MXPA03007751A - Life attenuated strains of prrs virus. - Google Patents

Abstract

The present invention realtes to live attenuated European PRRS viruses which are attenuated by nucleic acid mutations on specific sties of the viral protein coded by ORF 2, 3 and 5. The invention also pertains to nucleotide sequences coding said viruses, methods of generating such viruses and a pharmaceutical composition comprising said PRRS viruses and teh use of said PRRS virus in the manufacture of a vaccine for the prophylaxis and treatment of PRRSV infections.

Description

ATTENUATED LIVE SCRAPS OF PRRS VIRUS FIELD OF THE INVENTION The present invention relates to viruses PRRS European, live, attenuated, which are attenuated by mutations in the nucleic acids on specific sites. The invention also pertains to the nucleic acid sequences encoding said viruses, the methods for the generation of such viruses and a pharmaceutical composition comprising said PRRS viruses and the use of PRRS viruses in the manufacture of a vaccine for the prophylaxis and treatment of infections by PRRS.

BACKGROUND OF THE INVENTION Porcine Reproductive and Respiratory Syndrome (PRRS) is caused by a enveloped, positive-strand RNA virus from the family arteriviridae (Snijder EJ, Meulenberg JJM 1998. "The molecular biology of arteriviruses." Journal of General Virology 79 (5 ): 961-971). About 10 to 15 years ago, two different strains of PRRS virus appeared to appear independently in the United States of America and Europe. The disease is now endemic in many pig-producing countries in North America, Europe and Asia. This continues to be a major cause of reproductive loss and respiratory disease in pigs. In the United States, the prevalence of infection is estimated at up to 70%. The virus is transmitted by inhalation, ingestion, intercourse, bite wounds or needles. This is replicated in mucosal, pulmonary or regional macrophages. Subclinically, the disease results in persistent resolution or infection. Persistently infected animals spread the virus in oral / pharyngeal fluids, blood, feces, urine and semen. Clinical symptoms in female pigs refer to abortion or premature births with weak live born pigs, stillborn pigs and autolysates. Infected neonatal pigs have a high mortality or suffer from pneumonia. The rearing and subsequent development of the pigs is complicated by pneumonia, concurrent bacterial infections and increased mortality. Male adult pigs are prone to fever and morphological changes in semen. As for all arterioviruses, the PRRS virus genome is a simple positive-stranded RNA molecule of approximately 15 kilobases. Open reading structures (ORFs) la and Ib code for replicases, ORF's 2 to 5 which are putative glycoproteins (gp 1 to 4), ORF 6 a membrane protein (M) and ORF 7 codes for a Nucleocapsid protein (N). The original descriptions of PRRS infection in the United States (WO93 / 03760, ATCC isolate VR-2332, deposited on July 17, 1991 at the American Type Culture Collection in Rockville, Maryland, USA, NCBI Genebank Access No. U 87392 U00153) and Europe (W092 / 21375, isolated Lelystad Agent (CDI-NL-2.91), Accession No. CNCM 1-1102, filed on June 5, 1991 with the Pasteur Institute, Paris, -NCBI GeneBank Nos. Access M 96262 [gi: 11125727], NC 002533 [gi: 11138120]) identified viruses that had genomic and serological differences. The comparison showed that both had a common ancestor which had diverged before the clinical disease was described in the late 1980s. Full-length genomic sequences have been reported for a number of PRRS viruses and the regions coding for the complete structural protein thereof (Snijder et al., 1998, supra; Meulenberg JJM, Hulst, MM et al., 1993. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory sydrome ..., Virology 192, 62-72; Conzelmann K., Visser., Van Oensel P., Thiel HJ, 1993. Molecular characterization of reproductive reproductive and respiratory syndrome virus, a member of the arterivirus group, Virology 103, 329-339; Murtaugh MP., Elam MR , Kakach LT, 1995. Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus, Archives of Virology 140, 1451-1460; Kapur V., Elam M.R., Pawtovich T.M. Murtaugh M.P., 1996. Genetic variation in poxcine reproductive and respiratory syndrome virus isolates in the midwester United States, Journal of General Virology 77, 1271-1276). The PRRS virus can be replicated in vi tro in pig lung macrophages, monocytes, gill cells and two subpopulations of MA-104 cells (monkey embryonic kidney cells) known as CL-2621 and MARC-145 (KD Rossow, Porcine reproductive and respiratory syndrome, Vet Pathol 35: 1-20, 1998). Recombinant media for the generation of infectious PRRS clones are also available (European Patent EP-0839912). To protect the pigs, live attenuated PRRS vaccines (RespPRRS / lngelvac® PRRS MLV, Boehringer Ingelheim) are commercially available.

Dead vaccines (inactivated whole virus) or subunit vaccines (purified, heterologously expressed or conventionally purified viral proteins) are most frequently inferior to live vaccines in their efficacy to produce a complete protective immune response, even in the presence of adjuvants. For PRRS, it has been shown that compared to the currently available dead vaccines, attenuated vaccines induce an immunity against the disease that lasts longer and is more efficient (Snijder et al., Referred to above). The present PRRS live vaccines are conventionally attenuated by serial passage of the virus in appropriate host cells until pathogenicity is lost (American strain: EP-0529584; European strain: EP-0676467; EP-0835930). The present PRRS live vaccines still leave space for improvement. First, they do not prevent reinfection. Second, they do not allow serological discrimination between vaccinated animals and animals infected with the virus in the field. In addition, a live vaccine in principle has a theoretical risk of reversion to the non-attenuated phenotype. In particular, RNA viruses such as PRRS virus are considered to possess high mutation rates due to inaccurate replication of the A N genome resulting from a lack of reading of the test by the RNA replication enzyme. For conventionally derived attenuated viruses, obtained by conventional multiple passes, the molecular origin as well as the genetic stability remains unknown, and the characteristics of the reversals are unpredictable. Thus, the problem underlying the invention was to provide improved strains of PRRS virus, which can be used for the manufacture of vaccines that overcome the disadvantages of the prior art.

Legends of the Figures Figure 1: Lelystad wild type virus sequence comparison, published in GenBank, attenuated virus A (abbreviated Vir.A) and attenuated Lelystad virus B Genomic area: ORF 2 Number of nucleotides: 750 Bold and framed indicate mutations (non-synonymous core exchanges) according to the invention (see also claims 2 and 3).

Figure 2: Lelystad wild type virus sequence comparison, published in GenBank, attenuated virus A (abbreviated Vir.A) and attenuated Lelystad virus B Genomic area: ORF 3 Number of nucleotides: 798 Bold and framed indicate mutations (non-synonymous nucleotide exchanges) according to the invention (see also claims 2 and 3).

Figure 3: Lelystad wild type virus sequence comparison, published in GenBank, attenuated virus A (abbreviated Vir.A) and attenuated Lelystad virus B Genomic area: ORF 4 Number of nucleotides: 552 Figure 4: Sequence comparison of the wild-type Lelystad virus, published in GenBank, attenuated virus A (abbreviated Vir.A) and attenuated Lelystad virus B Genomic area: ORF 5 Number of nucleotides: 606 In bold and framed the mutations are indicated (non-synonymous nucleotide exchanges) according to the invention (see also claims 2 and 3).

DESCRIPTION OF THE INVENTION Prior to the embodiments of the present invention it should be noted that as used herein and in the appended claims, the singular forms "a, an", "an" and "the," include the plural reference, a be that the context clearly dictates otherwise. Thus, for example, the reference to "a PRRS virus" includes a plurality of such PRRS viruses, the reference to "cell" is a reference to one or more cells equivalent thereto known to those skilled in the art, and so on. Unless defined otherwise, all technical and scientific terms used herein have the same meanings that are commonly understood by a person skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods, devices and materials are now described. All publications mentioned herein are incorporated by reference herein for purposes of describing and detailing cell lines, vectors and methodologies that are reported in publications that may be used in connection with the invention. Nothing in the present should be considered as an admission that the invention is not to precede such a description by virtue of the prior invention. The present invention provides a European, attenuated PRRS virus encoded by a nucleic acid comprising 0RF1, 0RF2, 0RF3, 0RF4, 0RF5, ORF6 and ORF7 and subtypes thereof, such as ORFla and ORFlb or ORF2a and ORF2b, characterized in that: a) 0RF2 comprises at positions 11915-11935 at least one of the nucleotides as described in Table 1: at positions 12037-12057 at least one nucleotide as described in Table 2: t G T T A A C C T C C T T C C T T G C C C C C C C T C C C C C T A C A A A G T G G G G G A T G A G T G G A T G T T T and at positions 12058-12078 at least one of the nucleotides as described in Table 3: T A A C C C A T A C A A A C C G T G T A C C C T T C C C T C C C C T T A C C C C G G G A A A G G G A G G G G A A T G T G G and / or a deletion at or in the positions and / or ORF3 comprises at positions 12660-12680 minus one of the nucleotides as described in Table 4: and / or a deletion at the position (s) and / or c) ORF5 comprises at positions 13684-13704 at least one of the nucleotides as described in Table 5: c T G G A A A c A c G A A A T G G G C C A T c A A C C C T C T C C C C A A A T T C C G T G G A G G T G G T T A A G and / or a suppression in said position (s).

It has surprisingly been found that PRRS viruses comprise specific sites on individual viral proteins which, if mutated, lead to an attenuated phenotype in comparison to the original, virulent field strain. The evolutionary pressure on these from now on called "specific sites of virulence" or simply referred to as "sites of invention" or just "sites" is immense. It is assumed that these sites have a general involvement in the process that leads to the attenuation of vaccine strains similar to PRRSV Europeans. These sites described in Tables 1 to 5 are specific for European strains (Lelystad Agent C CM 1-1102 wild-type is considered to be the reference type strain) and not shared with the United States strains of PRRS viruses. In a further aspect, the present invention relates to an attenuated European PRRS virus wherein at least one of the sequential sections corresponding to SEQ ID NOs: 25, 26, 27, 28 or 29 is mutated into one, two, three or more positions, up to a maximum of ten mutations. The sites SEQ ID NOs: 25, 26 and 27 are located at 0RF2 (positions 130-150, 252-272 and 273-293, respectively, in Figure 1), the site SEQ ID NO: 28 is located at ORF3 (position 267-287 in Figure 3), and the site SEQ ID NO: 29 is located at ORF5 (position 201-221 in Figure 4). Said sequential sections represent the specific virulence sites according to the invention. Since the PRRS virus is a positive strand RNA virus, it will comprise a positive RNA sequence instead of the DNA sequences given in the sequence listings, for example it will contain uracil (U) at the positions indicated as thymine ( T) in the sequence listings, and ribose instead of deoxyribose. It is understood that an attenuated European PRRS virus, according to the invention, comprises genomic RNA containing sequential sections corresponding to the DNA sequences with SEQ ID NOs .: 25, 26, 27, 28 or 29, wherein at least one of said sequential sections differs from the sequences referred to by at least one mutation. In this context, "corresponding to" means that the virus of the invention contains sequential sections that can be aligned to the sequences referred to by a standard alignment algorithm such as BLAST (Altschul, SF, Gish, W., Miller,., Myers, EW &Lipman, DJ (1990) "Basic local alignment searc tool." J. Mol. Biol. 215: 403-410; Gish, W. &States, DJ (1993) "Identification of protein coding regions by datbase similarity search "Nature, Genet 3: 266-272; Madden, TL, Tatusov, RL &Zhang, J. (1996)" Applications of network BLAST server "Meth. Enzymol 266: 131-141; Zhang, J. &; Madden, TL (1997) "PowerBLAST: A new network BLAST application for interactive or automated seguence analysis and annotation" Genome Res. 7: 649-656; Altschul, Stephen F., Thomas L. Madden, Alexander A. Scháffer, Zhang Jinghui, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein datbase search programs ", Nucleic Acids Res. 25: 3389-3402). Sequence positions that are aligned by bases by such an algorithm "correspond to" one another. After such alignment, a respective mutated sequence section of the virus could then differ from the corresponding reference sequence ("site", for example either SEQ ID NO: 25, 26, 27, 28 or 29) in one, two, three, or more positions, up to a maximum of ten positions. Preferably, a virus of the invention is mutated in the five sequence sections cited (sites). Preferably, the mutation (s) is / are substitutions and / or deletions. In the mutation (s) it is / are substitutions and / or deletions. In preferred embodiments, the mutations result in a change of the amino acid sequence (s) of the protein (s) encoded by the respective ORF (s). In a further aspect, the present invention relates to a European, attenuated PRRS virus having a 0RF2 containing a sequential section corresponding to SEQ ID NO: 25, wherein the triplet corresponding to positions 10 to 12 of the SEQ ID NO: 25 is mutated. Preferably, said triplet does not code for phenylalanine. Preferably, the triplet codes for a different amino acid. In a preferred embodiment, the triplet encodes serine. More preferably, the nucleotide corresponding to position 11 of SEQ ID NO: 25 is a C. Therefore, in a preferred embodiment, the attenuated European PRRS virus has an ORF2 coding for a protein that does not have phenylalanine in the position 47 (or the corresponding position in a BLAST alignment).

Preferably, the protein has a serine instead.

In a further aspect, the present invention relates to an attenuated European PRRS virus having an 0RF2 containing a sequence section corresponding to SEQ ID NO: 126, wherein the triplet corresponding to positions 10 to 12 of SEQ ID NO: 26 is mutated. Preferably, said triplet does not code for valine. Preferably, the triplet codes for a different amino acid. In a preferred embodiment, said triplet encodes for phenylalanine. More preferably, the nucleotide corresponding to position 10 of SEQ ID NO: 26 is a T (or a U in the RNA, respectively). Therefore, in a preferred embodiment, the attenuated European PRRS virus has an ORF2 that codes for a protein that does not have valine at position 88 (or the corresponding position in a BLAST alignment). Preferably, the protein has a phenylalanine in this position. In a further aspect, the present invention relates to an attenuated European PRRS virus having an 0RF2 containing a sequential section corresponding to SEQ ID NO: 27, wherein the triplet corresponding to positions 11 to 13 of SEQ ID NO. : 27 is mutated. Preferably, said triplet does not code for phenylalanine.

Preferably, the triplet codes for a different amino acid. In a preferred embodiment, said triplet encodes for leucine. More preferably, the nucleotide corresponding to position 11 of SEQ ID NO: 27 is a C. Therefore, in a preferred embodiment, the attenuated European PRRS virus has a 0RF2 which codes for a protein that does not have phenylalanine in the position 95 (or the corresponding position in the BLAST alignment). Preferably, the protein has a leucine in this position. In a further aspect, the present invention relates to an attenuated European PRRS virus having an 0RP3 containing a sequential section corresponding to SEQ ID NO: 28, wherein the triplet corresponding to positions 11 to 13 of SEQ ID NO. : 28 is mutated. Preferably, said triplet does not code for serine. Preferably, the triplet encodes a different amino acid. In a preferred embodiment, said triplet encodes proline. More preferably, the nucleotide corresponding to position 11 of SEQ ID NO: 28 is a C. Therefore, in a preferred embodiment, the attenuated European PRRS virus has an ORF2 that codes for a protein that does not have serine in the position 93 (or the corresponding position in the BLAST alignment). Preferably, said protein rather has a proline in this position. In a further aspect, the present invention relates to an attenuated European PRRS virus having an ORF5 containing a sequential section corresponding to SEQ ID NO: 29, wherein the triplet corresponding to positions 11 to 13 of SEQ ID NO. : 29 is mutated. Preferably, said triplet does not code for serine. Preferably, the triplet codes for a different amino acid. In a preferred embodiment, said triplet encodes for phenylalanine. More preferably, the nucleotide corresponding to position 11 of SEQ ID NO: 29 is a T (or a U in the RNA, respectively). Therefore, in a preferred embodiment, the attenuated European PRRS virus has an ORF2 that codes for a protein that does not have leucine at position 71 (or the corresponding position in a BLAST alignment). Preferably, said protein has rather a phenylalanine in this position. Live PRRS vaccines based on European strains attenuated by mutations and / or defined deletions, allow to avoid the disadvantages of the present generation of attenuated vaccines. If the attenuated strain carries defined and known mutations, it can be conveniently analyzed for quality control and genetic stability. The possibility of introducing defined deletions and / or substitutions, particularly as double or multiple mutations, decreases the probability of reversion to the non-attenuated or virulent phenotype. An additional advantage of such attenuation mutations lies in their unique known molecular character, which allows use as distinctive markers for attenuated PRRS viruses and distinguish them from PRRS viruses from the field. With the identified sites of the present invention, safe and site-specific attenuated viruses can be generated. Such viruses are useful for the preparation of a safe live vaccine for use in the prevention and / or treatment of PRRS infections. The present invention is directed to the European, attenuated PRRS strains and to the methods for the production thereof. In this context, "attenuated" means that a virulent strain is or has been modified in a manner that is less virulent or pathogenic than before the modification. In particular, "attenuated" or "attenuated" means that the virus has a significantly reduced ability to cause clinical disease, while the latter is still capable of replicating in the host. Preferably, the virulence or pathogenicity is reduced to an amount that makes the virus acceptable for administration as a vaccine. In a preferred embodiment, the virus is attenuated to a degree such that it does not cause clinical disease, while still capable of replicating in the host. Such an attenuated strain is an ideal agent for vaccination, because replication in the host ensures the stimulation of a rapid and excellent immune response. In a preferred embodiment, an attenuated European PRRS virus is less virulent than the Lelystad Agent. In yet another embodiment, the attenuated virus is less virulent than the parent strain from which it is derived. The term "less virulent than Agent Lelystad of the PRRS virus" (or, likewise, the parent strain from which it is derived) must be understood in terms of a comparison of the clinical symptoms of the virus of interest with the Agent. Lelystad (CDI-NL-2.91 (CNCM I-1102), or the parental strain A preferred method of determining whether a PRRS virus is less virulent than Lelystad Agent, or likewise to determine whether a virus modified according to the invention is less virulent than its parent strain before modification, is described in Example 1.

