WO2002072802A2

WO2002072802A2 - Live attenuated strains of prrs virus

Info

Publication number: WO2002072802A2
Application number: PCT/EP2002/002486
Authority: WO
Inventors: Knut Elbers; Stefan Peschke; Bettina Schuetz
Original assignee: Boehringer Ingelheim Vetmedica GmbH
Current assignee: Boehringer Ingelheim Vetmedica GmbH
Priority date: 2001-03-09
Filing date: 2002-03-07
Publication date: 2002-09-19
Anticipated expiration: 2003-09-09
Also published as: CZ20032743A3; MXPA03007751A; EP1421184A2; KR20030082960A; RU2003129068A; BR0207988A; SK11282003A3; HUP0303431A2; WO2002072802A3; PL370541A1; JP2004534522A; CA2439254A1

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

Life attenuated strains of PRRS virus

Field of the invention

The present invention relates to live attenuated European PRRS viruses which are attenuated by nucleic acid mutations on specific sites. The invention also pertains to nucleotide sequences coding said viruses, methods of generating such viruses and a pharmaceutical composition comprising said PRRS viruses and the use of said PRRS virus in the manufacture of a vaccine for the prophylaxis and treatment of PRRS infections.

Background of the invention

Porcine reproductive and respiratory syndrome (PRRS), is caused by an enveloped positive-stranded RNA virus of the family arteriviridae (Snijder E.J., Meulenberg J.J.M. 1998. „The molecular biology of arteriviruses". Journal^' of General Virology 79(5) : 961- 971). About 10 to 15 years ago, two different PRRS virus strains emerged apparently independently in the USA and Europe. The disease is now endemic in many swine producing countries in North America, Europe and Asia. It continues to be a major cause of reproductive loss and respiratory disease in swine. In the USA the prevalence of infection is estimated to be up to 70 %.

The virus is transmitted by inhalation, ingestion, coitus, bite wounds or needles. It replicates in mucosal, pulmonary or regional macrophages. Subclinically, the disease results in resolution or persistent infection. Persistently infected animals shed virus in oral/pharyngeal fluids, blood, feces, urine and semen. Clinical symptoms in sows relate to abortion or premature farrowing with weak live-born pigs, stilborn pigs and autolyzed fetuses. Infected neonatal pigs have a high mortality or suffer from pneumonia. The subsequent nursery and growth of pigs is complicated by pneumonia, concurrent bacterial infections and increased mortality. Boars are prone to fever and morphological changes in semen.

Like for all arteriviruses, the PRRS virus genome is a single positive-stranded RNA molecule of about 15 kilobases. ORF's (open reading frame) la and lb code replicases, ORF's 2 to 5 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 USA (WO 93/03760, isolate ATCC VR- 2332, deposited July 18, 1991 at the American Type Culture Collection in Rockville, Maryland, USA, NCBI GeneBank Accession No. U 87392 U00153) and Europe (WO 92/21375, isolate Lelystad Agent (CDI-NL-2.91), Accession No. CNCM 1-1102, deposited June 5 1991 with the Institute Pasteur, Paris; NCBI GeneBank Accession Nos. M 96262 [gi:l 1125727], NC 002533 [gi:l 1138120]) identified viruses that had genomic and serological differences. Comparison demonstrated that both had a common ancestor which had diverged before the clinical disease was described in the late 1980's. Full length genomic sequences have been reported for a number of PRRS viruses and complete structural protein-coding regions thereof (Snijder et al. 1998, supra; Meulenberg J.J.M., Hulst, M.M. et al., 1993. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome ..., Virology 192, 62-72; Conzelmann K.K., Visser N., Van Woensel P., Thiel H.J., 1993. Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group, Virology 103, 329-339;. Murtaugh M.P., Elam M.R., Kakach L.T., 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 porcine reproductive and respiratory sydrome virus isolates in the midwestern United States, Journal of General Virology 77, 1271-1276).

PRRS virus can be replicated in vitro in pig lung macrophages, monocytes, glial cells and two MA-104 cell subpopulations (embryonic monkey kidney cell) known as CL-2621 and MARC-145 (K.D. Rossow, Porcine reproductive and respiratory syndrome, Vet Pathol 35:1-20, 1998). Recombinant means for generating infectious PRRS clones are also available (EP 0839912).

For protecting pigs, live attenuated PRRS vaccines are commercially available

(RespPRRS/Ingelvac^® PRRS MLV, Boehringer Ingelheim). Killed vaccines (inactivated whole virus) or subunit vaccines (conventionally purified or heterologously expressed purified viral proteins) are most often inferior to live vaccines in their efficacy to produce a full protective immune response even in the presence of adjuvants. For PRRS, it has been demonstrated that in comparison to the currently available killed vaccines, the attenuated vaccines induce an immunity against the disease which lasts longer and is more efficient (Snijder et al., referenced above). The present live PRRS vaccines are attenuated conventionally by serially passaging the virus in appropriate host cells until pathogenicity is lost (American strain: EP 0529584; European strain: EP 0676467; EP 0835930).

Present live PRRS vaccines still, leave room for improvement. For one, they do not prevent reinfection. Secondly, they do not allow serological discrimination between vaccinated animals and animals infected with the field virus. Furthermore, a live vaccine in principle has a theoretical risk of reversion to the non-attenuated phenotype. In particular, RNA viruses such as the PRRS virus, are considered to have high rates of mutation due to imprecise replication of the RNA genome resulting from a lack of proofreading by the RNA replication enzyme. For conventionally derived attenuated viruses obtained by conventional multiple passaging, the molecular origin as well as the genetic stability remains unknown, and the features of revertants are unpredictable.

Thus, the problem underlying the invention was to provide improved PRRS virus strains which can be used for the manufacture of vaccines which overcome the disadvantages of the prior art.

