Prrsv vaccine strain

Abstract

The present invention relates to a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain, the methods of producing such vaccine strains, vaccine compositions comprising such vaccine strains and their use thereof in the prevention or reduction of porcine reproductive and respiratory syndrome (PPRS) in pigs.

Description

[0001] PRRSV Vaccine Strain

[0002] Technical Field of the Invention

[0003] The present invention relates to a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain, the methods of producing such vaccine strains, vaccine compositions comprising such vaccine strains and their use thereof in the prevention or reduction of porcine reproductive and respiratory syndrome (PPRS) in pigs.

[0004] Background to the Invention

[0005] Porcine reproductive and respiratory syndrome (PPRS) is a highly contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is an enveloped RNA virus in the genus Arterivirus of the family Arteriviridae. PRRS has two distinct clinical phases; respiratory disease in pigs of any age, and reproductive impairment or failure in pregnant sows characterised by late miscarriage, stillbirth, mummified, and weak or tired piglets.

[0006] The genome of PRRSV is approximately 15kb in length and contains a 5’- unstranslated region (UTR), at least ten open reading frames (ORF lb, ORF lb, ORF2a, ORF2b, 0RF3, ORF4, ORF5a, and ORF5-7), and a 3’-UTR. ORFla and ORFlb encode at least 16 non-structural proteins and ORF2a, ORF2b, ORF5a, ORF3-7 encode eight viral structural proteins.

[0007] PPRS occurs worldwide in most major swine-raising countries, e.g. North and South America, Asia, Africa and Europe. There is considerable heterogeneity in the genome of PRRSV and there are significant genetic and antigenic differences between isolates reported from different countries. According to its antigenic differences, PRRSV can be divided into two subgroups: The European-type subgroup A (PRRSV- 1), and the North American-type Subgroup B (PRRSV-2). The nucleotide sequence similarity of the two subgroups ranges from 54% to 67%. In the past, the predominant strains in Europe were PRRSV-1, while in the United States and the Asia-Pacific region, PRRSV-2 predominated. However, PRRSV has broken its geographical constraints and now both genotypes have spread to various countries.

[0008] The variability between genotypes and the coexistence of PRRSV-1 and PRRSV-2 could cause problems for PRRSV diagnosis and management. More specifically, there is an increased difficulty of analysing the highly pathogenic PRRSV and identifying the molecular mechanisms of immunisation. As such, there are currently no vaccines on the market that protect against both PRRSV-1 and PRRSV-2

[0009] There are several modified live virus (MLV) vaccines against either PRRSV-1 or PRRSV-2 that are licensed in various countries depending on circulating viral genotypes. However, commercially available MLV vaccines against PRRSV-1 or PRRSV-2 elicit only relatively weak humoral and cell-mediated immune responses during infection with virulent PRRSV strains, which indicate all ML Vs currently used are ineffective and cannot meet practical needs.

[0010] There are also a number of inactivated PRRSV vaccines that have been licensed worldwide. However, since 2005 these vaccines are no longer available in the US due to their poor observed efficacy. The inactivated PRRSV vaccine failed to show statistically significant benefits in vaccinated herds against wild types.

[0011] New techniques have been developed in an attempt to improve the above vaccines including; new approaches for administration of MLV vaccines (such as co- administration with various adjuvants), use of DNA vaccines, subunit vaccines or virus- vectored vaccines incorporating other viruses. However, most vaccines developed using these techniques appear to be less effective than MLV vaccines. Consequently, vaccines based on new approaches are still far from ready for practical application and have little potential to replace MLV vaccines without a major technological breakthrough.

[0012] It is an aim of embodiments of the present invention the present invention to provide a PRRSV vaccine strain that is effective against both PRRSV-1 and PRRSV- 2.

[0013] It is also an aim of embodiments of the present invention to provide a PRRSV vaccine strain with improved efficacy against PRRSV-1 and PRRSV-2 compared to current approaches.

[0014] It is also an aim of embodiments to the invention to overcome or mitigate at least one problem of the prior art, whether disclosed herein or not.

[0015] Summary of the Invention

[0016] According to a first aspect of the invention there is provided, a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain comprising: at least a part of a nucleic acid sequence of a first PRRSV strain, and wherein a nucleic acid sequence encoding glycoprotein 5 (GP5) of a second PRRSV strain is inserted into the at least a part of the nucleic acid sequence of the first PRRSV strain after ORF lb and before ORF2b.

[0017] “ORF” is an open reading frame in the nucleic acid sequence. The PRRSV vaccine strain may be a recombinant chimeric PRRSV vaccine strain, comprising of one or more genes from a first PRRSV strain and one or more genes from a second PRRSV strain.

[0018] PRRSV strains are known in the art.

[0019] In some embodiments, the first PRRSV strain is a live attenuated PRRSV strain. A live attenuated strain is safer to use and could also provide improved protection compared to inactivated viruses.

[0020] In some embodiments, the first PRRSV strain is a PRRSV genotype 2 (PRRSV- 2) strain.

[0021] In some embodiments, the first PRRSV strain is a JXAl-like strain.

[0022] In some embodiments, at least a part of a gene may be deleted from the first PRRSV strain. The deletion of the at least a part of a gene from the first PRRSV strain may not substantially impact the immune response to PRRSV infection. Thus, the at least a part of a gene for deletion may be selected from any gene of the PRRSV strain in which it is known, or it is expected, does not substantially impact the immune response to PRRSV infection.

[0023] In some embodiments, at least a part of a gene encoding non-structural protein 2 (NSP2) is deleted from the first PRRSV strain. The deletion of at least a part of a gene encoding NSP2 does not impact the immune response to PRRSV infection.

[0024] In some embodiments, the entire gene encoding NSP2 is deleted from the first

[0025] PRRSV strain. In some embodiments, only a part of the NSP2 gene is deleted from the first PRRSV strain. The at least a part of the first PRRSV strain may lack between 150-250 amino acids in the NSP2 region of the first PRRSV strain. Preferably, the at least a part of first PRRSV strain may lack around 200 amino acids in the NSP2 region of the first PRRSV strain.

[0026] In some embodiments, at least a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 is deleted from the NSP2 gene of the first PRRSV strain. The nucleic acid sequence of SEQ ID NO: 1 may comprise one or more nucleic acid substitutions that does not alter the amino acid encoded (i.e. silent mutation).

[0027] In some embodiments, at least the nucleic acid sequence of SEQ ID NO: 1 is deleted from the NSP2 gene of the first PRRSV strain. Further nucleic acid sequences of the NSP2 gene that flank the sequence of SEQ ID NO: 1 may also be deleted from the first PRRSV strain. Preferably, only the nucleic acid sequence of SEQ ID NO: 1 is deleted from the NSP2 gene of the first PRRSV strain.

[0028] The deletion of at least a part of the gene encoding NSP2 results in the formation of an NSP2-deficient first PRRSV strain.

[0029] In some embodiments, the nucleic acid sequence of the NSP2-deficient first PRRSV strain has at least 99%, or 100% sequence identity with SEQ ID NO: 4. Preferably, the NSP2-deficient first PRRSV strain may have a nucleic acid sequence of SEQ ID NO: 2.

[0030] In some embodiments, the second PRRSV strain is a PRRSV genotype 1 (PRRSV-1) strain. In such embodiments, the GP5 may be PRRSV-1 GP5. The nucleic acid sequence encoding GP5 may have 100% sequence identity with SEQ ID NO: 3. The nucleic acid sequence encoding GP5 may have the nucleic acid sequence of SEQ ID NO: 3.

[0031] Insertion of the GP5 gene “after ORF lb” means that the GP5 gene is inserted into the at least a part of the nucleic acid sequence of the first PRRS V strain at any point following the ORFlb gene, when reading the nucleic acid sequence in a 5’-3’ direction. Similarly, insertion of the GP5 gene “before ORF2b” means that the GP5 gene is inserted into the at least a part of the nucleic acid sequence of the first PRRSV strain at any point preceding the ORF2b gene, when reading the nucleic acid sequence in a 5’- 3’ direction.

[0032] The PRRSV vaccine strain may be used in the prevention, elimination or reduction of PRRSV in pigs. The PRRSV vaccine strain may be suitable for use in the prevention, elimination or reduction of PRRSV-1 or PRRSV-2 in pigs. Beneficially, the PRRSV vaccine strain may be suitable for use in the prevention, elimination or reduction of PRRSV-1 and PRRSV-2 in pigs.

[0033] According to a second aspect of the invention there is provided, a method of constructing a PRRSV vaccine strain, the method comprising the following steps;

[0034] (i) providing at least a part of a nucleic acid sequence of a first PRRSV strain, and

[0035] (ii) inserting into the at least a part of the nucleic acid sequence of the first PRRSV strain a nucleic acid sequence encoding GP5 of a second PRRSV strain, wherein the nucleic acid sequence encoding GP5 is inserted into the at least a part of the nucleic acid sequence of the first PRRSV strain after ORF lb and before ORF2b. The PRRSV vaccine strain, the first and second PRRSV strains and their components may be as described herein above for the first aspect of the invention and may include any optional feature or combination of optional features described for the first aspect.