It may happen that not every single one of the possible mutations of amino acids in the specific site of virulence is involved in the reduction of virulence. The procedure of Example 1 provides an accurate and direct experimental protocol for determining whether a live PRRS virus according to the teaching of the invention is less virulent than its progenitor strain or a virulent field isolate such as Lelystad Agent. The present invention is directed to the European PRRSV strains which can be distinguished from the American strains as follows. Shortly after the virus was found, ensvoort et al. (J. Vet., Diagn. Invest. 4: 134-138 (1992) observed differences in antigenic characteristics between the American and European isolates, they produced sera against the American type and the European type in several pigs, and compared cross-reactivity. of anti-European (LV) and anti-American sera (VR-2332) with three isolates of American virus and 4 European isolates.Serums against PRRS viruses of the European serotype are significantly less reactive with American isolates than with European isolates. In addition, the sera produced against the American serotype viruses are less reactive, or even non-reactive at all, with the European virus isolates, ensvoort et al also showed the reactivity of the sera produced against the European and American isolates with the two reference virus CNCM 1-1102 (European) and ATCC VR-2332 (American) In this experiment, sera produced against European strains were not reactive at all. the American strain. In this way, two completely different serotypes of the virus exist. The American and European serotypes, which can be distinguished based on their serological properties. This can also be shown at the molecular level. Nelson et al. (74th Annual Meeting of the Conference of Research Workers in Animal Deseases, 8-9 November 1993) compared the sequences of the genes encoding the polymerase of various isolates. They showed that the polymerase genes show an 87-95% homology within the American group. However, a homology as low as 64-67% was found based on the nucleic acids between European serotypes and American serotypes. Mardassi published comparable results in the conference mentioned above, showing that the 530 3'-nucleic acids of the Quebec PRRS reference strain and the European isolates only show a homology as low as 59%. The results of the documents mentioned above make it more likely that the American and European serotypes diverged a long time ago, which could then easily explain their genetic differences and their non-serological relationship. The American and European serotypes can be easily discriminated in this way. European serotype viruses are characterized by reacting to a higher titre in Monolayer Peroxidase Assay with a panel of antisera against the European PRV virus LV (CNCM 1-1102) compared to a panel reaction of antisera against the virus American PRRS (ATCC VR-2332). If a panel of sera is used, obtained approximately 40 days after infection, and from different animals, any virus can be easily classified as belonging to either the American or European serotype. Typically, the reactivity of a European strain with a panel of antisera against other European strains is approximately 400 times higher than with a panel of antisera against the American strains. When a European strain is reacted with the antisera against the deposited European strain 1-1102 and the deposited American strain VR-2332, a typical difference in reactivity of about 55-fold is found (Wensvoort et al., Referenced above). "Mutation" means the substitution, deletion or insertion of a nucleotide or nucleic acid at a given position of a nucleotide or an amino acid sequence. A "substitution" is a replacement of one nucleotide or amino acid for another (for example, C for T). "Suppression" means the elimination of a nucleotide or amino acid. "Insertion" means that a nucleotide or amino acid is inserted at a given position. In an additional aspect, the present invention comprises a method or process of attenuation of a European PRRS virus, characterized in that: a) the nucleotide sequence of said virus is modified by site-directed mutagenesis in at least one of the positions of the ORF2 corresponding to the positions 130 to 150 and / or positions 252 to 272 and / or positions 273 to 293 of SEQ ID NO: 22; b) it is tested whether the resulting PRRS virus is attenuated or not. In a further aspect, the present invention comprises a method for attenuating a European PRRS virus, characterized in that: a) the nucleotide sequence of said virus is modified by site-directed mutagenesis in at least one of the 0RF3 positions corresponding to the positions 267 to 287 of SEQ ID NO: 23; b) it is tested whether the resulting PRRS virus is attenuated or not. In a further aspect, the present invention comprises a method for attenuating a European PRRS virus, characterized in that: a) the nucleotide sequence of said virus is modified by site-directed mutagenesis in at least one of positions 201 to 221 of ORF5 of according to SEQ ID NO: 24; b) it is tested whether the resulting PRRS virus is attenuated or not. In this context, the term "corresponding to" has a meaning as described above, for example, that the two positions corresponding to each other could be aligned as a pair in a sequence alignment such as BLAST. Preferably, the modification according to the invention results in a change of the amino acid sequence of the encoded protein. In preferred embodiments, the modifications are deletions and / or substitutions, preferably double or multiple mutations. In a preferred embodiment, two or more of the aforementioned modifications are combined. In a further preferred embodiment, the sequence of each 0RF2, 0RF3 and 0RF5 is modified. Preferably, the sequence of 0RF2 is modified in at least two, preferably at least three positions. Preferably, the aforementioned method comprises (a) the one or more modifications that result in one or more of the following characteristics: 0RF2 encoding a protein having the amino acid (s) at one or more of the amino acid sequence positions corresponding to positions 47, 88 and / or 95 of SEQ ID NO: 22 substituted or deleted; an ORF3 coding for a protein having the amino acid at the amino acid sequence position, corresponding to position 93 of SEQ ID NO: 23 substituted or deleted; and / or an ORF5 which codes for a protein having the amino acid at the amino acid sequence position corresponding to position 71 of SEQ ID NO: 24, substituted or deleted. Preferably, all the above-mentioned positions are mutated.

Preferably, the method according to the invention comprises (a) the modification or modifications that result in one or more, preferably all of the following characteristics: an 0RF2 encoding a protein that does not have phenylalanine at the position of the amino acid sequence corresponding to position 47 of SEQ ID NO: 22, an 0RF2 coding for a protein that does not have valine at the position of the amino acid sequence corresponding to position 88 of SEQ ID NO: 22, a 0RP2 that does not have phenylalanine at the position of the amino acid sequence corresponding to position 95 of SEQ ID NO: 22, a 0RF3 having no serine at the position of the amino acid sequence corresponding to position 93 of SEQ ID NO: 23, and / or a 0RF5 that does not have leucine at the position of the amino acid sequence corresponding to position 71 of SEQ ID NO: 24. Preferably, the method according to the invention comprises (a) the one or more modifications that result in one or more, preferably all of the following characteristics: an 0RF2 encoding a protein having serine at the position of the amino acid sequence corresponding to position 47 of SEQ ID NO: 22, a 0RF2 encoding a protein having phenylalanine at the position of the amino acid sequence corresponding to position 88 of SEQ ID NO: 22, a 0RF2 having leucine at the position of the amino acid sequence corresponding to the position 95 of SEQ ID NO: 22, a 0RF3 having proline at the position of the amino acid sequence corresponding to position 93 of SEQ ID NO: 23, and / or a 0RF5 having phenylalanine at the position of the sequence of amino acids corresponding to position 71 of SEQ ID NO: 24. Preferably a method according to the invention comprises (a) the modification or modifications that result in one or more, preferably t all the following characteristics: a 0RF2 having a C in the position corresponding to the position 140 of the SEQ ID NO: 22, an ORF2 having a T in the position corresponding to the position 262 of the SEQ ID NO: 22, a ORF2 having a C in the position corresponding to position 283 of SEQ ID NO: 22, an ORF3 having a C in the position corresponding to position 277 of SEQ ID NO: 23, and / or a 0RF5 having a T in the position corresponding to position 211 of SEQ ID NO: 24.

In addition, the present invention relates to an attenuated European PRRS virus obtainable by one of the aforementioned methods. In a further aspect, the present invention comprises a nucleic acid containing the coding information of a European PRRS virus, attenuated as defined above. As described above, this may be a ribonucleic acid (RNA). Said nucleic acid may also be a cleic deoxyribon acid which is complementary to such ribonucleic acid, for example a cDNA, or any other type of DNA. In a preferred embodiment, such cDNA is infectious. This means that if such a cDNA is introduced into a suitable host cell, said cell will start to generate viral particles. The present invention further relates to an RNA, cDNA or other DNA comprising any, or a plurality of the mutations described above. In a further aspect, the invention comprises a vaccine comprising a European PRRS virus, attenuated as described in combination with a pharmaceutically acceptable carrier. In a further aspect, the present invention comprises a method of vaccinating a pig against PRRS, characterized in that an efficient amount of the aforementioned vaccine is administered to said pig. In a further aspect, the present invention relates to the use of a European, attenuated PRRS virus, as described for the manufacture of a PRRS vaccine. According to the present invention, the numbering of the nucleotides or amino acids is according to the Lelystad Agent publicly available as described above. However, as shown in Figures 1 to 4, the ORFs ("open reading structures = ORF" for their acronym in English) are numbered separately since some of them are overlapped. The nucleotides of the specific sites in which the European PRRS virus, attenuated from the virulent wild type, are different are ORF2 nucleotides 11915-11935 Table 1 corresponding to numbers 130 to 150 in Figure 1), 12037-12057 (Table 2 numbers 252-272 in Figure 1), 12058-12078 (Table 3 numbers 273-293 in Figure 1); from 0RF3 12660-12680 (Table 4, numbers 267-287 in Figure 3), and / or from ORF5 13684-13704 (Table 5, numbers 201-221 in Figure 4). By mutating the Lelystad Agent in one or more of the above-mentioned positions for any of the nucleotides indicated in said tables and / or the deletion of the nucleotides in said positions, the attendant in the art will obtain the attenuated European vaccine strains, of the PRRS virus. Mutations such as replacement or deletion at such sites are only limited by the condition that the virus must be able to replicate, for example it must still be a living virus. This can be determined without undue experimentation. The skilled person can, based on the publicly available Lelystad Agent and the teachings according to the invention, introduce for example a simple mutation within each described site based on the tables provided, for example at position 11915 he can enter a A, C or G o can suppress the nucleotide in that position (which is a T). An unrestrained example is shown in Figures 1 to 3 (0RF2, 0RF3 and 0RF5 of Lelystad B and Attenuated Virus A) and in Example 1. The invention comprises such viruses wherein only one nucleotide is mutated, but also several nucleotides, or even all nucleotides, for example one or more triplets that code for one or more amino acids at said positions. However, the sequence will not be mutated to the extent that the virus is not able to replicate further, for example according to the invention, a live virus is encoded. Additional nucleic acids outside of said regions can also be mutated, however, this is not essential for the invention (see Figure 1, for example position 426 of 0RF2 of Lelystad B which is a C instead of a T). Such mutations can be carried out by standard genetic engineering methods known in the art, in particular by site-directed mutagenesis. In the context of the invention "site-directed mutagenesis" means a genetic engineering technique that allows direct mutation of the preselected sites of a nucleic acid. In general, such a technique requires at least partial knowledge of the sequence of such a nucleic acid. The classical method of attenuation by the serial phase dose does not allow the mutation of preselected sites. Such genetic engineering methods are well known in the art (see for example Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Chapter 15; (ed.) (1993), Methods in Enzymology Vol. 217, pp. 173-285).

As an initial material for the introduction of mutations, a cDNA clone of a virulent PRSSV strain, for example the Lelystad Agent (CNC I-1102), can be generated according to procedures known in the art (Boyer et Haenni. transcripts and cDNA clones of RNA viruses Virology (1994) 198: 415-426) For example, an infectious copy of PRRSV can be prepared as described (WOOO / 53787; Meulenberg, et al., Adv. Exp. Biol. 1998) 440: 199-206; Meulenberg et al., J. Virol. (1998) 72 (1): 380-387) which can then be mutated according to the invention. The references cited provide detailed procedures of how to obtain infectious PRRSV cDNA clones, and how to create the mutants thereof. The mutated clone or the infectious transcripts thereof generated in vi tro can be transfected into appropriate host cells which will then generate attenuated viruses. In particular, it was surprisingly found that sites 11925, 12047 and 12068 of ORF2 (see Figure 1), · 12670 of 0RF3 (see Figure 2) and / or 13694 of ORF5 (see Figure 4) are consistently changed compared to Lelystad Agent of the field strain, virulent. Therefore, another preferred embodiment of the present invention is an attenuated European PRRS virus according to the invention characterized in that: a) ORF2 comprises a C, A or G at position 11925 and / or a C, T or A in the position 12047 and / or an A, C or G at position 12068 or a suppression at said position or said positions and / or b) ORF3 comprises an A, C or G at position 12670 or a deletion at said position and / or c) 0RF5 comprises a G, A or T at position 13694 or a deletion at that position. Even more particularly, it was found that site 11925 of 0RF2 is consistently changed to a C, site 12047 of 0RF2 is consistently changed to T, and site 12068 of ORF2 is consistently changed to C (see Figure 1); site 12670 of ORF3 is consistently changed to C (see Figure 2) and / or 13694 of 0RF5 is consistently changed to T (see Figure 4) compared to Agent Lelystad of the virulent field strain. Thus, another more preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention, wherein the nucleic acid is further characterized in that: a) the ORF comprises a C at position 11925 and / or a T at position 12047 and / or a C at position 12068 or a suppression at said position (s) and / or b) ORF3 comprises a C at position 12670 or a suppression at said position and / or c) 0RF5 comprises a T at position 13694 or a deletion at that position. Another more preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention, designated attenuated virus A in the figures, wherein the nucleic acid is further characterized in that: a) ORF2 comprises the nucleic acid as defined in SEQ ID NO: 1 and / or) 0RF3 comprises the nucleic acid as defined in SEQ ID NO: 2 and / or c) ORF4 comprises the nucleic acid as defined in SEQ ID NO: 3 and / or ) ORF5 comprises the nucleic acid as defined. in SEQ ID NO: 4, or a fragment, allelic variant, functional variant, variant based on the code of the degenerative nucleic acid, fusion molecule or a chemical derivative thereof. A "fragment" according to the invention is any immunogenic subunit of a PRRS virus or an ORF according to the invention, for example any polypeptide subgroup, characterized by being encoded by a shorter nucleic acid molecule than the one described above. , nevertheless it still conserves its activity. A "functional variant" of the PRRS or ORF virus according to the invention is a PRRS virus or an ORF possessing a biological activity (either functional or structural) that is substantially similar to the PRRS or ORF virus according to the invention. The term "functional variant" also includes "a fragment", "an allelic variant", "a functional variant", "variant based on the degenerative nucleic acid code" or "chemical derivatives". Such a "functional variant" can, for example, carry one or several point mutations, one or more exchanges, deletions or insertions of nucleic acids, or one or more exchanges, deletions or insertions of amino acids. Said functional variant still retains its biological activity, for example the function as a vaccine strain, at least in part or even together with an improvement in biological activity. A "variant based on the degenerative genetic code" is a variant due to the fact that a certain amino acid can be encoded by several different nucleotide triplets. Said variant still retains its biological activity, at least in part or even together with an improvement in biological activity. A "fusion molecule" can be the PRRS virus or ORF according to the invention, fused for example to a reporter such as a radiolabel, a chemical molecule such as a fluorescent label or any other molecule known in the art. As used herein, a "chemical derivative" according to the invention is a PRRS or ORF virus according to the invention chemically modified or containing additional chemical moieties that are not normally part of the molecule. Such portions may improve the solubility, absorption, biological half-life of the molecule, etc. A molecule is "substantially similar" to another molecule if both molecules have substantially similar biological structures or activity. Thus, with the proviso that the two molecules have a similar activity, these are considered variants as that term is used in the present, even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical.

Another more preferred embodiment of the present invention, is a European PRRS virus, attenuated according to the invention (designated Virus A attenuated in the figures), wherein the nucleic acid is characterized in that: a) 0RF2 consists of the nucleic acid as defined in SEQ ID NO: 1 and / or) 0RF3 consists of the nucleic acid as defined in SEQ ID NO: 2 and / or c) ORF4 consists of the nucleic acid as defined in SEQ ID NO: 3 and / or d) the ORF5 consists of the nucleic acid as defined in SEQ ID NO: 4. Another preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention (designated virus A attenuated in the figures), wherein the nucleic acid is characterized in that it comprises the nucleic acid as defined in SEQ ID NO: 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Another more preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention (designated attenuated virus A in the figures), wherein said nucleic acid is characterized by nucleic acid as defined in SEQ ID NO. : 5. Another preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention (LELYSTAD-B in the figures), wherein said nucleic acid is further characterized in that: a) ORP2 comprises the nucleic acid as is defined in SEQ ID NO: 6 and / or b) 0RF3 comprises the nucleic acid as defined in SEQ ID NO: 7 and / or c) 0RF4 comprises the nucleic acid as defined in SEQ ID NO: 8 and / od) 0RF5 comprises the nucleic acid as defined in SEQ ID NO: 9 and / or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative of the same. Another more preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention (LELYSTAD-B in the figures), wherein the nucleic acid is further characterized by: a) 0RF2 consists of the nucleic acid as defined in SEQ ID NO: 6 and / or) 0RF3 consists of the nucleic acid as defined in SEQ ID NO: and / or c) 0RF4 consists of the nucleic acid as defined in SEQ ID NO: 8 and / or) 0RF5 consists of the nucleic acid as defined in SEQ ID NO: 9. Another important embodiment of the present invention is a European, attenuated PRRS virus (attenuated virus in the figures), wherein the PRRS virus is characterized by: a) ORF1 comprises the amino acid as defined in SEQ ID NO: 10 and / or) ORFlb comprises the amino acid as defined in SEQ ID NO: 11 and / or c) ORF2 comprises the amino acid as defined in SEQ ID NO: 12 and / or d) ORF3 comprises the amino acid as defined in SEQ ID NO: 13 and / or e) O RF4 comprises the amino acid as defined in SEQ ID NO: 14 and / or) ORF5 comprises the amino acid as defined in SEQ ID NO: 15 and / or g) ORF6 comprises the amino acid as defined in SEQ ID NO. : 16 y / o) ORF7 comprises the amino acid as defined in SEQ ID NO: 17. or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative of the same. Another more preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention (attenuated virus A in the figures), wherein the PRRS virus is characterized in that: a) ORFla consists of the amino acid as defined in SEQ. ID NO: 10 and / or) ORFlb consists of the amino acid as defined in SEQ ID NO: 11 and / or c) 0RF2 consists of the amino acid as defined in SEQ ID NO: 12 and / or d) 0RF3 consists of the amino acid as defined in SEQ ID NO: 13 and / or e) ORF4 consists of the amino acid as defined in SEQ ID NO: 14 and / or) ORF5 consists of the amino acid as defined in SEQ ID NO: 15 and / or g) ORF6 consists of of the amino acid as defined in SEQ ID NO: 16 and / oh) 0 F7 consists of the amino acid as defined in SEQ ID NO: 17. Another very important embodiment of the present invention is a European PRRS virus., attenuated (LELYSTAD-B in the figures), wherein the PRRS virus is characterized in that: a) 0RF2 comprises the amino acid as defined in SEQ ID NO: 18 and / or b) 0RF3 comprises the amino acid as defined in SEQ. ID NO: 19 and / or c) 0RF4 comprises the amino acid as defined in SEQ ID NO: 20 and / or d) ORF5 comprises the amino acid as defined in SEQ ID NO: 21. or a fragment, allelic variant, functional variant , variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Another more preferred embodiment of the present invention is a European PRRS virus, attenuated according to the invention (LELYSTAD-B in the figures), wherein the PRRS virus is characterized in that: a) ORF2 consists of the amino acid as defined in SEQ. ID NO: 18 and / or b) 0RF3 consists of the amino acid as defined in SEQ ID NO: 19 and / or c) 0RF4 consists of the amino acid as defined in SEQ ID NO: 20 and / or) ORF5 consists of the amino acid as defined in SEQ ID NO: 21. Another important embodiment of the present invention is a nucleotide sequence coding for a virus according to the invention as described above. Such nucleotide sequences include the sequences described in the sequence listing (for example with identification numbers 1 through 9 (given under <; 400 > = SEQ ID NO :) The invention further relates to the proteins described in SEQ ID NOs: 10 to 21. Another preferred embodiment of the present invention is a nucleotide sequence according to the invention, wherein the nucleotide sequence has been modified to encode a marker of virulence and / or a serological marker. As mentioned in the introductory pages, it is important for the health management of pigs to be able to distinguish between the less virulent live vaccine strain of the pharmaceutical composition, and the virulent, wild-type virus infections. This is often difficult, especially when the clinical symptoms of an infection in the field are not that specific or superimposed by other infections, or the period of time for observation and evaluation is short. The recombinant generation of the viruses of interest allows the introduction of modifications in the genetic code, which establishes a serological marker and / or a virulence marker. A serological marker refers to an antigenically detectable molecule such as a peptide, a protein, glycoprotein that can be isolated from infected cells of bodily fluids, such as, but not limited to pharyngeal or nasal fluids or urine. A virulence marker should be understood as a marker in the genetic code that can be identified by recombinant analytical methods such as, but not limited to, PCR and conventional sequencing. Therefore, in a preferred embodiment, the present invention relates to a nucleotide sequence according to the invention, wherein the nucleotide sequence has been modified to encode a virulence marker and / or a serological marker. Particularly, mutations or deletions introduced for the purpose of attenuating viruses are useful as markers of virulence and serological markers. By monitoring these mutations at the specific virulence sites described, it is possible to predict the emergence of potentially virulent cassettes at an early stage. Another preferred embodiment of the present invention is a nucleotide sequence according to the invention, wherein the nucleic acid encoding said marker is located within any of the open reading structures that code for the viral structural proteins. In still another aspect, the invention relates to a method for the generation of a European, attenuated, live, infectious PRRS virus, said method comprises the production of a recombinant nucleic acid comprising at least one full-length DNA copy or a copy of RNA transcribed in vi tro, or a derivative of said DNA or RNA, whereby the nucleotide sequence is a nucleotide sequence according to the invention. Thus, according to the invention, the method leads to the PRRS virus as described above. The person skilled in the art can employ site-directed mutagenesis (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Chapter 15; u (ed. ) (1993), Methods in Enzymology Vol. 217, p 173-285) or any other method for inserting one or more mutations, to specifically insert one or more mutations, to obtain a PRRS virus as described above. The method described in the following preferred aspect will lead (at the genomic level) to the PRRS virus according to the invention as described above. Thus, in another preferred aspect, the invention relates to a method as described for the generation of a live, infectious, attenuated PRRS virus, said method is characterized by the following steps: a) a PRRS virus according to the invention is used to infect a suitable cell line b) the PRRS virus is further attenuated via cell passages of culture. In another preferred aspect, the invention relates to a method as described, wherein the cell line is an embryonic kidney cell of mono or preferably an Marc cell or a derivative thereof (see below). In another preferred aspect, the invention relates to a method as described, wherein the PRRS virus is or are the viruses according to the invention as described above.