Figure Legends

Figure 1: Sequence comparison of wild type Lelystad Virus, published in GenBank, Attenuated virus A (abbreviated Vir. A) and attenuated Lelystad Virus B Genomic area: ORF 2

Number of nucleotides : 750

In Bold and boxed are indicated the mutations (non-synonymous nucleotide exchanges) according to the invention (s^"ee also claims 2 and 3)

Figure 2: Sequence comparison of wild type Lelystad- Virus, published in GenBank, Attenuated virus A (abbreviated Vir .A) and attenuated Lelystad- Virus B Genomic area: ORF 3

Number of nucleotides: 798 In Bold and boxed are indicated the mutations (non-synonymous nucleotide exchanges) according to the invention (see also claims 2 and 3)

Figure 3: Sequence comparison of wild type Lelystad Virus, 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 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 boxed are indicated the mutations (non-synonymous nucleotide exchanges) according to the invention (see also claims 2 and 3)

Disclosure of the invention

.. Before the embodiments of the present invention it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a PRRS virus" includes a plurality of such PRRS viruses, reference to the "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. The present invention provides an attenuated European PRRS virus encoded by a nucleic acid comprising ORF1, ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7 and subtypes thereof such as ORFla and ORFlb or ORF2a and ORF2b, characterized in that:

a) ORF2 comprises at positions 11915 - 11935 at least one of the nucleotides as set out in table 1 :

and/or a deletion at said position(s) and/or b) ORF3 comprises at positions 12660 - 12680 at least one of the nucleotides as set out in table 4:

and/or a deletion at said position(s) and/or c) ORF5 comprises at positions 13684 - 13704 at least one of the nucleotides as set out in table 5:

and/or a deletion at 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 compared to the original virulent field strain. The evolutionary pressure on these from now on called "virulence specific sites" or simply referred to as "sites of the invention" or just "sites" is immense. It - is assumed that these sites have a general involvement in the process leading to the attenuation of European PRRSV like vaccine strains. These sites disclosed in tables 1 to 5 are specific for European strains (wild type Lelystad Agent CNCM I-l 102 is regarded as reference type strain) and not shared with US strains of PRRS viruses.

In a further aspect, the present invention relates to an attenuated European PRRS virus wherein at least one of the sequence sections corresponding, to SEQ ID NOs: 25, 26, 27, 28, or 29 is mutated at one, two, three, or more positions, up to a maximum often

. mutations. The sites SEQ ID NO's: 25, 26, and 27 are located in ORF2 (positions 130-150, ; 252-272, and 273-293, respectively, in Fig 1), the site SEQ ID NO: 28 is located in ORF3 ^' ^■ (position 267-287 in Fig. 3), and the site SEQ ID NO: 29 is located in ORF5 (position 201- 221 in Fig. 4). Said sequence sections represent the virulence specific sites according to the invention. As PRRS virus is a positive-stranded RNA virus, it will comprise a respective RNA sequence instead of the DNA sequences given in the sequences listings, i.e. it will contain uracil (U) at positions indicated to be thymin (T) in the sequence listings, and ribose instead of deoxyribose.

It is to be understood that an attenuated European PRRS virus according to the invention comprises genomic RNA containing sequence sections corresponding to the DNA sequences with SEQ ID NOs: 25, 26, 27, 28, or 29, wherein at least one of said sequences sections differs from the referred sequences by at least one mutation. In this context, "corresponding to" means that the virus of the invention contains sequence sections which can be aligned to the referred sequences by a standard alignment algorithm like BLAST (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. (1993) "Identification of protein coding regions by database similarity search." Nature Genet. 3:266-272; Madden, T.L., Tatusov, R.L. & Zhang, J. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141; Zhang, J. & Madden, T.L. (1997)

"PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation." Genome Res. 7:649-656; Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Sequence positions which are aligned pairwise by such an algorithm "correspond to" each other.

Upon such an alignment, a respective mutated sequence section of the virus then would differ from the corresponding reference sequence ("site", i.e. either SEQ ID NO: 25, 26, 27, 28, or 29) at one, two, three, or more positions, up to a maximum often positions. Preferably, a virus of the invention is mutated at all five of the cited sequence sections (sites). Preferably, the mutation(s) is/are substitution(s) and/or a deletion(s). In preferred embodiments, the mutations result in a change of the amino acid sequence(s) of the protein(s) coded by the respective ORF(s).

In a further aspect, the present invention relates to an attenuated European PRRS virus having an ORF2 containing a sequence section corresponding to SEQ ID NO: 25, wherein the triplet corresponding to positions 10 to 12 of SEQ ID NO: 25 is mutated. Preferably, said triplet does not code for phenylalanine. Preferably, said triplet codes for a different amino acid. In a preferred embodiment, said triplet codes for serine. More preferably, the nucleotide correponding to position 11 of SEQ ID NO: 25 is a C. Hence in a preferred embodiment, the attenuated European PRRS virus has an ORF2 coding for a protein which has not phenylalanine at position 47 (or the corresponding position in a BLAST alignment). Preferably, said protein has a serine at this position instead.

In a further aspect, the present invention relates to an attenuated European PRRS virus having an ORF2 containing a sequence section corresponding to SEQ ID NO: 26, wherem the triplet corresponding to positions 10 to 12 of SEQ ID NO: 26 is mutated. Preferably, said triplet does not code for valine. Preferably, said triplet codes for a different amino acid. In a preferred embodiment, said triplet codes for phenylalanine. More preferably, the nucleotide correponding to position 10 of SEQ ID NO: 26 is a T (or an U in the RNA, respectively). Hence in a preferred embodiment, the attenuated European PRRS virus has an ORF2 coding for a protein which has not valine at position 88 (or the conesponding position in a BLAST alignment). Preferably, said protein has a phenylalanine at this position instead.

In a further aspect, the present invention relates to an attenuated European PRRS virus having an ORF2 containing a sequence 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, said triplet codes for a different amino acid. In a preferred embodiment, said triplet codes for leucine. More preferably, the nucleotide coπeponding to position 11 of SEQ ID NO: 27 is a C. Hence in a prefeπed embodiment, the attenuated European PRRS virus has an ORF2 coding for a protein which has not phenylalanine at position 95 (or the conesponding position in a BLAST alignment). Preferably, said protein has a leucine at this position instead.

In a further aspect, the present invention relates to an attenuated European PRRS virus having an ORF3 containing a sequence section corresponding to SEQ ID NO: 28, wherein the triplet conesponding to positions 11 to 13 of SEQ ID NO: 28 is mutated. Preferably, said triplet does not code for serine. Preferably, said triplet codes for a different amino acid. In a preferred embodiment, said triplet codes for proline. More preferably, the nucleotide coπeponding to position 11 of SEQ ID NO: 28 is a C. Hence in a prefeπed embodiment, the attenuated European PRRS virus has an ORF2 coding for a protein which has not serine at position 93 (or the conesponding position in a BLAST alignment). Preferably, said protein has a proline at this position instead.