[0036] In some embodiments, the method further comprises the following step:

[0037] (iii) deleting at least a part of a gene encoding NSP2 from the at least a part of the nucleic acid sequence of the first PRRSV strain.

[0038] The gene deletion step, i.e. step (iii), may precede or follow step (i). The gene deletion step, i.e. step (iii), may precede or follow step (ii). The gene deletion step, i.e. step (iii), may follow step (i) but precede step (ii). Preferably, the gene deletion step follows step (i) and precedes step (ii).

[0039] According to a third aspect of the invention there is provided, a vaccine composition comprising the PRRSV vaccine strain of the first aspect of the invention and a pharmaceutically acceptable carrier or excipient.

[0040] The PRRSV vaccine strain may be as described herein above for the first aspect of the invention and may include any optional feature or combination of optional features described for the first aspect.

[0041] Suitable pharmaceutically acceptable carriers or excipients are known in the art. The vaccine composition may comprise at least one of a diluent, preservative, additive, or adjuvant.

[0042] The vaccine composition may be suitable for administration to a pig. The vaccine composition may stimulate an immune response in the pig. The vaccine composition may be suitable for intranasal, subcutaneous and intramuscular administration. The vaccine composition may be suitable for oral administration.

[0043] The vaccine composition may be used in the prevention, elimination or reduction of PRRSV in pigs. The vaccine composition may be used in the prevention, elimination or reduction of PRRSV-1 or PRRSV-2 in pigs. Beneficially, the vaccine composition may be used in the prevention, elimination or reduction of PRRSV-1 and PRRSV-2 in pigs. This is a key benefit over the vaccines of the prior art, which target only one of the two genotypes of PRRSV.

[0044] According to a fourth aspect of the invention there is provided, a method of preventing, eliminating or reducing porcine reproductive and respiratory syndrome (PRRS) in a pig comprising administering to the pig a vaccine composition of the third aspect of the invention.

[0045] The vaccine composition may be as described herein above for the third aspect of the invention and may include any optional feature or combination of optional features described for the third aspect.

[0046] The method may include intranasal, subcutaneous, oral, or intramuscular administration. Preferably, the method may include administering the vaccine composition intramuscularly.

[0047] A therapeutically effective amount of the vaccine composition may be administered. A “therapeutically effective amount” refers to an amount that is effective for an intended therapeutic purpose. A therapeutic effective amount for administration may be around 105TCID o / mL.

[0048] The therapeutically effective amount of vaccine composition administered may depend on the route of administration, the frequency and dose of administration, as well as the age, weight, sex and physical condition of the pig.

[0049] The vaccine composition may be administered in a single dose, or in multiple doses. Preferably, the vaccine composition may be administered in a single dose. The vaccine composition may be administered monthly, or yearly. In some embodiments, the vaccine composition is administered once a year, twice a year, three times a year, four times a year, five times a year, six times a year, or more than six times a year. In some embodiments, the vaccine composition is administered every two years, every three years, every four years, every five years, every six years, every seven years, every eight years, every nine years, every ten years, or more than every ten years. The vaccine composition may be administered as needed.

[0050] According to a fifth aspect of the invention there is provided, a vector comprising the PRRSV vaccine strain of the first aspect of the invention.

[0051] The PRRSV vaccine strain may be as described herein above for the first aspect of the invention and may include any optional feature or combination of optional features described for the first aspect.

[0052] The vector may be a plasmid, or a viral vector. The vector may be a cloning vector.

[0053] In specific embodiments, the vector may be a pCMS plasmid. The plasmid may contain multiple cloning sites (for example, notl and pmel). According to a sixth aspect of the invention there is provided, a host cell comprising the vector of the fifth aspect of the invention.

[0054] The vector may be as described herein above for the fifth aspect of the invention and may include any optional feature or combination of optional features described for the fifth aspect.

[0055] Detailed Description of the Invention

[0056] In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

[0057] Figure 1 illustrates the construction of the PRRSV vaccine strain of an embodiment of the first aspect of the invention.

[0058] Figure 2 shows the CPE of Marc-145 cells after transfection with the pCMSJ521euGP5 plasmid of an embodiment of the fifth aspect of the invention after 4 days.

[0059] Figure 3 shows immunofluorescence images for the identification of PRRSV-1 GP5 protein expression. 48 hours after inoculation with the PRRSV vaccine strain of an embodiment of the first aspect of the invention in Marc-145 cells, PRRSV-1 GP5 gene expression was detected with GP5 specific antibody (left) and cell negative control (right).

[0060] Figure 4 shows a graph of the levels of specific antibodies of PRRSV-1 GP5 in piglet blood serum after immunisation with the PRRSV vaccine strain of an embodiment of the first aspect of the invention (compared to immunisation with a control, PBS).

[0061] Figure 5 shows a graph of a neutralising antibody titer with PPRSV-1 Lee virus challenge after immunisation with the PRRSV vaccine strain of an embodiment of the first aspect of the invention (compared to immunisation with a control, PBS).

[0062] Figure 6 shows a graph of piglet body temperature after PRRSV vaccine strain challenge.

[0063] Example 1 - Construction o f the PRRSV vaccine strain

[0064] 1) RNA extraction and cDNA preparation

[0065] Marc-145 cells were plated in a 6-well plate (lxlO6 / mL). When cell density reached 80%, 50 pL PRRSV-1 or PRRSV-2 virus was added to each well and replaced with the maintenance medium containing 2% serum and cells were cultured for 48-72 hours. The supernatant of the virus-infected wells was discarded, and the monolayer of cells was collected. The collected cells were transferred into a centrifuge tube and resuspended in 300 pL PBS. The total RNA was then extracted using RNeasy Kit (Qiagen).

[0066] After the RNA was successfully prepared, cDNA was prepared using QuantiTect Reverse Transcription Kit (Qiagen).

[0067] 2) Construction of recombinant pCMSJ521euGP5 plasmid:

[0068] The vector pCMS was used to construct a recombinant plasmid, pCMSJ521euGP5. pCMS contains multiple cloning sites of notl and pmel. The primers of full-length construction were designed according to the NCBI PRRSV reference sequence (NCBI

[0069] GENE ID: EFl 12445.1) (Table 1).

[0070] Although particular strains of PRRSV-1 (Lee) and PRRSV-2 (JM2019, JXAl-like) were used in the examples herein, other strains may work equally well.

[0071] The template PRRSV-2 cDNA was amplified with primers P1 / P2, P3 / P4, P5 / P6, and P7 / P8, and the target gene fragments were recovered and fused to create the first gene segment. The first gene segment was connected to the notl and pmel sites of the pCMS vector using non-cloning method to form the pCMSJ521 plasmid (SEQ ID NO: 2).

[0072] The second gene segment was then prepared for construction. PRRSV-2 cDNA was amplified with P9 / P10, Pl 1 / P12, P15 / P16, and P17 / P18 and the target gene fragments were recovered. PRRSV-1 Lee cDNA was amplified with primers P13 / P14 to recover the PRRSV-1 GP5 gene. The recovered gene fragment of P11 / P12 and the recovered gene fragment of P13 / P14 (PRRSV-1 GP5 gene) were fused by fusion PCR to recover the fusion gene P24. The recovered gene fragments P9 / P 10, P24, P15 / P16, and P17 / P18 fragments were recovered to create the second gene segment. The second gene segment was cloned into the pCMSJ521 plasmid behind the first gene fragment using the seamless cloning method to form the pCMSJ521euGP5 plasmid. Sequencing was performed to verify that the pCMSJ521euGP5 plasmid was constructed correctly.

[0073] Table 1 :

[0074] Primer Primer sequence (5'— >3') SEQ ID NO. Size

[0075] (bp)

[0076] Pl ATGACGTATAGGTGTTGGCTCTATG SEQ ID NO: 4 25

[0077] P2 AGCAGCAATCCTCAATAACTTGACA SEQ ID NO: 5 25 P3 TGTCAAGTTATTGAGGATTGCTGCT SEQ ID NO: 6 25

[0078] P4 CTGGGTCGGACACAGGCTTGAACAG SEQ ID NO: 7 25

[0079] P5 CTGTTCAAGCCTGTGTCCGACCCAG SEQ ID NO: 8 25

[0080] P6 AGGGCACAAAGATCTGAAGGCACCT SEQ ID NO: 9 25

[0081] P7 AGGTGCCTTCAGATCTTTGTGCCCT SEQ ID NO: 10 25

[0082] P8 CGGCTAGCAGTTTAAACACTGCTCC SEQ ID NO: 11 25

[0083] P9 TAAGGAGCAGTGTTTAAACTGCTAGCCGCC SEQ ID NO: 12 30

[0084] PIO CCCCTGATGCCACGCCTAACGGAGA SEQ ID NO: 13 25

[0085] P 11 TCTCCGTTAGGCGTGGC ATCAGGGG SEQ ID NO: 14 25

[0086] P12 AAAGAGTGGCCGCAGCCATATCAATTCAGGCCTAAAG SEQ ID NO: 15 44

[0087] TTGGTTC

[0088] P13 TAGGCCTGAATTGATATGGCTGCGGCCACTCTTT SEQ ID NO: 16 34

[0089] P14 TGCATAGACCCCATTTCATTCATATTGCCAAGAGAATG SEQ ID NO: 17 38

[0090] P15 CATTCTCTTGGCAATATGAATGAAATGGGGTCTATGCA SEQ ID NO: 18 38

[0091] GACTGCGTAAATGCTACTCAAGACA

[0092] P16 TGTCTTGAGTAGCATTTACGCAGTC SEQ ID NO: 19 25

[0093] P17 GCCGCTCACTAGTAACGGCCTTTTTTTTTTTTTTTTTTT SEQ ID NO: 20 25

[0094] P18 TTTTTTTTAATTACGGCCGCATGGTTCTCGC SEQ ID NO: 21 70

[0095] 3) Cell transfection:

[0096] Marc-145 cells were plated in a 6-well plate (lxlO6 / mL). When the cell density reached 80%, plasmid transfection was performed using Lipofectamine 3000 Transfection Reagent. The ratio of pCMSJ521euGP5 plasmid to Lipofectamine 3000 Transfection Reagent was 1:3 for transfection complex configuration. After 15 minutes incubating at room temperature, the transfection reagent was added dropwise to the 6- well plate. The culture was continued in a 37 °C incubator for 72-96 hours and the cytopathic effect (CPE) was observed every day. The supernatant of the virus-infected wells was discarded, and the monolayer of cells was collected. The collected cells were transferred into a centrifuge tube and resuspended in 300 pL PBS.