In a further aspect, the present invention relates to a cell line comprising a PRRS virus according to the invention. Examples for such cells include permanent cell lines known to the person skilled in the art, preferably porcine, monkey or human cell lines, such as the human embryonic kidney (HEK) 293, BH, GH3, H4, U373, NT2, PC12 , Yeast, COS, CHO, Ltk ", fibroblasts, myelomas, neuroblastomas, hybridomas, oocytes, embryonic stem cells), insect cell lines (for example, using baculoviral vectors such as pPbac or pMbac (Stratagene, La Jolla, USA)), yeast (eg, Pichia pastoris or using yeast expression vectors such as pYESHIS (Invitrogen, San Diego, USA)), and fungi In a further preferred aspect, the present invention relates to a cell line according to the invention, wherein the cell line is an embryonic monkey kidney cell, or preferably an Marc cell or a derivative thereof In a further aspect, the present invention comprises a method or process of Tension of a European PRRS virus, characterized in that: a) the nucleotide sequence of said virus is modified by site-directed mutagenesis in at least one of the 0RF2 positions corresponding to positions 130 to 150 and / or positions 252 to 272 and / or positions 273 to 293 of SEQ ID NO: 22; b) it is tested if the PRRS virus is attenuated. In this context, a position within a nucleic acid or amino acid sequence "corresponding to" a position of another sequence will mean that, if the two sequences have sufficient structural similarity to be aligned with a standard alignment algorithm such as BLAST (Altschul, SF, Gish, W., Miller, W., Myers, EW &Lipman, DJ (1990) "Basic local alignment search tool", J. Mol. Biol. 215: 403-410; Gish,. & DJ (1993) "Identification of protein coding regions by datbase similarity search." Nature Genet 3: 266-272; Madden, TL, Tatusov, RL &Zhang, J. (1996) "Applications of network BLAST server" Meth. Enzymol 266: 131-141; Zhang, J. &Madden, TL (1997) "Po erBLAST: A New network BLAST application for automated or automated sequence analysis and annotation" Genome Res. 7: 649-656; Altschul, Stephen F., Thomas L. Madden, Alejandro A. Scháffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), " Gapped BLAST and PSI-BLAST: a new generation of protein datbase search programs ", Nucleic Acids Res. 25: 3389-3402), and such alignment is performed, the two positions will be aligned as a pair. In a further embodiment, the present invention relates to a method for attenuating a European PRRS virus, characterized in that: a) the nucleotide sequence of said virus is modified by site-directed mutagenesis in at least one of the corresponding ORF3 positions to positions 267 to 287 of SEQ ID NO: 23 b) it is tested whether the resulting PRRS virus is attenuated. In a further embodiment, the present invention relates to a method for attenuating a European PRRS virus, characterized by: a) the nucleotide sequence of the virus is modified by site-directed mutagenesis in at least one of the corresponding positions 201 a 221 of ORF5 according to SEQ ID NO: 24; b) it is tested whether the resulting PRRS virus is attenuated or not. Preferably, a modification as described above results in a change in the amino acid sequence of the encoded protein.

Preferably, the modification is a deletion or a substitution. In a preferred embodiment, the sequence of each ORF2, ORF3 and 0RF5 is modified. In another preferred embodiment, the sequence of 0RF2 is modified by at least two, preferably at least three positions. Preferably, the modification results in one or more of the following characteristics: an 0RF2 encoding a protein having the amino acid substituted or deleted at one or more positions in the amino acid sequence, corresponding to positions 47, 88 and / or 95 of the amino acid sequence encoded by SEQ ID NO: 22; an 0RF3 coding for a protein having substituted or deleted the amino acid corresponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23; and / or an 0RF5 coding for a protein having the amino acid corresponding to position 71 of the amino acid sequence encoded by SEQ ID NO: 24, substituted or deleted. More preferably, the modification made by such method results in a more preferably all of the following characteristics: 0RF2 encoding a protein having serine at the corresponding position 47 of the amino acid sequence encoded by SEQ ID NO position: 22, an ORF2 encoding a protein having phenylalanine in the position corresponding to position 88 of the amino acid sequence encoded by SEQ ID NO: 22, a 0RF2 having leucine in the position corresponding to position 95 of the amino acid sequence encoded by SEQ ID NO: 22, a 0RF3 having proline in the position corresponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23, and / or a 0RF5 having ne phenylalanine in the position corresponding to position 71 of the amino acid sequence encoded by SEQ ID NO: 24. In preferred embodiments, the modification results in one or more, preferably all of the following characteristics: an 0RF2 having a C in the position corresponding to the position 140 of the SEQ ID NO: 22, a 0RF2 having a T in 'the position corresponding to the position 262 of the SEQ ID NO: 22, an ORF2 having a C in the position corresponding to the position 283 of SEQ ID NO: 22, an ORF2 having a C in the position corresponding to position 277 of SEQ ID NO: 23, and / or a 0RF5 having a T in the position corresponding to position 211 of SEQ ID NO: 24 In a further embodiment, the present invention relates to a European, attenuated PRRS virus obtainable by a method described above. In a further embodiment, the present invention relates to an attenuated European PRRS virus having an 0RF2 that differs from SEQ ID NO: 22 in one or more of positions 130 to 150, and / or in one or more of the positions 252 to 272, and / or in one or more of positions 273 to 293. In a further embodiment, the present invention relates to an attenuated European PRRS virus having an 0RF3 that differs from SEQ ID NO: 23 in one or more of positions 267 to 287. In a further embodiment, the present invention relates to an attenuated European PRRS virus having an ORF5 that differs from SEQ ID NO: 24 in one or more of positions 201 to 221 In a further embodiment, the present invention relates to a vaccine comprising a European PRRS virus, attenuated, as described above, in combination with a pharmaceutically acceptable carrier. In a further embodiment, the present invention relates to a method of vaccination of a pig against PRRS, characterized in that an efficient amount of such vaccine is administered to said pig. Alternatively, the present invention relates to the use of a European PRRS virus, attenuated as described, for the manufacture of a PRRS vaccine. Preferably, the PRRS virus, attenuated, alive, can be used for the treatment, prophylaxis or diagnosis of diseases caused by the wild-type PRRS virus. Such diseases and use are exemplified in Example 1. A further aspect of the invention relates to the use of the viruses of the invention. Their defined molecular basis of attenuation makes them superior to viruses known in the art. Especially, the use of viruses according to the invention comprising deletions at specific sites of virulence is preferred, since deletions are less likely to be reversed. Another preferred embodiment of the present invention is a pharmaceutical composition comprising one or more PRRS viruses according to the invention, and a pharmaceutically acceptable carrier. A preferred aspect is a pharmaceutical composition comprising not only the European PRRS virus according to the invention, but also a PRRS virus from the United States, attenuated such as the virus which is sold in a pharmaceutical composition under the trade name RespPRRS / lngelvac ® PRRS MLV, Boehringer Ingelheim. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds which act, for example, to stabilize or increase absorption or form part of a slow release of the PRRS virus according to the invention. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (see also, for example, Remington's Pharmaceutical Sciences (1990), 18th ed. Mack Publ., Easton). A person skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition. More preferably, the composition is formulated by obtaining the supernatant containing the virus (tissue culture fluid) from a culture of infected cells, and lyophilizing said supernatant, optionally after the addition of a stabilizer. The lyophilized composition can be reconstituted with water before administration. In yet another example, a suitable carrier could be a physiological saline solution. Preferably, the pharmaceutical composition is injected intramuscularly or intradermally. The vaccine according to the invention can be administered to pigs depending on the vaccination history of the female pigs at 1, 3, 6 or 10 weeks of age, to female pigs before intercourse and / or until 6 weeks before parturition (booster vaccination), or to male pigs every half year (booster). Another preferred embodiment of the present invention is the use of a PRRS virus according to the invention, in the manufacture of a vaccine for the prophylaxis and treatment of PRRS infections. The following example serves to further illustrate the present invention; but it should not be considered as limiting the invention described herein.

Example 1 Establishment of attenuation This example provides a clear guide for the comparison of the virulent character of two different strains of PRRS virus. As a reference strain for a typical virulent European strain, a cell culture pass (or more than 5) of the Lelystad Agent (CDI-NL-2.91) can serve. At least 10 young sows per group are included in each trial, which are derived from a PR S free farm. The animals are tested free of antibodies in serum specific for PRRS virus and negative for PRRSV. All the animals included in the test are from the same source and created and come from a farm historically free of any PRRSV infection. The assignment of the animals to the group is random. The challenge is carried out at days 85-90 of pregnancy with intranasal application of 1 ml of PRRSV with 105 TDCID50 (third pass) per nostril. There are at least three groups for each test structure: One group for the challenge with Agent Lelystad (CDI-NL-2.91); a test group for the challenge with the possibly attenuated virus; and a strict control group. The validity of the study is given when strict controls remain negative PRRS in the course of study time, and at least 25% fewer live healthy piglets are born compared to the strict controls in the group challenged with Agent Lelystad.