In a further aspect, the present invention relates to an attenuated European PRRS virus having an ORF5 containing a sequence section conesponding to SEQ ID NO: 29, wherein the triplet conesponding to positions 11 to 13 of SEQ ID NO: 29 is mutated. Preferably, said triplet does not code for leucine. Preferably, said triplet codes for a different amino acid. In a prefened embodiment, said triplet codes for phenylalanine. More preferably, the nucleotide coneponding to position 11 of SEQ ID NO: 29 is a T (or an U in the RNA, respectively). Hence in a prefened embodiment, the attenuated European PRRS virus has an ORF2 coding for a protein which has not leucine at position 71 (or the conesponding position in a BLAST alignment). Preferably, said protein has a phenylalanine at this position instead.

Live PRRS vaccines based on European strains attenuated by defined mutations and/or 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 analysed conveniently for quality control and genetic stability. The possibility to introduce defined deletions and/or substitutions, in particular as double or multiple mutations decreases the probability of reversion to the non-attenuated or virulent phenotype. A further advantage of said attenuating mutations lies in their known molecular uniqueness which allows for use as distinctive labels for attenuated PRRS viruses and to distinguish them from PRRS viruses from the field. With the sites identified in the present invention, safe and site- specifically 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 attenuated European PRRS strains and methods of production thereof. In this context, "attenuated" means that a virulent strain is or has been modified in a way that he is less virulent or pathogen than before the modification. In particular, "attenuated" means that the virus has a significantly reduced ability of causing clinical disease, while he is still able to replicate in the host. Preferably, virulence or pathogenicity is reduced to an amount which makes the virus acceptable for administration as a vaccine. In a prefeπed embodiment, the virus is attenuated to an extent that is does not cause clinical diesease while still being able to replicate in the host. Such an attenuated strain is an ideal agent for vaccination because replication in the host ensures stimulation of a rapid and excellent immune response. In one prefeπed embodiment, an attenuated European PRRS virus is less virulent than the Lelystad Agent. In another embodiment, the ^■ attenuated virus is less virulent than the parent strain from which it s derived. The term „less virulent than the PRRS virus Lelystad Agent" (or, likewise, the parent strain from which it is derived) is to be understood in terms of a comparison of clinical symptoms of the virus of interest with Lelystad Agent (CDI-NL-2.91/ CNCM I-l 102), or the parent strain. A prefeπed procedure for determining if a PRRS virus is less virulent than Lelystad Agent, or likewise for determining if a virus modified according to the invention is less virulent than its parent strain before the modification, is disclosed in example 1. It may be that not each and every possible nucleic acid mutation at the virulence specific site is implicated in reducing virulence. The procedure of example 1 provides a precise and straight forward experimental setup for determining whether a live PRRS virus according to the teaching of the invention is less virulent than its parent strain or a virulent field isolate like Lelystad Agent.

The present invention is directed to European PRRSV strains which can be distinguished from American strains as follows. Soon after the virus was found, Wensvoort et al. (J. Vet. Diagn. Invest 4: 134-138 (1992) observed differences in antigenic characteristics between American and European isolates. They raised sera against both the American type and the European type in several pigs, and compared the cross-reactivity of the anti-European (LV) and anti- American (VR-2332) sera with three American virus isolates and 4 European isolates. Sera against PRRS viruses of the European serotype are significantly less reactive with American isolates than with European isolates. Furthermore, sera raised against viruses of the American serotype are less reactive, or even not reactive at all, with European virus isolates. Wensvoort et al. also showed the reactivity of sera raised against European and American isolates with the two reference viruses CNCM I-l 102 (European) and ATCC VR-2332 (American). In this experiment, sera raised against European strains were not reactive at all with the American strain. Thus, two fully different serotypes of the virus exist: the American and the European serotypes, which may be distinguished based : y on their serological properties.

This can also be shown on the molecular level. Nelson et al: (74th Annual Meeting of the ^• Conference of Research Workers in Animal Diseases, November 8-9, 1993) compared sequences of polymerase-encoding genes of various isolates. They demonstrated that ^• ■ polymerase genes show a 87-95% homology within the American group. However, a ' homology as low as 64-67% based on nucleic acids was found between European :^•- serotypes and American serotypes. Mardassi published comparable results at the _; Conference mentioned above, showing that the 3'-terminal 530 nucleic acids of the Quebec PRRS Reference strain and European isolates only show a homology as low as 59%. The results from the papers mentioned above make it most likely that the American and European serotypes have diverged a long time ago, which would then easily explain their genetic differences and their serological unrelatedness.

American and European serotypes thus may be easily discriminated. Viruses of the European serotype are characterised in that they react to a higher titer in an Immunoperoxidase Monolayer Assay with a panel of antisera against the European PRRS virus LV (CNCM I-l 102) compared with a reaction with a panel of antisera against the American PRRS virus (ATCC VR-2332). If a panel of sera is used, obtained about 40 days post infection, and from different animals, any virus can easily be classified as belonging either to the American or European serotype. Typically, the reactivity of a European strain with a panel of antisera against other European strains is about 400 times higher than with a panel of antisera against American strains. When a European strain is reacted with antisera against the deposited European strain I-l 102 and the deposited American strain VR-2332, a typical difference in reactivity of about 55 times is found (Wensvoort et al, referenced above).

"Mutation" means the substitution, deletion, or insertion of a nucleotide or amino acid at a given position of a nucleotide or amino acid sequence. A "substitution" is a replacement of a nucleotide or amino acid by another (e.g. C for a T). "Deletion" means the removal of a , nucleotide or amino acid. "Insertion" means that a nucleotide or amino acid is inserted at a given position. ^{' ■ ■}

. In a further aspect, the present invention comprises a method or process of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions of ORF2 conesponding 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.

In a further aspect, the present invention comprises a method of attenuation of a European : PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions of ORF3 conesponding to positions 267 to 287 of SEQ ID NO: 23; b) it is tested whether the resulting PRRS virus is attenuated.

In a further aspect, the present invention comprises a method of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions conesponding to positions 201 to 221 of ORF5 according to SEQ ID NO: 24; b) it is tested whether the resulting PRRS virus is attenuated.