[0097] 4) Recombinant virus passage and titer determination: The rescued virus was inoculated with Marc- 145 cells at a ratio of 5%, and the virus was collected after 4 days. The virus titer was measured when stable CPE appeared.

[0098] 5) Identification of recombinant virus:

[0099] The virus was identified by immunofluorescence assay (IF A) and PCR, and the target gene fragment of PCR amplification was recovered and sequenced for identification.

[0100] Figure 2 shows that the PRRSV-1 GP5 gene was successfully inserted between ORF1 and GP2 of the pCMSJ521 plasmid. After transfection of the recombinant plasmid (pCMSJ521euGP5) into Marc-145 cells for 4 days, obvious pathological characteristics such as cell aggregation and shedding could be observed (Figure 3). After identification with PRRSV-1 GP5 polyclonal antibodies, significant fluorescence of the cells can be observed (Figure 4).

[0101] Example 2 In vivo testing of PRRSV vaccine strain

[0102] Piglets without PRRS, porcine circovirus (PCV), porcine epidemic diarrhea virus (PEDV) and other pathogens were randomly divided into two groups, one group was vaccinated with the PRRSV vaccine strain constructed as described in Example 1 above, and the other group was treated with PBS (control). After 35 days of immunisation, all piglets were challenged with the PRRSV-1 Lee strain. During the immunisation period, the changes in the piglet's body temperature were measured daily, the piglet's status was observed, and blood was collected every week to separate the piglet's serum for antibody testing.

[0103] Specific antibodies and virus-neutralising antibodies were gradually increased after immunisation with the PRRSV vaccine strain (Figures 5 and 6), and the titer of neutralising antibodies was 1 :18 at 35 days post vaccination. After challenge with PRRSV-1 Lee virus, the average body temperature of piglets of the vaccinated group remained about 39.3 °C (Figure 6). Since the normal body temperature of piglet is 39.2 °C, the experimental group was more stable than the control group, which indicates that the constructed PRRSV vaccine strain could provide effective protection against PRRSV-1 Lee infection. As the construction of the PRRSV vaccine strain in Example 1 is based on a PRRSV-2 strain, the PRRSV vaccine strain will be generally effective against PRRSV-2 infection. Thus, the PRRSV vaccine strain constructed in Example 1 is effective against both PRRSV-1 and PRRSV-2 infection. The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention.

[0104] SEQUENCE LISTINGS

[0105] SEQ ID NO: 1 (sequence ofNSP2 deletion)

