide, can be used to interfere with the specific binding between the polypeptide and the test antibody in the sample. The competitive binding can be detected to provide information of the test antibody in the sample.

[00077] In certain embodiments, the specific binding between the polypeptide and the test antibody in the sample can be detected by Enzyme-Linked Immuno-Sorbent Assay (ELISA), an immunofluorescence-based method, a chemo-immunoluminescence-based method, an immunoradioactivity-based method, an immunochromatography-based method, or an immunofiltration-based method.

[00078] In certain embodiments, the test antibody in the sample is detected by ELISA. For example, the polypeptide or the detecting reagent provided herein can be immobilized on a solid support, and then contacted with the sample (such as, blood or serum from a pig suspected of PR S V infection) to allow sufficient formation of a capture molecule - test antibody complex. An enzyme-labeled detecting antibody (e.g., goat anti-pig antibody) is subsequently added to allow sufficient formation of a triple-complex of capture molecule - test antibody - detecting antibody. Enzyme substrate is added and converted to a detectable product under a suitable condition. The change in detectable signal after addition of the substrate indirectly indicates the presence and/or amount of the test antibody in the sample. [00079] In certain embodiments, the enzyme labeled on the detecting antibody can catalyze a substrate and produce a color change of the substrate. The color change can be detected by a spectrophotometer, or a microplate reader, etc. The enzyme labeled on the detecting antibody may change the fluorescence of a substrate or produce luminescence through a chemical reaction, which can be detected by a luminescence detector or a fluorescence detector. Exemplary useful enzymes include, without limitation, luciferase (such as firefly luciferase, renilla luciferase, bacterial luciferase), peroxidase (such as horseradish peroxidase), alkaline phosphatase, β-galactosidase, malate dehydrogenase, urease, saccharifying enzyme, lysozyme, saccharide oxidase (such as glucose oxidase, galactose oxidase, etc.), heterocycloxygenase, etc. An enzyme can be labeled on a detecting antibody by any suitable techniques known in the art, for example, see P. Tijssen, Practice and theory of enzyme immunoassays, Volume 15, Chapter 11 , published by Elsevier, 1985; O'SuUivan et al., Methods for the Preparation of Enzyme- Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), published by Academic press, New York, 73:147-166 (1981).

[00080] In certain embodiments, an immunofluorescence-based method can be used to detect the test antibody in the sample. For example, the detection can be based on sandwich ELISA, which allows formation of a triple complex of capture molecule - test antibody - detecting antibody, in which the detecting antibody is labeled with a fluorescent molecule. The fluorescent signal emitted by the triple complex can indirectly indicate the presence and/or amount of the test antibody in the sample. The fluorescence signal can be detected by any suitable methods known in the art, for example, without limitation, by using a fluorescence microscope or a fluorescence detector etc. Exemplary useful fluorescent molecules includes, without limitation, fluorescein isothiocyanate (FITC), rhodamine (TRITC), fluorescein, dichlorotrazinylaminofluorescein (DATF), Cy2, Cy3, Cy5, AMCA, LRSC (lissamine-rhodamine sulfonyl chloride), dansyl chloride, Texas red, R-phycoerythrin, etc. Fluorescent molecules can be labeled on a detecting antibody by any suitable method, for example, see Wulf Storch, Immunofluorescence in clinical immunology: a primer and atlas, published by Birkhauser, Chapters 2 and 3 (2000).

[00081] In certain embodiments, a chemo-immuno luminescence-based method can be used to detect the test antibody in the sample. For example, a sandwich ELISA can be used to form a triple complex of capture molecule - test antibody - detecting antibody, wherein the detecting antibody is labeled with a chemo luminescent molecule. The light signal emitted by the chemo luminescent molecules in the triple complex can indirectly indicate the presence and/or amount of the test antibody in the sample. The chemoluminescent signal can be detected, for example, by introducing a catalyst and/or an oxidant to induce oxidation of the chemoluminescent molecules and generation of luminescence, which can be detected by a chemoluminescence detector. Exemplary chemoluminescent molecules include, without limitation, luminol and derivatives thereof, iso-luminol, acridinium ester and derivatives thereof, and terpyridyl ruthenium, etc. Chemoluminescent molecules can be labeled on the detecting antibody by any suitable methods known in the art, for example, see J. Stuart Woodhead et al, Pure & Appl. Chem., 57(3):523-529 (1985); D. M. Kemeny, ELISA and other solid phase immunoassays: theoretical and practical aspects, published by John Wiley and Sons, 1988; Aldo Roda, Chemiluminescence and Bio luminescence: Past, Present and Future, published by Royal Society of Chemistry, 2010. [00082] In certain embodiments, an immunoradioactivity-based method can be used to detect the test antibody in the sample. For example, the polypeptide provided herein can be labeled with a radioactive substance, and then contacted with the sample to allow sufficient formation of a complex between the polypeptide and the test antibody. The complex is separated for detection and/or measurement of the radioactivity, which indicates the presence and/or amount of test antibody in the sample. Radioactivity can be detected by any suitable methods known in the art, for example, without limitation, by using a radioactivity counter, scintillation counter, and γ counter, etc. Exemplary radioactive substances include, without limitation, ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ^mIn, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, and ⁸⁶Y, etc. Radioactive substances can be labeled on the polypeptides provided herein by a suitable method known in the art, for example, see M. Holtzhauer, Basic methods for the biochemical lab, Springer Lab Manuals, Chapter 6, 2006.

[00083] In certain embodiments, an immunochromatography-based method can be used to detect the test antibody in the sample. For example, the polypeptide provided herein can be immobilized on a solid support of an immunochromatographic device, in which the sample containing the test antibody flows along the solid support and towards a label reagent and the polypeptide which are immobilized on separate regions. The test antibody become labeled after contacting with the label reagent, and is then captured by the immobilized polypeptide through specific binding, thereby forming a visible blot or line at the capture region. Exemplary label reagents useful for an immunochromatographic method include, without limitation, a detecting antibody labeled with colloidal gold or colloidal silver, and etc. The label reagents can be prepared by any suitable methods known in the art, for example, a detecting antibody can be mixed with a metal colloidal solution under a suitable condition, followed by separation of the metal colloid labeled antibody (see, for example, G. T. Hermanson, Bioconjugate Techniques, published by Academic Press, page 919-936, 2008).

[00084] In certain embodiments, an immunofiltration-based method can be used to detect the tested antibody in the sample. For example, the polypeptide provided herein can be immobilized on a solid filter, and a sample containing a test antibody is added to allow formation of a polypeptide-antibody complex, which is then contacted with a label reagent. The complex is washed with a buffer to remove un-bound substances while the complex of polypeptide - antibody - label is trapped on the solid filter. Detection of the label in the complex can indirectly indicate the presence and/or amount of the test antibody in the sample. Exemplary label reagents include, without limitation, metal colloid labeled protein or antibody, radioactive ligand, and enzyme labeled protein or antibody, etc. The label can be detected by a suitable method, for example, by direct visual observation when the labeled reagent is a colloidal gold labeled antibody, or by radioactivity measurement when the labeled reagent is a radioactive ligand.

[00085] In another aspect, the present disclosure provides a method for distinguishing a pig immunized by an attenuated PR SV from a pig infected with a wild type PRRSV, comprising: contacting a sample of the pig with the polypeptide provided herein, detecting in the sample for the presence of an antibody that specifically binds to the polypeptide, wherein the presence of the antibody in the sample indicates that the sample is from a pig infected with the wild type PRRSV. The absence of the antibody in the sample can indicate that the sample is from an uninfected pig or a pig immunized with an attenuated PRRSV.

[00086] In certain embodiments, the attenuated PRRSV is an attenuated American type PRRSV. In certain embodiments, the attenuated PRRSV comprises a polynucleotide molecule having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homology to SEQ ID NO: 5, wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 1. In certain embodiments, the attenuated PRRSV comprises a polynucleotide molecule having at least 80%) homology to SEQ ID NO: 5, wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide at least 80% homologous to SEQ ID NO: 1.

[00087] In certain embodiments, the attenuated PR SV is an attenuated PR SV live vaccine. In certain embodiments, the attenuated PRRSV live vaccine comprises a PRRSV having a Deposit Number of CGMCC No. 3121.

[00088] In certain embodiments, the wild type PRRSV is an American type PRRSV. In certain embodiments, the wild type PRRSV comprises a polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the wild type PRRSV contains a polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1.

[00089] In certain embodiments, the sample can be any pig sample suspected of containing a test antibody, including without limitation, blood, plasma, serum, and body fluid, etc. In certain embodiments, the sample is the blood or plasma of a pig.

[00090] In another aspect, the present disclosure further provides uses of the polypeptides provided herein in the preparation of a detecting reagent.

[00091] In certain embodiments, the detecting reagent can be used to detect an antibody in the sample which is from a pig suspected of PRRSV infection. Any suitable methods can be used for the detection, for example, the methods may include contacting the sample with the isolated polypeptide provided herein, and detecting the specific binding of the polypeptide with the antibody in the sample.

[00092] In certain embodiments, the detecting reagent can be used to distinguish a pig infected with an attenuated PRRSV from a pig infected with a wild type PRRSV. Any suitable methods can be used for the methods. For example, the sample of a pig can be contacted with the polypeptide provided herein, and the presence of an antibody that specifically binds to the polypeptide can be detected, wherein the presence of an antibody that specifically binds to the polypeptide provided herein indicates that the sample is from a pig infected with the wild type PRRSV. Antibody

[00093] In another aspect, the present disclosure further provides isolated antibodies, which can specifically binds to an immunogenic fragment of a polypeptide provided herein.

[00094] The term "antibody" as used herein, encompasses a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a bispecific (bivalent) antibody, as well as an antigen binding fragment, for example, without limitation, Fab, Fab', F(ab')₂, Fv fragment, Fv fragment stabilized with a disulfide bond (dsFv), single-chain antibody (scFv), scFv dimer (bivalent bi-functional antibody), a bivalent single-chain antibody (BsFv), a camelized single domain antibody, a nanobody, a domain antibody, or any other antibody fragments that is capable of specific binding to an antigen yet without a complete antibody structure.

[00095] The isolated antibodies can be prepared by an skilled artisan using any suitable methods known in the art.

[00096] For example, to prepare a polyclonal antibody, an antigen composition containing a polypeptide provided herein can be inoculated (e.g. by injection) to an animal (e.g. mouse, rabbit, goat, pig, and bovine, etc.) at a suitable dosing frequency or interval. Polyclonal antibodies can be isolated from the serum collected from the immunized animals. The polyclonal antibodies may be further separated and purified (e.g. by immunoaffinity chromatography) to identify antibodies that specifically bind to the polypeptide or that have a desired purity. Methods for preparation and purification of polyclonal antibodies are well-known in the art, for details please see: Harlow et al., Antibodies: A laboratory manual, Cold Spring Harbor, 1988.