Attenuation, in other words less virulence, is defined as the significant statistical change of one or more parameters that determine reproductive functioning: The significant reduction in at least one of the following parameters for the test group is preferred -. frequency of stillbirths abortion on or before day 112 of pregnancy number of mummified piglets number of live and weak piglets pre-weaning mortality or in addition a significant increase in one of the following parameters for the test group is preferred: weaned by sow • number of live healthy piglets born per sow • compared to the group infected with Agent Lelystad In an exemplary manner the following results can be obtained in a clinical trial according to the description given with the cell culture pass 147 of the Agent Lelystad: Live / healthy level Live / weak Born dead Mummified Average pass live baits per sow 5 30.5% 45.5% 17.9% 6.2% 11 147 71.9% 10.5% 17.54% 0 11.4 The reproductive performance data of the individual sows are given, from the sows that are inoculated as described above with the Lelystad agent of passage 5 (group 1) and the cell culture pass 147 of the Lelystad agent (group 2). In addition groups of 3 animals served as strict controls and were only inoculated with the free cell culture supernatant PRRSV: Group Level Duration Total number of Live / Live / Born Mummified pass of weak healthy piglets dead of gestation born inoculum on days 1 5 115 15 5 7 2 1 1 5 111 7 5 1 1 0 1 5 113 6 2 2 1 1 1 5 110 14 2 10 2 0 1 5 115 13 0 8 4 1 Prome 11 2.8 5.6 2.0 0.6 gave group 1"or 100 30.5 45.5 17.9 6.2 2 147 115 13 11 1 1 0 2 147 113 12 12 0 0 0 2 147 115 10 0 4 6 0 2 147 115 8 8 0 0 0 2 147 115 14 10 1 3 0 Prome 11.4 8.2 1.2 2 0 gave group 2% 100 71.93 10.53 17.54 0 3 117 12 11 1 0 0 3 '115 9 8 1 0 0 3 116 14 9 5 0 0 3 115 6 6- 0 0 0 3 115 14 11 3 0 0 3 116 15 14 1 0 0 Prome 11.7 9.8 1.8 0 0 gave group 3% 100 86.1 13.9 0 0 LIST OF SEQUENCES < 110 > Boehringer Ingelheim Vetmedica GmbH < I20 > Live strains of the PRRS virus attenuated <30 > X-117S < 260 > 29 < 170 > Patentiza Ver. 2.1 < 210 > 1 · < 211 > 750 | < 212 > ADH: < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 1 atgcaatggg gttactgtgg agtaaaatca gccagctgtt cgtggacgcc ttcaefcgagt 60 ·. "Tccttgttag tgtggttgat attgttattt tccttgccat actgtttggg ttcaccgtcg 120" caggatggtt actggtcttc cttctcagag tggtttgctc cgogcttctc cgttcgcgct 180- ctctcccgaa ctgccafctca ctatcgaagg tcctatgaag gcttgttgcc caactgc'aga 24; 0 ccggatgtcc cacaatttgc attcaagcac ccattgggta tgctttggca catgcgagtt 300. tcccaattka tfcgatgagat ggtctctcgt cgcatttacc agaccatgga acattcaggt 360 caagcggcct ggaagcaggo ggttggfcgag gccactctca cgaagctgtc aaggctcgat 420 attfcccaaca atagtfcactc cctggccgca gtagaggcgg a'ttcttgccg ctfctctcage 480 tcacgactcg tgatgctaaa aaatcttgcc gttggcaatg tgagcctaca gtacaácacc 540 acgttggacc gcgttgagct catcttcccc acgccaggta cgaggcccaa gttgaccgac 600 ttcagacaat ggctcatcag tgtgcacgct tccatttttt cctctgtggc ttcatctgtt 660 accttgttca tagtgctttg gcfctcgaatt ccagctctae gctatgtttt tggtttccat -720. tggcccacgg caacacatca ttcgagctga. · 750- < 210 > 2 < 2U > 798 < 212 > DNA < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 2 atggctcatc agtgtgcacg cttccatttt ttcctctgtg gcttcatctg ttacctfcgtt 60 catagtgctt fcggcttcgaa ttccagctct acgctatgtt tttggttfccc attggcccac 120 ggcaacacat cattcgagct gaccatcaac tacaccatat gcatgccctg ttctaccag ISO caagcggctc gccaaaggct cgagcccggt cgtaacatgt ggtgcaaaat agggtatgac 240 aggtgtgagg agcgfcgacca tgatgagttg tfeaafcgccca tcccgtecgg gtacgacaac 300 ctcaaacttg agggttatta tgcttggctg gcttttttgt ccttttccta cgcggcccaa 360 ttccatccgg agttgttcgg gat & gggaat gtgtcgcgcg tcttcgtgga caagcgacac 420 gtgccgagca cagttcattt tgatggacag aattcaaccg fcatctaccgg acacaacatc 480 tecgcattat atgcggcata ttaecaceac caaatagacg ggggcaattg gttcaatttg 540 gaatggctgc ggecactctt ttcctcctgg ctggtgctca atatatcatg gtttetgagg 600 cgttcgcctg taagccctgt ttctcgacgc atctatcaga tattgagacc aacacgaccg 6S0 cggctgccgg tttcatggtc cttcaggaca tcaattgtct ccgacctcac ggggtctcag 720 cagcgcaaga ggaaattccc ttcggaaagt cgtcccaatg tcgtgaagcc gtcggtactc 780 798 cacgataa cccagtacat < 210 > 3 < 211 > 552 < 212 > DNA < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 3 atggctgcgg ccac ctttt cctcctzggct ggtgctcaat atatcatggt ttctgaggcg 60 ttcgcctgta agcccfcgttt ctcgacgcat ctatcagata ttgagaccaa cacgaccgcg 120 gcfcgccggtt; tcatggfccct tcaggacatc aattgtctcc gacctcacgg ggtctcagca 180 gcgcaagagg aaattccctt cggaaagtcg tcccaatgtc gtgaagccgt cggtactccc 240 cagtacafcca cgataaeggc taacgfcgacc gacgaatcat acttgtacaa cgcggacttg 300 ctgatgcttt ctgcgtgcct tttceacgce tcagaaatga gcgagaaagg cttcaaagtt 360 atgtctctgg atcttfcggga gettgtgtca cgttgtttcc attfccacaga fctafcgtggcc 420 catgtgaccc aacataceea gcagcatcafc ttggtaafctg atcacattccr gttgctgcat 480 ttcctgacac catctgcaat gaggtgggct acaaccattg cttgtttgtt cgecattctc 540 ttggcgatat ga 552 < 210 > i. < 211¾ 606 < 212 > DNA -. < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 4 .atgagatgtt ctcacaaatt ggggcgtfctc ttgactccgc actcttgctt cfcggtggttt 60 ·· ttttfcgctgt gtaccggctt gtccfcggfcqc fcttgccgatg gcaacggcaa cagetcgaca 120 taccaataca..tatataacct gacgatatgc gagcfcgaatg ggaccgactg gttgtccagc 180 cattttggfct gggcagtcga gacctttgtg fctttacccgg tatcctctca ttgccacfcca -240. ctgggttt-tc tcacaacaag ccatttfcfcfct gacgcgetcg, gtctcggcgc- tgtatccact.300 .gcaggatttg ttggcgggcg -gtatgtactc tgcagtígtct. cggcgcttg tgctttcgca 360 gcgttcgtat gtt'ttgtcat ccgtgctgct aaaaat-tgca- tggcetgccg ctatgcccgt 4.20 acccggttta ccaactfccat tgtagacaac cgggggagag ttcatcgatg gaagtcfccca 480 atagtggfcag aaaaattggg caaagccgaa gtcgacggca acctcgtcac catcaaacat 540 aaggggttaa gtcgfccctcg agctcaaccc ttgacgagga cttcggctga gcaatgggag gcctag 600 605 < 210 > 5 < 211 > 14815 < 212 > ¾DN: 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 5 gganacatac acgacacttc tagtgtfctgt gtaecttgga ggcgtgggta cagccccgcc 60 • ccaccccttg gcccctgttc tagcccaaca ggtatccttc tctctcgggg cgagfcgcgcc 120 gcctgctgct cccttgcagc gggaaggacc tcccgagtat ttccggagag cacctgcttt 180 acgggatcfcc caccctttaa ccatgtctgg gacgttctcc cggtgcafcgt gcaccccggc 240 tgcccgggta ttttggaacg ccggccaagt cttttgcaca egg gtctca gtgcgcggtc 300 tcttctctct ccagagcttc aggacactga cctcggtgea gttggctfcgt t tacaagcc 350 tagggacaag cttcaetgga aagtccctafc cggcatccct caggtggaat gtactccatc 420 cgggtgctgt tggctctcag ctgttfctccc tttggcgcgt atgacctccg gcaafccacaa 480- cttcctccaa cgacttgtga aggttgctga tgttfctgtac cgtgacggtt gcttggcacc 540 tcgacacctfc cgtgaactcc aagtfctacga gcgcggctgc aacfcggtacc cgatcacggg 600 gcccgtgccc gggatgggtt tgtttgcgaa ctccatgcac gtatccgacc agccgttccc 660 tggtgccacc catgtgttga ctaacfccgcc tttgcctcaa caggcttgtc ggcagccgtt 720 ctgfcccattfc gaggaggctc attctagcgt gfcacaggtgg aagaaatttg tggttttcac 780 ggactcctcc ctcaacggtc gatctcgcat gatgtggacg ccggaafcccg atgattcage 840 cgccctggag gtactaccgc ctgagttaga acgtcaggtt gaaatcctca ttcggagttfc 900 tccfcgctcat caccctgtcg accfcggccga ctgggagctc actgagtccc ctgagaacgg 960 tttttccttc aacacgtctc attcttgcgg tcaccttgtc caaaaccccg acgtgtttga 1020 tggcaagtgc tggctctcct gctttttggg ccagtcggcc gaagtgcgcfc gccatgagga 1080 acatcfcagct gacgccttcg gttaccaaac caagtggggc gfcgcatggfca agtacctcea 1140 gcgcaggctt caagtfccacg gcatfccgtgc tgtagtcgat cctgacggtc ccattcacgt 1200 tgaagcgctg tcttgccccc agtcttggat caggcacctg actctgaatg atgatgfccac 12S0 cccaggafcfcc gttcgcctga catcccttcg cattgtgccg aacacagagc cfcaccacttc 1320 ccggatctfct cggtttggag cgcataagtg gtatggcgct gccggcaaac gggctcgtgc 1380 taagcgtgcc gctaaaagfcg agaaggattc ggctcccacc cccaaggttg ccctgcctgt 14"40 ggaafcfcacca ccccacctgt cctactctcc accgacagac gggtcttgtg gttggcatgt 1500 0 ccttgecgcc ataatgaacc ggatgataaa tggtgacttc acgtcccctc tgactcagta 1560 caacagacca gaggatgatt gggcfctctga ttatgatctt gttcaggcga- ttcaatgtct 1620 acaactgcct gctacegtgg ttcggaatcg cgcctgtcct aacgccaagt accttataaa 1680 acttaacgga gttcactggg aggtagaggt gaggtctgga atggctcctc gctcccttcc 1740 tcgtgaatgt gtggttggcg tttgctctga aggctgtgt c geaccgcctt atecagcaga 1800 5 cgggctacct aaacgfcgcac tcgaggcctt ggcgtctgct tacagactac cctccgattg 18SO tgttagctct ggtafctgctg actttcttgc taatccacct cctcaggaat tctggaccct 1920 cgacaaaafcg ttgacntccc cgtcaccaga gcggtccggc ttctctagtfc tgtafcaaatt 1980 actattagag gttgttecgc aaaaatgcgg tgccacggaa ggggctttca tctatgctgt 2040 -tgagaggatg ttgaaggatt gtccgagctc caaacaggcc atggcccttc tggcaaaaat 2100 0 taaagttcca tcctcaaagg ccccgtctgfc gtccctggac gggtgtttcc ctacggatgt 2160 tccagccgac ttcgagecag catctccgga aaggccagct ggtctaatta acctggtagg 2220 cgggaatttg tccccctcag actccatgaa agaaaacatg ctcaatagcc gggaagacga 2280 accadtggat ttgtcccaac cagcaccagc tgccacaacg aeecfctgfcga gagagcaaac 2340 acccgacaac ceaggfcfcctg afcgccggfcgc -cctccccgtc accgfctcgag- aatfctgtccc 2400 5 gacggggcct atacfcccgtc atgttgagca ctgcggcacg gagtcgggcg. acagcagttc 24S0- gcctttggac cagtctgatg cgcaaaccct -ggaccagcct ttaaatctat ccctggccgc 2520". fetggccagfeg agggccaccg -cgtctgaccc tggctgggtc cacggfcaggc gcgagcctgt 25-80- '• ttttgtaaag- cctcgaaatg ctttctctga tggcgattca gcccttcagt tcggggágct 2640-, ttctgaatcc agccctgtca tcgagfctfcga ccggacaaaa gatgctccgg tggttgacgc.2700 0 -ccctgtcgac ttgacgactt cgaacgaggc ccfcctctgta gfccgaccctt tcqaat'fcfcgc 276.0 - cgaactcaag cgcccgcgtt tctccgcaca agccttaatt. gaccgaggcg gtccacttgc 2820 egafcgtccat gcgaaaataa agaaccgggt atatgaacag tgcctccaag cttgtgagcc 2880 'cggtagtcgt gcaaccccag ccaccaggga gtggctcgac aaaatgtggg atagggtgga 2940 catgaaaact tggcgctgca cctcgcagtt ccaagctggt cgcattcttg cgtccctcaa 3000 5 attcctccct gacatgafcte aagacacacc gcctcctgtt cccaggaaga ac.cgagctag 3060 tgacaatgcc ggcctgaagc aaccggtggc acagtgggat aggaaattga gtgtgacccc 3120 ccggttgggc ccccccaaaa ccagaccgtc cagtgcttga cggatatcca cctccgccta 3180 gcaagaagat gtcaccccct ccgatgggcc accccatgcg ccggattttc ctagtcgagt 3240 gagcaegggc gggagttgga aaggcctcat gctttccggc acecgtctcg cggggtctat 3300 Q cagtcagcgc ctcatgacat gggtttttga agttttctcc cacctcccag cttttatgct 3360 cacacttttc tcgccacggg gctctatggc tccaggtgat tggttgtttg caggtgtcgt 3420 • -tttactcgot ctcttgctct gtcgttctta cccgatactc ggatgccttc ccctattggg 3480 tgtcttttct ggttctttgc ggcgtgttcg tctgggtgtt tttggttctt ggatggcttt 3540 tgctgtattt ttattcfccga ctccatccaa cccagtcggt tcttcttgtg accacgattc 3600 gccggagtgt catgctgagc ttttggctct tga gcagcgc caactttggg aacctgtgcg 3660 cggccttgtg gtcggcccet "cgggcctctt atgtgtcatt cttggcaagt tactcggtgg 3720 gtcacgttat ctctggcatg ttttcctacg tttatgcatg cttgcggatt tggccctttc 3780 fccttgtttat gtggtgtccc aggggcgttg tcacaagtgt tggggaaagt gtataaggac 3840 agctcctgcg gaggtggctc ttaatgtatt tcctttcttg cgcgccaccc gtgcctctct 3900 Q tgtatccttg tgtgatcgat tccaaacgcc aaaaggggtt gatcctgtgc acttggcaac 3960 gggttggcgc gggtgctggc gtggtgagag tcccattcat caaccacacc aaaagcccat 4020 agcttatgcc aatttggatg aaaagaaaat atctgctcaa acggtggttg ctgtcccata 4080 caggctgtca cgatcccagt aatgcctgaa agttctgcag gcgggagggg ctatcgtgga 4140 ccagcctaca cctgaggtcg ttcgcgtgtc cgagatcccc ttctcagccc catttttccc 4200 5 aaaagttcca gtcaacccag attgcagggt tgtggtagat tcggacactt- ttgtggctgc 4260 ggtccgcfcgc ggttactcga cagcacaact ggtcctgggc cggggcaact ttgccaagtt 4320 aaatcagacc ccccccagga actctatcec caccaaaacg actggtgggg cctcttacac 4380 ccttgctgtg gchcaagtgt etgcgtggac tctfcgttcat ttcatcctcg gtetttggtt 4440 cacatcacct caagfcgcgbg gccgaggaa c cgctgacaca ggtgttcaa atccttfcttc 4500 atatectacc tatggccccg gagttgtgtg ctcccctcga ctttgtgtgt ctgccgacgg 45S0 ggtcacccfcg ccaftfcgttcfc 'cagccgtggc acaactctcc ggtagagagg tggggatttt 4620 ta tttggtg ctcgtctcct tgactgcfctt ggcccaccgc atggctctta aggcagacat 4580 gttagtgatc ttttcggctt tcfcgtgctta cgcctggccc atgagctccfc ggttaa-ctg 4740 cttctfctcct atactcttga agtgggttac ccttcaccct ctcactatgc tttgggfegea 4800 ctcattcttg gtgtfcttgtc tgccagcagc cggcatcctc tcactaggga taactggcct 4850 tctetgggca attggccgct ttacccaggt tgccggaatt attacacctt atgacatcca 4920 ccagtacacc tctgggccac gtggtgeagc tgctgtggcc acagccccag aaggcactta 4980 .tatggccgcc gtccggagag ctgctttaac tgggcgaact ttaatcttca ccccgfccfcgc 5040 .agfctggatcc cttctegaag gtgcttfccag ccctgcctta gactcataaa acaccgtgaa 5100 tgttgtaggc tcttccettg gttccggagg ggttttc 5160 cc attgatggca gaagaactgt egteactgct gcccatgtgt tgaacggega gtcaccggcg cacagctaga actcctacaa 5220 actfctcaaga ccgcatgcac ccaatggtga ttatgcctgg tcccatgctg atgactggca 5280 • gggcgtfegcc cctg tggtca · aggttgcgaa ggggtaccgc ggtcgtgccfc actggcaaac 5340 atcaactggt gtcgaacccg gtatcattgg ggaagggttc gccttctgtt ttactaáctg- 5"400 • tggcgattcg gggtcacccg tcatctcaga atctggtgat cttattggaa tccacaccgg 5460 ttcaaacaaa cttggttctg gtctfcgtgac aacccctgaa ggggagacct gcaccatcaa 5520 • agaaaccaag ctctctgacc - ttccagaca ccaagcgttc ttfctgcaggc ctcttgggga 5580 · cattaaattg agfcccggcca tcatccctga tgtaacatcc attccgagtg acttggcatc -5640 · Gctcctagcc tccgtccctg cagfcggsagg cggcctctcg accgttcaac fctttgtgfcgt 5700 ctttttcctt cfcetggcgca tgatgggcca tgcctggaca. cccafctgfctg ccgfcgggctfc 5760 ctttttgctg aatgaaattc- ttccagcagt - tttggtccga gccgtgtttt cttttgcact 5820 gcatgggcca ctttgtgctt cccectggtc tgcacaggtg ttgatgatta gactcctcac 5880 ·· .ggcatctctc aaccgcaaca agcfctfcctct ggcgttctac gcactcgggg gtgtcgtcgg -5S40 · .tttggccgct gaaafccggga cttttgctgg cagattgtct gaattgtctc aagctóttfcc.6000· Gacatacfcga ttcttaccta gggfccctfcgc tafcgaccagt tgtgttccca ccatcatcat 6060 · cggtggactc cataccctcg gtgtgattct gtggttattc aaataccggt 6-120 gcctccacaa "eatgctggfct-ggtgatggga gtttctcaag cgccttcttc ctacggtatt ttgcagaggg 6180. fcaatcfccaga aaaggtgtfct cacagfccctg tggcatgaat aacgagtccc taacggctgc 6240 tttagcttgc aagttgtcac aggctgacct tgattttttg tccagcttaa cgaacfctcaa 6300 tctgct gtgctttgta acatgaaaaa caa tgctgccggc cagtacattg aagcagcgta 6360 tgccaaggcc ctgcgccaag agttggcctc tctagttcag attgacaaaa tgaaaggagt 6420 ttfcgtccaag ctcgaggcct ttgctgaaac agccaccccg tcccttgaca taggtgacgt S48D gattgttctg cttgggcaac atcctcacgg atccatcctc gatatfcaatg fcgggga'cfcga 6540 aaggaaaact gtgtccgtgc aagagacccg gagcctaggc ggctccaaat tcagtgtttg 5600 fcactgtcgtg tccaacacac ecgtggacgc cttaaccggc atcccactcc agacaccaac 6660 ccctcttttt gagaatggtc cgcgtcatcg cagcgaggaa gacgafccfcta aagtcgagag 6720 gafcgaagaaa cactgtgtat ccctcggctt ccacaacatc aatggcaaag tttactgcaa 6780 aatttgggac aagtctaccg gtgacacctt ttacacggafc gatt cccggt acacccaaga 5840 ccatgctttt caggacaggt cagccgacta cagagacagg gactatgagg gtgtgcaaac 6900 cgccccccaa cagggatttg atccaaagtc tgaaaccccg g tggcaccg ttg gafccgg 6960 cggtattscg tataacaggt atctgatcaa aggtaaagag gttctggttc ccaagcctga 7020 gaagctgcca caactgcctt agctgtccct tgagcaagct ctcgcfcggga tgggccaaae 7080 ttgcgacctt acagctgccg aggtggaaaa gctaaagcgc atcattagtc aactccaagg 7140 cttgaccact gaacaggctt taaactgtta gccgccageg gcttgacccg ctgtggocgc 7200 ggcggcct & g ttgtaactga aaaafctataa aacggcggta aataccacag cagaactttc 7260 accttaggcc ctttagacct aaaagtcact tccgaggtgg aggtaaagaa atcaactgag 7320 cagggccacg ctgttgtggc aaacttatgt tccggtgtca tctfcgatgag acctcaccca 7380 ccgtcccttg ttgacgttct tctgaaacec ggacttgata caacacccgg cattcaacea 7440 gggcatgggg ccgggaatat gggcgtggac ggttctattt gggattttga aaccejcaccc 7500 acaaaggcag aactcgagtt atccaagcaa ataatccaag catgtgaagt taggcgcggg 7560 gacgccccga acctccaact cccttacaag ctctatcctg ttagggggga tcctgagcgg 7620 cataaaggcc gccttatcaa caccaggttt ggagatttac c ttacaaaac tcctcaagac 7680 accaagtccg caatccacgc ggettgttgc ctgcacccca acggggcccc agtgtctgat 7740 cacfcaggfcac ggtaaafccca cactcttcaa cafcggtttcg agctfctatgt ccctactgtg 7800 tcatggagta ecctatagtg cettgafctca cgccctgaca cccctfcttat gfcgfcactaaa 78S0 cafcggcactfc ccaaggatgc fcgcagaggac