In this context, the term "conesponding to" has a meaning as outlined above, i.e. that the two positions conesponding to each other would be aligned as a pair in a sequence aligmnent like BLAST. Preferably, the modification according to the invention results in a change of the amino acid sequence of the encoded protein. In prefened embodiments, the modifications are deletions and/or substitutions, preferably double or multiple mutations. In a prefened embodiment, two or more of the aforementioned modifications are combined. In a further prefened embodiment, the sequence of each of ORF2, ORF3, and ORF5 is modified. Preferably, the sequence of ORF2 is modified at least at two, preferably at least at three positions.

Preferably, the aforementioned method comprises (a) modification(s) resulting in one or ; more of the following features: an ORF2 encoding a protein having the amino acid(s) at one or more of amino acid sequence positions conesponding to positions 47, 88 and/or 95 of SEQ ID NO: 22 substituted or deleted; an ORF3 encoding a protein having the amino acid at amino acid sequence position conesponding to position 93 of SEQ ID NO: 23 substituted or deleted; and/or an ORF5 encoding a protein having the amino acid at amino acid sequence position conesponding to position 71 of SEQ ID NO: 24 substituted or deleted. Preferably, all of the aforementioned positions are mutated.

Preferably, the method according to the invention comprises (a) modification (s) resulting in one or more, preferably all of the following features: an ORF2 encoding a protein having not phenylalanine at amino acid sequence position conesponding to position 47 of ■ SEQ ID NO: 22, an ORF2 encoding a protein having not valine at amino acid sequence - position conesponding to position 88 of SEQ ID NO: 22, an ORF2 having not phenylalanine at amino acid sequence position conesponding to position 95 of SEQ ID NO: 22, an ORF3 having not serine at amino acid sequence position conesponding to position 93 of SEQ ID NO: 23, and/or an ORF5 having not leucine at amino acid sequence position conesponding to position 71 of SEQ ID NO: 24.

Preferably, the method according to the invention comprises (a) modification (s) resulting in one or more, preferably all of the following features: an ORF2 encoding a protein having serine at amino acid sequence position conesponding to position 47 of SEQ ID NO: 22, an ORF2 encoding a protein having phenylalanine at amino acid sequence position conesponding to position 88 of SEQ ID NO: 22, an ORF2 having leucine at amino acid sequence position conesponding to position 95 of SEQ ID NO: 22, an ORF3 having proline at amino acid sequence position conesponding to position 93 of SEQ ID NO: 23, and/or an ORF5 having phenylalanine at amino acid sequence position conesponding to position 71 of SEQ ID NO: 24.

Preferably, a method according to the invention comprises (a) modification (s) resulting in one or more, preferably all of the following features: an ORF2 having a C at the position conesponding to position 140 if SEQ ID NO: 22, an ORF2 having a T at the position conesponding to position 262 of SEQ ID NO: 22, an ORF2 having a C at the position conesponding to position 283 of SEQ ID NO: 22, an ORF3 having a C at the position conesponding to position 277 of SEQ ID NO: 23, and/or an ORF5 having a T at the position conesponding to position 211 of SEQ ID NO: 24.

' Furthermore, the present invention relates to an attenuated European PRRS virus obtainable by any one of the aforementioned methods.

In a further aspect, the present invention comprises a nucleic acid containing the coding information of an attenuated European PRRS virus as described above. As outlined above, this may be a ribonucleic acid (RNA). Such nucleic acid may furthermore be a deoxyribonucleic acid which is complementary to such a ribonucleic acid, i.e. a cDNA, or any other type of DNA. In a prefeπed embodiment, such a cDNA is infectious. This means that if such a cDNA is introduced into a suitable host cell, said cell will start generating virus particles. The present invention furthermore relates to a RNA, cDNA, or other DNA comprising any one,' or a multitude of the mutations outlined above.

In a further aspect, the invention comprises a vaccine comprising an attenuated European PRRS virus as described in combination with a pharmaceutically acceptable carrier.

In a further aspect, the present invention comprises a method of vaccination of a pig against PRRS, characterised in that an efficient amount of the aforementioned vaccine is adminstered to said pig.

In a further aspect, the present invention relates to the use of an attenuated European PRRS virus as described for the manufacture of a vaccine against PRRS. According to the present invention, the numbering of nucleotides or amino acids is according to the publicly available Lelystad agent as disclosed above. However, as disclosed in figures 1 to 4, the ORFs ("open reading frames = ORF") are numbered separately as some of them are overlapping.

The nucleotides of the specific sites at which the attenuated European virus is different from the virulent wild type are of ORF 2 the nucleotides 11915 - 11935 (table 1 conesponding 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); of ORF 3 12660 - 12680 (table 4; numbers 267-287 in figure 3), and/or of ORF5 13684 - 13704 (table 5; numbers 201-221 in figure 4). By mutating the Lelystad Agent at one or several of the before-mentioned positions to any one of the nucleotides indicated in said tables and/deleting said nucleotides at said positions, the artisan will obtain life attenuated European vaccine strains of PRRS virus. The mutations such as replacement or deletion at said locations are only limited by the condition that the virus must still be able to replicate, i.e. it must still be a live virus. This can be determined without undue experimentation. The skilled person can, on the basis of the publicly available Lelystad Agent and the teachings according to the invention, introduce e.g. one single mutation into every site disclosed on the basis of the provided tables, e.g. at position 11915 he may introduce an A, C or G or he may delete the nucleotide at that position (which is a T). A non-limited example is shown in figures 1 to 3 (ORF2, ORF 3 and ORF 5 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 e.g. one or several triplets encoding one or several amino acids at said positions. However, the sequence shall not be mutated to the extend that the virus is not capable of replicating anymore, i.e. according to the invention, a live virus is encoded.

Additional nucleic acids outside said regions may also be mutated, however, this is not essential to the invention (see figure 1, e.g. position 426 of ORF2 of Lelystad-B wich is a C instead of a T). Said mutations may 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 which allows directed mutation of preselected sites of a nucleic acid. In general, such a technique requires at least partial knowledge of the sequence of such nucleic acid. The classical attenuation method by serial passaging dose not allow mutation of preselected sites. Such genetic engineering methods are well-known in the art (see e.g. Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Chapter 15; Wu (ed.) (1993), Methods in Enzymology Vol 217, p. 173-285).