[00097] For another example, to prepare a monoclonal antibody, an antigen composition containing a polypeptide provided herein can be inoculated (e.g. by injection) to an animal (e.g. mouse, rabbit, goat, pig, and bovine, etc.) at a suitable dosing frequency or interval. Spleen cells of the immunized animals are collected and fused with myeloma cells to prepare hybridoma cells that produce monoclonal antibodies (see, for example, Harlow et al, Antibodies: A laboratory manual, Cold Spring Harbor, 1988; Monoclonal Antibody production, published by National academies Press, 1999; J. Eryl Liddell et al, A practical guide to monoclonal antibodies, published by John Wiley and Sons, 1991). The monoclonal antibodies can be screened for desirable characteristics, e.g., high affinity and good specificity, etc., using methods known in the art, such as for example, by ELISA method, Western blotting, and immunohistochemical method, etc. (see, for example, J. Eryl Liddell et al, A practical guide to monoclonal antibodies, published by John Wiley and Sons, 1991). Monoclonal antibodies that can specifically bind to the polypeptide provided herein can be screened and identified by, e.g., phage display technique, see, for example, Philippa M. O'Brien et al., Antibody phage display: methods and protocols, published by Humana Press, 2002; Kay et al, Phage display of peptides and proteins: A laboratory manual, San Diego: Academic Press, 1996.

[00098] For another example, an antigen binding fragment can be prepared by a suitable method known in the art, for example, without limitation, by digesting a full antibody with a protease (e.g. pepsin, papain, etc.), or by recombinant expression of a polynucleotide encoding the antigen binding fragment in a suitable host. [00099] The antibodies can be purified using a suitable method known in the art, for example, by hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ethanol precipitation, reversed phase HPLC, silica gel chromatography, heparin sepharose chromatography (such as poly aspartic acid column) which is based on anion or cation exchange resin, chromatofocusing, polyacrylamide gel electrophoresis (SDS-PAGE), and ammonium sulfate precipitation, etc., see, for example, James W. Goding, Monoclonal antibodies: principles and practice, published by Academic Press, 1996. After preliminary purification, the antibody composition can be further treated with hydrophobic interaction chromatography at low pH to remove remaining impurities, using an elution buffer with a pH of about 2.5-4.5 and preferably at a low salt concentration (e.g., the salt concentration from about 0 to 0.25M).

[000100] In certain embodiments, the antibody as provided herein further comprises a conjugate (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). A conjugate can be linked to the antibodies provided herein by any suitable methods, for example, without limitation, by covalent binding, affinity binding, embedding, coordinate binding, complexation, binding, mixing, or association, etc. In certain embodiments, specific sites useful for conjugation but do not affect epitope binding can be further engineered on the antibodies provided herein. For example, such sites can contain one or more reactive amino acid residues, such as a cysteine residue, a histidine residue, which can form a covalent linkage with a conjugate or a label. In certain embodiments, the antibodies can be indirectly linked to a conjugate, or can be conjugated through an additional linker. For example, the antibody or antigen binding fragment thereof can be conjugated with biotin, and then indirectly conjugated to a second conjugate which is linked to avidin.

[000101] In certain embodiments, the conjugate is a label. In certain embodiments, the label can be a fluorescent molecule, a chemiluminescent molecule, a radioactive label, an enzyme label or a colored substance. Exemplary fluorescent molecules include, without limitation, fluorescein isothiocyanate (FITC), rhodamine (TRITC), fluorescein, dichlorotrazinylamino fluorescein (DATF), Cy2, Cy3, Cy5, AMCA, LRSC (lissamine-rhodamine sulfonyl chloride), dansyl chloride, texas red, R- phycoerythrin. Exemplary chemoluminescent molecules include, for example, without limitation, luminol and derivatives thereof, iso-luminol, acridinium ester and derivatives thereof, terpyridyl ruthenium. Exemplary radioactive labels include, without limitation, ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ^mIn, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, etc. Exemplary enzyme labels include, without limitation, luciferase (such as firefly luciferase, renilla luciferase, bacterial luciferase, etc.), peroxidase (such as horseradish peroxidase), alkaline phosphatase, β-galactosidase, malate dehydrogenase, urease, saccharifying enzyme, lysozyme, saccharide oxidase (such as glucose oxidase, galactose oxidase, etc.), and heterocycloxygenase, etc. Exemplary colored substance include, without limitation, colloidal gold and colloidal silver, etc.

Detecting reagents and detection methods containing the antibody herein [000102] In another aspect, the present disclosure also provides a detecting reagent comprising an isolated antibody provided herein. The detecting reagent can comprise a suitable amount of the isolated antibody in a suitable purity and/or at a suitable concentration, such that the antibody in the detecting reagent is suitable for the detection purpose. In certain embodiments, the antibody in the detecting reagent is in a freeze-dried form or is dissolved in a suitable vehicle.

[000103] In another aspect, the present disclosure also provides detecting devices, comprises the detecting reagent comprising the antibody, wherein the detecting reagent is attached to a solid support. The solid support can be any material that is suitable for detection purpose and attachment of a detecting reagent. The detecting reagent can be attached to the solid support in any appropriate manners, using any suitable methods, for example, but without limitation, by immobilization, coating, adsorption, spray painting, and printing, etc.

[000104] In another aspect, the present disclosure also provides methods for detecting the presence of a PR SV in the sample, wherein the sample is from a pig suspected of PRRSV infection, comprising contacting the sample with an isolated antibody as provided herein, and detecting in the sample for the presence of an antigen that specifically binds to the antibody.

[000105] In certain embodiments, the PRRSV comprises a polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the PRRSV comprises a polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO:l . [000106] The specific binding of the antibody and an antigen of the PRRSV in the sample can be detected by any suitable detection methods known in the art, for example, without limitation, methods that are based on Sandwich method, direct detection method and competitive binding method. For example, a person skilled in the art can detect the specific binding of the antibody herein and the PRRSV antigen in the sample by using ELISA, immunofluorescence-based methods, chemo-immunoluminescence-based methods, immunoradiactivity-based methods, immunochromatography-based methods, immunofiltration-based methods and immunohistochemistry methods, as described above (see, for example, Frederick A. Murphy et al, Veterinary virology, published by Elsevier, 1999).

[000107] In certain embodiments, the presence of PRRSV in the sample is detected by a sandwich-based ELISA method. For example, an unlabeled antibody provided herein can be used to capture the PRRSV antigen in the sample. An enzyme-labeled detecting antibody is subsequently added to allow sufficient formation of a triple-complex of capture molecule - antigen - detecting antibody. Enzyme substrate is added and is converted to a detectable product under a suitable condition. The change in detectable signal after addition of the substrate indirectly indicates the presence and/or amount of the PRRSV antigen in the sample. The presence of a PRRSV in the sample may also be detected by a direct detection method. For example, the antibody provided herein which is conjugated with a label can be contacted with a sample to allow sufficient formation of the antigen - antibody complex, and the signal produced by the label can be subsequently determined. For another example, the antibody herein which is conjugated with biotin can be contacted with a sample to allow formation of a complex between the biotin-conjugated antibody and the PRRSV antigen in the sample. An avidin labeled enzyme can be further added to allow specific binding to the biotin labeled antibody, followed by addition of a substrate. The signal of the substrate produced upon reaction with the enzyme can indicate the presence of a PRRSV antigen in the sample.

[000108] In certain embodiments, the presence of a PRRSV antigen in the sample can be detected by immunofluorescence-based methods, chemo- immunoluminescence-based methods, and immunoradioactivity-based methods. They work in a similar way as ELISA except that they use different labels, for example, fluorescent molecule, chemoluminescent material or radioactive substance, which in turn require different detection methods and devices, such as a fluorescence microscope, a fluorescence detector, a chemiluminescence detector, or a γ counter.

[000109] In certain embodiments, an immunochromatography-based method can be used to detect the presence of a PR SV antigen in the sample. For example, the antibody provided herein can be immobilized on a solid support of an immunochromatographic device, in which the sample containing a PRRSV antigen flows along the solid support and towards a label reagent and the antibody that are immobilized on separate regions. The PRRSV antigen is labeled after contacting with the label reagent, and is then captured by the immobilized antibody through specific binding, thereby forming a visible blot or line at the capture region. A positive control may be used in an immunochromatography-based method. It is also possible to use immunofiltration-based methods, which work in a similar fashion to that of an immunochromatography method, except that the antigen - antibody complex is separated by filtration but not by chromatography.

[000110] In certain embodiments, the presence of a PRRSV antigen in the sample can be detected by an immunohistochemisty-based method. For example, the test sample can be fixed (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology," 3^rd edition (1960) Lee G. Luna, HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, New York), and then contacted with an antibody provided herein to allow specific binding. If a labeled antibody is used, then the presence of PRRSV antigen in the sample can be directly detected through the label. If a un-labeled antibody is used, then a labeled detecting antibody specifically binding to the un-labeled antibody can be added to form a triple complex of antigen - antibody - detecting antibody. The presence of a PRRSV antigen in the sample can be determined by detecting the triple complex. [000111] In another aspect, the present disclosure also provides a method for distinguishing a pig immunized by an attenuated PRRSV from a pig infected with a wild type PRRSV, comprising: contacting a sample of the pig with an antibody provided herein, detecting in the sample for the presence of an antigen that specifically binds to the antibody, wherein the presence of the antigen in the sample indicates that the sample is from a pig infected with the wild type PRRSV. The absence of the antibody in the sample can indicate that the sample is from an uninfected pig or a pig infected with an attenuated PRRSV.

[000112] In certain embodiments, the attenuated PRRSV is an attenuated American type PRRSV. In certain embodiments, the attenuated PRRSV comprises polynucleotide molecule having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homology to SEQ ID NO: 5, wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the attenuated PRRSV comprises a polynucleotide molecule having at least 80%) homology to SEQ ID NO:5, wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide having at least 80%> homology to SEQ ID NO: l . [000113] In certain embodiments, the attenuated PRRSV is an attenuated PRRSV live vaccine. In certain embodiments, the attenuated PRRSV live vaccine contains PRRSV having a Deposit Number of CGMCC No. 3121.

[000114] In certain embodiments, the wild type PRRSV is an American type PRRSV. In certain embodiments, the wild type PRRSV comprises one polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the wild type PRRSV comprises one polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1.

[000115] In certain embodiments, the sample can be any pig sample suspected of containing a PRRSV antigen, including without limitation, blood, plasma, body fluid, secretion, excreta, tissue sample, etc. In certain embodiments, the sample of a pig is the blood, plasma, respiratory secretions and excreta of a pig.

[000116] In another aspect, the present disclosure further provides uses of an antibody herein in the preparation of a detecting reagent.