ctccaaaaat acgacctafcc cacccaagga 7920 tttgtcctgc ctggggtcct acgcctagta egcagattca tctttggcca tattggtaag 7980 gcgccgccat tgtfccctccc abcaacttafc cccgccaaga actetafcggc agggateaat 8040 ggccagaggfc tcccaacaaa ggacgttcag agcatacctg aaattgatga aatgfcgtgcc 8100 cgcgccgtca aggagaattg gcaaactgtg acaccttgta ccctcaagsa acagtactgt 8160 tccaagccca aaaccaggac catcetgggc accaacaact ttattgcctfc ggctcacaga 8220 tcggcgcfcca gtggtgtcac ccaggcafcfcc atgaagaagg cttggaagtc cccaattgcc 8280 acaaattcaa ttggggaaaa ggagctgcat tgoactgtcg ccggcagatg tcttgaggcc 8340- gacttggcct cctgtgaccg cagcaccccc gccattgtaa gatggtttgt tgccaacctc ctgtafcgaac 8400 fctgcaggatg tgaagagtac tfcgcctagct atgtgctfcaa fctgctgccafc 846? gacetcgfcgg caacacagga tggtgccfctc acaaaacgcg gtggcctgfcc gtccggggac 8520 gtgtgtccaa cccgtcaoca tcactggtaa caccgtatat gcacatggta tttatgccca 8580 ttgtcggcct tgaaaatggg tcatgaaatt ggtcttaagt tcctcgagga acagctcaaa 8640 ttcgaggacc fcccfctgaaat teagcctatg ttggtatact ctgatgaccfc fcgtcttgfcac 870D gctgaaagac ccacct-TTCC caabtaccac bggfcgggtcg agcaccttga cctgatgcfcg 8760 -ggfctteagaa- cggacccaaa gaaaaccgtc ataactgata aacccagcbt cctcggctgc 8820. agaattgagg cagggcgaca gcbagtcccc aatcgcgaoc gcabcctggc tgcbcbfcgca 8880. tatcac &fcga aggcgcagaa cgcctcagag fcattabgcgfc ctgcbgccgc aatccfcgatg -8940- • gattcatgtg- cttgcattga ccatgaccct gagtggbatg aggacctcat cbgcggtatt- 9000 > gcccggtgcg -cccgccagga tggttafcagc ttcccaggte cggcattfctt cafcgfcccabg "9050 • .tgggagaagc tgagaagtca fcaatgaaggg aagaagtfccc gccacfcgcgg catcfcgcgac 9120. gccaaagc.cg actatgcgtc cgcctgfcggg cttgatfctgfc gtttgfctcca fcfccgcacttt 91SQ- catcaacact- gcccbgfccac tctgagefcgc ggtcaccatg ccggtbcaaa ggaatgttcg 924 · 0 > --cagtgtcagfc cacctgtfcgg ggcfcggcaga tcaccfccfctg atgccgtgct aaaacaaatt 9300 -ccatacaaac ctcctcgcac tgtcafccatg aaggtgggta ataaaacaac- ggecctcgafc-, 9360 'ccggggaggt -accagtcccg bcgaggtcbc gttgcagbca agaggggtat tgcaggcaat -9½20' -gaagtfcgatc ttfccfcgafcgg agactaccaa gtggtgcctc 9480 · ttttgccgac ttgcaaagac. taaacafcgg tgaaggfcggc ctactcagca ttgcaatgta agggccacca agttcatagt 9540; ggtfcocggaa agaccaccbg gcbactgagt caagtccagg acgafcgatgt catttacaca 9500 | cccacccafcc-agacfcafcgfct tgatatagfcc agtgctcfcca aagfcbbgcag gfcafctccgbfc 9SSQ ~ ccaggagccfc caggactccc ttfccccaeca cctgccaggt ccgggccgtg ggtbaggcbt 9720 attgccageg ggcacgfcccc tggccgagba tcataccbcg atgaggcbgg abafctgtaat 9780 cafcctggaea ttctfcagact gcfcttccaaa acaccccbbg tgtgfcfctggg tgacctfccag 9840 caactfccacc ctgtcggcfct tgattcctrc tgttafcgtgfc tcgatcagat gcctcagaag 9900 cagctgacea ctafcttacag atttggccct aacafcfcfcgcg eagecatcca gecfctgtfcan 9960 agggaaaaac tbgaatcbaa ggctaggaac accagggbgg tttfcfcaccac ccggcctgfcg 10020 gccfcfcfcggtc aggtgotgac accataccat aaagatcgca tcggctctgc gataaccafca 1008O gatfccatccc agggggccac ctttgatafct gtgacattgc atctaccatc gccaaagtcc ioi40 ctaaataaat cccgagcact tgtagccatc actcgggcaa gacacgggtt gttcatttat 10200 accagctcca gaccctcata ggagtttttc aatctaaccc ctgagcgcac tgattgfcaac 102SO gctgtgggga cfcfcgtgfctca tgagcfcggta gfctcfcgaatg cggafcaatgc aghcacaact 10320 gtagcgaagg ccctaga gac aggtccatct cgatttcgag tatcagaccc gaggtgcaag 10380 tctctcttag ccgcttgttc ggccagtctg gaagggagct gtatgccacfc accgcaagtg 10440 gcacataacc tggggtttfca efctfctecccg gacagtccag tatfctgcacc tctgccaaaa ????? gagttggcgc cacattggcc agtggttacc caccagaaca atcgggcgtg gcctgatnga IOBSO cttgtcgcta gtatgcgccc aattgatgcc cgctacagca agccaatggt cggtgcaggg 10520 -tatgtggteg ggccgtccac ctttcfctggt actcctggcg fcggtgtcata loseo ctatctcaca ctatacatca ggggfcgaacc tcaggecttg ccagaaacac tcgtttcaac aggacgtata xo'740 gccacagatt gtcgggagta tctcgacgcg gctgaggaag aggcagcaaa agaactcccc IOBOO cacgcattca ttggcgafcgt caaaggtacc acggtggggg ggtgtcatca cattacatca 10860 aaatacctac ctaggtccct gcctaaggae fcctgttgccg fcagtfcggagt aagttcgccc 10920 ggcagggctg ctaaagccgt gtgcactctc accgatgtgt acctccccga actccggcca 103B0 tatctgcaac ctgagacggc atcaaaatgc tggaaactca aattagactt cagggacgtc 11040 cgactaatgg tctggaaagg agccaccgcc tatttccagt tggaagggct tacatggtcg moo gcgctgcccg aetatgccag gtttatfccag ctgcccaagg atgccgttgt atacafetgat MSO ccgtgtatag gaccggcaac agccaaccgt aaggtcgtgc gaaccacaga ctggcgggcc 112-20 gacctagcag tgacacogta tgattacggc gcccagaaca ttttgacaac agcctggttc 11280 5 -gaggaccfccg ggccgcagtg gaagattttg gggttacagc cctttag GCG agcatttggc 11340 tttgaaaaea ctgaggattg ggcaatcctt gcacgccgta tgaatgacgg caaggactac 11400 actgactafca actggaactg tgbtcgagaa cgcceaeacg ccafcctacgg gcgtgctcgt 11460 gaccatacgfc atcattttgc ccctggcaca gaattgcagg tagagctagg taaaccecgg 11520 ctgccgccfcg ggcaagtgcc .gt.gaa.ttcgg agtgatgcaa tggggttact gtggagtaaa 11580 • JO atcagccagc fcgttcg gga cgccttcact gagttccttg ttagtgfcggt tgatattgtt 11640 'atttfccc FCG ccateacfcgtt tgggtteacc gtcgcaggat ggttactggt cttccttctc 1170D - gagtggttt- gctccgcgct tctccgttcg cgctctgcca ttcactctcc cgaactatcg 11760 aaggtccfc-afc gaaggcttgt tgcccaactg cagaccggat gtcccacaat ttgcafctcaa 11820 gcacccattg gg £ atgcttt ggcacatgcg agtttcccaa ttaattgatg agatggtctc 11880 15 tcgtcgcatt taccagacca tggaacattc aggtcaagcg gcctggaagc aggcggttgg 11940 tgaggccact ctcaegaagc tgteaaggct cgatatagtt actcatttcc aacacctggc 12000 cgcagtagag gcggattctt gccgctttct cagctcacga ctcgtgatgc taaaaaatct 12060 .tgccgttggc aatgtgagcc tacagtacaa caccacgttg gaccgcgttg agctcatctt 12120 ccccacgcca g gtacgaggc cgacttcaga ccaagttgac caatggctca tcagtgtgca 12180 20 -cgcttecatt tttt-cctctg tggcttcatc fcgttaccttg ttcatagtgc tttggcttcg 12-240 • aattccagct ctacgctatg tttttggttt ccattggccc .acggcaacac atcattcgag 12300 |ctgaccafcpa- ctacaccat atgcatgccc tgttctacca gtcaagcggc tcgccaaagg 123S0 ctcgagcccg gtcgtaacat gtggtgcaaa atagggtatg acaggfcgtga ggagcgtgac 124-20 • catgatgagfc tgfctaatgcc. catcccgtcc gggtacgaca, acptcaaacfc tgagggttat. 124: 30-25. tggctttttt-tatgcttggc gtcctfcttcc tacgeggccc -aattccatcc ggagttgttc 12540 gggataggga- -atgfcgfccgcg · cgtcttcgtg gacaagcgac accagttcat ttgtgccgag 12600 • catgatggac.-agaattcaac cgtatctacc ggacacaaca-.tctccgcatt atatgcggca -.tattaccaec 12560 --- accaaataga cgggggcaat cggttccatt. fcggaatggc gcggccactc- 127-20 -ttttcctcnt ggctggtgct -caatatatca tggtttctga- ggcgttcgcc tgtaagcc'ct 12780 30 -.gtttctcgac igeatc.fcatca gatattgaga ccaacacgac cgcggctgcc ggtttcatgg 12840: tccfctcagga catcaattgt -ctccgacctc acggggtctc agcagcgcaa. gaggaaattc 12900.-cettcggaaa gtcgtcccaa- .tgfccgtgaag ccgtcggtac tccccagtac atcacgataa 12960 tcggctaacgt gaccgacgaa tcatacttgt acaacgcgga cttgctgatg ctttctgcgt 13020 gcctfcttcca cgcctcagaa atgagcgaga aaggcttcaa agttatcttt gggaatgtct l3 & Ao 35: ctggcgttgt ttccgcfctgt gtcaafcfcfcca cagattatgt ggcccatgtg acccaacata 13140 tcatttggta .cccagcagca -attgatcaca tcggttgct gcattfccctg acaccafcctg 13200 caatgaggtg ggctacaacc- attgctfcgtt tgttcgccat fcctcttggcg atatgagatg 13260 ttctcacaaa fcfcggggcgfcfc tcttgactcc gcacfccttgc ttctggfcggt tttttfctgct 13320 gtgtaccggc ttgtcctggt ccfcttgccga tggcaacggc aacagctcga cataccaata 13380 40 .catatataac ttgacgatat gcgagctgaa tgggaccgac tggttgtcca gccattttgg 13440 ttgggcagtc gagacctttg tgttttaccc ggttgccact catatcctct cactgggttt 13500 tctcacaaca agccattttt ttgacgcgct cggtctcggc gctgtatcca ctgcaggatt 13S60 tgttggcggg cggtatgtac tctgcagcgt ctacggcgct tgtgctttcg cagcgttcgt 13620 afcgtttfcgfcc atccgtgctg ctaaaaattg catggcctgc cgctatgccc gtacceggtt 13680 45 taccaacttc attgtagaca accgggggag agttcatcga tggaagtctc caatagtggt 13740 .agaaaaafctg ggcaaagccg aagtcgacgg caacctcgtc accatcaaac afegtcgfccct 13800 cgaaggggtt aasgctcaac ccttgacgag gactbcggct gagcaatggg aggcctagac 13660 gatttttgca acgat-cctat c gccgcacaa aagctcgtgc fcagccfcttag catcacatae 13920 .acacctataa tgatatacgc ccttaaggtg tcacgcggcc gaetcctggg gctgttgcac 13980 50 atcctaatat ttctgaactg ttcctttaca ttcggataca tgacatatgt gcattfctcaa 1404th • tccaccaacc gfcgtcgcact taccctgggg gctgttgtcg cccttctgtg gggtgttfcac 14100 agcttcacag aatcatggaa gtttatcact tceagatgca gattgtgttg ccttggccgg 14160 tggcccctgc cgatacattc gaaagtgctg ccatcacgta ttcaatctca caggtctcca 14220 accgagcata gcgtctggta cgctgtgaga aagcceggac taacatcagt gaacggcact 14280 55 ctagtaccag gacttcggag cctcgtgctg ggcggcaaac gagctgttaa acgaggagtg 14340 gttaacctcg tcaagtatgg ccggtaaaaa ccagagccag aagaaaaaga aaagtacagc 14400 tccgatgggg aafcggccagc cagtcaatca actgtgccag ttgctgggtg caafcgafcaaa 1 eo .gtcccagcgc ggggaggaca cagcaaccfca ggccaaaaag aaaaagcctg agaagccaca 14520 .fc-tttccccfcg gctgcfcgaag afegacafcccg gcaccaectc acccagactg aacgcteccfc 145S0 etgcfctgeaa cggcttfccaa tcgatccaga tcaaggcgca ggaactgcgfc cgctfctcafcc 14540 cagcgggaag gtcagttttc agg tgagtt tatgctgccg gttgctcata cagcgcgcct 14700 gattcgcgfcg acttctacat ccgccagtoa gggtgcaagt taatttgaca gtcaggtgaa 14760; tggccgcgafc tggcgfcgtgg -cetcfcgagtc acctattcaa ttagggcggt catag: 14815 . < 2ll > 75.0 < 212 > ADK - < 213 > Porcine reproductive and respiratory syndrome virus |, < 4D0 > 6 ~. • .afcgcaatggg gtcactgtgg agtaaaatca gccagctgtt cgtggacgcc ttcactgagt 60. -fcectfcgfet-ag fcgfcggbfcgat. attgccafctfc- tccfctgccafc actgfcttggg fctcaccgtcg 120. ..caggafcggtt actggtcttt cttctcagag tggfctfcgctc cgcgcttctc cgfctcgcgct -180 • ctgccafctca ctcfceccgaa ctatcgaagg tcctatgaag gcttgttgcc caactgcaga 240- ccggafcgtcc cacaafcfcfcgc "agtcaagcac tgfcfcfctggca ccattgggya catgagagtt -300 'tcccacfctga ttgafcgagat · ggtctctcgt cgcatttacc agaccatgga acafctcaggt -. 360 caagcggccfc ggaagcaggt ggtfcggtgag gccactctca cgaagctgtc agggctcgat 42C- atagttactc- atfctecaaca ccfcggccgca gtggaggcgg attcttgccg cfcfctcfccagc -480 fcgabgetaaa fceacgacfceg "aaafccfctgcc gtfcggcaatg tgagcctaca- gtacaacacc, 540 / acgfcfcggax: -gcgttgagct catcttcccc acgccaggta cgaggcccaa gtfcgacegafc .600. vtteagacaafc ggcfccafccag tgtgcacgct tccatttttt- cctctgtggc · ttcatctgtfc ^ 650 · • accttgbtca tagtgctfctg- gcttcgaafcfc ccagctctac gctatgtfcfct. tggfcttccafc -720 tggcccacgg caaca.catca - fc cgagctga 750 · ; < 210 >; 7 • .í211 > 7-9.8 212 > DNA < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 7 afcggctcafcc agtgfcgcacg cttccafcfctt tfccctcfcgtg gctfccafccfcg fcfcacctfcgfct SO- catagtgctt tggcttcgaa ttccagcfcct acgctatgtt tttggttfccc attggcccac 120 ggeaacacat cattcgagct gaccatcaac tacaccatat gcatgccctg fcfccfcaccagt 180 caagcggctc gccaaaggct cgagcccggt catagcatgt ggtgcaaaafc agggcafcgac 240 ag tgtgagg agcgfcgacca tgafcgagttg ttaafcgccca tcccgtccgg gfcacgacaac 300 ctcaaact-tg agggttatta tgcfcfcggctg gcfcttttfcgt cctttfcccta "egcggeecaa 360 ttccatccgg agttgttcgg gatagggaafc gtgtcgcgcg tcttcgtgga caagcgacac 420 gfcgcegagca cagttcafctt tgafcggacae aafcfccaaecg tgfccfcaccgg acacaacatc 480 tccgcattat afcgcggcata ttaccaccac caaafcagacg ggggcaattg gfcfcceatfcfcg 540 gaafcggctgc ggccactctt ttcttcctgg cfcggtgctca acafcafccatg gfcfcfcctgagg 600 cgttcgcctg taagcccfcgt fctctcgacgc atctatcaga tatfcaagacc aacacgaccg 660 cggctgccgg fcfcfccafcggfcc ctfccaggaca tcaafctgttfc ccgacc cac ggggtctcag 720 - cagcgcaaga gaaaattfccc ttcggaaagt cgtcccaafcg fccgtgaagcc gtcggtactc 780 cccagtaeat cacgata to 798 < 211 > 55 < 212 DNA < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 8 afcggctgcgg ccactcfctfct cttcctggct ggtgctcaac atatcatggt ttctgaggcg 60 ttcgcctgta agccctgttt ctcgacgeat cfcatcagata tfcaagaccaa- cacgaacgcg 120 gctgccggtt tcatggtcct tcaggacatc aattgtttcc gacetcacgg ggtctcagca 180 gcgceagaga aaatttcctt cggaaagtcg tcccaatgtc gtgaagccgt cggtactccc 240 cagtacacca cgataaoggc taacgtgacc gacgaatcat acttgtacaa cgcggacctg 300 cfcgatgcttt ctgcgtgcct tttcfcacgcc tcagaaatga gcgagaaagg cttcaaagtc 360 atctttggga atgtctctgg cgttgttfcct gcttgtgtca afcfctcacaga ttatgtggcc 420 catgtgaccn aacstaccca gcagcatcat ctggtagttg atcacattcg gttgctgcat 480 ttcctgacac catctgcaat gaggtgggct acaaccattg cttgtttgct cgccat ctc 540 ttggcnatat ga · • 552 < 210 > S < 211 > 606 .c212 > DNA < 213 > porcine reproductive and respiratory syndrome virus; c400 > 9 atgagatgtt ctcacaaatt ggggcgttec ttgactccgc actettgctt ctggtggctt- 60. tttttgctgt gtaccggctt gtcctggtcc -tttgccgatg gcaacggcga-- cagctcgaca 120 · "taccaataca tatatgactfc gacgatatgc gagctgaatg ggaccgactg gttgtccagc 180 catttfcggtt gggcagfccga gacctttgtg · tttfcacccgg tatcctctca tfcgccactca 240 • ctgggttttc.fccacaacaag ccatfcttttt gacgcgctcg gtctcggcgc tgfcafcecact-300. Gcaggatttg ' ttggcgggcg gtacgtactc tgcagcgtct acggcgcttg tgctttcgca; 360 gcgttcgtat gttttgtcat ccgtgctgct aaaaattgca tggcctgccg etatgcccgt 420- acccggttta ccaaettcat fcgfcggacgac-; cgggggagag ttcatcgatg gaagtctaca- 480 atagtggtag aaaaattggg caaagccgaa gtcgatggca acctcgtcac catcaaacat 540 aaggggttaa gtcgtcctcg fctgacgagga agctcaaccc cttcggctga gcaatgggag gcctag 600 - - = 506 < 210 > 10 < 211 > 2396 < 212 > PRX < 213 > Porcine reproductive and respiratory syndrome virus <;: 400 > 10 Met Ser GLy hr Phe Ser Srg Cys ¾et Cye Thr Pro Ala Ala Arg Val. i s 10 15 Phe Trp Asn. Ala Gly Gln Val Phe. Cys ¾br Arg Cys Le Ser Wing Arg 20 25 30 Ser Leu ie Ser Ero Glu Leu Gln Asp Thr Asp Leu Gly Wing Val Gly • 35 4? 45 Leu Phe Tyr Lya Pro Arg Asp Lys Leu His Trp Lys Val Pro lis Gly 50 55 60 lie Pro Gln al Glu Cys Thr Pro Ser Gly Cys Cys Trp Leu Ser wing 65 70 '75 80 Val Phe Pro Leu Ala Arg Met Thr Ser Gly Asn His Asn Phe Leu Gln 85 90 95 Arg Leu Val Lys Val Wing Asp Val Leu Tyr Arg Asp Gly Cys Leu Wing 100 105 * ~ 110 • Pro Arg His Leu Arg Glu Leu Gln Val Tyr Glu Arg Gly Cys Asn Trp 115 - 120 125 yr Pro lie Thr Gly Pro Val Pro Gly Met Gly Leu Phe illa Asn Ser 130 135 140 Met Hxs- Val Ser Asp Gis. Pro Piie Pro Gly Ala Thr His Val Leu Thr 145 - 150 155 1S0 Asn Ser Pro Leu Pro Gln. Glu Ala Cys Arg Gln Pro Phe Cys Pro P > he 155 170 175 Glu Glu Wing His Ser Ser Val Tyr Arg Trp Lys Lys Phe Val Val Phe 180 185 190 Tur Asp Ser Ser Leu Asn Gly Arg Ser Arg Met Met Trp Thr Pro Glu G -195 · 200 205. Be Asp Asp Be Ala Ala Leu Glu Val Leu Ero Pro Glu Leu Glu Arg 210 215 220 · Gln Val Glule Leu lie- Arg Ser- Phe. Pro Ala His His Pro Val Asp • -225 230 235 240 -Leu Ala Asp Trp Glu Leu Tlrr Glu-. Ser -Pro Glu. Asn Gly Phe Ser Phe -245 250. - 255 Asn Thr Ser His Ser Cys Gly His Leu Val Gln Asn Pro Asp Val Phe 260 265 270 Asp Gly Lys Cys Trp Leu Ser Cys Phe Leu Gly Gln Ser Wing Glu Val 275 280 285 Arg Cys His Slu Glu His Leu Wing Asp Wing Phe Gly Tyr Gln Thr Lys 290 2S5 300 Trp Gly Val His Gly Lys Tyr Leu Gln Arg Arg Leu Gln Val His Gly 305 310 315 320 lie Arg Ala Val Val Asp Pro Asp Gly Pro lie His Val Glu Ala Leu 325 330 335 Ser Cys Pro Gln Ser Trp lie Arg His Leu Tr Leu Asn Asp Asp Val 340 345 35 Thr Pro Gly Phe Val Arg Leu Thr Ser Leu Arg lie Val Pro Asn Thr 355 360 365 Glu Pro Thr Thr Ser Arg lie Phe Arg Phe Gly Ala His Lys Trp Tyr 370 375 380 Gly Ala Ala Gly Lys Arg Ala Arg Ala Lys Arg Ala Ala Lys Ser Glu 385 320 395 400 Lys Asp Ser Wing Pro Thr Pro Lys Val Wing Leu Pro Val Pro Thr Cys 405 410 415 Gly lie Thr Tfar Tyr Ser Pro PrD Thr Asp Gly Ser Cys Gly Trp His 420 425 430 Val Leu Ala Ala lie Met Asn Arg Met lie Asn Gly Asp Phe Thr Ser 435 440 445 Pro Leu Thr Gln Tyr Asn Arg Pro Glu Asp Asp Trp Ala Ser Asp Tyr 450 455 460 Asp Leu Val Gln Ala lie Gln Cys Leu Gln Leu Pro Ala Thr Val Val 465 470 475 480 Arg Asn Arg Ala Cys Pro Asn Ala Lys Tyr Leu lie Lys Leu Asn Gly 485 490 4S5 Val His Trp Glu. Val Glu Val Arg Ser Gly Met Pro Wing -Arg Ser Lsu 500 505 510 Pro Axg Glu Cys Val Val Gly Val Cys Ser Glu- Gly Cys Val Wing Pro 515 520 · 525 Pro Tyr Pro Wing Asp Gly Leu Pro Lys Arg Wing Leu Glu Ala Leu Ala 530 - 535 540. . Be Wing Tyr Arg Leu Pro Be Asp Cys Val Ser Ser Gly lie Wing Asp 545 550 555 5S0 Phe Leu Wing Asn Pro Pro Pro Gln Glu Phe Trp Thr Leu ASO Lys Met 565.575"" 575 Leu Thr Ser