[0106] GCCGGAAAGAGAGCAAGGAAAACACGCTCTGGTGCGACTACTATG

[0107] GTCGCTCATCACGCTTCGTCCGCTCATGAAACCCGGCAGGCCACGA

[0108] AGCACGAGGGTGCCGGCGCTAATAAGGCTGAGCATCTCAAGCGCTA

[0109] CTCTCCGCCTGCCGAAGGGAACTGTGGTTGGCACTGCATTTCCGCCA

[0110] TCGCCAACCGGATGGTGAATTCCAACTTTGAGACCACCCTTCCTGAA

[0111] AGAGTAAGGCCTTCAGATGACTGGGCCACTGACGAGGATCTTGTGA

[0112] ACACCATCCAAATCCTCAGGCTCCCTGCGGCCTTGGACAGGAACGG

[0113] CGCTTGCGGTAGCGCCAAGTACGTGCTTAAACTGGAGGGTGAGCAT

[0114] TGGACTGTCTCTGTGATCCCTGGGATGTCCCCTACTTTGCTCCCCCTT

[0115] GAATGTGTTCAGGGTTGTTGTGAGCATAAGGGCGGTCTTGTTTCCCC

[0116] GGATGCGGTCGAAATTTCCGGATTTGATCCTGCCTGCCTTGACCGAC

[0117] TGGCTAAGGTAATGCACTTGCCTAGCAGTACCATCCCAGCCGCTCTG

[0118] GCCGAATTGTCCGACGACTCCAACCGTCCGGTTTCCCCGGCCGCTAC

[0119] TACGTGGACTGTTTCGCAATTCTATGCTCGTCATAGAGGAGGAGATC

[0120] ATCATGACCAGGTGTGCTTGGGGAAAATCATCAGCCTTTGTCAAGTT

[0121] ATTGAGGATTGCTGCTGCCATCAGAATAAAACCAACCGGGCTACTC

[0122] CGGAAGAGGTCGCGGCAAAGATTGATCAGTACCTCCGTGGCGCAAC

[0123] AAGTCTTGAGGAATGCTTGGCCAAACTTGAGAGAGTTTCCCCGCCG

[0124] AGCGCTGCGGACACCTCTTTTGATTGGAATGTTGTGCTTCCTGGGGT

[0125] TGAGGCGGCGAATCAGACAACCGACCAACCTCACGTCAACTCATGC TGCACCCTGGTCCCTCCCGTGACTCAAGAGCCTTTGGGCAAGGACTC

[0126] GGTCCCTCTGACCGCCTTCTCACTGTCCAATTGCTATTACCCTGCAC

[0127] AAGGTGACGAGGTTCATCACCGTGAGAGGTTAAATTCCGTACTCTCT

[0128] AAGTTGGAAGAGGTTGTCCTGGAAGAATATGGGCTCATGTCCACTG

[0129] GACTTGGCCCGCGACCCGTGCTGCCGAGCGGGCTCGACGAGCTTAA

[0130] AGACCAGATGGAGGAGGATCTGCTACAACTAACCAACACCCAGGCG

[0131] ACTTCAGAAATGATGGCCTGGGCGGCTGAGCAGGTCGATTTAAAAG

[0132] CTTGGGTCAAAAGCTACCCGCGGTGGACACCACCACCCCCTCCACC

[0133] AAGAGTTCAACCTAGAAGAACAAAGTCTGTCAAAAGCTTGCCAGAG

[0134] GGCAAGCCTGTCCCTGCTCCGCGCAGGAAGGTCAGATCCGATTGCG

[0135] GCAGCCCGGTTTTGATGGGCGACAATGTCCCTAACGGTTCGGAAGA

[0136] AACTGTCGGTGGTCCCCTCAATTTTCCGACACCATCCGAGCCGATGA

[0137] CACCTATGAGTGAGCCCGTACTTGTGCCCGCGTCGCGACGTGTCGGT

[0138] TCAGTGGCCACCGAGGATGTTCCACGCATCCTCGGGAAAATAGGAG

[0139] ACACTGACGAGCTGCTTGACCGGGGTCCCTCGGCTCCCTCCAAGGG

[0140] AGAACCGGTCTGTGACCAACCTGCCAAAGATCCCCGGATGTCGCCA

[0141] CGGGAGTCTGACGAGAGCATGATGGCTCCGCCCGCAGATACAGGTG

[0142] GTGTCGGCTCATTCACTGATTTGCCGTCTTCAGATGGTGTGGATGTG

[0143] GACGGGGGGGGGCCGTTAAGAACGGTAAAAACAAAAGCAGGAAGG

[0144] CTCTTAGACCAACTGAGCTGCCAGGTTTTTAGCCTCGTTTCCCATCT

[0145] CCCTATTTTCTTCTCACACCTCTTCAAATCTGACAGTGGTTATTCTCC

[0146] GGGTGATTGGGGTTTTGCAGCTTTTACTCTATTTTGCCTCTTTCTATG TTACAGTTACCCATTCTTCGGTTTTGCTCCCCTCTTGGGTGTATTTTC

[0147] TGGGTCTTCTCGGCGTGTGCGAATGGGGGTTTTTGGCTGCTGGTTGG

[0148] CTTTTGCTGTTGGTCTGTTCAAGCCTGTGTCCGACCCAGTCGGCACT

[0149] GCTTGTGAGTTTGACTCGCCAGAGTGTAGGAACGTCCTTCATTCTTT

[0150] TGAGCTTCTCAAACCTTGGGACCCTGTCCGCAGCCTTGTTGTGGGCC

[0151] CCGTCGGTCTCGGCCTTGCCATTCTTGGCAGGTTACTGGGCGGGGCA

[0152] CGCTACATCTGGCACTTTTTGCTTAGGCTTGGCATTGTTGCAGACTG

[0153] TATCTTGGCTGGAGCTTATGTGCTTTCTCAAGGTAGGTGTAAAAAGT

[0154] GCTGGGGATCTTGTGTAAGAACTGCTCCTAATGAGATCGCCTTCAAC

[0155] GTGTTCCCTTTTACACGTGCGACCAGGTTGTCACTCATCGACCTGTG

[0156] CGATCGGTTCTGCGCACCAAAAGGCATGGACCCCATTTTTCTCGCCA

[0157] CTGGGTGGCGTGGGTGCTGGACCGGCCGGAGTCCCATTGAGCAACC

[0158] TTCTGAAAAACCCATCGCGTTCGCCCAGCTGGATGAGAAGAGGATT

[0159] ACGGCTAGAACTGTGGTCACTCAGCCTTATGATCCCAACCAGGCCG

[0160] TAAAGTGCTTGCGGGTATTACAGGCGGGCGGGGCGATGGTGGCCGA

[0161] GGCAGTCCCAAAAGTGGTCAAAGTTTCCGCCATTCCATTCCGAGCTC

[0162] CTTTCTTTCCCGCTGGAGTGAAAGTTGATCCTGAGTGCAGAATCGTG

[0163] GTTGATCCCGATACTTTTACTACAGCCCTCCGGTCTGGCTATTCCAC

[0164] CGCGAACCTCGTCCTTGGTACGGGGGACTTTGCCCAGCTGAATGGA

[0165] CTAAAGATCAGGCAAATTTCCAAGCCTTCAGGGGGA

[0166] SEQ ID NO: 2 (pCMSJ521, NSP 2 -deficient PRRSV-2 JM2019 strain) ATGACGTATAGGTGTTGGCTCTATGCCACGGCATTTGTATTGTCAGGAGCT