[000117] In certain embodiments, the detecting reagent can be used to detect a PRRSV antigen in the sample which is from a pig suspected of PRRSV infection. Any suitable methods can be used for the detection, for example, the methods may include contacting the sample with the isolated antibody provided herein, and detecting the specific binding of the antibody with the antigen in the sample.

[000118] In certain embodiments, the detecting reagent can be used to distinguish a pig immunized by an attenuated PRRSV from a pig infected with a wild type PRRSV. Any suitable methods can be used for the methods. For example, the sample of a pig can be contacted with the antibody provided herein, and the presence of a PRRSV antigen that specifically binds to the antibody can be detected, wherein the presence of the PRRSV antigen that specifically binds to the antibody indicates that the sample is from a pig infected by a wild type PRRSV. Kit

[000119] In another aspect, the present disclosure further provides kits comprising an isolated polypeptide provided herein, wherein the polypeptide is attached to a solid support.

[000120] In certain embodiments, the kit comprises a polypeptide comprising one or more immunogenic fragments of an Nsp2 protein sequence, wherein the immunogenic fragment is absent in the Nsp2 protein sequence of an attenuated PRRSV, but present in the Nsp2 protein sequence of a wild type PRRSV. In certain embodiments, the wild type PR SV is a highly-pathogenic PR SV. In certain embodiments, the kit comprises a polypeptide comprising one or more immunogenic fragments of a polypeptide fragment having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the immunogenic fragment comprises at least six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acids.

[000121] The solid support can be any materials that are suitable for detection and for attachment of the detecting reagent. Examples include, without limitation, plastic dish, plastic plate, nitrocellulose filter, glass fiber membrane, glass plate, and bead, etc. The polypeptide can be attached to an appropriate solid support by any suitable methods, for example, without limitation, by immobilization, coating, adsorption, spray painting, and printing, etc. The polypeptide attached to the solid support is suitable for the detection purpose, for example, the attachment is sufficiently strong, and the attached amount is sufficient for detection, etc. A person skilled in the art can choose a suitable attachment method as appropriate according to the detection method to be used.

[000122] In certain embodiments, the kit further comprises a detecting antibody. The detecting antibody can be provided in a suitable form, for example, in freeze-dried powders or in a solution, etc. In certain embodiments, the detecting antibody is an anti-pig antibody, for example, without limitation, mice anti-pig antibody, rat anti-pig antibody, rabbit anti-pig antibody, goat anti-pig antibody, chicken anti-pig antibody, and donkey anti-pig antibody, etc.

[000123] The detecting antibody can further labeled with, for example, without limitation, an enzyme, a fluorescent molecule, a chemoluminescent molecule, a radioactive substance, a colored reagent, etc. For example, the labeled detecting antibody can be an enzyme-labeled antibody, a fluorescently-labeled detecting antibody, a chemoluminescent substance labeled detecting antibody, a radioactive substance labeled detecting antibody, a colloidal gold labeled antibody, etc. Optionally, in certain embodiments, the kit can further comprise a detection reagent for the label, for example, a substrate of an enzyme, a substrate chromogenic solution, and a reaction termination buffer, etc.

[000124] Optionally, the kit can further comprise one or more reagents useful in the detection, for example, without limitation, sample diluents, wash buffer, positive control serum, and negative control serum, etc. Optionally, the kit can further comprise one or more reagents useful for the storage, for example, without limitation, a drying agent, etc. The kit can also comprise an instruction, which specifies the usage, storage condition or precautions of the kit.

[000125] In certain embodiments, the kit can also further comprise a preparation of an attenuated PR SV. The attenuated PRRSV can be an attenuated American type PRRSV. In certain embodiments, the attenuated PRRSV comprises a polynucleotide molecule having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homology to SEQ ID NO: 5, wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the attenuated PRRSV comprises a polynucleotide molecule having at least 80%) homology to SEQ ID NO:5, wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide having at least 80%> homology to SEQ ID NO: 1.

[000126] In certain embodiments, the attenuated PRRSV is an attenuated PRRSV live vaccine. In certain embodiments, the attenuated PRRSV is attenuated from a highly-pathogenic PRRSV. In certain embodiments, the PRRSV attenuated live vaccine contains a PRRSV having a deposit number of CGMCC No. 3121. [000127] The attenuated PRRSV can be provided in a form that is suitable for animal immunization. In certain embodiments, the attenuated PRRSV is provided in a sterile composition. In certain embodiments, the attenuated PRRSV is provided as a freeze-dried sterile composition. In certain embodiments, the attenuated PRSV further comprises a cryoprotectant. A cryoprotectant can keep the attenuated PRRSV in good stability and reduce the damage to the biological activity of the attenuated PRRSV during the process of lyophilization. Exemplary cryoprotectants include, without limitation, sucrose, L-sodium glutamate or lactalbumin hydrolysate, and gelatin, etc.

[000128] The attenuated PRRSV in the kit can be prepared by the methods known in the art. For example, the attenuated PRRSV can be propagated in the cells that support its growth (such as Marc- 15 cells), and the virus solution can be harvested when the cultured cells show a pre-determined degree of cytopathic effect. Optionally, the harvested attenuated PRRSV can be further mixed with an appropriate excipient, for example, without limitation, a cryoprotectant and an antibiotic, etc. The excipients can be sterile.

Oligonucleotide primer

[000129] In another aspect, the present disclosure further provides oligonucleotide primers useful in detecting the presence of a PRRSV in a sample. The oligonucleotide primers can be used to amplify a polynucleotide template (such as, a cDNA template) derived from the sample, and the amplification product can be analyzed for determination of the presence of a PRRSV in the sample, or for determination of certain deletions in the PRRSV present in the sample. [000130] "Primer" as used herein refers to an oligonucleotide molecule with a length of 10 - 38 nucleotides, preferably 15-30 nucleotides, or 15-25 nucleotides, or 17-25 nucleotides. For example, the primer can an oligonucleotide having a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Primers are usually used in the amplification of a DNA sequence by polymerase chain reaction (PCR). For a DNA template sequence to be amplified, a pair of primers can be designed at its 5' upstream and its 3' downstream sequence, i.e. 5 ' primer and 3 ' primer, each of which can specifically hybridize to a separate strand of the DNA double strand template. 5' primer is complementary to the anti-sense strand of the DNA double strand template; and 3' primer is complementary to the sense strand of the DNA template. As known in the art, the "sense strand" of a double stranded DNA template is the strand whose sequence is identical to the mRNA sequence transcribed from the DNA template (except that "U" in RNA corresponds to "T" in the DNA) and encodes for a protein product. In the present disclosure, SEQ ID NOs: 5-6, 8-19 are all sense strand DNA. The complementary sequence of the sense strand is the "anti-sense strand," and thus the sequences to which SEQ ID NOs: 5-6, 8-19 are complementary are anti-sense strand of the DNA template.

[000131] "Hybridize to" as used herein refers to the process of hydrogen bond formation between a primer and a template DNA through base pairing. "Specifically hybridize to" as used herein means that a sufficient number of hydrogen bonds are formed between the primer and the template DNA, so that the template DNA can be amplified by PCR. In certain embodiments, at least 70%, 75%, 80%, 85%, 90%, 95%), 97%o, 98%o, 99%o or 100% nucleotides of the primer form hydrogen bonds with the template DNA. In certain embodiments, the primer is complementary to the template DNA. The term "complementary" as used herein can be used interchangeably with the term "specifically hybridize to".

[000132] The primers can be used to amplify the DNA template sequence through PCR reaction. PCR usually includes multiple reaction cycles, each of which includes a denaturation step, an annealing step and an extension step, etc. During the denaturation, the reaction is heated to a high temperature and the DNA double strand melt into single-stranded DNA molecules. The temperature is lowered to the annealing temperature, at which the 5 ' primer and 3 ' primer specifically hybridize to their target sequences respectively on each of the single strands of the DNA template. The DNA template sequence hybridized with the 5' primer is referred to as 5' upstream sequence, and the DNA template sequence hybridized with the 3' primer is referred to as the 3' downstream sequence. During the extension step, the DNA polymerase elongates the primers at its 3' end by adding nucleotides that are complementary to the DNA template in 5 ' to 3 ' direction, thereby synthesizes a new DNA strand that is complementary to the DNA template strand. The newly synthesized DNA chain can serve as DNA template in subsequent reaction cycles. In this way, the DNA template sequence between the 5 ' upstream sequence and the 3 ' downstream sequence (i.e. the sequence to be amplified) can be exponentially amplified through PCR, and the DNA product obtained thereof is usually referred to as an amplified product. Typically, the length of an amplified product is longer than or at least equal to the sum of the lengths of 5 ' primer and 3 ' primer. The amplified product has a sense strand whose 5 ' end sequence is the sequence of the 5 ' primer, and an anti-sense strand whose 5 ' end sequence is the sequence of the 3 ' primer. [000133] In certain embodiments, the sequence to be amplified using the oligonucleotide primers as provided herein comprises at least partial coding sequence of an Nsp2 protein, wherein the coding sequence is absent in the Nsp2 coding sequence of an attenuated PRRSV, but is present in the Nsp2 coding sequence of a wild type PRRSV. [000134] In certain embodiments, the oligonucleotide primer as provided herein can amplify the nucleotide sequence of a classical type PRRSV. A "classical type PRRSV" as used herein, refers to the PRRSV whose Nsp2 coding sequence contains substantially no deletion when compared to that of VR-2332, the standard strain of American type PRRSV. "Substantially no deletion" as used herein, means that the number of nucleotides that is absent is less than or equal to 10, 9, 8, 7, 6, 5, 4, or 3. A classical type PRRSV can be a PRRSV whose sequence is known, for example, but without limitation, PRRSV VR-2332 strain and 16244B strain (Genbank Accession No.: AF046869).

[000135] In certain embodiments, the oligonucleotide primers as provided herein can be used to identify a highly-pathogenic PRRSV. A highly-pathogenic PRRSV comprises a Nsp2 coding sequence that lacks 90 discontinuous nucleotides when compared to that of VR-2332 (see, for example, Figure 1, Figure 2, Tian et al, PLoS ONE 2(6): e526, (2007) doi: 10.1371). There're substantially no additional deletions in the Nsp2 coding sequence of a highly-pathogenic PRRSV. In certain embodiments, the oligonucleotide primers as provided herein can be used to identify the presence of the 90 discontinuous nucleotides in a PRRSV in a sample, and therefore can be useful in determining the pathogenicity of the PRRSV that is present in the sample. [000136] In certain embodiments, the oligonucleotide primers provided herein can be used to identify an attenuated PRRSV. As described above, an attenuated PRRSV contains a sequence deletion in the Nsp2 coding sequence that reduces its pathogenicity. In certain embodiments, the Nsp2 coding sequence of the attenuated PRRSV lacks a 360-nucleotide fragment which encodes a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In certain embodiments, the Nsp2 coding sequence of the attenuated PRRSV contains substantially no additional sequence deletion. In certain other embodiments, the Nsp2 coding sequence of the attenuated PRRSV further lacks additional nucleotide sequences, for example, further lacks the 90 discontinuous nucleotides as disclosed above. In certain embodiments, the attenuated PRRSV is a PRRSV having a Deposit No. of CGMCC No. 3121 (i.e. PRRSV TJM strain).