Pro Ser Pro Glu Arg Ser Gly Phe Ser Ser Leu Tyr Lys 580 585 530 Leu Leu Leu Glu Val Val Pro Gln Lys Cys Gly Wing Thr Glu Gly Wing 595 600 605 Phe lie Tyr Wing Val Glu Arg Tlet Leu Lys Asp Cys Pro Ser Ser Lys 610 515 620 Gln Ala Met Ala Leu Leu Ala Lys lie Lys Val Pro Ser Ser Lys Ala 625 630 535 640 Pro Ser Val Ser Leu Asp Gly Cys Phe Pro Thr Asp Val Pro Wing Asp 645 650"655 Phe Glu pro Wing Pro Pro Glu Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 660 665 670 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 675 680 685 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa € 90 695 700 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 7OS 710 715 720 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 725 730 735 Xaa Xaa Xaa Ala Gly Leu lie Asn Leu Val Gly Gly Asn Lsu Ser Pro 740 745 750 Ser A.sp Ser Met Lys Glu Asn Met Leu Asn Ser Arg Glu Asp Glu Pro 755 760 765 Asp Leu Ser Gln Pro Ala Pro Ala Ala Thr Thr Thr Leu Val Arg 770 775 780 Glu Glu Thr Pro Asp Asn. P or Gly Ser Asp Ala Gly Ala Lsu Pro Val 785 '790 795 - 800 .Thr Val Arg Glu Plie Go! Pro Thr Gly Pro lie Leu Arg His Val Glu 805 810 815 His Cys Gly Thr Glu Ser Gly Asp Ser Ser Pro Leu Asp Gln Ser 820 825 · 830 Asp Wing Glu Thr Leu Asp Glu Pro Leu Asn Leu Ser Leu Wing Wing Trp 835 840 845 | Pro Val Arg Wing Thr Wing Ser Aap Pro Gly Trp Val His Gly Arg Arg 850 855 860 Glu Pro Val Pha Val Lys Pro Arg Asn Wing Phe be Asp Gly Asp be 865 870 875 880 Ala Leu Gln Phe Gly Glu Leu Ser Glu Ser Ser Pro Val lie Glu Phe 885 8S0 895 Asp Arg Thr Lys Asp Ala Pro Val Val "Asp Ala Pro Val Asp Leu Thr 900 905 910 Thr Ser Asn Glu Ala Leu Ser Val Val Asp Pro Phe Glu Phe Wing Glu 915 920 925 Leu Lys Arg Pro Arg Phe Be Wing Gln Wing Leu lie Asp Arg Gly Gly 930 935 940 Pro Leu Wing Asp Val His Wing Lys lie Lys Asn Arg Val Tyr Glu Gln 945 950 955 960 Cys Leu Gln Wing Cys Glu Pro Gly Ser Arg Wing Thr Pro Wing Thr Arg 965 ~ 970 975 Glu Trp Lsu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg 980 S85 990 Cys Thr Ser Gla Phe Gln Wing Gly Arg lie leu Wing Ser Leu 1_ys Phe 995 1000 1005 Leu Pro Asp Met Lie Glu Asp Thr Pro Pro Pro Val Pro Arg Lys Handle 1010 ~ 1015 10g Arg Wing Ser Asp Asn Wing Gly Leu Lys Gln Pro Val Wing Gln Trp Asp 1025 1030 1035 1040 Arg Lys Leu Ser Val Thr Pro Pro Pro Val Lys Pro Val Gly Pro Val leu 1045 1050 1055 Asp Gln Tiir Val Pro Pro Pro Tfar Asp lie Gln Gln Glu Asp Val Thr 1060 1065 1070 Pro Ser Asp Gly Pro Pro His Wing Pro Asp Phe Pro Ser Arg Val Ser 1075 1080 1085 Thr Gly Gly Ser Trp Lys Gly Leu Met Leu Ser Gly Thr Arg Leu Wing 1090 10S5 1100 Gly Ser lie Ser Gln Arg Leu Met Th Trp Val Phe Glu Val Phe Sér- 1105 1110 1115 1120 His T.RII PT-O Ala Phe Met Leu Thr Leu Phe Ser Pro Arg Gly Ser Met 1125 1130 1135 Wing Pro Gly Asp Trp Leu Phe Wing Gly Val Val Leu Leu Wing Leu Leu 1140 1145 1150 Leu Cys Arg Ser Tyr Pro lie Leu Gly Cys Leu Pro Lsu Leu Gly Val 1155 1160 1165 Phe Ser Gly Ser Leu Arg Arg Val Arg Leu Gly Val Phe Gly Ser Tp 1170 1175 11B0 Met Ala Phe Ala Val Phe Leu Phe Ser Thr Pro Ser Asn Pro Val Gly 1185 1190 1195 1200 Ser Ser Cys Asp His Asp Ser Pro Glu Cys Eis Ala Glu leu Leu Ala 1205 1210 1215 Leu Glu Gln Arg Gln Leu Trp Glu Pro Val Arg Gly Leu Val Val Gly 1220 1225 1230 Pro Ser Gly leu Leu Cys Val lie Leu Gly Lys Leu Leu Gly Gly Ser 1235 1240 1245 Arg Tyr leu Trp His Val Phe Leu Arg leu Cys Met Leu Ala Asp leu 1250 1255 1260 Wing Leu Ser ie Val Tyr Val Val Ser Gln Gly Arg Cys His Lys Cys 1265 1270 1275 12S0 Trp Gly Lys Cys lie Arg Thr Ala Pro Ala Glu Val Ala J-.eu Asn Val Phe Pro Phe Leu Arg Ala Thr Arg Ala Ser Lau Val Ser Leu Cys Asp 1300 1305 1310 Arg Phe Gln Thr Pro Lys Gly Val-Asp Pro Val His Leu Wing Thr Gly 1315 1320 1325 Trp Arg Gly Cys Trp Arg Gly Glu Pro Pro lie His Gln Pro His Gln 1330 1335 1340 Lys Pro lie Wing Tyr Wing Asa Leu Asp Glu Lys Lys lie Wing Gln 1345 1350 1355 1360 Thr Val Val Wing Val Pro Tyr Asp Pro Ser Gln Wing Val Bys Cys Leu 13S5 1370 1375 Lys Val Leu Gln Ala Gly Gly Ala lie Val Asp Gln Pro Thr Pro Glu 1380 1385 1350 Val Val Arg Val Ser Glu lie Pro- Phe Ser Wing Pro Phe Phe Pro Lys 1395 1400 1405 Val Pro -Val Asn Pro Asp Cys Arg Val Val Val Asp Ser Asp Thr Phe 1410 1415 1420 Val Ala Ala Val Arg Cys Gly Tyr Ser Thr Ala Gla Leu Val Leu Gly 1425 1430 · 1435 1440 Arg Gly Asn Phe Ala Lys Leu Asa Gln Thr Pro Pro Arg Asn Ser lie 1445 1450 1455 Be Thr Lys Th Thr Gly Gly Wing Ser Tyr Thr Leu Wing Val Wing Gln 1450 1465 1470 Val Ser Wing Trp Thr Leu Val His Phe lie Leu Gly Leu Trp Phe Thr 1475 ~ 1480 1485 Ser Pro Gln Val Cys Gly Arg Gly Thr Wing Asp Pro Trp Cys Ser Asn 1490 14S5 1500 Pro Phe Ser Tyr Pro Thr Tyr Gly Pro Gly Val Val Cys Ser Ser Arg 1505 1510 1515 1520 Leu Cys Val Ser Wing Asp Gly Val Thr Leu Pro Leu Phe Ser Val Wing 1525 1530"1535 Ala Gln. Leu Ser Gly Arg Glu Val Gly lie Phe lie Leu Val Leu Val 1540 1545 1550 Ser Leu Thr Ala Leu Ala His Arg Het Ala Leu Lys Ala Asp Ket Leu 1555 1550 1565 Val lie Phe Ser Ala Phe Cys Ala Tyr Ala Trp Pro Met Ser Ser Trp 1570 1575"~ 158Q e lie Cys Phe Phe Pro lie Leu Leu Lys Trp Val Tiir Leu His Pro 585 15S0 1555 1600 Leu Thr Met Leu Txp Val His Ser Phe Leu Val Phe Cys jjeu Pro Wing 1605 1610 ~ 1615 Ala Gly lie Leu Ser Leu. Gly lie Thr Gly Leu Leu Trp Wing lie Gly 1620 1625 1630 Arg Phe Thr Gln Val Wing Gly lie lie Thr Pro Tyr Asp lie Eis Gln 1635 1640 1645 Tyr Thr Ser Gly Pro Arg Gly Wing Wing Wing Val Wing Thr Wing Pro Glu 1650 1655 1660 Gly Thr Tyr Met Wing Wing Val Arg Arg Wing Wing Leu Thr Gly Arg Thr 1665 1670 1675 1CS0 'Leu lie Phe Thr Pro Ser Wing Val Gly Ser Leu Leu Glu Gly Wing Phe 1685 1690 1695 Arg Thr His lys Pro Cys Leu Asn Thr Val Asn Val Val Gly Ser Ser 1700 1705 1710 Leu Gly Be Gly Gly Val Phe Thr lie Asp Gly Arg Arg Thr Val Val 1715 1720 1725- Thr Ala Ala His Val Leu Asn Gly Asp Thr Ala Arg Val Thr Gly Asp 1730. 1735 1740 Ser Tyr Asn Arg Met His Thr Phe Lys Thr Asn Gly Asp Tyr Wing Trp 1745 1750 1755 1750 Ser His Wing Asp Asp Trp Gln Gly Val Wing Pro Val Val Lys Val Wing 1765 1770 1775 Lys Gly Tyr Arg Gly Arg Wing Tyx Trp Gln Thr Ser Thr Gly Val Glu 1780 1785 1790 Pro Gly lie lie Gly Gly Phe Wing Phe Cys Phe Thr Asn Cys Gly 1795 1800 1805 Asp Ser Gly Ser Pro Val lie Ser Glu Ser Gly Asp Leu lie Gly lie IB10 1815 1820 His Thr Gly Ser A.sn Lys Leu Gly Ser Gly Leu Val Thr Thr Pro Glu 1825 1830 1835 1840 Gly Glu Thr Cys Thr lie Lys Glu Thr Lys Leu Ser Asp Leu Ser Arg 1845 1850 1855 His Phe Wing Gly Pro Ser Val Pro Leu Gly Asp lie Lys Leu Ser Pro 1860 1865 ~ 1870 Ala lie lie Pro Asp Val Thr Ser lie Pro Ser Asp Leu Ala Ser Leu 1875 ~ 1880 1885 Leu Ala Ser Val Val Val Val Glu Gly Gly Leu Ser Th Val Gln Leu 1890 1895 1900 Leu Cys Val Phe Phe Leu Leu Trp Arg Met Met Gly His Wing Trp Thr 1905 1310 1915 1920 Pro lie Val Wing Val Gly Phe Phe Leu Leu Asn Glu lie Leu Pro Wing 1925 1930 1935 Val Leu to Arg Ala Val Phe Ser Phe Ala Leu Phe Val Leu Ala Trp 1940 1945 1950 Wing Thr Pro Trp Ser Wing Gln Val Leu Met He Arg Leu Leu Thr Wing 1955 1960 1965 Ser Leu Asn Arg Asn. Lys Leu Ser Leu Ala Phe Tyr Ala Leu Gly Gly 1970 1975 1980 Val Val Gly Leu Ala Al Glu lie Gly Thr Phe Ala Gly Arg Leu Ser 1985 1990 1995 2000 Glu Leu Ser Gln Ala Leu Ser Thr Tyr Cys Phe Leu Pro Arg Val Leu 2005 2010 2015 Wing Met Thr Ser Cys Val Pro Thr lie lie lie Gly Gly Leu His Thr 2020 2025 2030 Leu Gly Val lie Leu Trp Leu Phe Lys Tyr Arg Cys Leu Eis Asn Met 2035 2040 2045 Leu Val .Gly Asp Gly Ser Phe Ser Be Wing Phe Phe Leu Arg Tyr Phe 2050 ~ 2055 2060 Wing Glu Gly Asn Leu Arg Lys Gly Val Ser Gln Ser Cys Gly Met Asn 2055 2070 2075 2080 Asn Glu Be Leu Thr Wing Wing Leu Wing Cys Lys Leu Ser Gln Wing Asp 2085 2090 2095 Leu Asp Phe Leu Ser Ser Leu Thr Asn Phe Lys Cys Phe Val Ser Wing 2100 2105 2110 Ser Asn Met Lys Asn Wing Wing Gly Gln Tyr He Glu Ala Ala Tyr Ala 2115 2120. 2125 Lys Wing Leu Arg Gln Glu Leu Wing Being Leu Val Gln lie Asp Lys Met 2130 2135 2140 Lys Gly Val Leu Ser Lys Leu Glu Wing Phe Wing Glu Thr Wing Thr Pro 2145 2150 2155 2160 Ser Leu Asp lie Gly Asp Val lie Val Leu Leu Gly Gln His Pro Eis 2165"2170 2175 Gly Ser lie Leu Asp lie Asn Val Gly Thr Glu Arg Lys Thr Val Ser 2180 218S 2190 val Gln Glu Thr Arg Ser Leu Gly Gly Ser Lys Phe Ser Val Cys Thr 2ZS5 2200 2205 val Val Ser Asn Thr Pro Val Asp Ala Leu Thx Gly lie Pro Leu Gln 2210 2215 2220 Thr Pro Thr Pro Leu Phe Glu Asn Gly Pro Arg His Arg Ser Glu Glu 2225 2230 2235 2240 Asp Asp Leu Lys Val Glu Arg Met Lys Lys His Cys Val Ser Leu Gly 2245 2250 2255 Phe His Asn xie Asn Gly Lys Val Tyr Cys Lys lie Trp Asp Lys Ser 22S0 2265 2270 Thr Gly Asp Thr Phe Tyr Thr Asp. Asp Ser Arg Tyr Thr Gln Asp His 2275 2280 2285 Wing Phe Gln Asp Arg Wing Asp Tyr Arg Asp Arg Asp Tyr Glu Gly 2290 2295 2300 Val Gln Thr Ala Pro Gln Gln Gly Phe Asp Pro Lys Ser Glu Thr Pro 2305 2310 ~ 2315 2320 .Val Gly Thr Val Val lie Gly Gly lie Thr Tyr Asn Arg Tyr Leu Xle 2325 2330 2335 Lys Gly Lys Glu Val Leu Val Pro Lys Pro Asp Clan Leu Glu Wing 2340 2345 2350 Wing Lys Leu Ser Leu Glu Gln Ala Wing Ala Gly Met Gly Gln Thr Cys 2355 2350 2365 Asp Leu Thr Wing Wing Glu Val Gln Lys Leu lys Arg lie lie Ser Gln 2370 2375 2380 Leu Gln Gly Leu Thr Glu Gln Ala Leu Asn Cys 2385 2390 2395 < 210 > 11 < 211 > 14S3 < 212 > PKT < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 11 Thr Gly Phe Lys Leu Leu Wing Wing Ser Gly Leu Thr Arg Cys Gly Arg 1 5 10 15 Gly Gly Leu Val Val Thr Glu Thr Ala Val Lys lie lie Lys Ty His 20 25 30 Ser Arg Thr Phe Thr Leu Gly Pro Leu Asp Leu Lys Val Thr Ser Glu 35 40"45 Val Gln Val Lys Lys Ser Thr Glu Gln Gly His Ala Val Val Ala Ala 50 - 55 SO Xeu Cys Ser Gly Val lie ieu.Meb Arg Pro His Pro Pro Ser Leu Val 65 70 75 · 80 Asp Val Leu Leu. Bys Pro Gly Leu Asp Thr Thr Pro Gly lie Gln Pro 85. 90 55 Gly His Gly Ala Gly Asn Met. Gly Val Asp Gly Ser lie Trp Asp Phe 100 105 110 Glu Thr Wing Pro Thr Lys Wing Glu Leu Glu Leu Ser Lys Gln lie lie. 115 120 125 Gln Ala Cys Glu Val Arg Arg Gly Asp Ala Pro Asn Leu Gln Leu Pro 130: 135 - 140 Tyr Lys Leu Tyr Pro Val Arg Gly Asp Pro Gln Arg His Lys Gly Arg 145 - · - 150 155,. 160 Leu lie Asn Thr Arg Phe Gly Asp Leu Pro Tyr Lys Thr Pro Gln Asp 165 ~ 1-70 175 Thr Lys Ser Ala lie His Ala Ala Cys Cys Leu His Pro Asn Gly Ala 180 185. 190 Pro Val Ser Asp. Gly Lys Ser Thr Leu Gly Thr Thr Leu Gln His Gly 195 200 - 205 Phe Glu Leu Tyr Val Pro Thr Val Pro Tyr Ser Val Met Glu Tyr Leu 210 215 220 Asp Ser Arg Pro Asp Thr Pro Phe Mst Cys Thr Lys His Gly Thr Ser 225 -| '230 235. 240 Lys Ala Ala Ala Glu Asp Leu Gln Lys Tyr Asp Leu Ser Thr Gln Gly 245 250 255 Phe Val Leu Pro Gly Val leu Arg Leu Val Arg Arg Phe Phe Gly 250 265 270 His Gly Lys Ala Pro Pro ieu Phe Leu Pro Ser Thr Tyr Pro Wing 275 280 2S5 Lys Asn Ser Mefe Wing Gly Asn Gly Gln Arg Phe Pro Thr Lys Asp 290 295 300 Val Gln Ser Pro Glu Asp Glu Met. Cys Ala Arg Ala Val Bys 305 310 315. 320 Glu Asn Trp Gln Thr Val Thr Pro Cys Thr Leu Lys Lys Gln Tyr Cys 325 330 335 Ser Lys Pro Lys Thr Arg Thr Leu Gly Thr Asn Asn Phe Al 340 345 350 Leu Ala His Arg Ser Ala Leu Ser Gly Val Thr Gln Wing Phe Het Lys 355 360 365 Lys Wing Trp Bys Ser Pro Wing Leu Gly Lys Asn Lys Phe Lys Glu 370 375 380 Leu His Cys Thr Val Wing Gly Arg Cys Leu Glu Wing Asp Leu Wing Ser 385 3S0 395 400 Cys Asp Arg Ser Thr Pro Ala Val Arg Trp Phe Val Wing Asn Leu 405 410 415 Leu Tyr Glu Leu Wing Gly Cys Glu Glu Tyr Leu Pro Ser Tyr Val Leu 420 425 430 Asn Cys Cys His Asp Leu Val Wing Thr Gln Asp Gly Wing Phe Thr Lys 435 440 445 Arg Gly Gly Leu Ser Gly Asp Pro Val Thr Se Val Ser Asn Thr 450 455 460 Val Tyr Ser Leu Val Tyr Ala Glai His Mefc Val Leu Ser Ala Leu 4S5 470 475 480 Lys Me Gly His Glu Gly Leu Lys Phe Leu Glu Glu Gln Leu Lys 485 490 435 Phe Glu Asp Leu Leu Glu Gln Pro Met Leu Val Tyr Ser Asp Asp 500 505 510 Leu Val Leu Tyr Ala Glu Arg Pro Thr Phe Pro Asn Tyr His Trp Trp 515. 520 525 Val Glu His Leu Asp Leu Met Leu Gly Phe Arg Thr Asp Pro Lys Lys 530 535 540 Thr Val Thr Asp Lys Pro Ser Phe Leu Gly Cys Arg Glu Ala 545 55Q 555 560 Gly Arg Gln Leu Val Pro Asn Arg Asp Arg Leu Ala Ala Leu Ala 565 570 575 Tyr His Met Lys Wing Gln Asn Wing Ser Glu Tyr Tyr Wing Wing Wing Wing 580 585 590 Wing Wing Leu Met Asp Ser Cys Wing Cys Asp His Asp Pro Glu Trp 595 600 605 Tyr Glu Asp Leu Cys Gly Wing Arg Cys Wing Arg Gln Asr > Gly 610 615 620 Tyr Ser Phe Pro Gly Pro Wing Phe Ehe Met Ser Met Trp Glu Lys Leu 625 630 635 640 Arg Ser nis Asn Glu Gly Lys Lys Phe Arg His Cys Gly Cys Asp 645 eSO "655 Ala lys Ala Asp Tyr Ala Ala Ala Cys Gly Leu Asp Leu Cys Leu Phe 560 665 670 His Ser His Phe His Gln His Cys Pro Val Thr Leu Ser Cys Gly His 675 680 685 His Ala Gly Ser I »ys Glu Cys Ser Glu Cys Gln Ser Pro Val Gly Wing 690 695 700 Gly Arg Ser Pro Leu Asp Wing Val Leu Lys Gln Pro Tyr Lys Pro 705 710 715 720 Pro Arg Thr Val Met Lys Val Gly Asn Lys Thr Thr Ala Leu Asp 72S 730 735 Pro Gly Arg Tyr Gln Ser Arg Arg Gly Leu Val Wing Val Lys Arg Gly 745 750 Wing Gly Asn Glu Val Asp Leu Ser Asp Gly Asp Tyr Gln Val Val 755 760 765 Pro Leu Leu Pro Thr Cys Lys Asp Asn Met Val Lys Val Ala Cys 770 '775 780 Asn Val Leu Leu Ser Lys Phe Val Gly Pro Pr Gly Ser Gly Lys 7B5 790 755 - 800 Thr Thr Trp Leu Leu Ser Gln Val Gln Asp Asp Asp Val Tyr Thr 805 810 815 Pro Thr His Gln Thr Met Phe Asp Val Be Wing Leu lys Val Cys 820 825 830 Arg Tyr Ser 'Val Pro Gly Wing Ser Gly Leu Pro Phe Pro Pro Pro Wing 835 840 845 Arg Ser Gly Pro Trp Val Arg Leu Wing Ser Gly His Val Pro Gly 850 855 860 Arg Val Ser Tyr Leu Asp Glu Wing Gly Tyr Cys Asn His Leu Asp 865 870 875 680 Leu Arg Leu Leu Ser Lys Thr Pro Leu Val Cys Leu Gly Asp Leu Gln 885 890 395 Gln Leu His Pro Val Gly Phe Asp Ser Xaa Cys Tyr Val Phe Asp Gln 900 905 910 Met Pro Gln Lys Gln Leu Thr Thr Tyr Arg Phe Gly Pro Asn 315 920 925 Cys Ala Ala Gln Pro Cys Tyr Arg Glu Lys Leu Glu Ser Lys Wing 930 935 940 Arg Asn Thr Arg Val Val Phe Thr Thr Arg Pro val Wing Phe Gly Gln 94H 950 955 950 Val Leu Thr Pro Tyr His lys Asp Arg Gly Ser Ala Thr 9S5"970 575 Asp Being Ser Gln Gly Wing Thr Phe Asp Val Thr Lau His Leu Pro 980 985 990 Ser Pro Lys Ser Leu Asn Lys Ser Arg Ala Leu Val Ala Thr A 995 1000 1005 Wing Arg His Gly Leu Phe Tyr Asp Pro His Asn Gln Leu Gln Glu 1010 1015 1020 Phe Phe Asn Leu Thr Pro Glu Arg Thr Asp Cys Asn Leu Val Phe Ser 1025 1030 1035 1040 Cys Gly Asp Glu Leu Val Val Leu Asn Wing Asp Asn Wing Val Thr Thr 1045 1050 · 1055 Val Ala Lys Ala Leu Glu Thr Gly Pro Ser Arg Phe Arg Val Ser Asp xoeo ao65 xo7o Pro Axg Cys Lys Ser Leu Leu Ala Ala Cys Ser Ala Ser Leu Glu Gly 1075 10S0 · 1085 Ser Cys Met Pro Leu Pro Gln Val Ala His Asn Leu Gly Phe Tyr Phe 1030 1095 - 1100 Ser Pro Asp Ser Pxo Val Phe Wing Pxo Leu Pro Lys Glu Leu Wing Pronos 1110 1115 1120 His Txp Pro Val Val Thr His Gln Asn Asn Arg Wing Trp Pro Asp Arg 1125 1130 1135 Leu Val Ala Ser Met Arg Pro lie Asp Wing Arg Tyr Ser Lys Pro Met 1140 1145 1150 Val Gly Wing Gly Tyx Val Val Gly Pxo Ser Thr Phe Leu Gly Thr Pro 1155 1160 11S5 Gly Val Val Ser Tyr Tyr Leu Thr Leu Tyr lie Axg Gly Glu Pxo Gln 1170 1175 1180 Wing Leu Pro Glu Thr Leu Val Ser Thr Gly Arg lie Wing Thr Asp Cys 11B5 1190"ll95 1200 Arg Glu Tyx Leu Asp Ala Ala Glu Glu Glu Ala Ala Lys Glu Leu Pro 1205 1210 1215 His Wing Phe lie Gly Asp Val Lys Gly Thr Thr Val Gly Gly Cys His 1220 1225 1230 His lie Thr Ser Lys Tyr Leu Pro Arg Ser Leu Pro Lys Asp Ser Val 1235 1240 1245 Wing Val Val Gly Val Ser Ser Pro Gly Arg Ala Wing Lys Ala "val Cys 1250 1255 1260 Thr Leu Thr Asp Val Tyr Leu Pro Glu Leu Arg Pro Tyr Leu Gln Pro 1255 1270 1275 1280 Glu Thr Wing Ser Lys Cys Trp Lys Leu Lys Leu Asp Phe Arg Asp Val 1285 1290 - 1295 Arg Leu Met Val Trp Lys Gly Wing Thr Wing Tyr Phe Gln Leu Glu Gly 1300 ~ 1305 1310 Leu Thr Trp Ser Ala Leu Pro Asp- Tyr Ala Arg Phe lie Gln Leu Pro 1315 1320 1325 Lys Asp Ala Val Val Tyr lie Asp Pro Cys lie Gly Pro Ala Th Ala 1330 1335 1340 Asn Arg Lys Val Val Arg Thr Thr Asp Trp Arg Ala Asp Leu Ala Val 1345 1350 1355 ~ 1360 Bir Pxo Tye Asp Ty Gly Ala. Gln Asn lie- Leu Thr Thr Ala Trp Phe "l365 1370 1375 Glu Asp Leu Gly Pro Gln. Trp Lys lie Leu Gly Leu Glu. Pro Phe Arg 1380 1385 - 1390 Arg Wing Phe Gly Phe Glu Asn Thr- Glu Asp Trp Wing lie Leu Wing Arg 13S5 1400 | 1405 Arg Me -Asn Asp Gly Lys Asp Tyr Thr Asp Tyr Asn Trp Asn Cys Val 1410 1415 1420 Arg Glu Arg Pro His Ala lie Tyr Gly Arg Ala Arg Asp His Thr Tyr 1425 1430 1435 1440 His Phe Wing Pro Gly Thr Glu Leu Gln. Val Glu Leu Gly Lys Pro Arg 1445 1450 1455 Le Pro Pro Gly Gln Val Pro 1460 < 210 > 12 < 211 > -249 < 212 > PRT < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 12 Met Gln Trp Gly Tyr Cys Gly Val Lys Ser Wing Ser Cys Ser Trp Thr 1 '5 10 15 Pro Ser Leu Be Ser Leu Leu Val Trp Leu lie Leu Leu Phe Ser Leu 20 25 30 Pro Tyr Cys Leu Gly Ser Pro Ser Gln Asp Gly Tyr Trp Ser Ser Phe 35 40 45 Ser Glu Trp Phe Wing Pro Arg Phe Ser Val Arg Wing Leu Pro Phe Thr 50 S5 60 Leu Pro Asn Tyr Arg Arg Ser Tyr Glu Gly Leu Leu Pro Asn Cys Arg 65 70 75 80 Pro Asp Val Pro Gln Phe Wing Phe Lys His Pro Leu Gly Met Leu Trp 85 90. 