[0167] GTGACCATTGGCACAGCCCAAAACTTGCTGCACGGGAACACCCTCCTGTG

[0168] ACAGCCCTCTTCAGGGGGATTAGGGGTCTGTCCCTAACACCTTGCTTCCGG

[0169] AGTTGCACTGCTTTACGGTCTCTCCACCCCTTTAACCATGTCTGGGATACT

[0170] TGATCGGTGCACGTGTACCCCCAATGCCAGGGTGTTTGTGGCGGAGGGCC

[0171] AGGTCTACTGCACACGATGTCTCAGTGCACGGTCTCTCCTTCCTCTGAATC

[0172] TCCAAGTTCCTGAGCTTGGGGTGCTGGGTCTATTTTATAGGCCCGAAGAGC

[0173] CACTCCGGTGGACGTTGCCACGTGCATTCCCCACTGTCGAGTGCTCCCCCG

[0174] CCGGGGCCTGCTGGCTTTCTGCGATCTTTCCGATTGCACGAATGACTAGTG

[0175] GAAACCTGAACTTTCAACAAAGAATGGTGCGGGTCGCAGCTGAAATCTAC

[0176] AGAGCCGGCCAACTCACCCCTACAGTTCTAAAGACTCTACAAGTTTATGA

[0177] ACGGGGTTGTCGTTGGTACCCCATTGTCGGGCCCGTCCCTGGGGTGGGCGT

[0178] TTACGCCAACTCCCTGCATGTGAGTGACAAACCTTTCCCGGGAGCTACTCA

[0179] TGTGTTAACCAACTTGCCGCTCCCGCAGAGGCCCAAACCTGAGGACTTTTG

[0180] CCCTTTTGAGTGTGCTATGGCTGACGTCTATGACATTGGTCGTGGCGCCGT

[0181] CATGTATGTGGCCGGAGGAAAGGTCTCTTGGGCCCCTCGTGGTGGGAATG

[0182] AAGTGAAATTTGAACCTGTCCCCAAGGAGTTGAAGTTGGTTGCGAACCGA

[0183] CTCCACACCTCCTTCCCGCCCCATCACGTAGTGGACATGTCCAAGTTTACC

[0184] CTCATGACCCCTGGGAGTGGTGTCTCTATGCGGGTTGAGTACCAACACGG

[0185] CTGCCTCCCCGCTGACACTGTCCCTGAAGGAAACTGCTGGTGGCGCTTGTT

[0186] TGACTCGCTCCCACCGGAAGTTCAGTACAAAGAAATTCGCCATGCTAACC

[0187] AATTTGGCTATCAAACCAAGCATGGTGTCCCTGGCAAGTACCTACAGCGG

[0188] AGGCTGCAAGTTAATGGTCTTCGAGCAGTGACCGACACACATGGACCTAT

[0189] CGTCATACAGTACTTCTCTGTTAAGGAGAGTTGGATCCGCCACTTGAAGTT GGTGGAAGAACCCAGCCTCCCCGGGTTTGAGGATCTCCTCAGAATCAGGG

[0190] TTGAGCCCAATACGTCACCACTGGCTGGAAAGGATGAGAAGATTTTCCGG

[0191] TTTGGCAGTCATAAGTGGTACGGTGCCGGAAAGAGAGCAAGGAAAACAC

[0192] GCTCTGGTGCGACTACTATGGTCGCTCATCACGCTTCGTCCGCTCATGAAA

[0193] CCCGGCAGGCCACGAAGCACGAGGGTGCCGGCGCTAATAAGGCTGAGCA

[0194] TCTCAAGCGCTACTCTCCGCCTGCCGAAGGGAACTGTGGTTGGCACTGCAT

[0195] TTCCGCCATCGCCAACCGGATGGTGAATTCCAACTTTGAGACCACCCTTCC

[0196] TGAAAGAGTAAGGCCTTCAGATGACTGGGCCACTGACGAGGATCTTGTGA

[0197] ACACCATCCAAATCCTCAGGCTCCCTGCGGCCTTGGACAGGAACGGCGCT

[0198] TGCGGTAGCGCCAAGTACGTGCTTAAACTGGAGGGTGAGCATTGGACTGT

[0199] CTCTGTGATCCCTGGGATGTCCCCTACTTTGCTCCCCCTTGAATGTGTTCAG

[0200] GGTTGTTGTGAGCATAAGGGCGGTCTTGTTTCCCCGGATGCGGTCGAAATT

[0201] TCCGGATTTGATCCTGCCTGCCTTGACCGACTGGCTAAGGTAATGCACTTG

[0202] CCTAGCAGTACCATCCCAGCCGCTCTGGCCGAATTGTCCGACGACTCCAA

[0203] CCGTCCGGTTTCCCCGGCCGCTACTACGTGGACTGTTTCGCAATTCTATGC

[0204] TCGTCATAGAGGAGGAGATCATCATGACCAGGTGTGCTTGGGGAAAATCA

[0205] TCAGCCTTTGTCAAGTTATTGAGGATTGCTGCTGCCATCAGAATAAAACCA

[0206] ACCGGGCTACTCCGGAAGAGGTCGCGGCAAAGATTGATCAGTACCTCCGT

[0207] GGCGCAACAAGTCTTGAGGAATGCTTGGCCAAACTTGAGAGAGTTTCCCC

[0208] GCCGAGCGCTGCGGACACCTCTTTTGATTGGAATGTTGTGCTTCCTGGGGT

[0209] TGAGGCGGCGAATCAGACAACCGACCAACCTCACGTCAACTCATGCTGCA

[0210] CCCTGGTCCCTCCCGTGACTCAAGAGCCTTTGGGCAAGGACTCGGTCCCTC

[0211] TGACCGCCTTCTCACTGTCCAATTGCTATTACCCTGCACAAGGTGACGAGG

[0212] TTCATCACCGTGAGAGGTTAAATTCCGTACTCTCTAAGTTGGAAGAGGTTG TCCTGGAAGAATATGGGCTCATGTCCACTGGACTTGGCCCGCGACCCGTG

[0213] CTGCCGAGCGGGCTCGACGAGCTTAAAGACCAGATGGAGGAGGATCTGCT

[0214] ACAACTAACCAACACCCAGGCGACTTCAGAAATGATGGCCTGGGCGGCTG

[0215] AGCAGGTCGATTTAAAAGCTTGGGTCAAAAGCTACCCGCGGTGGACACCA

[0216] CCACCCCCTCCACCAAGAGTTCAACCTAGAAGAACAAAGTCTGTCAAAAG

[0217] CTTGCCAGAGGGCAAGCCTGTCCCTGCTCCGCGCAGGAAGGTCAGATCCG

[0218] ATTGCGGCAGCCCGGTTTTGATGGGCGACAATGTCCCTAACGGTTCGGAA

[0219] GAAACTGTCGGTGGTCCCCTCAATTTTCCGACACCATCCGAGCCGATGAC

[0220] ACCTATGAGTGAGCCCGTACTTGTGCCCGCGTCGCGACGTGTCCCCAAGC

[0221] TGATGACACCTTTGAGTGGGTCGGCACCAGTTCCTGCACCGCGTAGAACT

[0222] GTGACAACAACGCTGACGCACCAGGATGAGCCTCTGGATTTGTCTGTGTC

[0223] CTCACAGACGGAATATGAGGCTTTCCCCCTAGCACCATTGCAGAACATGG

[0224] GCATCCTGGAGGCGAGGGGGCAAGAAGTTGAGGGAGTCCTGAGTGAAAT

[0225] CTCGGATATACTAAATGACACCAACCCTGCACCTGTGTCATCAAGCAGCT

[0226] CCCTGTCAAGTGTTAAGATCACACGCCCAAAATACTCAGCTCAAGCCATC

[0227] ATCGACTCTGGCGGGCCTTGCAGTGGGCATCTCCAAAAGGAAAAAGAAGC

[0228] ATGCCTCAGCATCATGCGTGAGGCTTGTGATGCGTCCAAGCTTGGTGATCC

[0229] TGCTACGCAGGAGTGGCTCTCTCGCATGTGGGATAGGGTTGACATGCTGA

[0230] CTTGGCGCAACACGTCTGCTTACCAGGCGTTTCGCATCTTAAATGGCATGT

[0231] TTGAGTTTCTCCCAAAGATGATTCTCGAGACACCGCCGCCCCACCCGTGCG

[0232] GGTTTGTGATGTTACCTCGCACGCCTGCACCTTCCGTGAGTGCAGAGAGTG

[0233] ACCTCACCATTGGTTCAGTGGCCACCGAGGATGTTCCACGCATCCTCGGG

[0234] AAAATAGGAGACACTGACGAGCTGCTTGACCGGGGTCCCTCGGCTCCCTC

[0235] CAAGGGAGAACCGGTCTGTGACCAACCTGCCAAAGATCCCCGGATGTCGC CACGGGAGTCTGACGAGAGCATGATGGCTCCGCCCGCAGATACAGGTGGT

[0236] GTCGGCTCATTCACTGATTTGCCGTCTTCAGATGGTGTGGATGTGGACGGG

[0237] GGGGGGCCGTTAAGAACGGTAAAAACAAAAGCAGGAAGGCTCTTAGACC

[0238] AACTGAGCTGCCAGGTTTTTAGCCTCGTTTCCCATCTCCCTATTTTCTTCTC

[0239] ACACCTCTTCAAATCTGACAGTGGTTATTCTCCGGGTGATTGGGGTTTTGC

[0240] AGCTTTTACTCTATTTTGCCTCTTTCTATGTTACAGTTACCCATTCTTCGGT

[0241] TTTGCTCCCCTCTTGGGTGTATTTTCTGGGTCTTCTCGGCGTGTGCGAATGG

[0242] GGGTTTTTGGCTGCTGGTTGGCTTTTGCTGTTGGTCTGTTCAAGCCTGTGTC

[0243] CGACCCAGTCGGCACTGCTTGTGAGTTTGACTCGCCAGAGTGTAGGAACG

[0244] TCCTTCATTCTTTTGAGCTTCTCAAACCTTGGGACCCTGTCCGCAGCCTTGT

[0245] TGTGGGCCCCGTCGGTCTCGGCCTTGCCATTCTTGGCAGGTTACTGGGCGG

[0246] GGCACGCTACATCTGGCACTTTTTGCTTAGGCTTGGCATTGTTGCAGACTG

[0247] TATCTTGGCTGGAGCTTATGTGCTTTCTCAAGGTAGGTGTAAAAAGTGCTG

[0248] GGGATCTTGTGTAAGAACTGCTCCTAATGAGATCGCCTTCAACGTGTTCCC

[0249] TTTTACACGTGCGACCAGGTTGTCACTCATCGACCTGTGCGATCGGTTCTG

[0250] CGCACCAAAAGGCATGGACCCCATTTTTCTCGCCACTGGGTGGCGTGGGT

[0251] GCTGGACCGGCCGGAGTCCCATTGAGCAACCTTCTGAAAAACCCATCGCG

[0252] TTCGCCCAGCTGGATGAGAAGAGGATTACGGCTAGAACTGTGGTCACTCA

[0253] GCCTTATGATCCCAACCAGGCCGTAAAGTGCTTGCGGGTATTACAGGCGG

[0254] GCGGGGCGATGGTGGCCGAGGCAGTCCCAAAAGTGGTCAAAGTTTCCGCC

[0255] ATTCCATTCCGAGCTCCTTTCTTTCCCGCTGGAGTGAAAGTTGATCCTGAG

[0256] TGCAGAATCGTGGTTGATCCCGATACTTTTACTACAGCCCTCCGGTCTGGC

[0257] TATTCCACCGCGAACCTCGTCCTTGGTACGGGGGACTTTGCCCAGCTGAAT

[0258] GGACTAAAGATCAGGCAAATTTCCAAGCCTTCAGGGGGAGGCCCACACCT CATTGCTGCCTTGCATGTTGCCTGCTCGATGGCGTTACACATGCTTGCTGG

[0259] TGTTTATGTAACTGCAGTGGGGTCCTGCGGTACCGGTACTAACGATCCGTG

[0260] GTGCACTAACCCGTTTGCCGTCCCTGGCTACGGACCTGGCTCTCTTTGCAC