[000137] In certain embodiments, the present application provides a first pair of isolated oligonucleotide primers, comprising a 5 ' primer and a 3 ' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NOs: 8-10 and sequences having at least 80%> homology to any one of SEQ ID NOs: 8-10; the 3' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 13-19 and sequences having at least 80% homology to any one of SEQ ID NOs: 13-19; the 5' primer and the 3' primer each has a length of between 10 and 38 nucleotides; the 5' primer and the 3' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or at least equals to the combined lengths of the 5' primer and the 3' primer. The relative position of the primers on the Nsp2 coding sequence is shown in Figure 1 (b). [000138] In certain embodiments, the present application provides methods for detecting a PRRSV in a biological sample using the first pair of the isolated oligonucleotide primers, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using the first pair of the isolated oligonucleotide primers, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample. If the amplified product is absent in the test sample but present in the control sample, then it indicates that the test sample does not contain PRRSV or contains a PRRSV which is attenuated.

[000139] In certain embodiments, the determining of said difference comprises comparing the molecular weight of the amplified product of the test sample with that of the control sample, wherein the amplified product of the control sample has a molecular weight comparable to the amplified product of a classical type PRRSV by using the same pair of primers. If the amplified product as detected in the test sample is about 90 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PRRSV which is highly-pathogenic. If the amplified product as detected in the test sample is about 360 or 450 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PRRSV which is attenuated. If the amplified product as detected in the test sample has a molecular weight similar to that of the control sample, then it indicates that the test sample contains a PRRSV which is of classical type.

[000140] "Control sample," when used with respect to PCR detection methods herein, can be an amplified product of a classical type PRRSV using the primers provided herein (i.e. the amplified product obtained using a cDNA which is reverse transcribed from a classical type PRRSV), or can be any polynucleotide fragment having the same or comparable molecular weight of the amplified product of the classical type PRRSV. [000141] It would be understood by a person skilled in the art that, if a PRRSV is absent in the sample, the primer pair cannot produce an amplified product by PCR because there's no DNA template for the primer pair. If the PRRSV present in the sample lacks SEQ ID NO: 13, then the 3' primer complementary to SEQ ID NO: 13 would not specifically hybridize to the DNA template, and therefore the corresponding DNA single strand cannot be synthesized, and consequently no double strand amplified product would be produced. If the PRRSV present in the sample lacks SEQ ID NO: 13, then an amplified product can be obtained through PCR amplification using the 3 ' primer complementary to 3 ' downstream region of SEQ ID NO: 13 and one of the above-mentioned 5' primers. Since this amplified product does not contain SEQ ID NO: 13, and therefore theoretically it would be about 360bp shorter than the amplified product of a classical type PRRSV. Similarly, if the PRRSV present in the sample lacks both the 90 discontinuous nucleotides and SEQ ID NO: 13, then theoretically the amplified product obtained by the primer pair should be about 450bp shorter than the amplified product of a classical type PRRSV. If the PRRSV present in the sample only lacks the 90 discontinuous nucleotides, then theoretically the amplified product would be about 90bp shorter than the amplified product of a classical type PRRSV. By detecting the presence of and/or analyzing the molecular weight of the amplified product, people in the art would know the absence or presence and/or the size of any deletions in the Nsp2 nucleotide sequence of a PRRSV in the sample. The pathogenicity of the PRRSV in the sample can be determined based the corresponding biological meanings of these deletions. For example, with respect to the positive control (e.g. the amplified product of a classical type PRRSV), a PRRSV whose amplified product lacks 90bp only is probably a highly-pathogenic PRRSV, a PRRSV whose amplified product lacks 360bp only is probably an attenuated PRRSV, and a PRRSV whose amplified product lacks 450bp is also probably an attenuated PRRSV. The tested pigs can be appropriately grouped according to the pathogenicity of the PRRSV in the pig samples, such that pigs infected with a highly-pathogenic PRRSV or a wild type PRRSV can be isolated from those un-infected or immunized, thereby prevent further transmission of pathogenic virus, and reduce potential economic loss.

[000142] In certain embodiments, the present disclosure provides a second pair of isolated oligonucleotide primers, containing a 5' primer and a 3' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NO: 11 and the sequence having at least 80% homology to SEQ ID NO: 11, and the 3' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 13-19 and sequences having at least 80% homology to any one of SEQ ID NOs: 13-19; the 5' primer and the 3' primer each has a length of between 10 and 38 nucleotides; the 5' primer and the 3' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or at least equals to the combined lengths of the 5 ' primer and the 3 ' primer.

[000143] In certain embodiments, the present disclosure provides methods for detecting a PRRSV in a biological sample using the second pair of the isolated oligonucleotide primers, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using the second pair of the isolated oligonucleotide primers, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample. If the amplified product is absent in the test sample but present in the control sample, then it indicates that the test sample does not contain PRRSV or contains a PRRSV which is attenuated.

[000144] In certain embodiments, the determining of said difference comprises comparing the molecular weight of the amplified product of the test sample with that of the control sample, wherein the amplified product of the control sample has a molecular weight comparable to the amplified product of a classical type PRRSV by using the same pair of primers. If the amplified product as detected in the test sample is about 360 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PR SV which is attenuated. If the amplified product as detected in the test sample has a molecular weight similar to that of the control sample, then it indicates that the test sample contains a PRRSV which is of classical type. [000145] In certain embodiments, the present application provides a third pair of isolated oligonucleotide primers, comprising a 5 ' primer and a 3 ' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NOs: 11-13 and the sequences having at least 80% homology to any of SEQ ID NOs: 11-13, and the 3' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 14-16 and sequences having at least 80% homology to any one of SEQ ID NOs: 14-16; the 5' primer and the 3' primer each has a length of between 10 and 38 nucleotides; the 5' primer and the 3' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or at least equals to the combined lengths of the 5 ' primer and the 3 ' primer.

[000146] In certain embodiments, the present disclosure provides methods for detecting a PRRSV in a biological sample using the third pair of the isolated oligonucleotide primers, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using the third pair of the isolated oligonucleotide primers, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample. If the amplified product is absent in the test sample but present in the control sample, then it indicates that the test sample does not contain PRRSV or contains a PRRSV which is attenuated.

[000147] In certain embodiments, the determining of said difference comprises comparing the molecular weight of the amplified product of the test sample with that of the control sample, wherein the amplified product of the control sample has a molecular weight comparable to the amplified product of a classical type PR SV by using the same pair of primers. If the amplified product as detected in the test sample is about 360 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PRRSV which is attenuated. If the amplified product as detected in the test sample has a molecular weight similar to that of the control sample, then it indicates that the test sample contains a PRRSV which is of classical type.

[000148] In certain embodiments, the present application provides a fourth pair of isolated oligonucleotide primers, comprising a 5 ' primer and a 3 ' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of: SEQ ID NO: 12 and sequences having at least 80% homology to SEQ ID NO: 12; the 3' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 17-19 and sequences having at least 80% homology to any one of SEQ ID NOs: 17-19; the 5' primer and the 3' primer each has a length of between 10 and 38 nucleotides; the 5' primer and the 3' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or at least equals to the combined lengths of the 5' primer and the 3 ' primer.

[000149] In certain embodiments, the present application provides methods for detecting a PRRSV in a biological sample using the fourth pair of isolated oligonucleotide primer, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using the fourth pair of the isolated oligonucleotide primers, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample. If the amplified product is absent in the test sample but present in the control sample, then it indicates that the test sample does not contain PRRSV or contains a PRRSV which is attenuated. [000150] In certain embodiments, the determining of said difference comprises comparing the molecular weight of the amplified product of the test sample with that of the control sample, wherein the amplified product of the control sample has a molecular weight comparable to the amplified product of a classical type PR SV by using the same pair of primers. If the amplified product as detected in the test sample is about 360 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PRRSV which is attenuated. If the amplified product as detected in the test sample has a molecular weight similar to that of the control sample, then it indicates that the test sample contains a PRRSV which is of classical type.

[000151] In certain embodiments, the present disclosure provides a fifth group of isolated oligonucleotide primers, comprising a ' primer and a 3 ' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NOs: 8-10 and sequences having at least 80% homology to any of SEQ ID NOs: 8-10; the 3' primer is complementary to a sequence selected from the group consisting of SEQ ID NO: 13 and sequences having at least 80% homology to any one of SEQ ID NO: 13; wherein the 5' primer and the 3' primer respectively have a length between 10 and 38 nucleotides; the 5' primer and the 3' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or equals to the combined lengths of the ' primer and the 3 ' primer.

[000152] In certain embodiments, the present application provides methods for detecting a PRRSV in a biological sample using the fifth pair of isolated oligonucleotide primers, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using the fifth pair of the isolated oligonucleotide primers, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample. If the amplified product is absent in the test sample but present in the control sample, then it indicates that the test sample does not contain PRRSV or contains a PRRSV which is attenuated.

[000153] In certain embodiments, the determining of said difference comprises comparing the molecular weight of the amplified product of the test sample with that of the control sample, wherein the amplified product of the control sample has a molecular weight comparable to the amplified product of a classical type PRRSV by using the same pair of primers. If the amplified product as detected in the test sample is about 90 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PRRSV which is highly-pathogenic. If the amplified product as detected in the test sample has a molecular weight similar to that of the control sample, then it indicates that the test sample contains a PRRSV which is of classical type.

[000154] In certain embodiments, the present application provides a sixth group of isolated oligonucleotide primers, comprising a 5' primer and a 3' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NO: 13 and sequences having at least 80% homology to SEQ ID NO: 13; the 3' primer is complementary to a sequence selected from the group consisting of SEQ ID NO: 13 and sequences having at least 80% homology to any one of SEQ ID NO: 13; wherein the 5' primer and the 3' primer respectively have a length between 10 and 38 nucleotides; the 5' primer and the 3' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or equals to the combined lengths of the ' primer and the 3 ' primer. [000155] In certain embodiments, the present disclosure provides methods for detecting a PRRSV in a biological sample using the sixth pair of the isolated oligonucleotide primers, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using the sixth pair of the isolated oligonucleotide primers, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample. If the amplified product is absent in the test sample but present in the control sample, then it indicates that the test sample does not contain PRRSV or contains a PRRSV which is attenuated.