95 Kis Met Arg Val Ser Gln. Leu lie Asp Glu Met Val Ser Arg Arg I-le 100 X05. · Xio Ty Gln Thr Met Glu His Ser Gly Gln Wing Wing Trp Lys Gln Wing Val 115 120 125 Gly Glu Wing Thr Leu Thr Lys Leu Ser Arg Leu Asp lie Val Thr Hís 130 - 135 140 Phe Gln His Leu Ala Ala Val Glu Ala Asp Ser Cys Arg Phe Leu Ser '145 · - 150 - · 155 - -. 1S0 Being Arg Leu Val Met Leu Lys Asn Leu Wing Val Gly Asn Val Being Leu- • 1S5 170. . 175 Gln Tyr Asn Thr Thr Leu Asp Arg Val Glu Leu Iie one Pro Thr Pro 180 185 1S0 Gly Thr Arg Pro Lys Leu Thr Asp Phe Arg Gl Trp Leu lie Ser Val 1S5 200: 205 His Wing Ser lie Phe Ser Ser Val Wing Ser Ser Val Thr Leu Phe lie 210 215 · 220 Val Leu Trp Leu Arg lie Pro Ala Leu Arg Tyr Val Phe Gly Pie His 225 230 235 240 Trp Pro Thr Ala Thr His Bis Ser Ser 245 < 210 > 13 < 211 > 265 < 212 > par < r213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 13 Met Ala His Gln Cys Ala Arg Phe His Phe Phe Leu Cys Gly Phe lie 1 - 5 10 X5 Cys Tyr Leu Val Eis Ser Ala Leu Ala Ser Asn Ser Ser Thr Leu 20 25 30 Cys P s Trp Phe Pro Leu Wing His Gly Asn Thr Ser Phe Glu. Leu Thr '35 40 45 lie Asn Tyr Thr lie Cys Ket Pro Cys Ser Thr Ser Gln Ala. Ala Arg 50 - 55 SO Gln Arg Leu Glu Pro Gly Arg Asn Met Trp Cys Lys lie Gly Tyr A.SP 65 70 75 80 Arg Cys Glu Glu Arg Asp His As Glu Leu Leu Met Pro lie Pro S¾r 85 90 95 Gly Tyr Asp Asn Leu Lys Leu Glu Gly Tyr Tyr Wing Trp Leu Wing Phe - "100 105 * 1X0 Leu Ser Phe Ser Tyr Wing Wing Glm Phe His Pro Glu Leu Phe Gly lie • 115 120 - 125. Gly Asn Val Ser Arg Val Phe Val Asp Lys Arg His Gln Phe lie Cys 130 · - 135 140 - Wing Glu His Asp Gly Gln Asn Ser Thr Val Ser Thr Gly His Asn lie 145 '150 155 160 Be Ala Leu Tyr Ala Ala Tyr Tyr His His Gln lie Asp Gly Gly Asn 165 170 175 Trp Phe His Leu Glu Trp Leu Arg Pro Leu Phe Ser Ser Trp Leu Val 180 185 - < -190 Leu Asn lie Ser Trp Phe Leu Arg Arg Ser Pro Val Ser Pro Val Ser- 195 200 205 Arg Arg lie Tyr Gln lie Leu. Arg Pro Thr Arg Pro Arg Leu Pro Val 2X0 | 215 220 Ser Trp Ser Phe Arg Thr Ser XIe Val Ser Asp Leu Thr Gly Ser Gln 225 230 235 240 Gln Arg Lys Arg Lys Phe Pro Ser Glu Ser Arg Ero Asn Val Val Lys 245 250 255 Pro Ser Val Leu Pro Ser Thr Ser Arg 260 265 < 210 > 14 < 211 > 1B3 c212 > PJT < : 213 > Porcine Reproductive and Respiratory Syndrome Virus 400 > 14 Met Ala Ala Ala Thr Leu Phe Leu Leu Ala Gly Ala Gln Tyr He Mefc 1 5 10 15 Val Ser Glu Wing Phe Wing Cys Lys Pro Cys Phe Ser Thr His Leu Ser 20 25 30 Asp JLle Glu Thr Asn Thr Thr Wing Wing Wing Wing Phe Mefc Val Leu Gln 35 40 45 Asp lie Asn Cys Leu Arg Pro His Gly Val Ser Wing Ala Gln Glu Glu 50 55 60 lie Pro Phe Gly Lys Ser Ser Gln Cys Arg Glu Wing Val Gly Thr Pro 65 - 70 75 80 Gln Tyr lie Thr lie Thr Wing Asn Val Thr Asp Glu Ser Tyr Leu Tyr 85 90 95 Asn. Wing Asp Leu Leu Met Leu Be Wing Cys Leu Phe His Wing Ser Glu 100 - 105 · 110 Mefc Ser Glu Lys Gly Phe Lys Val lie Phe Gly Asn Val Ser Gly Val 115 120 | 125 Val Ser Wing Cys Val Asn Phe Thr Asp Tyr Go Ala His Val Thr Gln 130 .-, 135 ··. 140 His Thr Gln Gln His His Leu Val lie Asp His lie Arg Leu Leu His 1 ^ 5 - 150 ·. 155 150 Phe Leu Thr Pro Ser Wing Mefc Arg Trp Wing Thr Thr lie Wing Cys Leu 165 170 175 Phe Ala lie Leu Leu Ala lie 180 «210 > 15"<211> 201 <212> PRT <213> Porcine Reproductive and Respiratory Syndrome Virus <400 =» 15 Met Arg Cys Ser His Lys Leu Gly Arg Phe Leu Thr Pro His Ser Cys 1 5 10 15 Phe Trp Tr Phe Phe Leu Leu Cys Thr Gly Leu Ser Trp Ser Phe Wing 20 25 30 Asp Gly Asn Gly Asn Being Ser Thr Tyr Gln Tyr lie Tyr Asn Leu Thr 35"40 45 lie Cys Gln Leu Asn Gly Thr Asp Trp Leu Ser Ser His Phe Gly Trp 50 55 60 ¾la Val Glu Thr Phe Val Phe Tyr Pro Val Wing Thr His lie Leu Ser S5 70 75 80 Leu Gly Phe Leu Thr Thr Ser His Phe Phe Asp Wing Leu Gly Leu Gly Wing Val Ser Thr Wing Gly Phe Val Gly Gly Arg Tyr Val Leu Cys Ser 100 105 110 Val Tyr Gly Ala Cys Ala Phe Ala Ala Phe Val Cys P e Val lie Arg 115 120 · 125 · Ala Ala L s Asn Cys Met Ala Cys Arg Tyr Ala Arg Thr Arg Phe Thr 130 135 140 Asn Phe lie Val Asp 3n Arg Gly Arg Val His Arg Trp Lys Ser Pro 145 - 150 155 · ISO lie Val Val Glu Lys Leu Gly Lys Wing Glu Val Asp Gly Asn Leu Val 165 170 175 Thr lie Lys His Val Val Leu Glu Val Val Lys Gln Pro Pro Leu Tbr 180 185 190 Arg Thr Ser Ala Glu Gln Trp Glu Ala 195 200 - < 210 > 1 & . T211 > 173 < 212 > PRT < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 16 Met Gly Gly Leu Asp Asp Ehe Cys Asa Asp Pro lie Ala Wing Gln Lys 1 5 10 15 Leu Val Leu Wing Phe Se lie Thr Tyr Thr Pro lie Met lie Tyr Wing 20 _ 25 30 Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His lie Leu lie 35 40. · 45 Phe Leu Asn Cys Ser Phe Thr Phe Gly Tyr Met Thr Tyr Val His Phe 50 55 eo Gln Ser Thr Asn Arg Val Ala Leu Thr Leu Gly Ala Val Val Ala Leu 65 70 75 80 Leu Trp Gly Val Tyr Ser Phe Thr Glu Ser Trp Lys Phe lie Thr Ser 85 SO - 95 Arg Cys Arg Leu Cys Cys Leu Gly Arg Arg Tyr lie Leu Ala Pro Wing 100 105 no His His Val Glu Be Ala Wing Gly Leu His Ser lie Ser Wing Be Gly 115 120 125 Asn Arg Ala Tyr Ala Val Arg Lys Pro Gly Leu Thr Ser Val Asn Gly 130 135 140 Thr lien Val Pro Gly leu Arg Ser Leu Val Leu Gly Gly Lys Arg Ala 145 150 155 160 Val Lys Arg Gly Val Val Asn. Leu Val Lys Tyr Gly Arg 165 no < 210 > 17 < 211 > 128 < 212 > PRT < 213 > Porcine reproductive and respiratory syndrome virus < 400 > 17 Met Ala Gly Lys Asn Gln Ser Gln Lys Lys Lys Ser Thr Ala Pro '1 5 10 15 Met Gly sn Gly Gln Pro Val Asn Gln Lea Cys Gln Leu Leu Gly Wing 20 25 30 I lie Lys Ser Gln Arg Gln Gln Pro Arg Gly Gly Gln Ala Lys Lys 35 40 45 Lys Lys Pro Gln Lys Pro His Phe Pro Leu Ala Wing Gln Asp Asp lie 50 55 60 Arg His His Leu Thr Gln Thr Glu Arg Ser Leu Cys Leu Gln Ser lie 65 · 70 - - 75 ~. 80 Gln Thr Wing Ene Asn Gln Gly Wing Gly Thr Wing Being Leu Being Ser- 85 90 95 Gly Lys Val Ser Phe Gln Val Glu Phe Met Leu Pro Val Wing His Thr 100 IOS 110 Val Arg Leu lie Arg Val Thr Ser Thr Ser Ala Ser Gln Gly Ala Ser 115 120 125 < 210 > 18 -r211 > 245 < 212 > PRT < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 18 Thr Pro Ser Leu Ser Ser Leu Leu Val Trp Leu lie Leu Ser Phe Ser Lau 20 25 30 Pro Tyr Cys Leu Gly Ser Pro Ser Gln Asp Gly Tyr Trp Ser Ser Phe 35 40 45 ser Glu Trp Phe Wing Pro Arg Phe Ser Val Arg Ala Leu Pro? He Thr 50 55 60 leu Pro Asn Tyr Arg Arg Ser Tyr Glu Gly Leu Leu Pro Asn Cys Arg 65 70 75 80 Pro Asp Val Pro Gln Phe Wing Phe Lys His Pro Leu Gly Met Leu Trp 85 90 95 Eis Met Arg Val Ser His Leu He Asp Glu Met Val Ser Arg Axg He 100 105 110 Tyr Gln Thr Met Glu His Ser Gly Gln Ala Wing Trp Lys Gln Val Val 115 120 ~ 125 Gly Glu. Wing Thr Leu Thr Ly3 Leu Ser Gly Leu Asp He Val Thr His | 130 135 140 -Phe Gln. Xaa Leu Ala Ala Val Glu Ala Asp Ser Cys Axg Phe Leu Ser 145 150 155 160 Ser Arg Leu Val ¾et Leu Lys Asn Leu Wing Val Gly Asn Val Ser Leu 155 170 175 Gln Tyr -Asn Thr Thu Leu Asp Arg Val Glu Leu He Phe Pro Thr Pro 180 185 190 Gly Thr Arg Pro Lys Leu Thr Asp Phe Arg Gln Trp Leu He Ser Val 195 20.0 ~ 205 | · • His Wing Ser He Phe Ser Ser Val Ala Ser Ser Val Thr Leu Phe He 210 215 220 Val Leu Trp Leu Arg Ha Pro Ala Leu Arg Tyr Val Phe Gly Phe His 225 230 235 240 Trp Pro Thr Ala Thr His His Ser Ser 245 < 210 > 19 < 211 > 265 < 212 > PRT: 213 > Porcine reproductive and respiratory syndrome virus. < 400 > 13 Met Ala His Gln Cys Ala. Arg Phe His Phe Phe Leu Cys Gly Phe I-le 1 5 10 15 Cys Tyr Leu Val His Ser Wing Leu Wing Being Asn Being Ser Thr Leu 20 25 30 Cys Phe Trp Phe Pro Leu Wing His Gly Asn Thr Ser Phe Glu Leu Thr 35 40 45 He Asn Tyr Thr He Cys Met Pro Cys Ser Thr Ser Gln Ala Ala Arg 50 55 SO Gln Arg Leu Glu Pro Gly His Ser Ket Trp Cys Lys He Gly His Asp 65 70 - 75 80 Arg Cys Glu Glu Arg Asp His Asp Gl Leu Leu! -5at Pxo He Pro Ser 85 90 95 Gly Tyr Asp Asn Leu Lys Leu Glu Gly Tyr Tyr ¾la Trp Leu Ala Phe - '100 105 -. 110 Leu Ser Phe Ser Tyr Ala Ala Gln Phe His Pro Glu Leu Phe Gly lie: -115 - 120 '125 Gly A & Val Ser Arg Val Phe Val Asp Lys Arg His Gln Phe He Cys 130 135 140 Ala Glu His Asp Gly His Asn Ser Thr Val Ser Thr Gly His Asn He - 145 - '· 150 155 -. ISO Be Ala Leu Tyr Ala Ala lyr Tyr His His Gln He Asp Gly Gly Asn 1S5 · 170 - »· 175 Trp Phe His Leu Glu Trp Leu Arg Pro Leu P e Ser Ser Trp Leu Val. - 180 | - 185 190 Leu Asn lie Ser Trp Phe Leu Arg Arg Ser Pro Val Ser Pxo Val Ser 195 200. · 205 Arg He lyr Gln He Leu Arg Pro Thr Arg Pro Arg Leu Pro Val 210, 215 · 220 Ser Trp Ser Phe Axg Tlix Ser lie Val Ser Asp Leu Thr Gly Ser Gln -225 230. . 235 240 Gln Arg Bys Arg Lys Phe Pro Ser Glu Ser Arg Pro Asn Val Val Lys 245 250 255 Pro Ser Val Leu. Pro Ser Thr Ser Arg 260 2S5 < 210 > 20 < 211 183 < 212 > PRT < 213 > Porcine Reproductive and Respiratory Syndrome Virus < 400 > 20 Ket Ala Ala Ala Tlir Leu Phe Phe Leu Ala Gly Ala Gln His He Met 1 5 10 - 15 Val Ser Glu Ala Phe Ala Cys Lys Ero Cys Phe Ser Thr His Leu Ser 20 25 30 Asp He Lys T r Asn Thr Thr Ala Ala Ala Gly Phe ffefc Val Leu Gln 35 40 4H Asp He Asa Cys Pee Arg Pro His Gly Val Ser Ala Ala Gln Glu Lys 50 55 60 lie Ser Phe Gly Lys Ser Ser Gln Cys Arg Glu Ala Val Gly Thr Pro 65 · 70 75 - 80 Gln Tyr lie Thr lie Thr Ala Asa Val Thr Asp Glu Ser Tyr Leu Tyr 85 - 90 95 Handle Wing Asp Leu Leu Mst Leu Be Wing Cys Leu Phe Tyr Wing Ser Glu 100 - 105 · · 110 Met Ser Glu Lys Gly Phe Lys Val lie Phe Gly Asn Val Ser Gly Val • 115 120 125 Val Ser Wing Cys Val Asn Phe Thr Asp Tyr Val Wing His Val Thr Gln 130 135 140 His Thr Gln Gln His His Leu Val Val Asp His lie Arg Leu Leu His l S |||| 150 -.- 155. · - 160 Phe Leu Thr Pro Ser Wing Mefc Arg Trp Wing Thr Thr lie Wing Cys Leu 165 - 170 | > VIS Leu Ala xle Leu Leu Ala lie 180 < 210 > 21- < 211 > 201 < 212 > PRT < 213 > Porcine reproductive and respiratory syndrome virus < 400 > 21 Met Arg Cys Ser His Lys Leu Gly Arg Ser Lsu Thr Pro His Ser Cys i - s 10 15 Phe Trp Trp Leu Phe Leu Leu Cys Thr Gly Lsu Se Trp Ser Phe Wing 20 25 30 Asp Gly Asn Gly Asp Ser Ser Thr Tyr Gln Tyr lie Tyr Asp Leu Thr 35 40 45 lie Cys Glu Leu Asn Gly Thr Asp Trp Leu Ser His Phe Gly Trp 50 55 60 Wing Val Glu Thr Phe Val Phe Tyr Pro Val Ala Thr His lie Leu Ser 65 > 70 75 '80 Leu Gly Phe Leu Thr Thr Ser His Phe Phe Asp Ala Leu Gly Leu Gly - 85 90 95 Ala Val Ser Thr Ala Gly Phe Val Gly Gly Arg Tyr Val Leu Cys Ser 100 105 ~ 110 Val Tyr Gly Ala Cys Ala Phe Ala Ala Phe Val Cys Phe Val lie Arg 115 120 125 Ala Ala Lys Asn Cys Hefc Ala Cys Arg Tyr Ala Arg hr Arg Ehe T r 130 135 140 Asn Phe lie Val Asp Asp Arg Gly Arg Val fíis Arg Trp Lys Ser Pro 145"150 155 160 lie Val Val Glu Lys Leu Gly Lys Wing Glu Val Asp Gly Asn Leu Val 1S5 170 175 Tbxr lie Lys His Val Val Leu Glu Gly Val Lys Ala Gln Pro Leu Xhr 180 185 - 190 Arg Xiir Ser Ala Glu Gln Xrp Glu Ala 135 - 200 27 <210> 22 - < 211> 750 · · 21 = > DNA < 213 > OKE 2 Agent Lelystad PRRSV < 400 >?. 22 | ATGCAAIGGG GXCACTGTGG AGXAAAAXCA GCCAGCXGXX CGXGGACGCC XTCACxGAGX 60 TCCTXGTXAG XGXGGXIGAX AXTGCCAXXT XCC XGCCAX ACEGTXIGGG XXCACCGXCG 1-20 CAGGAXGGIX ACXGGXCXXX CTTCTCAGAG TGGTTTGCTC CGCGCTXCXC CGIXCGCGCX 180-CTGCCAXXCA CXCTCCCGAA CXATCGAAGG TCCTA GAAG GCXTGTTGCC CAACTGCAGA 240 CCGGAXGXeC C & CLSATTXGC AGXCAAGCAC CCAXXGGGyA IGX TTXGGCA CAXGCGAGTX 300 TCCCACXXGA TTGAXGAGAX GGTCTCXCGI CGCATTTACC AGACCATGGA A.CAXXCAGGX 3 SO 'CA & GCGGCCX GGAAGCAGGX GGTXGGIGAG GCCACTCTCA CGAAGCXGIC AGGGCTCGAT 420' AXAGI ACXC ???? CCAACA CCTGGCCGCA GIGGAGGCGG AxXCXXGCCG CTTTCTCAGC 480'-TCACGACTCG TGAXGCXAAA AAATCXTGCC GTTGGCAATG TGAGCCTACA GTACASGACC 540 ACGTXGGACC GCGXXGAGCI CAXCTTCCCC ACGCCAGGT CGAGGCCC & A GTTGACCGAT € 00 XXCAGACAAX GGCTCATCAG TGXGCACGCX XCCAIXXIXX CCXCXGXGGC XXCATCTGTX 6S0 ACCTTGTXCA XAGXGCITXG GCXTCGAAXX CCAGCXCXAC GCTAIGXXII TGGxITCCAI 720 XGGCCCACGG CAACACATCA TXCGAGCXGA · 750 < 210 > 23 < : 211 > 7S8 < 212 > AD1I < 213 > 0RF3 of Agent Lelystad of PRE.SV < 400 > 23 ATGGCTCATC AGXGIGCACG CXXCCAXXTX TTCCTCTGTG GCXXCATCXG XXACCTTGTX 60 CATAGTGCTT XGGCCTCGAA TTCCAGCXCI ACGCXAXGXX 2TXGGTXXCC AXXGGCCCAC 120 GGCAACACAI CA ICGAGCT GACCATCAAC TACACCATAT GCAXGCCCTG rTCXACCAGT 180"CAAGCGGCTC OCCAAAGGCI CGAGCCCGG CGTAACR! Gx GGTGCAAAAX SGGGCATGAC 240 AGGTGXGAGG AGCGxGACCA TGAXGAGXXG XTAAXGXCCA XCCCGXCCGG GXACGGACAA 300 CXCAAACXXG AGGGTXAXXA XGCXIGGCXG GCXXOTXTGX CCTXTXCCXA CGCGGCCCAA 360 XTCCAXCCGG AGXXGXXCGG GAXAGGGAAX GXGTCGCGCG XCXXCGIGGA CAAGCG & CAC 420 CAGTXCAXTX GTGCCGAGCA IG & TGGACAC AAXTCA & CCG XATCXACCGG ACACAACAXC 480 TCCGCATXAT AXGCGGC ATA TTACCACCAC CAAATAGACG GGGGCSAXXG GXXCCATTXG 540 GAAXGGCIGC GGCCACTCTT TTCTXCCXGG CTGGTGCTCA ACAXAXCAXG GXXICXGAGG 500 CGTTCGCCIG TAAGCCCXGX XxCICGACGC AXCTATCAGA TAXXGAGACC AACACGACCG 660 CGGCTGCCGG TTXCATGGTC CTSCAGGACA T ¾¾TIGTTT CCGACCTCAC GGGGTCTCAG 720 CAGCGCa &amp!; GA GAAa &TT CC TTCGGS & aGT CGTCCCAATG TCGTGAAGCC GICGG7ACTC 780 CCCAGTACAT CACGATAA - 798 < 210 > 24 < 211 > 506 < 2X2 > < 2X3 > ORF5 of the Lelystad Agent of PRRSV < 400 > 24 ATGAGATGTT CTCACAAATT GGGGCGTTTC TTGACTCCGC ACTCTIGCTT CTGGTGGCTT 60 TTTTTGCTGÍ GTACCGGCTS GTCCTGGTCC TTSGCCGATG GCAACGGCGA CAGCTCGACA 120 TACCAATACA TATATAACTT GACGATATGC GAGCTGAATG GGACCGACTG GTTGTCCAGC 80 · CAXTTTGGTT GGGCAGTCGA GACCXTTGTG CTIXACCCGG TTGCCftCTCA TATCCTCTCA 240 CTGGGTTÜTC TCACAACAAG CCATTTTTT GACGCGCTCG GTCTCGGCGC TGTATCCACT 300 GCAGGA3TTG XTGGCGGOCG GTACGTACTC TGCAGCGTCT ACGGCGCTTG TGCTCTCGCA 3S0 GCGTTCGTAT GTXTTGXCAT CCGTGCTGCT AAAAAT GCA TGGCCTGCCG CTATGGCCGT TA £ 420 ACCCGG2 Ca¾ACXTCAT IGTGGACGAC CGSGGGAGAG TTCATCGRTG GSAGTCTCCA 480 ATAGTGGTAG AAAAATIGGG CAA¾GCCGAA GTCGATGGCA ACCTCGTCAC CATCA¾ACA1 540 GTCGTCCTCG AAGGGGTTAA AGCTC & ACCC TTGACGAGGA CTTCGGCTGA GCAATGGGAG 500 GCCTAG SOS 210 > 25 < 211 > 21 < 212 > DNA s213 =. ORF2 of Site I of PRRSV Attenuation < 4G0 > 25 TACTGGTCXT TCTTCTCAGA G < 210 > 26 < 211 > 21 < 212 > DNA < 213 > ORF2 of Site II of PRRSV Attenuation < 400 > 26 ACAATTTGCA GTCAAGCACC C < = 210 > 27 < 211 > 21 < 212 > DNA < 213 > 0RF2 of Site III of PRRSV Attenuation < 400 > 27 ATXGGGyTG TT TGGCACA T 21 ' < 21G > 28 < 211 > 21 < 212 > DNA < 213 > 0RF3 of the PRRSV Attenuation Site < 400 > 28 GTIGTAAT GTCCATCCCG! T C < 210 > 29 < 211 > 21 < 2X2 > < 213 > 0RF5 of the PRRSV Attenuation Site • < 400¾ 29 GACCTTTGTG CTTTACCCGG T