[0261] GTCTAGATTGTGCATCTCCCAACACGGCCTCACCTTGCCCTTGACAGCACT

[0262] TGTGGCGGGATTCGGCCTTCAAGAGATTGCCTTGGTCGTTTTGATTTTTGT

[0263] CTCCATCGGAGGCATGGCTCATAGGTTGAGTTGTAAGGCTGACATGTTGT

[0264] GCATCTTACTCGCAATCGCTAGTTATGTTTGGGTACCTCTTACCTGGTTGCT

[0265] TTGTGTGTTTCCTTGTTGGTTGCGCTGGTTCTCTTTGCACCCCCTCACCATC

[0266] CTGTGGTTGGTGTTTTTCTTGATTTCTGTAAATATACCCTCGGGAATCTTGG

[0267] CCGTGGTGTTATTGGTTTCTCTCTGGCTTCTAGGTCGTTATACTAACATTGC

[0268] TGGTCTCGTCACCCCCTATGACATTCATCATTACACCAGTGGCCCCCGCGG

[0269] TGTTGCCGCCTTGGCCACCGCACCAGATGGAACCTACTTGGCTGCCGTCCG

[0270] CCGTGCTGCGCTGACTGGTCGTACCATGCTGTTCACCCCGTCTCAGCTCGG

[0271] GTCCCTCCTTGAGGGCGCTTTCAGAACTCAAAAGCCCTCACTGAACACCG

[0272] TCAATGTGGTCGGGTCCTCCATGGGCTCTGGCGGAGTGTTCACTATTGACG

[0273] GGAAAATCAAGTGCGTGACTGCCGCACATGTCCTTACGGGTAACTCAGCT

[0274] AGGGTTTCCGGGGTCGGCTTCAATCAAATGCTTGACTTTGATGTAAAAGG

[0275] GGACTTCGCCATAGCTGATTGCCCGAATTGGCAAGGGGTTGCTCCCAAGG

[0276] CCCAGTTCTGCGAGGATGGGTGGACTGGTCGCGCCTATTGGCTGACATCCT

[0277] CTGGCGTTGAACCCGGTGTTATTGGGAATGGGTTCGCCTTCTGCTTCACCG

[0278] CGTGTGGCGATTCTGGATCCCCAGTGATTACCGAAGCCGGTGAGCTTGTC

[0279] GGCGTTCACACAGGATCAAACAAACAAGGAGGAGGCATTGTCACGCGCC

[0280] CCTCAGGCCAGTTTTGTAATGTGAAGCCCATCAAGCTGAGCGAGTTGAGT

[0281] GAATTCTTCGCTGGACCTAAGGTCCCGCTCGGTGATGTGAAAATTGGCAG TCACATAATTAAAGACACATGCGAGGTGCCTTCAGATCTTTGTGCCCTGCT

[0282] TGCTGCCAAACCCGAACTGGAAGGAGGCCTTTCCACAGTTCAACTTCTGT

[0283] GTGTGTTTTTCCTCCTGTGGAGAATGATGGGGCATGCCTGGACGCCCTTGG

[0284] TTGCTGTGGGGTTTTTCATCCTGAATGAGATTCTCCCAGCTGTCCTGGTCC

[0285] GGAGTGTTTTCTCCTTTGGGATGTTTGTGCTATCTTGGCTCACACCATGGTC

[0286] TGCGCAAGTCCTGATGATCAGGCTTCTGACAGCAGCCCTTAACAGAAACA

[0287] GATGGTCTCTTGGTTTTTACAGCCTTGGTGCAGTAACCAGTTTTGTCGCAG

[0288] ATCTTGCGGTAACTCAAGGGCATCCGTTACAGGTGGTAATGAACTTAAGC

[0289] ACCTATGCCTTCCTGCCCCGGATGATGGTTGTGACCTCGCCAGTCCCAGTG

[0290] ATCGCGTGTGGTGTTGTGCACCTCCTTGCCATAATTTTGTACTTGTTTAAGT

[0291] ACCGCTGCCTTCACAATGTCCTTGTTGGCGATGGGGTGTTCTCTTCGGCTT

[0292] TCTTCTTGCGATACTTTGCCGAGGGAAAGTTGAGGGAAGGGGTGTCGCAA

[0293] TCCTGCGGGATGAGTCATGAGTCGCTGACTGGTGCTCTCGCCATGAGACTC

[0294] ACTGACGAGGACTTGGATTTCCTTACGAAATGGACTGATTTTAAGTGCTTT

[0295] GTTTCTGCGTCCAACATGAGGAATGCAGCGGGCCAATTTATCGAGGCTGC

[0296] TTATGCAAAAGCACTAAGAATTGAACTTGCTCAGTTGGTACAGGTTGATA

[0297] AGGTCCGAGGCACCATGGCCAAACTCGAGGCTTTTGCCGATACCGTGGCA

[0298] CCCCAACTCTCGCCCGGTGACATTGTTGTTGCCCTTGGCCACACGCCTGTT

[0299] GGCAGCATCTTCGACCTAAAGGTTGGTAGCACCAAGCATACTCTCCAAGC

[0300] CATTGAGACTAGAGTCCTTGCCGGGTCCAAAATGACTGTGGCGCGTGTCG

[0301] TTGACCCAACTCCCGCACCCCCACCCGTACCTGTGCCCATCCCTCTCCCAC

[0302] CGAAAGTTCTGGAGAACGGTCCCAATGCCTGGGGGGATGAGGACCGTTTG

[0303] AGCAAGAAGAAGAGGCGCAGGATGGAAGCCGTCGGCATTTTTGTCATGG

[0304] ACGGGAAAAAGTACCAGAAATTTTGGGACAAGAATTCCGGTGATGTGTTT TATGAGGAGGTCCATACTAGCACAGACGAGTGGGAGTGCCTTAGAACTGG

[0305] CGACCCTGTCGACTTTGATCCTGAGACAGGGATTCAGTGTGGGCATATCA

[0306] CCATTGAAGATAAGGTTTACAATGTCTTCACCTCCCCATCTGGTAGGAGAT

[0307] TCTTGGTCCCCGCCAACCCCGAGAATAGAAGAGCTCAGTGGGAAGCCGCC

[0308] AAGCTTTCCGTGGAGCAAGCCCTTGGTATGATGAACGTCGACGGCGAACT

[0309] GACTGCCAAAGAACTGGAGAAACTGAAAAGAATAATTGACAAACTCCAG

[0310] GGCCTGACTAAGGAGCAGTGTTTAAACTGCTAGCCGCCAGCGGCTTGACC

[0311] CGCTGTGGTCGCGGCGGCTTAGTTGTTACTGAGACAGCGGTAAAAATAGT

[0312] CAAATTTCACAACCGGACCTTCACCCTAGGACCTGTGAACTTAAAAGTGG

[0313] CCAGTGAGGTTGAGCTAAAAGACGCGGTTGAGCACAACCAACATCCGGTT

[0314] GCCAGACCGGTTGATGGTGGTGTTGTGCTCCTGCGCTCTGCAGTTCCTTCG

[0315] CTTATAGATGTCTTGATCTCCGGCGCTGATGCATCTCCTAAGTTACTCGCC

[0316] CGCCACGGGCCGGGAAACACTGGGATTGATGGCACGCTTTGGGATTTTGA

[0317] GGCCGAGGCTACTAAAGAGGAAGTTGCACTCAGTGCGCAAATAATACAG

[0318] GCTTGTGATATTAGGCGCGGCGACGCGCCTGAAATTGGTCTCCCTTACAA

[0319] GTTGTACCCTGTTAGGGGCAACCCTGAGCGGGTAAAAGGAGTTTTACAGA

[0320] ATACAAGGTTTGGAGACATACCTTACAAAACCCCTAGTGACACTGGAAGC

[0321] CCGGTGCACGCGGCTGCCTGCCTCACGCCTAATGCTACTCCGGTGACTGAT

[0322] GGGCGCTCCGTCTTGGCTACAACCATGCCCTCTGGCTTTGAGTTGTATGTG

[0323] CCGACCATTCCAGCGCCCGTCCTTGATTATCTTGATTCTAGGCCTGACTGC

[0324] CCTAAACAGTTAACAGAGCACGGTTGTGAGGATGCTGCATTAAGAGACCT

[0325] CTCCAAGTATGATTTGTCCACCCAAGGCTTTGTTTTGCCTGGAGTTCTTCG

[0326] CCTCGTGCGGAAGTACCTGTTCGCCCACGTGGGTAAGTGCCCGCCCGTTC

[0327] ATCGGCCTTCCACTTACCCTGCTAAGAATTCTATGGCTGGAATAAATGGGA ACAGGTTTCCAACCAAGGACATTCAGAGCGTCCCTGAAATCGACGTTCTG

[0328] TGCGCACAGGCTGTGCGAGAAAACTGGCAAACTGTTACCCCTTGTACCCT

[0329] CAAGAAACAGTACTGTGGGAAGAAGAAGACTAGGACAATACTTGGCACC

[0330] AATAACTTCATTGCGTTGGCCCATCGGGCAGCGTTGAGTGGTGTTACCCAG

[0331] GGCTTCATGAAAAAAGCGTTCAACTCGCCCATCGCCCTCGGGAAAAACAA

[0332] ATTTAAGGAGCTACAAGCCCCGGTCCTAGGCAGGTGCCTTGAAGCTGATC

[0333] TTGCGTCCTGCGATCGATCCACACCTGCAATTGTCCGCTGGTTTGCCGCCA

[0334] ATCTTCTTTATGAACTCGCCTGTGCTGAGGGGCATCTACCGTCGTACGTGC

[0335] TTAACTGCTGCCACGACTTACTGGTCACGCAGTCCGGCGCGGTGACTAAG

[0336] AGAGGTGGCCTGTCGTCTGGCGACCCGATTACCTCTGTGTCAAACACCATT

[0337] TACAGCTTAGTGATATATGCACAGCACATGGTGCTCAGTTACTTCAAAAGT

[0338] GGTCACCCTCATGGCCTTCTGTTTCTGCAAGACCAGCTAAAGTTTGAGGAC

[0339] ATGCTCAAGGTTCAACCCCTGATCGTCTATTCCGACGACCTTGTGCTGTAT

[0340] GCCGAGTCTCCCTCCATGCCAAACTACCACTGGTGGGTTGAACATCTGAAT

[0341] CTTATGCTGGGTTTCCAGACGGATCCAAAGAAGACAACCATCACAGACTC

[0342] ACCATCATTCCTAGGTTGCAGGATAATAAATGGGCGCCAGCTAGTCCCTA

[0343] ACCGTGACAGGATCCTCGCGGCCCTCGCCTACCACATGAAGGCAAGTAAT

[0344] GTTTCTGAATACTACGCCTCGGCGGCTGCAATACTCATGGACAGCTGTGCT

[0345] TGTTTAGAGTATGATCCTGAATGGTTTGAAGAGCTCGTAGTTGGGATAGC

[0346] GCAGTGCGCCCGCAAGGACGGCTACAGCTTTCCTGGCCCACCGTTCTTCTT

[0347] GTCCATGTGGGAAAAACTCAGGTCCAATCATGAGGGGAAGAAGTCCAGA

[0348] ATGTGCGGGTACTGCGGGGCCCCGGCTCCGTACGCCACTGCCTGTGGCCT

[0349] CGATGTCTGTGTTTACCACACCCACTTCCACCAGCATTGTCCTGTTATAAT

[0350] CTGGTGTGGCCACTCGGCGGGTTCTGGTTCTTGTAGTGAGTGCGAACCCCC CCTAGGAAAAGGCATAAGCCCTCTAGATGAGGTGTTAGAACAAGTTCCGT

[0351] ACAAGCCTCCGCGGACTGTGATCATGCATGTGGAGCAGGGTCTCACCCCT

[0352] CTTGACCCAGGTAGATACCAGACTCGCCGCGGATTAGTCTCCGTTAGGCG

[0353] TGGCATCAGGGGAAATGAAGTCGACCTACCAGACGGTGATTACGCTAGTA

[0354] CCGCCTTGCTCCCTACTTGTAAAGAGATCAACATGGTCGCTGTTGCCTCTA