[000156] In another aspect, the present disclosure further provides isolated DNA fragments comprising an amplified product, wherein the amplified product is obtained through polymerase chain reaction in the presence of a sample containing a PRRSV and a pair of 5 ' primer and 3 ' primer as provided herein. In certain embodiments, the 5' primer and 3' primer are the first pair of the isolated oligonucleotide primers, the second pair of the isolated oligonucleotide primers, the third pair of the isolated oligonucleotide primers, the fourth pair of the isolated oligonucleotide primers, the fifth pair of the isolated oligonucleotide primers, or the sixth pair of the isolated oligonucleotide primers.

[000157] As is known in the art, a suitable primer hybridization position and a primer sequence can be selected according to the common knowledge of a skilled artisan. For example, the position of primer hybridization can be selected according to the length of the sequence to be amplified. For example, if the position of the 5' primer is determined, then the hybridization position of the 3 ' primer can be selected based on the expected size of amplified product and the hybridization position of 5' primer, and vice versa.

[000158] A person skilled in the art can determine a suitable length of the amplified product according to the common knowledge in the art. In certain embodiments, the length of an amplified product is less than or equals to 6000 base pair (6000bp), 5500bp, 5000bp, 4500bp, 4000bp, 3500bp, or 3000bp. In certain embodiments, the length of an amplified product can range from, for example, about lOObp to about 3000bp, about lOObp to about 2500bp, about lOObp to about 2000bp, about lOObp to about 1900bp, about lOObp to about 1800bp, about lOObp to about 1700bp, about lOObp to about 1600bp, about lOObp to about 1500bp, about lOObp to about 1400bp, about lOObp to about 1300bp, about lOObp to about 1200bp, about lOObp to about l lOObp, about lOObp to about lOOObp, or about lOObp to about 800bp, etc. In certain embodiments, a suitable length of the amplified fragment can be selected based on the length of the deletion fragment of interest. For example, the length of the amplified product can be selected such that the presence or absence of an interested deletion in the amplified product can be revealed by electrophoresis. For example, if the interested deletion fragment is 360bp, then the corresponding amplified product can be less than or equal to 6000bp, 5500bp, 5000bp, 4500bp, 4000bp, 3500bp or 3000bp, and if the interested deletion fragment is 90bp, then the corresponding amplified product can be less than or equal to 3000bp, 2500bp, 2000bp.

[000159] Once the positions for primer hybridization are selected and/or the length and sequence of amplified fragment are determined, the primer sequence and PCR protocols can be readily designed using methods known in the art, see, for example, J. Bartlett et al, PCR Protocols, published by Humana Press, 2003 ;A. Yuryev, PCR primer design, published by Humana Press, 2007.

[000160] In certain embodiments, the 5' primer, which is complementary to SEQ ID NO: 8, is 5'-ATGTCCCTAACAGTTGGAA-3'. In certain embodiments, the 3' primer, which is complementary to SEQ ID NO: 15, is 5 '-CGCCGAGAAG ACCC AGA-3 '.

[000161] In certain embodiments, the present disclosure provides a pair of primers in which the 5' primer is 5 '- ATGTCCCTAAC AGTTGGAA-3 ', and the 3' primer is 5 '-CGCCGAGAAG ACCC AGA-3 '. [000162] In certain embodiments, the biological samples provided herein are from a pig suspected of PRRSV infection. The biological samples can be, for example, without limitation, blood, plasma, body fluid, secretion (such as respiratory secretion), excreta (such as urine, feces, etc.), tissue sample (such as lymphatic tissue, pulmonary tissue, and muscular tissue), etc.

[000163] In certain embodiments, the reverse transcribed products can be obtained through reverse transcription PCR (RT-PCR). For example, a sample from a pig suspected of PRRSV infection can be routinely processed for RNA isolation (for example, using Trizol), and then the RNA is reverse transcribed using RT-PCR to obtain the reverse transcribed product of the RNA (for example, by using a random hexamer primer and a reverse transcription polymerase, or by using primers designed specifically for a PRRSV sequence and a reverse transcription polymerase, see also, for example, J. O'Connell, RT-PCR protocols, Methods in Molecular Biology, Vol 193, published by Humana Press, 2002).

[000164] The reverse transcribed product can be used as the DNA template and amplified using a pair of primers provided herein. In certain embodiments, the reverse transcribed product is amplified by PCR. A person skilled in the art can select and optimize the PCR reaction conditions , for example, the type of polymerase, the temperatures, the number of reaction cycles, and reaction volume used in PCR, etc., based on methods known in the art (see, for example, J. Bartlett et al, PCR Protocols, published by Humana Press, 2003).

[000165] The presence of an amplified product can be detected by any suitable methods known in the art, for example, without limitation, by agarose gel electrophoresis. Suitable detection molecules can be selected for the detection, for example, but without limitation, DNA double strand intercalative dye (such as ethidium bromide, fluorescent stains that bind to DNA double strand, etc.).

[000166] Optionally, the molecular weight of the amplified product can be further measured. For example, a DNA molecular weight marker can be used in the electrophoresis, and the electrophoresis band of the amplified product is compared with that of a DNA molecular weight marker. For another example, the amplification product for a classical type PRRSV can be used as a positive control, since the length of its amplified product can be predicted based on its known sequence. EXAMPLES

[000167] The present disclosure is described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention, which is defined by the claims which follow thereafter.

Example 1: Establishment of an indirect ELISA diagnostic method for PRRSV

[000168] PRRSV TJ strain was isolated in the inventors' laboratory. A sequence deletion was found in the Nsp2 gene when the strain was passaged to the 92nd passage, and the virulence was found to be attenuated. The attenuated strain PRRSV TJM-F92 (i.e. PRRSV TJM strain) was prepared and developed as a PRRS attenuated live vaccine, characterized in that its Nsp2 nucleotide sequence was 2490bp in full length. The Nsp2 nucleotide, when compared with that of PRRSV TJ strain, lacked 360 continuous nucleotides (see Figure 3) encoding for the 598^th to the 717^th amino acids of the Nsp2 protein of PRRSV TJ strain (see Figure 4, SEQ ID NO: l). The Nsp2 protein of PPRSV TJM strain, when compared with that of PRRSV VR-2332 strain, lacked a total of 150 amino acids including the 481^s amino acid, the 537^th to the 565^th amino acid and the 628^th to the 747^th amino acids of that of PRRSV VR-2332. [000169] Preferred B cell epitopes were identified by analyzing the deleted 120-amino acid sequence (i.e. SEQ ID NO: 1) in the Nsp2 protein of PRRSV TJM strain, and an ELISA method was established for differentiation of a PRRSV virulent strain and PRRSV TJM vaccine strain. Preferred B cell antigen epitopes specific for SEQ ID NO: 1 were identified (the amino acid sequences shown in Figure 5) and synthesized in the inventors' laboratory. They can be used in large-scale detection of PRRSV antibody, and therefore facilitate the wide use of PRRSV vaccine (e.g. TJM strain) and provide a convenient method for eradication of PRRS.

[000170] 1. Experimental materials [000171] 1.1 Coating antigen: synthetic B cell epitope polypeptides shown in Figure 5 were synthesized.

[000172] 1.2 Test serum and test animals: PRRSV (TJ strain) positive serum and PRRSV (TJM strain) immunized serum were both prepared by the inventors by conventional methods using pigs negative for PRRS antibody. Standard negative serum was the serum that was detected as negative by IDEXX PRRS antibody kit (purchased from Beijing IDEXX Yuanheng Biotech. Co., Ltd.).

[000173] 2. General experimental method and optimization of conditions

[000174] Indirect ELISA method was used in the experiments, and the detection was performed on the microtiter plate according to conventional methods. Generally, the synthetic B cell epitopes provided herein were diluted with 0.05M pH9.6 buffer bicarbonate, and then were coated on the microtiter plate and placed under 4°C overnight. The microtiter plate was washed on the next day, and blocked with a blocking buffer for 2h. After washing the plate, the test serum was added and the plate was placed in a 37°C incubator to react for 45 min. The plate was washed before addition of an enzyme-conjugated antibody at a suitable concentration, and then placed in a 37°C incubator to react for lh. The plate was washed, and the substrate chromogenic solution was added. After reaction for 10-15 min at 37°C, 2M H₂SO₄ was added to terminate the reaction. The OD₄₅onm value was determined by a microplate reader. The experiment included a positive serum, a negative serum and an immunized serum as controls.

[000175] A series of experiments were performed to optimize the indirect ELISA diagnostic method with respect to the following conditions: antigen concentration and fold of dilution for the serums; blocking reagents and blocking time; fold of dilution and reaction time of the primary antibody; fold of dilution and reaction time of the enzyme-labeled secondary antibody; and substrate reaction time. The optimal conditions of the ELISA methods were established with the following details.

[000176] 2.1 Determination and synthesis of B cell epitope [000177] The full-length sequence of the Nsp2 protein of PR SV TJ strain was aligned to that of PRRSV T JM-F92 vaccine strain by biological software DNAStar, to identify the missing nucleotide sequence (see Figure 3) from which the missing amino acid sequence was derived (see Figure 4). [000178] The missing amino acid sequence in PRRSV TJM strain was analyzed using the Kyte-Doolittle program in DNAStar to predict its hydrophilicity, the Emini program to predict the surface accessibility of the protein, and the Jameson- Wolf program to predict the antigenicity index. Suitable B cell epitopes were selected (see Figure 5) based on the comprehensive analysis and the assessment of the predicted epitopes according to the antigenicity index of 20 amino acid residues. The epitopes were synthesized by Beijing SBS Genetech Co., Ltd.

[000179] 2.2 Characterization of preferred B cell epitopes

[000180] The B epitope polypeptide was dissolved and diluted in 1 : 100 with 0.05M pH9.6 buffer bicarbonate (CBS), and then was coated on a microplate at ΙΟΟμΙ,ΛνεΙΙ at 4°C overnight. The solution was removed and the plate was washed with 0.02mol/L PBS (PBST, pH 7.2) containing 0.05% Tween-20 for 5 min x 4 times. PBST containing 10% fetal calf serum was added at lOOfxL/well, and reacted at 37°C for lh. The plate was washed, and reacted with rabbit-anti-pig secondary antibody diluted in 1 :10000 at ΙΟΟμΙ,ΛνεΙΙ at 37°C for lh. The plate was washed and reacted with freshly prepared TMB substrate solution at ΙΟΟμίΛνεΙΙ at 37°C for 15 min. 2M H₂S0₄ solution was added at 50μΕΛνε11 to terminate the reaction, and the OD₄₅₀ value was measured on a microplate reader. Each sample was tested in two replicates, and the average value was determined. Polypeptides were selected that provided a relatively high OD value and produced similar results for the immune serum of PRRSV TJM vaccine and for the negative serum. The three epitope polypeptides shown in Figure 5 all showed an OD value that was qualified for development of an ELISA kit. The results are shown in Table 1.