Claims (42)

1. The attenuated European PRRS virus encoded by a nucleic acid comprising ORF1, 0RF2, 0RF3, 0RF4, 0RF5, 0RF6 and 0RF7, characterized in that: a) ORF2 comprises at positions 11915-11935 at least one of the nucleotides as described in Table 1: at positions 12058-12078 at least one of the nucleotides as described in Table 3: T A C C C C C T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C G G G A A G G G A G G G G A A T G T G G and / or a deletion at or in the positions and / or b) 0RF3 comprises at positions 12660-12680 at least one of the nucleotides as described in Table 4: and / or a deletion in said position (s) and / or c) ORF5 comprises at positions 13684-13704 at least one of the nucleotides as described in Table 5: and / or a deletion in said position (s).

2. The European PRRS virus, attenuated according to claim 1, characterized in that: a) 0RF2 comprises a C, A or G at position 11925 and / or a C, T or A at position 12047 and / or an A, C or G at position 12068 or a suppression at said position (s) and / or b) 0RF3 comprises an A, C or G at position 12670 or a deletion at dich (s) position (s) and / or c) 0RF5 comprises a G, A or T at position 13694 or a deletion at that position.

3. The European PRRS virus, attenuated according to claim 1 or 2, wherein the nucleic acid is further characterized in that: a) the 'ORF comprises a C at position 11925 and / or a T at position 12047 and / or a C at position 12068 or a deletion at said position (s) and / or b) the ORF3 comprises a C at position 12670 or a deletion at said position (s) and / or c) the 0RF5 comprises a T at position 13694 or a deletion at that position.

4. The European PRRS virus, attenuated according to any of claims 1 to 3, wherein the nucleic acid is further characterized in that: a) the 0RF2 comprises the nucleic acid as defined in SEQ ID NO: 1 and / or b) the ORF3 comprises the nucleic acid as defined in SEQ ID NO: 2 and / or c) 0RF4 comprises the nucleic acid as defined in SEQ ID NO: 3 and / or d) 0RF5 comprises the nucleic acid as defined in SEQ ID NO: 4, or a fragment, allelic variant, functional variant, variant based on the code of the degenerative nucleic acid, fusion molecule or a chemical derivative thereof.

5. The European PRRS virus, attenuated according to any of claims 1 to 4, wherein the nucleic acid is characterized in that: 'a) the ORF2 consists of the nucleic acid as defined in SEQ ID NO: 1 and / or b) the 0RF3 consists of the nucleic acid as defined in SEQ ID NO: 2 and / or c) ORF4 consists of the nucleic acid as defined in SEQ ID NO: 3 and / or d) ORF5 consists of the nucleic acid as defined in SEQ ID NO: 4.

6. The European PRRS virus, attenuated according to any of claims 1 to 5, wherein the nucleic acid is characterized in that it comprises the nucleic acid as defined in SEQ ID NO: 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a derivative chemical thereof.

7. The European PRRS virus, attenuated according to any of claims 1 to 6, wherein the nucleic acid is characterized in that it consists of the nucleic acid as defined in SEQ ID NO: 5.

8. The European PRRS virus, attenuated according to any of claims 1 to 3, wherein the nucleic acid is further characterized in that: a) 0RF2 comprises the nucleic acid as defined in SEQ ID NO: 6 and / or b) 0RF3 comprises the nucleic acid as defined in SEQ ID NO: 7 and / or c) ORF4 comprises the nucleic acid as defined in SEQ ID NO: 8 and / or d) 0RF5 comprises the nucleic acid as defined in SEQ ID NO: 9 and / or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

9. The European PRRS virus, attenuated according to any of claims 1 to 3 or 8, wherein the nucleic acid is further characterized in that: a) the 0RF2 consists of the nucleic acid as defined in SEQ ID NO: 6 and / or ) 0RF3 consists of the nucleic acid as defined in SEQ ID NO: 7 and / or c) 0RF4 consists of the nucleic acid as defined in SEQ ID NO: 8 and / or d) 0RF5 consists of the nucleic acid as defined in SEQ ID NO: 9.

10. The European, attenuated PRRS virus, wherein the PRRS virus is characterized in that: a) ORFla comprises the amino acid as defined in SEQ ID NO: 10 and / or b) ORFlb comprises the amino acid as defined in SEQ ID NO: 11 and / or c) ORF2 comprises the amino acid as defined in SEQ ID NO: 12 and / or d) ORF3 comprises the amino acid as defined in SEQ ID NO: 13 and / or e) ORF4 comprises the amino acid as defined in SEQ ID NO: 14 and / or) ORF5 comprises the amino acid as defined in SEQ ID NO: 15 and / or g) ORF6 comprises the amino acid as is defined in SEQ ID NO: 16 and / oh) 0RF7 comprises the amino acid as defined in SEQ ID NO: 17, or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion or a chemical derivative thereof.

11. The European PRRS virus, attenuated according to claim 10, wherein the PRRS virus is characterized in that: a) ORFla consists of the amino acid as defined in SEQ ID NO: 10 and / or) ORFlb consists of the amino acid as defined in SEQ ID NO: 11 and / or c) ORF2 consists of the amino acid as defined in SEQ ID NO: 12 and / or d) ORF3 consists of the amino acid as defined in SEQ ID NO: 13 and / or e) ORF4 consists of the amino acid as defined in SEQ ID NO: 14 and / or) 0RF5 consists of the amino acid as defined in SEQ ID NO: 15 and / or g) ORF6 consists of the amino acid as defined in SEQ ID NO: 16 and / oh) 0RF7 consists of the amino acid as defined in SEQ ID NO: 17.

12. The European, attenuated PRRS virus, wherein the PRRS virus is characterized in that: a) 0RF2 comprises the amino acid as defined in SEQ ID NO: 18 and / or b) 0RF3 comprises the amino acid as defined in SEQ ID NO : 19 and / or c) 0RF4 comprises the amino acid as defined in SEQ ID NO: 20 and / or d) 0RF5 comprises the amino acid as defined in SEQ ID NO; 21 and / or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

13. The European PRRS virus, attenuated according to claim 14, wherein the PRRS virus is characterized in that: a) ORF2 consists of the amino acid as defined in SEQ ID NO: 18 and / or b) 0RF3 consists of the amino acid as defined in SEQ ID NO: 19 and / or c) 0RF4 consists of the amino acid as defined in SEQ ID NO: 20 and / or d) 0RF5 consists of the amino acid as defined in SEQ ID NO: 21.

14. A nucleotide sequence coding for a virus according to any of claims 1 to 13.

15. A nucleotide sequence according to claim 14, wherein the nucleotide sequence has been modified to encode a virulence marker and / or a serological marker.

16. A nucleotide sequence according to claim 14 or 15, wherein the nucleic acid encoding the marker is located within any of the open reading structures that code for the structural viral proteins.

17. Method for the generation of a European PRRS virus, attenuated, alive, infectious, the method comprises the production of a recombinant nucleic acid comprising at least one copy of full-length DNA or a copy of RNA transcribed in vit.ro or a derivative of any of them, whereby said nucleotide sequence is a nucleotide sequence according to any of claims 14 to 16.

18. Method for the generation of a European PRRS virus, attenuated, alive, infectious, the method is characterized by the following steps: a) a PRRS virus according to any of claims 1 to 13 is used to infect a suitable cell line. b) PRRS virus is attenuated by means of cell culture passages.

19. Method according to claim 18, wherein the cell line is a Marc cell or a derivative thereof.

20. Method according to any of claims 18 or 19, wherein the PRRS virus is the virus according to any of claims 1 to 13.

21. Method according to any of claims 18 to 20, wherein the PRRS virus is the virus according to any of claims 5, 9, 11 or 13.

22. Cell line comprising a PRRS virus according to any of claims 1 to 13.

23. Cell line according to claim 22, wherein the cell line is a Marc cell or a derivative thereof.

24. Pharmaceutical composition comprising a PRRS virus according to any of claims 1 to 13, and a pharmaceutically acceptable carrier.

25. Use of a PRRS virus according to any one of claims 1 to 13, in the manufacture of a vaccine for the prophylaxis treatment of PRRS infections.

26. Method of attenuation of a European PRRS virus, characterized in that: a) the nucleotide sequence of the virus is modified by site-directed mutagenesis in at least one of the ORF2 positions corresponding to positions 130 to 150 and / or positions 252 to 272 and / or positions 273 to 293 of SEQ ID NO: 22; b) it is tested whether the resulting PRRS virus is attenuated or not.

27. Method of attenuation of a European PRRS virus, characterized in that: a) the nucleotide sequence of the virus is modified by site-directed mutagenesis in at least one of the ORF3 positions corresponding to positions 267 to 287 of SEQ ID NO: 23; b) it is tested whether the resulting PRRS virus is attenuated or not.

28. Method of attenuation of a European PRRS virus, characterized in that: a) the nucleotide sequence of said virus is modified by mutagenesis in at least one of the positions corresponding to positions 201 to 221 of ORF5 according to SEQ ID NO: 24; b) it is tested whether the resulting PRRS virus is attenuated or not.

29. Method according to any of claims 26 to 28, wherein the modification results in a change of the amino acid sequence of the encoded protein.

30. Method of compliance with any of claims 26 to 29, wherein the modification is a deletion or a substitution.

31. Method according to any of claims 26 to 30, wherein the sequence of each of ORF2, ORF3 and ORF5 is modified.

32. Method according to claim 31, wherein the sequence of ORF2 is modified to at least two, preferably at least three positions.

33. Method according to any of claims 26 to 32, wherein the modification results in one or more of the following characteristics: an ORF2 that codes for a protein having the amino acid substituted or deleted in one or more of the amino acid sequences , corresponding to positions 47, 88 and / or 95 of the amino acid sequence encoded by SEQ ID NO: 22; an ORF3 coding for a protein having substituted or deleted the amino acid corresponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23; and / or an F5 O coding for a protein having substituted or deleted the amino acid corresponding to position 71 of the amino acid sequence encoded by SEQ ID NO: '24.

34. Method according to claim 33, wherein the modification results in one or more, preferably all of the following characteristics: an ORF2 coding for a protein having serine in the position corresponding to position 47 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF2 coding for a protein having phenylalanine in the position corresponding to position 88 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF2 having leucine in the position corresponding to position 95 of the amino acid sequence encoded by SEQ ID NO: 22, an 0RF3 having proline at the position corresponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23, and / or an ORF5 having phenylalanine in the position corresponding to position 71 of the amino acid sequence encoded by SEQ ID NO: 24.

35. Method according to claim 34, wherein the modification results in one or more, preferably all of the following characteristics: an ORF2 having a C in the position corresponding to the position 140 of the SEQ ID NO: 22, a 0RF2 which has a T in the position corresponding to position 262 of SEQ ID NO: 22, an ORF2 having a C in the position corresponding to position 283 of SEQ ID NO: 22, an ORF3 having a C in position corresponding to position 277 of SEQ ID NO: 23, and / or an ORF5 having a T in the position corresponding to position 211 of SEQ ID NO: 24.

36. The European, attenuated PRRS virus obtainable by the method according to any of claims 26 to 35.

37. The attenuated European PRRS virus having an ORF2 which differs from the SEQ ID NO: 22 in one or more of the positions 130 to 150, and / or in one or more of the positions 252 to 272, and / or in one or more from positions 273 to 293.

38. European, attenuated PRRS virus having an ORF3 that differs from SEQ ID NO: 23 in one or more of positions 267 to 287.

39. European, attenuated PRRS virus having an ORF5 which differs from SEQ ID NO: 24 in one or more of positions 201 to 221.

40. Vaccine comprising a European PRRS virus, attenuated according to any of claims 36 to 39, in combination with a pharmaceutically acceptable carrier.

41. Method of vaccination of a pig against PRRS, characterized in that the pig is administered an efficient amount of the vaccine according to claim 40.

42. Use of a European PRRS virus, attenuated according to any of the preceding claims, for the manufacture of a virus against PRRS.