[0355] ACGTGTTGCGTAGCAGGTTTATCATCGGCCCACCCGGTGCTGGGAAAACA

[0356] CACTGGCTTCTTCAACAAGTCCAGGATGGTGATGTCATTTACACGCCAACT

[0357] CACCAGACCATGCTCGACATGATTAGGGCTTTGGGGACGTGCCGGTTCAA

[0358] CGTTCCAGCAGGTACAACGCTGCAATTCCCTGCCCCCTCCCATACCGGCCC

[0359] ATGGGTTCGCATCTTGGCCGGCGGTTGGTGTCCTGGCAAGAACTCCTTCCT

[0360] GGATGAAGCGGCGTATTGCAATCACCTTGATGTCTTGAGGCTTCTCAGTAA

[0361] AACAACTCTCACTTGCCTAGGGGACTTCAAACAACTCCACCCTGTGGGTTT

[0362] TGACTCCCATTGCTAAGTATTTGACATCATGCCTCAGACCCAATTAAAGAC

[0363] CATTTGGAGGTTTGGGCAGAATATTTGTGATGCCATTCAACCAGATTACAG

[0364] GGACAAACTTATGTCCATGGTCAACACGACCCGTGTGACCTACGTAGAAA

[0365] AACCTGTCAGGTATGGGCAAGTCCTCACCCCCTACCACAGGGACCGAGAG

[0366] GACGGCGCCATTACTATTGACTCCAGTCAAGGCGCCACATTTGATGTGGTT

[0367] ACACTGCATTTGCCCACTAAAGATTCACTCAACAGGCAAAGAGCTCTTGTT

[0368] GCTATCACCAGGGCAAGACATGCTATCTTCGTGTATGACCCACACAGGCA

[0369] ATTGCAGAGCATGTTTGATCTCCCCGCGAAAGGCACACCCGTCAACCTCA

[0370] CAGTGCACCGTGACGAACAGCTGATCGTATTAGACAGAAACAACAGAGA

[0371] AATCACGGTTGCTCAGGCTCTAGGCAATGGAGATAAATTCAGGGCCACAG

[0372] ATAAGCGCGTTGTAGATTCTCTCCGCGCTATTTGCGCAGACCTGGAAGGGT

[0373] CGAGCTCCCCGCTCCCCAAGGTCGCGCATAACTTGGGATTCTATTTCTCAC CTGATTTGACTCAGTTTGCTAAACTCCCGGCAGAACTTGCACCCCACTGGC

[0374] CCGTGGTGACAACCCAGAACAATGAAAGGTGGCCAGATCGGTTGGTAGCC

[0375] AGCCTTCGCCCTATCCATAAATATAGCCGCGCGTGCATTGGTGCCGGCTAT

[0376] ATGGTGGGCCCCTCGGTGTTTTTAGGCACCCCTGGGGTTGTGTCATACTAT

[0377] CTCACAAAATTTGTTAGAGGCGAGGCTCAAGTGCTTCCGGAGACAGTCTT

[0378] CAGCACCGGCCGAATTGAGGTAGATTGTCGAGAGTATCTTGATGATCGGG

[0379] AGCGAGAAGTTGCTGAGTCCCTCCCACATGCCTTCATCGGCGATGTCAAA

[0380] GGTACCACCGTTGGGGGATGTCATCACGTTACCTCCAAATACCTTCCGCGC

[0381] TTCCTTCCCAAGGAATCAGTTGCGGTGGTCGGGGTTTCGAGCCCCGGGAA

[0382] AGCCGCGAAAGCAGTTTGTACATTGACGGATGTGTACCTCCCAGACCTTG

[0383] AAGCGTACCTCCACCCAGAGACCCAGTCCAGGTGCTGGAAAATGATGTTG

[0384] GACTTTAAGGAGGTTCGACTGATGGTATGGAAAGACAAGACGGCCTATTT

[0385] TCAACTTGAAGGCCGCCATTTTACCTGGTACCAACTTGCAAGCTACGCCTC

[0386] ATACATCCGAGTTCCTGTTAATTCTACTGTGTACTTGGACCCCTGCATGGG

[0387] CCCTGCTCTTTGCAACAGAAGGGTTGTCGGGTCCACCCATTGGGGAGCTG

[0388] ACCTCGCAGTCACCCCTTATGATTACGGTGCCAAAATTATTCTGTCTAGTG

[0389] CATACCATGGTGAAATGCCTCCAGGTTACAAAATTCTGGCGTGCGCGGAG

[0390] TTCTCGCTTGATGATCCAGTAAGGTACAAACACACCTGGGGATTTGAATC

[0391] GGATACAGCGTATCTGTACGAGTTTACTGGAAATGGTGAGGACTGGGAGG

[0392] ATTACAATGATGCGTTTCGGGCGCGCCAGAAAGGGAAAATTTATAAAGCT

[0393] AATGCCACCAGCATGAGGTTTCATTTTCCCCCGGGCCCTGTCATTGAACCA

[0394] ACTTTAGGCCTGAATTGAAATGAAATGGGGTCTATGCAAAGCCTCTTTGA

[0395] CAAAATTGGCCAACTTTTTGTGGATGCTTTCACGGAGTTTCTGGTGTCCAT

[0396] TGTTGATATCATCATATTTTTGGCCATTTTGTTTGGCTTCACAATTGCCGGT TGGCTGGTGGTCTTTTGCATCAGACTGGTTTGCTCCGCGGTACTCCGTGCG

[0397] CGGTCTACCGTTCACCCTGAGCAATTACAGAAGATCTTATGAGGCCTTTCT

[0398] TTCTCAGTGTCAGGTGGACATTCCCACCTGGGGCGTCAAACACCCTTTGGG

[0399] GGTGCTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGC

[0400] GTCGAATGTACCGCGTCATGGAAAAAGCAGGGCAGGCTGCCTGGAAACA

[0401] GGTGGTGAGCGAAGCTACATTGTCTCGCATCAGTGGTTTGGATGTGGTGG

[0402] CTCACTTTCAACATCTTGCCGCTATTGAAGCCGAGACTTGTAAATATTTGG

[0403] CTTCCCGGCTACCCATGCTGCACAACCTGCGCTTGACAGGGTCAAATGTA

[0404] ACCATAGTGTATAATAGTACTTTGGATCAGGTGTTTGCCATTTTCCCAACC

[0405] CCTGGTTCCCGGCCAAAGCTTAATGATTTTCAGCAATGGCTAATAGCTGTA

[0406] CATTCCTCCATATTTTCCTCCGTTGCAGCTTCTTGTACTCTTTTTGTTGTGCT

[0407] GTGGTTGCGAATTCCAATGCTACGTTCTGTTTTTGGTTTCCGCTGGTTAGG

[0408] GGCAACTTCTCTTTTGAACTCATGGTGAATTACACGGTATGCCCGCTTTGC

[0409] CCAACCCGGCAGGCAGCCGCTGAGATCCTTGAACCCGGCAAGTCTTTTTG

[0410] GTGCAGGATAGGGAATGACCGATGTAGTGAGAACGATCATGACGAACTA

[0411] GGGTTCATGGTTCCGCCTGGCCTCTCCAGCGAAGGCCACTTGACCAGTGTT

[0412] TACGCCTGGTTGGCGTTCCTGTCCTTCAGCTACACGGCCCAGTTCCATCCC

[0413] GAGATATTTGGGATAGGGAATGTGAGTCAAGTTTATGTTGACATCAAGCA

[0414] CCAATTCATCTGCGCTGTTCACGACGGGGATAACGCCACCTTGCCTCGCCA

[0415] TGACAATATTTCAGCCGTATTTCAGACCTACTACCAACACCAGGTCGACG

[0416] GCGGCAATTGGTTTCACCTGGAATGGCTGCGTCCTTTCTTTTCCTCTTGGTT

[0417] GGTTTTAAATGTTTCGTGGTTTCTCAGGCGTTCGCCTGCAAGCCATGTTTC

[0418] AGTTCGAGTCTTTCGGACATCAAAACCAACACCACCGCAGCATCAGGCTT

[0419] CGTTGTCCTCCAGGACATCAGCTGCCTTAGGCATGGCGACTCGTCCTCTCC GACAATTCGCAAGAGTTCTCAGTGCCGCACGGCGATAGGGACGCCCGTGT

[0420] ACATCACCATCACTGCCAATGTCACAGATGAAAATTATCTACATTCTTCTG

[0421] ATCTCCTCATGCTTTCTTCTTGCCTTTTCTATGCTTCCGAGATGAGTGAAAA

[0422] GGGATTCAAAGTGGTGTTTGGCAATGTGTCAGGCATCGTGGCTGTGTGCG

[0423] TCAACTTTACCAGCTACGTCCAACACGTCAAGGAGTTTACCCAACGCTCCT

[0424] TAGTGGTCGATCATGTGCGACTGCTTCATTTCATGACACCTGAGACCATGA

[0425] GGTGGGCAACCGTTTTAGCCTGTCTTGTTGCCATCCTACTGGCAATTTGAA

[0426] TGTTCAAGTATGTTGGGGAAGTGCTTGACCGCGTGCTGTTGCTCGCGATTG

[0427] CTTTTTTTGTGGTGTATCGTGCCGTTCTATCTTGCTGTGCTCGTCAACGCCA

[0428] GCAACAACAACAGCTCTCATATTCAGTTGATTTATAACTTAACGCTATGTG

[0429] AGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCAGTGGAG

[0430] ACTTTTGTCATCTTCCCCGTGTTGACTCACATTGTTTCCTATGGGGCACTCA

[0431] CCACCAGCCATTTCCTTGACACAGTTGGTCTGGCCACTGTGTCCACCGCCG

[0432] GATATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTC

[0433] TGGCTGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGTCCT

[0434] GGCGCTACTCTTGTACCAGATATACCAACTTCCTTCTGGACACTAAGGGCA

[0435] GACTTTATCGTTGGCGGTCGCCCGTCATTGTGGAGAAAAGGGGTAAGGTT

[0436] GAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTC

[0437] CGCGGCAACCCCTTTAACCAGAGTTTCAGCGGAACTATGGGGTCGTCTCT

[0438] AGACGACTTCTGCAATGATAGCACAGCTCCACAGAAGGTGCTTTTGGCGT

[0439] TTTCCATTACCTACACGCCAGTGATGATATATGCTCTAAAGGTAAGTCGCG

[0440] GCCGACTGCTAGGGCTTCTGCACCTTTTGATCTTTCTGAATTGTGCTTTTAC

[0441] CTTCGGGTACATGACATTCGTGCACTTTGAGAGCACAAATAGGGTCGCGC

[0442] TCACTATGGGAGCAGTAGTTGCACTTCTTTGGGGAGTGTACTCAGCCATAG AAACCTGGAAATTCATCACCTCCAGATGCCGTTTGTGCTTGCTAGGCCGCA

[0443] AGTACATTCTGGCCCCTGCCCACCACGTCGAAAGTGCCGCGGGCTTTCATC

[0444] CGATTGCGGCAAATGATAACCACGCATTTGTCGTCCGGCGTCCCGGCTCC

[0445] ACTACGGTCAACGGCACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGG

[0446] TGGCAGAAAAGCTGTTAAGCAGGGAGTGGTAAACCTTGTTAAATATGCCA