As a starting material for introduction of mutations, a cDNA clone of a virulent PRSSV i strain, e.g. Lelystad Agent (CNCM I-l 102), may be generated according to procedures known in the art (Boyer et Haenni. Infectious transcripts and cDNA clones of RNA viruses. Virology (1994) 198: 415-426). For example, an infectious PRRSV copy may be ' prepared as described (WO00/53787; Meulenberg. et al, Adv. Exp. Biol (1998) 440:199- . 206; Meulenberg et al., J. Virol. (1998) 72(1): 380-387) which then may be mutated , according to the invention. The cited references provide detailed procedures how to obtain infectious cDNA clones of PRRSV and to create mutants thereof. The mutated clone or infectious tanscripts thereof generated in vitro may then be transfected into appropriate host cells which will then generate attenuated virus.

In particular, it was surprisingly found that sites 11925, 12047, and 12068 of ORF2 (see figure 1); 12670 of ORF 3 (see figure 2) and/or 13694 of ORF5 (see figure 4) are consistently changed compared to the virulent field strain Lelystad Agent. Therefore, another prefeπed 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 at position 12047 and/or a A, C or G at position 12068 or a deletion at said position(s) and/or b) ORF3 comprises a A, C or G at position 12670 or a deletion at said position and/or c) ORF5 comprises a G, A or T at position 13694 or a deletion at said position.

Even more particular, it was found that found that site 11925 of ORF 2 is consistently changed to a C, site 12047 of ORF 2 is consistently changed to T, and site 12068 of ORF2 is consistently changed to C (see figure 1); site 12670 of ORF 3 is consistently changed to C (see figure 2) and/or 13694 of ORF5 is consistently changed to T (see figure 4) compared to the virulent field strain Lelystad Agent. Thus, another more prefeπed embodiment of the present invention is an attenuated European PRRS virus according to . the invention, wherein said nucleic acid is further characterized in that: a) said ORF2 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 i ■ b) said ORF3 comprises a C at position 12670 or a deletion at said position and/or c) said ORF5 comprises a T at position 13694 or a deletion at said position.

Another more prefeπed embodiment of the present invention is an attenuated European " ^■ PRRS virus according to the invention designated attenuated Virus A in the figures, i. i wherein said nucleic acid is further characterized in that: a) said ORF2 comprises the nucleic acid as defined in SEQ ID No. 1 and/or b) said ORF3 comprises the nucleic acid as defined in SEQ ID No. 2 and/or c) said ORF4 comprises the nucleic acid as defined in SEQ ID No. 3 and/or d) said ORF5 comprises the nucleic acid as defined in SEQ ID No. 4.

■ or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

A „fragment" according to the invention is any immunogenic subunit of a PRRS virus or , ORF according to the invention, i.e. any polypeptide subset, characterized in that it is encoded by a shorter nucleic acid molecule than disclosed above, however still retains its ^■ activity.

A „functional variant" of the PRRS virus or ORF according to the invention is a PRRS virus or ORF which possesses a biological activity (either functional or structural) that is substantially similar to the PRRS virus or ORF 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" e.g. may cany one or several point mutations, one or several nucleic acid exchanges, deletions or insertions or one or several amino acid exchanges, deletions or insertions. Said functional variant is still retaining its biological activity, e.g. function as a vaccine strain, at least in part or even going along with an improvement said biological activity. A „variant based on the degenerative of the genetic code" is a variant due to the fact that a< certain amino acid may be encoded by several different nucleotide tripletts. Said variant is still retaining its biological activity, at least in part or even going along with an improvement said biological activity.

A „fusion molecule" may be the PRRS virus or ORF according to the invention fused to e.g. 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 virus or ORF according to the invention chemically modified or containing additional chemical moieties not normally being part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life etc.

A molecule is ^substantially similar" to another molecule if both molecules have substantially similar structures or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein 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 most prefened embodiment of the present invention is an attenuated European PRRS virus according to the invention (designated attenuated Virus A in the figures), wherein said nucleic acid is characterized in that: a) said ORF2 consists of the nucleic acid as defined in SEQ ID No. 1 and/or b) said ORF3 consists of the nucleic acid as defined in SEQ ID No. 2 and/or c) said ORF4 consists of the nucleic acid as defined in SEQ ID No. 3 and/or d) said ORF5 consists of the nucleic acid as defined in SEQ ID No. 4.

Another prefeπed embodiment of the present invention is an attenuated European PRRS virus according to the invention (designated attenuated virus A in the figures), wherein said 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 most prefeπed embodiment of the present invention is an attenuated European PRRS virus according to the invention (designated attenuated virus A in the figures), wherein said nucleic acid is characterized in that it consists of the nucleic acid as defined in SEQ ID No. 5.

Another prefeπed embodiment of the present invention is an attenuated European PRRS virus according to the invention (LELYSTAD-B in the figures), wherein said nucleic acid is further characterized in that: a) said ORF2 comprises the nucleic acid as defined in SEQ ID No. 6 and or b) said ORF3 comprises the nucleic acid as defined in SEQ ID No. 7 and/or c) said ORF4 comprises the nucleic acid as defined in SEQ ID No. 8 and or d) said ORF5 comprises the nucleic acid as defined in SEQ ID No. 9 and/or or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

Another most prefened embodiment of the present invention is an attenuated European PRRS virus according to the invention (LELYSTAD-B in the figures), wherein said nucleic acid is further characterized in that: a) said ORF2 consists of the nucleic acid as defined in SEQ ID No. 6 and/or b) said ORF3 consists of the nucleic acid as defined in SEQ ID No. 7 and/or c) said ORF4 consists of the nucleic acid as defined in SEQ ID No. 8 and/or d) said ORF5 consists of the nucleic acid as defined in SEQ ID No. 9.

Another important embodiment of the present invention is an attenuated European PRRS virus (attenuated virus A in the figures), wherein said PRRS virus is characterized in that: a) ORF la 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 f) 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 and/or h) ORF7 comprises the amino acid as defined in SEQ ID No. 17. or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

Another more prefeπed embodiment of the present invention is an attenuated European PRRS virus according to the invention (attenuated virus A in the figures), wherein said PRRS virus is characterized in that: a) ORF la consists of the amino acid as defined in SEQ ID No. 10 and/or b) 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 f) ORF5 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/or h) ORF7 consists of the amino acid as defined in SEQ ID No. 17.