[000181] Table 1 Testing sample OD₄₅₀ Value

Polypeptide 1 Polypeptide 2 Polypeptide 3

(SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4)

Positive serum 0.357 0.401 0.448

Immune serum 0.190 0.275 0.312

Negative serum 0.189 0.219 0.278

[000182] 2.3 Determination of optimal coating amount of the B cell epitope polypeptide and the optimal serum dilution

[000183] The selected polypeptides were serially diluted to 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, and 0.390625 μg/mL, respectively, before they were coated on the plate and placed at 4°C overnight. The plate was washed and blocked before addition of the positive serums diluted in 50, 100, 200 and 400-fold, respectively. Checkerboard titration was used to determine the optimal antigen concentration and the optimal serum dilution. To provide for a maximum OD value yet economic use of the antigen, the optimal antigen coating concentration was determined as 12^g/mL, and the optimal serum dilution as 1 :200 which provided for the optimal concentration of the test serum for the reaction.

[000184] 2.4 Selection of a blocking buffer

[000185] To select a suitable blocking buffer, 10% fetal calf serum, 5% defatted milk and 1% bovine serum albumin were tested at 100 μΕΛνεΙΙ in an ELISA assay at 37°C for 2h. The results showed that the blocking buffer containing 10% fetal calf serum effectively blocked the residual active sites on the surface with a high specificity, and provided the optimal and satisfactory blocking effect. Therefore, PBS buffer containing 10% fetal calf serum was used as the blocking buffer in the examples provided herein, and the blocking period was 2h. The PBS buffer (0.01M pH7.4) was prepared by dissolving in 1L water with 8g NaCl, 0.2g KC1, 2.9g Na₂HP0₄ 12HO, and 0.5g KH₂P0₄.

[000186] 2.5 Determination of optimal reaction time of the test serum

[000187] The microplate was coated with the polypeptide, and reacted with optimally diluted PRRS negative serums and positive serums, respectively, following an indirect ELISA procedure at 37°C for 30, 45, 60 and 90 min, respectively. A reaction time of 45min provided for the highest OD value as well as the largest P/N ratio. Therefore, the optimal reaction time of the test serum was set as 45 min. [000188] 2.6 Determination of optimal dilution and reaction time of the enzyme-labeled secondary antibody

[000189] The microplate was coated with the polypeptide, and reacted with optimally diluted PRRS negative serums and positive serums, respectively, at 37°C for an optimal reaction time. The enzyme-labeled secondary antibody was diluted in 1 :5000, 1 : 10000, 1 :20000, and 1 : 40000, respectively. Each dilution was performed in four replicates, and each replicate was reacted for 30, 45, 60 or 90 min. The plate was washed before addition of freshly prepared substrate solution. A 1 : 10000 dilution of the secondary antibody and a reaction time of 60 min provided for the largest P/N ratio, and therefore were identified as the optimal dilution and the optimal reaction time.

[000190] 2.7 Determination of substrate action time

[000191] ELISA assay was performed under the above determined optimal conditions. The freshly prepared substrate solution was added to each assay well, and after 8, 10, 12, 15, 18, or 20 min, 2M Η₂8Ο₄50μΕ was added to the respective assay well, and the OD₄so value was measured on a microplate reader. A reaction time of 15 min provided for the largest P/N ratio, and therefore, the optimal substrate reaction time was determined as 15 min.

[000192] 2.8 Analysis of assay results

[000193] 50 porcine serums, which had been tested as PRRS negative by IDEXX PRRS antibody test kit, were assayed under the above optimal reaction conditions, and the S/P value was calculated using: S/P value = (sample OD value -negative control OD value)/(positive control OD value - negative control OD value). According to the statistical analysis results, the mean S/P of the negative samples is X=0.593, and the standard deviation SD=0.064. The upper limit of the confidence interval was defined as the lower limit for a positive serum, i.e. X+3SD=0.785. The suspected case is defined as X+2SD=0.721. According to statistics, if a sample has an S/P value >X+3SD, then the sample would be predicted as PR S positive sample with 99.7% reliability. Analysis criteria were determined accordingly, i.e. S/P value >0.785 would mean positive, S/P value <0.721 would mean negative, and a value somewhere in between would mean suspected.

Example 2: Preparation of the indirect ELISA diagnostic kit

[000194] 1 Preparation of the antigen-coated plate [000195] 1.1 Coating: The three preferred antigens (i.e. SEQ ID NOs: 2-4) were combined and were used to coat the ELISA microplate. Each testing well of the plate was coated with ΙΟΟμί of the antigen combination, in which each antigen was in a final concentration of 12^g/mL. The microplate was covered and incubated in a 4°C refrigerator overnight. [000196] 1.2 Washing: after overnight incubation, the coating solution in the plate was removed, and the plate was washed 3 times with a washing buffer (0.01M pH7.4 PBS with 0.05% Tween20).

[000197] 1.3 Blocking: the plate was blocked using a blocking buffer (PBS containing 10% fetal calf serum), and was covered and incubated in a 37°C incubator for 2h.

[000198] 1.4 Washing: the blocking buffer in the plate was removed and washed 3 times.

[000199] 1.5 Storage: the coated plate was stored at 4°C, and the shelf-life was determined as 6 months. [000200] 2 Preparation of negative serum

[000201] 2.1 Animals: PRRS negative pigs aged 4-6 weeks were used. [000202] 2.2 Negative control serum preparation: blood was collected from PR S negative pigs, and serum was separated and filtered through a 0.22μιη filter membrane, before being dispensed into 0.2 ml per vial under sterile conditions and stored at -20°C. The serum should appear yellowish or reddish clear liquid when in liquid status, and should be sterile.

[000203] 3 Preparation of infection positive serum

[000204] 3.1 Animals: PRRS negative pigs aged 4-6 weeks were used and raised in a negative pressure isolation house.

[000205] 3.2 Pathogen: PRRSV TJ strain was used to infect the animals. [000206] 3.3 Titration: the standard positive serum and serum as obtained from the infected animals were diluted in 1 :200 with 0.01M PBST buffer (pH7.4, PBS containing 0.05% Tween-20), and were assayed by ELISA. The S/P value of the standard positive serum was >0.785.

[000207] 3.4 Infection positive serum preparation: blood was collected from precaval veins of pigs having a qualified serum antibody titer, and serum was separated and filtered through 0.22μιη filter membrane, before being dispensed into 0.2 ml per vial under sterile conditions and stored at -20°C.

[000208] 4 Preparation of immunization positive serum

[000209] 4.1 Animals: PRRS negative pigs aged 4-6 weeks were used and raised in a negative pressure isolation house.

[000210] 4.2 Pathogen: PRRSV TJM strain was used to immunize the animals.

[000211] 4.3 Titration: the serum as obtained from the immunized animals was diluted and assayed by ELISA using both the IDEXX kit and the B cell epitope polypeptides provided in Example 1. The immunized serum showed positive results in IDEXX kit and negative results with the polypeptides of Example 1.

[000212] 4.4 Immunization positive serum preparation: blood was collected from precaval veins of pigs having a qualified serum antibody titer, and serum was separated and filtered through 0.22μιη filter membrane, before being dispensed into 0.2 ml per vial under sterile conditions and stored at -20°C.

[000213] 5. Preparation of the enzyme-labeled antibody [000214] Horseradish peroxidase (HRP) labeled goat-anti-pig IgG was purchased from Sigma-Aldrich (US). The HRP-labeled antibody was diluted in 1 : 10000 using 0.01M phosphate buffer (pH7.4), and filtered through 0.22μιη filter membrane.

[000215] 6. Preparation of substrate chromogenic solution

[000216] Chromogenic solution A was prepared by dissolving 10 mg of tetramethyl benzidine (TMB) in 1 ml of dimethyl sulfoxide (DMSO) followed by mixing. Chromogenic solution B was prepared by sequentially adding into the 700ml sterile deionized water with 10.3g citric acid H₂0, 35.8g Na₂HP0₄ 12H₂0, 1.0g hydrogen peroxide urea, and ΙΟΟμί of Tween-20. The solution was mixed and metered to the volume of 1L. Chromogenic solution A and B was mixed immediately before use at a ratio of 1 : 100.

[000217] 7. Preparation of sample diluting buffer

[000218] Sterile deionized water of 90% total volume was prepared, and 4g Na₂HP0₄ 12H₂0, 50g NaCl, 60g NaH₂P0₄ and 6g gentamicin were sequentially added and stirred at 18-25°C to make a homogeneous solution. The pH of the solution was measured and adjusted to 7.1 to 7.3 with 0.1 M HC1 or 0.1 M NaOH. The volume was metered to 30L with sterile deionized water, and was filtered through a 0.22μιη filter membrane.

[000219] 8. Preparation of washing buffer

[000220] Sterile deionized water of 80% total volume was prepared, and 300g Na₂HP0₄ 12H₂0, lOOg NaCl, 60g NaH₂P0₄ and 15 ml Tween-20 were sequentially added and stirred vigorously to allow complete dissolution. The volume was metered to 30L with sterile deionized water, and filtered through a 0.22μιη filter membrane.

[000221] 9. Preparation of termination buffer

[000222] Termination buffer was prepared by diluting sulfuric acid to 2M with deionized water. [000223] 10. Preparation of the kit

[000224] The qualified individual components were assembled into a kit according to the following table.

[000225] Table 2. Components of the kit

[000226] 11. Use of the kit [000227] The antigen-coated microplate was contacted with the diluted test serum, infection positive serum, immunization positive serum and standard negative serum, respectively, each was diluted with the sample diluting buffer. The microplate was incubated at 37°C for lh, and then washed with the washing buffer. The enzyme-labeled conjugate antibody was added, and incubated at 37°C for lh. The microplate was washed and then added with the chromogenic solutions A and B, followed by reaction at 37°C for 10-20 min (protected from light). Termination buffer was added to terminate the reaction. OD₄₅onm was measured on a microplate reader.

[000228] Samples with an S/P ratio >0.785 were determined as positive, and samples with an S/P ratio <0.72 was determined as negative. Samples with an S/P ratio in between were determined as suspected and were re -tested. If the re-tested S/P ratio was still within the suspected range, then the sample would be determined as positive.

Example 3: Differentiation of PRRSV TJ strain infected animal from PRRSV TJM strain vaccinated animal using the ELISA kit

[000229] An ELISA kit was prepared according the methods described in Example 2. The immunized serum was prepared by inoculating a PRRS negative pig with PRRSV TJM strain, according to conventional methods. The positive serum was prepared by challenging a PRRS negative pig with the PRRSV TJ strain, and blood was collected at day 20 for serum preparation. The negative serum was prepared from the serum of a PRRS negative pig. The PRRSV negative pigs used in the study were healthy piglets aged at 4 to 6 weeks. A commercialized kit, IDEXX PRRS antibody test kit (purchased from Beijing IDEXX Yuanheng Biotech. Co., Ltd.) was used in the Example to provide for comparison. [000230] 12 PRRSV negative piglets aged at 4 to 6 weeks were randomized into 4 groups. The first group (virus challenge group) received PRRSV TJ strain (F3, the 3^rd passage) via intramuscular injection in the neck muscle, and each pig was inoculated with 1 ml virus at a dose of 10^5'°TCID₅o/ml. Blood was collected from precaval veins every two days for separation of serum. The second group (control group) was injected with virus-free cell culture medium of Marc- 145 cells, and each pig was inoculated with 1 ml culture medium. Blood was collected from precaval vein every two days for separation of serum. Serum samples from both groups were tested using either the ELISA kit as prepared, or using the IDEXX PRRS antibody detection kit, according to the procedures described in Examples 1 and 2. The results were shown in Figure 6.