[0447] AATAACAACGGCAAGCAGCAAAAGAAAAAGAAGGGGAATGGCCAGCCA

[0448] GTCAATCAGCTGTGCCAAATGCTGGGTAAGATCATCGCCCAACAAAACCA

[0449] GTCCAGAGGCAAGGGACCGGGGAAGAAAAATAGGAAGAAAAACCCGGA

[0450] GAAGCCCCATTTCCCTCTAGCGACTGAAGATGACGTCAGGCATCACTTTAC

[0451] CCCTAGTGAGCGGCAATTGTGTCTGTCGTCGATCCAGACTGCCTTCAATCA

[0452] GGGCGCTGGAACTTGTGCCCTGTCAGATTCAGGGAGGATAAGTTACACTG

[0453] TGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTCTGATCCGCGCCACAG

[0454] CATCACCCTCAGCATGATGGGCTGGCATTCTTTGGCACCTCAGTGTTAGAA

[0455] TTGGGAGAGTGTGTGGTGAATGGCACTGATTGACACTGTGCCTCTAAGTC

[0456] ACCTATTCAATTAGGGCGACCGTGTGGGGGCAAAGTTTGATTGGCGAGAA

[0457] CCATGCGGCCGTAATT

[0458] SEQ ID NO: 3 (PRRSV-1 Lee GP5)

[0459] ATGAGATGTTCTCACACATTGGGGCGTTTCTTGATTCCTCACTCTTA

[0460] CTGTTGGTGGCTTTCTTTGCTGTGTATCGGCTTGTCTTGGTCCTTTGC

[0461] CGGTGGCAACGGCAACAGCTCGACATACCGATACATATACAACTTA

[0462] ACCATATGTGAGCTGAACGGGACCGAAGGGCTGTCCTCCCACTTTC

[0463] ATTGGGCAGTCGAAACCTTTGTCATGTACCCAGTGGCGACTCACATC

[0464] CTCTCACTGGGTTTCCTGACGACAAGCCACTTCTTTGACGCGCTCGG TCTAGGAGCTGTGTCTGTTGCTGGATTCACTAGTAAGCGGTACGTAC

[0465] TCAGCAGTGTGTACGGCGTTTGTGCATTAGCGGCGCTCGCGTGTTTT

[0466] GTCATCCGCGTTACTAAGAATTGTATGGCTTGCCGCTATGCTCGTAC

[0467] TCGTTTCACCAATTTTATTGTGGACGACCGGGGGAGAATCCATCGGT

[0468] GGAAGTCCCCAATAGTGGTGGAAAAACTAGGCAAAGCCGACGTTGG

[0469] CGGCGACCTTGTCACCATCAAGCACGTTGTCTTCGAGGGAGTTAAA

[0470] GCTCAACCCTTGACGAGGACTTCGGCCGAGCAATGGGAAGCCTAG

[0471] SEQ ID NO: 4 (Pl)

[0472] ATGACGTATAGGTGTTGGCTCTATG

[0473] SEQID NO: 5 (P2)

[0474] AGCAGCAATCCTCAATAACTTGACA

[0475] SEQID NO: 6 (P 3)

[0476] TGTCAAGTTATTGAGGATTGCTGCT

[0477] SEQID NO: 7 (P 4)

[0478] CTGGGTCGGACACAGGCTTGAACAG

[0479] SEQID NO: 8 (P5)

[0480] CTGTTCAAGCCTGTGTCCGACCCAG

[0481] SEQID NO: 9 (P6)

[0482] AGGGCACAAAGATCTGAAGGCACCT

[0483] SEQID NO: 10 (P7) AGGTGCCTTCAGATCTTTGTGCCCT

[0484] SEQID NO: 11 (P8)

[0485] CGGCTAGCAGTTTAAACACTGCTCC

[0486] SEQID NO: 12 (P9) TAAGGAGCAGTGTTTAAACTGCTAGCCGCC

[0487] SEQID NO: 13 (PIO)

[0488] CCCCTGATGCCACGCCTAACGGAGA

[0489] SEQID NO: 14 (P 11)

[0490] TCTCCGTTAGGCGTGGCATCAGGGG SEQID NO: 15 (Pl 2)

[0491] AAAGAGTGGCCGCAGCCATATCAATTCAGGCCTAAAGTTGGTTC

[0492] SEQID NO: 16 (Pl 3)

[0493] TAGGCCTGAATTGATATGGCTGCGGCCACTCTTT

[0494] SEQID NO: 17 (Pl 4) TGCATAGACCCCATTTCATTCATATTGCCAAGAGAATG

[0495] SEQID NO: 18 (Pl 5)

[0496] CATTCTCTTGGCAATATGAATGAAATGGGGTCTATGCAGACTGCGTAAAT

[0497] GCTACTCAAGACA

[0498] SEQID NO: 19 (Pl 6) TGTCTTGAGTAGCATTTACGCAGTC SEQIDNO: 20(P17)

[0499] GCCGCTCACTAGTAACGGCCTTTTTTTTTTTTTTTTTTT

[0500] SEQIDNO: 21 (P18)

[0501] TTTTTTTTAATTACGGCCGCATGGTTCTCGC

Claims

CLAIMS1. A porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain comprising: at least a part of a nucleic acid sequence of a first PRRSV strain, and wherein a nucleic acid sequence encoding glycoprotein 5 (GP5) of a second PRRSV strain is inserted into the at least a part of the nucleic acid sequence of the first PRRSV strain after ORF lb and before ORF2b.

2. The PRRSV vaccine strain as claimed in claim 1, wherein the first PRRSV strain is a PRRSV-2 strain.

3. The PRRSV vaccine strain as claimed in any preceding claim, wherein the first PRRSV strain is a JXAl-like strain.

4. The PRRSV vaccine strain as claimed in any preceding claim, wherein at least a part of a gene encoding non-structural protein (NSP2) is deleted from the first PRRSV strain.

5. The PRRSV vaccine strain as claimed in any preceding claim, wherein the first PRRSV strain lacks between 150-250 amino acids in the NSP2 region of the first PRRSV strain.

6. The PRRSV vaccine strain as claimed in any preceding claim, wherein at least a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 1 is deleted from the NSP2 gene of the first PRRSV strain.

7. The PRRSV vaccine strain as claimed in any preceding claim, wherein the second PRRSV strain is a PRRSV- 1 strain.

8. The PSSRV vaccine strain as claimed in any preceding claim, wherein the nucleic acid sequence encoding GP5 has the nucleic acid sequence of SEQ ID NO: 3.

9. A method of constructing a PRRSV vaccine strain, the method comprising the following steps; i. providing at least a part of a nucleic acid sequence of a first PRRSV strain, and ii. inserting into the at least a part of a nucleic acid sequence of the first PRRSV strain a nucleic acid sequence encoding GP5 of a second PRRSV strain, wherein the nucleic acid sequence encoding the GP5 is inserted into the at least a part of the nucleic acid sequence of the first PRRSV strain after ORF lb and before ORF2b.

10. The method as claimed in claim 9, wherein the method further comprises the step: iii. deleting at least a part of a gene encoding non- structural protein (NSP2) from the at least a part of a nucleic acid sequence of the first PRRSV strain.

11. The method as claimed in claim 10, wherein step (iii) is performed after step (i) but before step (ii).

12. A vaccine composition comprising the PRRSV vaccine strain as claimed in any one of claims 1 to 8 and a pharmaceutically acceptable carrier or excipient.

13. The vaccine composition as claimed in claim 12, or the PRRSV vaccine strain as claimed in any one of claims 1 to 8, for use in the prevention, elimination or reduction of PRRSV in pigs.

14. The vaccine composition as claimed in claim 12 or 13, or the PRRSV vaccine strain as claimed in any one of claims 1 to 8, for use in the prevention, elimination or reduction of PRRSV- 1 and / or PRRSV-2 in pigs.

15. A method of preventing, eliminating or reducing porcine reproductive and respiratory syndrome (PRRS) in a pig comprising administering to the pig the vaccine composition as claimed in any one of claims 12 to 14.

16. A vector comprising the PRRSV vaccine strain as claimed in any one of claims 1 to 8.

17. A host cell comprising the vector as claimed in claim 16.

Images