Another very important embodiment of the present invention is an attenuated European PRRS virus (LELYSTAD-B in the figures), wherein said PRRS virus is characterized in that: a) ORF2 comprises the amino acid as defined in SEQ ID No. 18 and/or b) ORF3 comprises the amino acid as defined in SEQ ID No. 19 and/or c) ORF4 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, glycosylation variant, fusion molecule or a chemical derivative thereof.

Another more prefeπed embodiment of the present invention is an attenuated European PRRS virus according to the invention (LELYSTAD-B in the figures), wherein said PRRS virus is characterized in that: a) ORF2 consists of the amino acid as defined in SEQ ID No. 18 and/or b) ORF3 consists of the amino acid as defined in SEQ ID No. 19 and/or c) ORF4 consists of the amino acid as defined in SEQ ID No. 20 and/or d) ORF5 consists of the amino acid as defined in SEQ ID No. 21. Yet 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 sequences disclosed in the sequence listing (e.g. with the identification numbers 1 to 9 (given under <400> = SEQ ID NO.). The invention further relates to the proteins disclosed in SEQ ID No. 10 to 21.

Yet another prefeπed embodiment of the present invention is a nucleotide sequence according to the invention, wherein the nucleotide sequence has been modified to encode a virulence marker 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 clinical symptoms of a field infection are not that specific or superimposed by other infections or the time period for observation and evaluation is short. The recombinant generation of the viruses of interest allows for the introduction of modifications in the genetic code that 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 or body fluids such as but not limited to pharyngeal or nasal fluids or urine. A virulence marker is to 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 prefeπed 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, the mutations or deletions introduced for the purpose of attenuating the virusare useful as virulence and serological markers. By monitoring these mutations in the disclosed virulence specific sites it is possible to predict the emergence of possibly virulent revertants at an early stage.

Yet another prefeπed 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 frames encoding structural viral proteins.

In another aspect, the invention relates to a method for the generation of an infectious live attenuated PRRS virus, said method comprising producing a recombinant nucleic acid comprising at least one full-length DNA copy or in vitro-transcribed RNA copy or a derivative of either said DNA or RNA whereby said nucleotide sequence is a nucleotide sequence according to the invention. Thus, according to the invention, the method leads to a PRRS virus as described above. The artisan may employ site-directed mutagenesis (Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.; Chapter 15; Wu (ed.) (1993), Methods in Enzymology Vol 217, p. 173-285) or any other method to insert mutation(s) to specifically insert mutation(s) to obtain a PRRS virus as disclosed above.

The method disclosed in the following prefeπed aspect will lead (at the genomic level) to the PRRS virus according to the invention as described supra. Thus, in another prefeπed aspect, the invention relates to a method as described for the generation of an infectious live 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) said PRRS virus is further attenuated via cell culture passages.

In another prefened aspect, the invention relates to a method as described, wherein said cell line is an embryonic monkey kidney cell or prefeπed a Marc cell or a derivative therof (see below).

In another prefeπed aspect, the invention relates to a method as described, wherein said PRRS virus is or are the virus(es) according to the invention as disclosed supra.

In a further aspect, the present invention relates to a cell line comprising a PRRS virus according to the invention. Examples for such cell lines include permanent cell lines known to the artisan, preferably porcine, monkey or human cell lines such as human embryonic kidney (HEK) 293, BHK, GH3, H4, U373, NT2, PC12, COS, CHO, Ltk^", fibroblasts, myelomas, neuroblastomas, hybridomas, oocytes, embryonic stem cells), insect cell lines (e.g., using baculovirus vectors such as pPbac or pMbac (Stratagene, La Jolla, USA)), yeast (e.g., Pichia pastoris or using yeast expression vectors such as pYESHIS (Invitrogen, San Diego, USA)), and fungi. In a further prefened aspect, the present invention relates to a cell line according to the invention, wherein said cell line is an embryonic monkey kidney cell or prefeπed a Marc cell or a derivative therof.

In a further aspect, the present invention comprises a method or process of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions of ORF2 conesponding 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.

In this context, a position within a nucleic acid or amino acid sequence "conesponding to" a position of another sequence shall mean that, if the two sequences have sufficient structural similarity to be aligned with a standard alignment algorithm like BLAST (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. (1993) "Identification of protein coding regions by database similarity search." Nature Genet. 3:266-272; Madden, T.L., Tatusov, R.L. & Zhang, J. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141; Zhang, J. & Madden, T.L. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation." Genome Res. 7:649-656; Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402), and such an alignment is done, the two positions would be aligned as a pair.

In a further embodiment, the present invention relates to a method of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions of ORF3 conesponding 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 of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions conesponding to positions 201 to 221 of ORF5 according to SEQ ID NO: 24; b) it is tested whether the resulting PRRS virus is attenuated.

Preferably, a modification as described above results in a change of the amino acid sequence of the encoded protein. Preferably, the modification is a deletion or a substitution. In a prefeπed embodiment, the sequence of each of ORF2, ORF3, and ORF5 is modified. In asnother prefeπed embodiment, the sequence of ORF2 is modified at least at two, preferably at least at three positions. Preferably, the modification results in one or more of the following features: an ORF2 encoding a protein having the amino acid at one or more of amino acid sequence positions conesponding to positions 47, 88 and/or 95 of the amino acid sequence encoded by SEQ ID NO: 22 substituted or deleted; an ORF3 encoding a protein having the amino acid conesponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23 substituted or deleted; and/or an ORF5 encoding a protein having the amino acid conesponding to position 71 of the amino acid sequence encoded by SEQ TD NO: 24 substituted or deleted.

More preferably, the modification made by such method results in one or more, preferably all of the following features: an ORF2 encoding a protein having serine at the position < conesponding to position 47 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF2 encoding a protein having phenylalanine at the position conesponding to position 88 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF2 having leucine at the postion conesponding to position 95 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF3 having proline at the position conesponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23, and/or an ORF5 having phenylalanine at the position conesponding to position 71 of the amino acid sequence encoded by SEQ ID NO: 24.