[000231] The third group (vaccine/challenge group) was first immunized with the PRRSV TJM strain, and was then challenged with PRRSV TJ strain at 28 day after the immunization. Blood was collected from precaval veins every 7 days for separation of serum. The fourth group (vaccine group) was immunized with the PRRSV TJM strain. Blood was collected from precaval veins every 7 days for separation of serum. The serum samples collected from the vaccine group, the vaccine/challenge group and the control group were tested using the ELISA kit as prepared herein, and also using the IDEXX PRRS antibody detection kit, according to the procedures described in Example 2. The results were shown in Figure 7.

[000232] As shown in Figures 6 and 7, the ELISA kit prepared herein detected the antibody against the PRRSV TJ strain in the sample, and did not show any cross-reaction with the samples from the pigs vaccinated with the PRRSV TJM strain. Therefore, the ELISA kit distinguished pigs infected with the attenuated PRRSV strain from pigs infected with the wild type PRRSV strain. The kit used antigen peptide instead of virus, and therefore would be of good bio-safety as it would not pose any threat of viral transmission.

Example 4: Sensitivity test of the indirect ELISA diagnostic kit [000233] An ELISA kit was prepared according the methods described in Example 2. A commercialized kit, IDEXX PRRS antibody test kit (purchased from Beijing IDEXX Yuanheng Biotech. Co., Ltd.) was used in the Example to provide for comparison. The PRRSV negative pigs used in the study were healthy piglets aged at 4 to 6 weeks. [000234] 5 PRRS negative pigs were each inoculated with PRRSV TJ strain at a dose of 10^{5 0}TCID₅₀/ml via intramuscular injection in the neck muscle. Blood was collected from precaval vein every other day, until the 28^th day. Serum samples were collected for the indirect ELISA assay, which was performed according to the procedures described in Example 2.

[000235] The serum samples were assayed by either the ELISA kit provided herein, or by the commercial IDEXX kit. The results were shown in Table 3.

[000236] Table 3

[000237] As shown in Table 3, one pig died on day 10 and day 14, respectively. The IDEXX kit detected the PRRSV antibodies from day 8 after the virus challenge, and the polypeptide ELISA kit detected the antibodies in all of the tested pigs from day 10 after the virus infection. The results obtained from the two kits were consistent and comparable. This demonstrated that the polypeptide ELISA kit was highly sensitive, and could provide early diagnosis for PRRSV infection.

[000238] The results suggested that the ELISA kit provided herein can identify antibodies produced by pigs infected with a PRRSV virulent strain. The coating antigen used in the kit was B cell epitope polypeptide, which was much safe than a whole virus that may pose a threat on virus escape and transmission. The ELISA kit provided herein thus can find a wide use in clinical practice.

Example 5: Specificity test of indirect ELISA diagnostic kit

[000239] The ELISA kit prepared according Example 2 was used in the study. Serum samples were prepared from pigs infected by classical swine fever virus (CSFV), pseudorabies virus (PRV), or swine influenza virus (SIV). The serum samples were diluted in 1 :200, and tested using the ELISA kit according to the procedure and optimal conditions as provided in Examples 1 and 2. [000240] The results showed that, the CSFV positive serum sample, the PRV positive serum sample and the SIV positive serum samples all showed an S/P ratio less than 0.72, indicating they were negative for PRRSV. The study showed that the ELISA kit provided herein was highly specific for PRRSV detection, and did not cross-react with other porcine virus antigens.

Example 6: Reproducibility study of the ELISA kit

[000241] Different batches of the ELISA kit were prepared and tested using different PRRS positive serum samples, so as to test the reproducibility of the testing results.

[000242] Four batches of the ELISA kit were prepared according to Example 2. 5 serum samples were prepared from pigs infected with PRRSV. A standard PRRSV positive serum was also used.

[000243] The 5 serum samples were tested using three ELISA kits prepared in one batch, according to the procedure and conditions as provided in Example 2. Each sample was tested in duplicate, and the results were shown in Table 4.

[000244] Table 4 Results of Reproducibility study using ELISA Kit of one batch

Serum No.l No.2 No.3 No.4 No.5 Negative

OD value 1.253 0.461 0.382 0.729 0.957 0.068

1.231 0.445 0.375 0.712 0.943 0.073

1.091 0.390 0.335 0.692 0.896 0.076

X Mean 1.192 0.432 0.364 0.711 0.932 0.072

S 0.054 0.026 0.021 0.014 0.044 0.005

C.V% 4.60% 6.30% 5.45% 1.79% 4.16% 7.21%

[000245] According to the results, each sample showed reproducible testing results using the ELISA kit, and the constant variance of the S/P value was 1.79-7.21% (less than 10%)), which indicated high reproducibility.

[000246] ELISA kits were prepared using antigens of four different batches, and were further tested with four positive serum samples having varying antibody levels, and also with one negative serum sample. Each sample was tested in duplicate. The OD value results were shown in Table 5.

[000247] Table 5 Results of Batch-to-Batch Reproducibility Study

Date

Serum 11.12 11.16 11.26 12.3 X Mean S C.V%

1 1.213 1.291 1.102 0.984 1.148 0.130 11.32%

2 0.441 0.432 0.398 0.450 0.431 0.026 6.03%

3 0.355 0.334 0.301 0.314 0.326 0.021 6.44%

4 0.721 0.675 0.725 0.718 0.711 0.015 2.11%

5 1.010 1.112 0.987 0.953 1.016 0.094 9.25%

Negative 0.074 0.068 0.070 0.072 0.071 0.006 8.455%

[000248] According to the results, each serum sample showed reproducible testing results on the ELISA kits containing different batches of the antigens, and the constant variance of the S/P value was 2.11-11.32% (less than 15%), which indicated high reproducibility with different batches of the ELISA kits. [000249] The ELISA kit disclosed herein provided a number of advantages. For example, the ELISA kit is very specific for detection and diagnosis of PRRSV highly virulent strain, whose infection in a pig results in production of antibodies that specifically binds to an antigenic fragment present in the virulent strain (e.g. PRRSV TJ strain) but absent in the attenuated strain (e.g. PRRSV TJM strain). If a sample is tested as positive using the ELlSAkit, then the sample can be identified as from a host infected with the PRRSV highly virulent strain. The ELISA kit is an important tool that allows differentiation of pigs vaccinated with the attenuated strain from those infected with the virulent strain, and therefore is advantageous in providing rapid and effective diagnostic methods for eradication of PRRS. Example 7: Detection of PRRSV using PCR

[000250] A PCR method was developed for detection of PRRSV and differentiation of wild type PRRSV from attenuated PRRSV.

[000251] The following primers were used in the study: Nsp2-F: 5'-ATGTCCCTAACAGTTGGAA-3'; and Nsp2-L: 5'-CGCCGAGAAGACCCAGA-3'. The pair of primer was designed to amplify the fragment starting from 2756th to 3975th nucleotide of the PRRSV genome.

[000252] The classical PRRSV VR2332 strain, highly-pathogenic PRRSV TJ strain and PRRSV TJM strain were tested as samples. Each of the strain was dissolved in 250μ1 sterile deionized water in a DEPC-pretreated vial. 250μ1 sterile deionized water was used as negative control.

[000253] RNA was extracted from each of the samples by Trizol Reagent. Briefly, 750μ1 Trizol Reagent lysis buffer was added to each of the samples, and was mixed well before incubation for 5 min at 15-30°C. 200μ1 chloroform was subsequently added, followed by vigorous shaking and incubation at 15-30°C for 2-3 minutes, and then centrifuged at 12000rpm at 2-8°C for 15 min. The water phase in the mixture was transferred to a new DEPC-pretreated vial, and 500μ1 isopropanol was added, followed by incubation at 15-30°C for 10 min and centrifugation at 12000rpm at 2-8°C for 10 min. The supernatant was discarded, 900μ1 of 75% DEPC ethanol was added to the precipitate, and then centrifuged at 12000rpm at 2-8°C for 10 min. The precipitate was dried and dissolved in 20μ1 DEPC water to obtain the extracted RNA solution for each virus sample.

[000254] The RNA samples were reverse transcribed using the following reaction conditions.

extracted RNA solution 5μ1

Primer 1 μΐ

2.5mmol/L dNTP 5μ1

5 xreverse transcription buffer 1 μΐ

RNA enzyme inhibitor 1 μΐ

DEPC water 2μ1

[000255] The reaction mixture was mixed well, placed at room temperature for 10 min, and incubated in a 42°C water bath for lh before further incubation in an ice bath for 2-3 min.

[000256] The reverse transcribed samples were used as cDNA templates for the PCR reaction, according to the following conditions.

cDNA 2.0μ1

10x Buffer 2.0μ1

2.5mmol/L dNTP 1.5μ1

2 Oumol/μΐ forward primer 0.5μ1

20umol/ μΐ reverse primer 0.5 μΐ

Taq polymerase 0.5 μΐ

Sterilized water 13 μΐ

[000257] The reaction mixture was reacted in a PCR cycler according to the following amplification procedures.

95°C 5min 35 cycles

72°C extending for lOmin

[000258] The PCR products were analyzed using agarose electrophoresis. The results were shown in Figure 8. According to Figure 8, the PCR amplification product of the classic PRRSV VR2332 strain showed a specific band at 1220bp, and that of PRRSV TJ strain showed a 1130 bp band, while that of PRRSV TJM strain showed a 770 bp band. No amplification product was found for the negative control. The PCR correctly distinguished the three different PRRSV strains, because the PRRSV TJ strain lacked 90 nucleotides relative to the PRRSV VR2332 strain, and the PRRSV TJM strain lacked 450 nucleotides relative to the PRRSV VR2332 strain.

Claims

1. An isolated polypeptide, comprising one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1, wherein the immunogenic fragment comprises at least 6 continuous amino acids.

2. The polypeptide of claim 1, wherein the immunogenic fragment comprises at least 9 continuous amino acids.

3. The polypeptide of claim 1, wherein the polypeptide is useful in producing an antibody that specifically binds to the immunogenic fragment.

4. The polypeptide of claim 1, wherein the polypeptide is useful in detecting in a sample for presence of an antibody that specifically binds to the immunogenic fragment.