In prefened embodiments, the modification results in one or more, preferably all of the following features: an ORF2 having a C at the position conesponding to position 140 if SEQ ED NO: 22, an ORF2 having a T at the position conesponding to position 262 of SEQ ID NO: 22, an ORF2 having a C at the position conesponding to position 283 of SEQ ID NO: 22, an ORF3 having a C at the position conesponding to position 277 of SEQ ID NO: 23, and/or an ORF5 having a T at the position conesponding to position 211 of SEQ ID NO: 24.

In a further embodiment, the present invention relates to an attenuated European PRRS virus obtainable by a method described above.

In a further embodiment, the present invention relates to an attenuated European PRRS virus having an ORF2 which differs from SEQ ID NO: 22 at one or more of positions 130 to 150, and/or at one or more of positions 252 to 272, and/or at one or more of positions 273 to 293.

In a further embodiment, the present invention relates to an attenuated European PRRS virus having an ORF3 which differs from SEQ ID NO: 23 at 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 which differs from SEQ ID NO: 24 at one or more of positions 201 to 221.

In a further embodiment, the present invention relates to a vaccine comprising an attenuated European PRRS virus 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, characterised in that an efficient amount of such vaccine is adminstered to said pig. Alternatively, the present invention relates to the use of an attenuated European PRRS virus as disclosed for the manufacture of a vaccine against PRRS.

Preferably, the live attenuated PRRS virus may be used for the treatment, prophylaxis or diagnosis of diseases caused by wild-type PRRS virus. Such diseases and uses 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 that comprise deletions in the virulence specific sites is prefened since deletions are less prone to revert.

Another prefened embodiment of the present invention is a pharmaceutical composition comprising one or several PRRS virus(es) according to the invention and a pharmaceutically acceptable carrier. A prefened aspect is a pharmaceutical composition comprising not only said European PRRS virus according to the invention, but also an attenuated US PRRS virus such as the virus which is sold in a pharmaceutical composition under the trade name RespPRRS/Ingelvac^® PRRS MLV, Boehringer Ingelheim. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption or form part of a slow release formulation 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 e.g. Remington's Pharmaceutical

Sciences (1990). 18th ed. Mack Publ., Easton). One 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. Most preferably, the composition is formulated by obtaining the virus-containing supernatant (tissue culture fluid) of an infected cell culture and freeze drying said supernatant, optionally after addition of a stabilisator. The freeze-dried composition may be reconstituted with water prior to administration. In another example, , a suitable carrier would be a physiological salt 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 sows at 1, 3, 6 or 10 weeks of age, to sows before mating and/or up to 6 weeks before fanowing (booster vaccination), or to boars each half a year (boosters).

Another prefened 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 the same should not be construed as limiting the scope of the invention disclosed herein. Example 1 Establishment of attenuation

This example provides a clear guidance for the comparison of the virulent character of two different strains of PRRS viruses. As reference strain for a typical virulent European strain can serve a low cell culture passage (not more than 5) of the Lelystad Agent (CDI-NL- 2.91).

At least 10 gilts per group are included in each trial, which are derived from a PRRS free farm. Animals are tested free of PRRS virus specific serum antibodies and negative for PRRSV. All animals included in the trial are of the same source and breed and from a farm historically free of any PRRSV infection. The animals allocation to the groups is randomized. Challenge is performed at day 85-90 of pregnancy with intranasal application of 1 ml PRRSV with 10⁵ TDC_D₅₀ (third passage) per nostril. There are at least three groups for each test setup:

One group for challenge Lelystad Agent (CDI-NL-2.91) ; one test group for challenge with the possibly attenuated virus; and one strict control group.

Validity of the study is given when the strict controls stay PRRS negative over the time course of the study and at least 25% less life healthy piglets are born compared to the the strict controls in the Lelystad agent challenged group.

Attenuation, in other words less virulence, is defined as the statistical significant change of one or more parameters determining reproductive performance:

Significant reduction in at least one of the following parameters for the test group is prefened:

^• frequency of stillborns * abortion at or before day 112 of pregnancy

^• number of mumified piglets

^• number of life and weak piglets

^• preweaning mortality or furthermore a significant increase in one of the following parameters for the test group is prefened:

^• number of piglets weaned per sow

^• number of life healthy piglets born per sow

^• compared to the Lelystad agent infected group.

In an examplary manner the following results can be obtained in a clinical trial according to the given description with the 147 cell culture passage of the Lelystad agent:

Individual sow reproductive performance data from sows being inocculated as described above with cell culture passage 5 Lelystad agent (group 1) and cell culture passage 147 of Lelystad agent (group 2)are' given. In addition group 3 animals served as strict controls and were only inocculated with PRRSV free cell culture supernatant:

Claims

1. Attenuated European PRRS virus encoded by a nucleic acid comprising ORFl, ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7, characterized in that: a) ORF2 comprises at positions 11915 — 11935 at least one of the nucleotides as set out in table 1 :

and/or a deletion at said position(s) and/or c) ORF5 comprises at positions 13684 — 13704 at least one of the nucleotides as set out in table 5:

and/or a deletion at said position(s).

2. Attenuated European PRRS virus according to claim 1, characterized in that: a) ORF2 comprises a C, A or G at position 11925 and/or a C, T or A at position 12047 and/or a A, C or G at position 12068 or a deletion at said position(s) and/or b) ORF3 comprises a A, C or G at position 12670 or a deletion at said position and/or c) ORF5 comprises a G, A or T at position 13694 or a deletion at said position.

3. Attenuated European PRRS virus according to claim 1 or 2, wherein said nucleic acid [ is further characterized in that: a) said ORF2 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) said ORF3 comprises a C at position 12670 or a deletion at said position and/or c) said ORF5 comprises a T at position 13694 or a deletion at said position.

4. Attenuated European PRRS virus according to any of claims 1 to 3, wherein said nucleic acid is further characterized in that: a) said ORF2 comprises the nucleic acid as defined in SEQ ID No. 1 and/or b) said ORF3 comprises the nucleic acid as defined in SEQ ID No. 2 and/or c) said ORF4 comprises the nucleic acid as defined in SEQ ID No. 3 and/or d) said ORF5 comprises the nucleic acid as defined in SEQ ID No. 4. or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

5. Attenuated European PRRS virus according to any of claims 1 to 4, wherein said nucleic acid is characterized in that: a) said ORF2 consists of the nucleic acid as defined in SEQ ID No. 1 and/or b) said ORF3 consists of the nucleic acid as defined in SEQ ID No. 2 and/or c) said ORF4 consists of the nucleic acid as defined in SEQ ID No. 3 and/or d) said ORF5 consists of the nucleic acid as defined in SEQ ID No. 4.

6. Attenuated European PRRS virus according to any of claims 1 to 5, wherein said 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.

7. Attenuated European PRRS virus according to any of claims 1 to 6, wherein said nucleic acid is characterized in that it consists of the nucleic acid as defined in SEQ ID

No. 5.

8. Attenuated European PRRS virus according to any of claims 1 to 3, wherein said nucleic acid is further characterized in that: a) said ORF2 comprises the nucleic acid as defined in SEQ ID No. 6 and/or b) said ORF3 comprises the nucleic acid as defined in SEQ ID No. 7 and/or c) said ORF4 comprises the nucleic acid as defined in SEQ ID No. 8 and/or d) said ORF5 comprises the nucleic acid as defined in SEQ ID No. 9 and/or or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

9. Attenuated European PRRS virus accordmg to any of claims 1 to 3 or 8, wherein said nucleic acid is further characterized in that: a) said ORF2 consists of the nucleic acid as defined in SEQ ID No. 6 and/or b) said ORF3 consists of the nucleic acid as defined in SEQ ID No. 7 and/or a) said ORF4 consists of the nucleic acid as defined in SEQ ID No. 8 and/or b) said ORF5 consists of the nucleic acid as defined in SEQ ID No. 9.

10. Attenuated European PRRS virus, wherein said PRRS virus is characterized in that: a) ORFl a 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 f) 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 and/or h) ORF7 comprises the amino acid as defined in SEQ ID No. 17. or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

11. Attenuated European PRRS virus according to claim 10, wherein said PRRS virus is characterized in that: a) ORF la consists of the amino acid as defined in SEQ ID No. 10 and/or b) 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 f) ORF5 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/or h) ORF7 consists of the amino acid as defined in SEQ ID No. 17.

12. Attenuated European PRRS virus, wherein said PRRS virus is characterized in that: a) ORF2 comprises the amino acid as defined in SEQ ID No. 18 and/or b) ORF3 comprises the amino acid as defined in SEQ ID No. 19 and/or c) ORF4 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, glycosylation variant, fusion molecule or a chemical derivative thereof.

13. Attenuated European PRRS virus according to claim 14, wherein said PRRS virus is characterized in that: a) said ORF2 consists of the amino acid as defined in SEQ No. 18 and/or b) said ORF3 consists of the amino acid as defined in SEQ No. 19 and/or c) said ORF4 consists of the amino acid as defined in SEQ No. 20 and/or d) said ORF5 consists of the amino acid as defined in SEQ No. 21.

14. A nucleotide sequence coding for a virus according to any one 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 said marker is located within any of the open reading frames encoding structural viral proteins.

17. Method for the generation of an infectious live attenuated PRRS virus, said method comprising producing a recombinant nucleic acid comprising at least one full-length DNA copy or in vitro-transcribed RNA copy or a derivative of either whereby said nucleotide sequence is a nucleotide sequence according to any one of claims 14 to 16.

18. Method for the generation of an infectious live attenuated PRRS virus, said 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) said PRRS virus is attenuated via cell culture passages.

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

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

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

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

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

24. Pharmaceutical composition comprising a PRRS virus according to any one 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 and treatment of PRRS infections.

26. Method of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions of ORF2 conesponding 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.

27. Method of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions of ORF3 conesponding to positions 267 to 287 of SEQ

^' ID NO: 23; b) it is tested whether the resulting PRRS virus is attenuated.

28. Method of attenuation of a European PRRS virus, characterised in that a) the nucleotide sequence of said virus is modified by site-directed mutagenesis at at least one of the positions conesponding to positions 201 to 221 of ORF5 according to SEQ ID NO: 24; b) it is tested whether the resulting PRRS virus is attenuated.

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

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

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

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

33. Method of any one of claims 26 to 32, wherein the modification results in one or more of the following features: an ORF2 encoding a protein having the amino acid at one or more of amino acid sequence positions conesponding to positions 47, 88 and/or 95 of the amino acid sequence encoded by SEQ ID NO: 22 substituted or deleted; an ORF3 encoding a protein having the amino acid conesponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23 substituted or deleted; and/or an ORF5 encoding a protein having the amino acid conesponding to position 71 of the amino acid sequence encoded by SEQ ID NO: 24 substituted or deleted.

34. Method of claim 33, wherein the modification results in one or more, preferably all of the following features: an ORF2 encoding a protein having serine at the position conesponding to position 47 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF2 encoding a protein having phenylalanine at the position conesponding to position 88 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF2 having leucine at the postion conesponding to position 95 of the amino acid sequence encoded by SEQ ID NO: 22, an ORF3 having proline at the position conesponding to position 93 of the amino acid sequence encoded by SEQ ID NO: 23, and/or an ORF5 having phenylalanine at the position conesponding to position 71 of the amino acid sequence encoded by SEQ ID NO: 24.

35. Method of claim 34, wherem the modification results in one or more, preferably all of the following features: an ORF2 having a C at the position conesponding to position 140 if SEQ ID NO: 22, an ORF2 having a T at the position conesponding to position

262 of SEQ ID NO: 22, an ORF2 having a C at the position conesponding to position 283 of SEQ ID NO: 22, an ORF3 having a C at the position conesponding to position 277 of SEQ ID NO: 23, and/or an ORF5 having a T at the position conesponding to position 211 of SEQ ID NO: 24.

36. Attenuated European PRRS virus obtainable by the method of any one of claims 26 to 35.

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

38. Attenuated European PRRS virus having an ORF3 which differs from SEQ ID NO: 23 at one or more of positions 267 to 287.

39. Attenuated European PRRS virus having an ORF5 which differs from SEQ TD NO: 24 at one or more of positions 201 to 221.

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

41. Method of vaccination of a pig against PRRS, characterised in that an efficient amount of the vaccine of claim 40 is adminstered to said pig.

42. Use of an attenuated European PRRS virus according to any one of claims for the manufacture of a vaccine against PRRS.