5. The polypeptide of claim 1, wherein the polypeptide comprises one or more immunogenic fragments of SEQ ID NO: 1.

6. The polypeptide of claim 5, wherein the immunogenic fragment is selected from the group consisting of: SVKITRPKYSAQAl (SEQ ID NO: 2), GHLQKEKEA (SEQ ID NO: 3) and PRTPAPSVSAESDLT (SEQ ID NO: 4).

7. The polypeptide of claim 6, wherein the immunogenic fragment is SVKITRPKYSAQAl (SEQ ID NO: 2).

8. The polypeptide of claim 1, wherein the polypeptide is conjugated to carrier molecule.

9. The polypeptide of claim 8, wherein the carrier molecule is a carrier protein.

10. The polypeptide of claim 8, wherein the carrier molecule is polymer.

11. A detecting reagent, comprising the isolated polypeptide of any of claims 1-10.

12. A detecting device comprising the detecting reagent of claim 11, wherein the detecting reagent is attached to a solid support.

13. A method for detecting an antibody in a sample obtained from a pig suspected of PRRSV infection, comprising contacting the sample to the polypeptide of any of claims 1-10, and detecting the specific binding of the polypeptide and antibody in the sample.

14. A method for distinguishing a pig immunized by an attenuated PRRSV from a pig infected with a wild type PRRSV, comprising: contacting a sample of the pig with the polypeptide of any of claims 1-10, and detecting in the sample for the presence of an antibody that specifically binds to the polypeptide, wherein the presence of the antibody in the sample indicates that the sample is from a pig infected with the wild type PRRSV.

15. The method of claim 14, wherein the attenuated PRRSV comprises a polynucleotide molecule having at least 80% homology to SEQ ID NO: 5, wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide at least 80% homologous to SEQ ID NO: 1.

16. The method of claim 15, wherein the wild type PRRSV comprises a polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1.

17. The method of claim 14, wherein the absence of the antibody in the sample indicates that the sample is from an uninfected pig or a pig immunized with an attenuated PRRSV.

18. The method of claim 14, wherein the attenuated PRRSV is an attenuated live PRRSV vaccine.

19. The method of claim 18, wherein the attenuated PRRSV is the PRRSV having a Deposit Number of CGMCC No. 3121.

20. An isolated antibody capable of specifically binding to the immunogenic fragment of the polypeptide of any of claims 1-10.

21. The antibody of claim 20, further comprising a label.

22. The antibody of claim 21, wherein the label is a fluorescent label, a luminescent label, a radioactive label, an enzyme label or a colored substance.

23. A detecting reagent comprising the antibody of any of claims 20-22.

24. A detecting device comprising the detecting reagent of claim 23, wherein the detecting reagent is attached to a solid support.

25. A method for detecting the presence of a PRRSV in a sample, wherein the PRRSV contains a polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1, comprising contacting the sample to the antibody of any of claim 20-22, and detecting the presence of an antigen that specifically binds to the antibody in the sample.

26. A method for distinguishing a pig infected by an attenuated PRRSV from a pig infected with a wild type PRRSV, comprising: contacting the sample of the pig with the antibody of any of claims 20-22, and detecting in the sample for the presence of an antigen that specifically binds to the antibody, wherein the presence of the antigen in the sample indicates that the sample is from a pig infected with the wild type PRRSV.

27. The method of claim 26, wherein the attenuated PRRSV comprises a polynucleotide molecule having at least 80%> homology to SEQ ID NO: 5, and wherein the polynucleotide molecule lacks a polynucleotide fragment encoding a polypeptide having at least 80%> homology to SEQ ID NO: 1.

28. The method of claim 26, wherein the wild type PRRSV comprises a polynucleotide molecule encoding one or more immunogenic fragments of a polypeptide fragment having at least 95% homology to SEQ ID NO: 1.

29. The method of claim 26, wherein the absence of the antigen in the sample indicates that the sample is from an uninfected pig or a pig infected with an attenuated PR SV.

30. The method of claim 26, wherein the attenuated PRRSV is an attenuated live PRRSV vaccine.

31. The method of claim 30, wherein the attenuated PRRSV is the PRRSV having a Deposit Number of CGMCC No. 3121.

32. A kit comprising the isolated polypeptide of any of claims 1-10, wherein the polypeptide is attached to a solid support.

33. The kit of claim 32, further comprising a detecting antibody.

34. The kit of claim 33, wherein the detecting antibody is conjugated to a label.

35. The kit of claim 33, wherein the detecting antibody is anti-pig antibody.

36. A pair of isolated oligonucleotide primers, comprising a 5' primer and a 3' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NOs: 8-10 and sequences having at least 80%> homology to any one of SEQ ID NOs: 8-10; wherein the 3 ' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 13-19 and sequences having at least 80%> homology to any one of SEQ ID NOs: 13-19; wherein the 5' primer and the 3' primer both have a length between 10 and 38 nucleotides; and wherein the 5 ' primer and the 3 ' primer are capable of amplifying the nucleotide sequence of a classical type PR SV, and the length of the amplified sequence exceeds or equals to the combined lengths of the 5 ' primer and the 3 ' primer.

37. A method for detecting PRRSV in a biological sample, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using a pair of the oligonucleotide primers of claim 36, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample.

38. The method of claim 37, wherein if the amplified product is absent in the test sample but present in the control sample, then it indicates that the test sample does not contain PRRSV or contains a PRRSV which is attenuated.

39. The method of claim 37, wherein the determining of said difference comprises comparing the molecular weight of the amplified product of the test sample with that of the control sample, wherein the amplified product of the control sample has a molecular weight comparable to the amplified product of a classical type PRRSV by using the same pair of primers.

40. The method of claim 39, wherein if the amplified product as detected in the test sample is about 90 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PRRSV which is highly-pathogenic.

41. The method of claim 39, wherein if the amplified product as detected in the test sample is about 360 or 450 nucleotides shorter than that of the control sample, then it indicates that the test sample contains a PRRSV which is attenuated.

42. The method of claim 39, wherein if the amplified product as detected in the test sample has a molecular weight similar to that of the control sample, then it indicates that the test sample contains a PRRSV which is of classical type.

43. A pair of isolated oligonucleotide primers, comprising a 5' primer and a 3' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NO: 11 and sequences having at least 80% homology to SEQ ID NO 11; wherein the 3 ' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 13-19 and sequences having at least 80%> homology to any one of SEQ ID NOs: 13-19; wherein the 5' primer or the 3' primer both have a length between 10 and 38 nucleotides; and wherein the 5 ' primer and the 3 ' primer are capable of amplifying the nucleotide sequence of a classical type PR SV, and the length of the amplified sequence exceeds or equals to the combined lengths of the 5 ' primer and the 3 ' primer.

44. A method for detecting PRRSV in a biological sample, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using a pair of the oligonucleotide primers of claim 43, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample.

45. A pair of isolated oligonucleotide primers, comprising a 5' primer and a 3' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NOs: 11-13 and sequences having at least 80%> homology to any of SEQ ID NOs: 11-13; wherein the 3 ' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 14-16 and sequences having at least 80% homology to any one of SEQ ID NOs: 14-16; wherein the 5' primer or the 3' primer both have a length between 10 and 38 nucleotides; and wherein the 5 ' primer and the 3 ' primer are capable of amplifying the nucleotide sequence of a classical type PR SV, and the length of the amplified sequence exceeds or equals to the combined lengths of the 5 ' primer and the 3 ' primer.

46. A method for detecting PRRSV in a biological sample, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using a pair of the oligonucleotide primers of claim 45, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample.

47. A pair of isolated oligonucleotide primers, comprising a 5' primer and a 3' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NO: 12 and sequences having at least 80% homology to SEQ ID NO: 12; wherein the 3 ' primer is complementary to a sequence selected from the group consisting of SEQ ID NOs: 17-19 and sequences having at least 80%> homology to any one of SEQ ID NOs: 17-19; wherein the 5' primer or the 3' primer both have a length between 10 and 38 nucleotides; and wherein the 5 ' primer and the 3 ' primer are capable of amplifying the nucleotide sequence of a classical type PR SV, and the length of the amplified sequence exceeds or equals to the combined lengths of the 5 ' primer and the 3 ' primer.

48. A method for detecting PRRSV in a biological sample, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using a pair of the oligonucleotide primers of claim 47, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample.

49. A pair of isolated oligonucleotide primers, comprising a 5' primer and a 3' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NOs: 8-10 and sequences having at least 80% homology to any of SEQ ID NOs: 8-10; wherein the 3 ' primer is complementary to a sequence selected from the group consisting of SEQ ID NO: 13 and sequences having at least 80% homology to any one of SEQ ID NO: 13; wherein the 5' primer or the 3' primer both have a length between 10 and 38 nucleotides; and wherein the 5 ' primer and the 3 ' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or equals to the combined lengths of the 5 ' primer and the 3 ' primer.

50. A method for detecting PRRSV in a biological sample, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using a pair of the oligonucleotide primers of claim 49, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample.

51. A pair of isolated oligonucleotide primers, comprising a 5 ' primer and a 3 ' primer, wherein the 5' primer is complementary to a sequence which is antisense to a sequence selected from the group consisting of SEQ ID NO: 13 and sequences having at least 80% homology to SEQ ID NO: 13; wherein the 3 ' primer is complementary to a sequence selected from the group consisting of SEQ ID NO: 13 and sequences having at least 80% homology to any one of SEQ ID NO: 13; wherein the 5' primer or the 3' primer both have a length between 10 and 38 nucleotides; and wherein the 5 ' primer and the 3 ' primer are capable of amplifying the nucleotide sequence of a classical type PRRSV, and the length of the amplified sequence exceeds or equals to the combined lengths of the 5 ' primer and the 3 ' primer.

52. A method for detecting PRRSV in a biological sample, comprising: amplifying a reverse transcribed product of an RNA in a test sample and in a control sample using a pair of the oligonucleotide primers of claim 51, and detecting the presence and/or molecular weight of the amplified products in both samples; and determining the difference between the amplified products of the test sample and the control sample, wherein the difference indicates the presence or absence or type of a PRRSV in the sample.

53. The oligonucleotide primers of claim 36, wherein the 5' primer, which is complementary to SEQ ID NO: 8, is 5'-ATGTCCCTAACAGTTGGAA-3'.

54. The oligonucleotide primers of claim 36, wherein the 3' primer, which is complementary to SEQ ID NO: 15, is 5'-CGCCGAGAAGACCCAGA-3'.

55. The oligonucleotide primers of claim 36, wherein the 5' primer is 5 '-ATGTCCCT AAC AGTTGGAA-3 ', and the 3' primer is 5 '-CGCCGAGAAG ACCC AGA-3 '.

56. An isolated DNA fragment comprising an amplified product obtained by polymerase chain reaction using a sample containing a PRRS V in the presence of a 5 ' primer and a 3' primer of any of claims 36, 43, 45, 47 and 51.