SK10932002A3 - Live attenuated prrs virus containing orf 1a, 1b and 2, a nucleotide sequence coding said virus, method of generating such virus, pharmaceutical composition comprising same and use thereof - Google Patents

Abstract

The present invention relates to live PRRS viruses which are attenuated by amino acid mutations in a specific site of the viral protein coded by the open reading frame (ORF) selected from the group of ORF 1a, ORF 1b and/or ORF 2. The invention also pertains to nucleotide sequences coding said viruses, methods of generating such viruses and their use for the preparation of a pharmaceutical composition for the prophylaxis and treatment of PRRS infections.

Description

Technical field

The present invention relates to live PRRS viruses that are attenuated through amino acid mutations at specific sites of the viral protein that are encoded by an open reading frame (ORF) selected from the group consisting of ORF 1a, ORF 1b and / or ORF 2. The invention also relates to nucleotide sequences encoding said viruses, methods for generating such viruses and their use for the preparation of a pharmaceutical composition for the prophylaxis and treatment of PRRS infections.

BACKGROUND OF THE INVENTION

The mysterious swine disease, later renamed to porcine reproductive and respiratory syndrome (PRRS), is caused by enveloped positive-stranded RNA virus of the arteriviridae family (E. J. Snijder, 1998). Approximately 10 to 15 years ago, two distinct PRRS virus strains appeared apparently independently in the US and Europe. The disease is currently endemic in many pig producing countries in North America, Europe and Asia. It is still a major cause of reproductive loss and respiratory disease in pigs. In the US, the prevalence of infection is estimated to be up to 70%.

The virus is transmitted by inhalation, digestion, coitus, bites caused by bites or needles. It replicates in mucosal, pulmonary or regional macrophages.

Subclinically, the disease leads to diffuse or persistent infection. Persistently infected animals secrete the virus into oral / pharyngeal fluids, blood, faeces, urine and semen.

Clinical symptoms in sows relate to abortion and premature litter with weak newborn pigs, stillborn pigs and

-2autolysed fruits. Infected new born piglets have high mortality or pneumonia. Follow-up care and breeding of piglets are complicated by pneumonia, parallel bacterial infections and increased mortality. Boars are susceptible to fever and morphological changes in semen.

As with all arteriviruses, the PRRS viral genome is an RNA with a single stranded strand of approximately 15 kilobases. ORF’s (open reading frames) 1a and 1b encode replicases, ORF’s 2 and 5 probable glycoproteins (gp 1-4), ORF 6 membrane protein (M) and ORF 7 encode nucleocapsid protein (N).

Original descriptions of PRRS infection in the US (ATCC VR-2332 isolate, deposited July 18, 1991 at the American Type Culture Collection in Rockville, Maryland, USA, Genebank U 87392 U00153) and in Europe (WO 92/21375, Lelystad Agent isolate (CDINL- 2.91), deposited on June 5, 19991 at the Pasteur Institute, Paris, Accession No. 1-1102), identified viruses that had genomic and serological aberrations. The comparison demonstrated that they both have a common ancestor who diverged before the disease was described in the late 1980s. Complete genomic sequences have been published for a number of PRRS viruses and their complete structural protein coding regions (Snijder et al. 1998, Meulenberg et al., 1993b; Conzelmann et al. 1993; Murtaugh et al., 1995; Kapur et al. 1996). PRRS virus can replicate in vitro in porcine lung macrophages, monocytes, glial cells and two MA-104 cell subpopulations (embryotic monkey kidney cells) known as CL-2621 and MAEC-145 (KD Rossow, Porcine reproductive and respirators syndrome, Vet Pathol 35: 1-20, (1998) Recombinant means for generating infectious PRRS clones are also available (EP 0 839 912 A1).

Live attenuated PRRS vaccines (RespPRRS / Ingelvac® PRS MLV, Boehringer Ingelheim) are commercially available for the protection of pigs.

Killed vaccines (inactivated whole virus) or subunit vaccines (conventionally purified or heterologously expressed purified viral proteins) are most often inferior to live vaccines in terms of their efficacy in producing a complete protective immune response, even in the presence of an adjuvant. For PRRS, it has been demonstrated that compared to the parallel killers available

-3vaccines induce attenuated vaccines immunity to disease that lasts longer and is more effective (Snijder et al., Supra). Current live PRRS vaccines are usually attenuated by serial passage of the virus in suitable host cells, unless there is a loss of pathogenicity (EP 0529584 B1). Current live PRRS vaccines still leave room for improvement. First, do not prevent reinfection. Secondly, they do not allow serological discrimination between vaccinated animals and animals infected with the field virus. But most important of all, live vaccines from complete microorganisms, although weakened, may be associated with serious safety problems. This is particularly true of RNA viruses, such as PRRS virus, which are considered to have a high rate of mutation due to inaccurate replication of the RNA genome due to a lack of RNA reading repair by the replication enzyme.

The potential reversion of attenuated live viruses can cause serious consequences for vaccinated animals. In conventional virus-derived viruses, where attenuation is achieved by conventional multiple passages, the molecular origin as well as the genetic stability remains unknown and it is not possible to predict when the onset of revertants will occur.

Therefore, the technical problem solved by the present invention was to provide PRRS viruses that are less likely to convert to wild-type viruses. SUMMARY OF THE INVENTION

The invention relates to live PRRS viruses that are attenuated by amino acid mutations at specific sites of the viral protein that are encoded by an open reading frame (ORF) selected from the group consisting of ORF 1a, ORF 1b and / or ORF 2. The invention also relates to nucleotide sequences encoding said viruses, methods for generating such viruses and their use in the preparation of a pharmaceutical composition for the prophylaxis and treatment of PRRS infections.

The solution to the above technical problem is achieved by the description and embodiments characterized in the claims.

Before describing the embodiments themselves, it should be noted that, as used herein and in the appended claims, the singular forms "a," "an" and "the" include plural referents.

-4number unless the context clearly dictates otherwise. For example, the designation of a PRRS virus includes a plurality of such PRRS viruses, the designation of a cell designates one or more cells and its equivalents known to one of ordinary skill in the art, and so on. 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 method and material similar or equivalent to those described herein can be used to practice or test the present invention, preferred methods, devices and materials will now be described. All publications cited herein are hereby incorporated by reference in order to describe cell lines, vectors, and methodologies that have been disclosed in publications that could be used in connection with the invention. However, nothing in them is to be considered as relevant to the prior art with respect to the present invention.

Surprisingly, it has been found that PRRS viruses include specific genomic sites in some open reading frames that at a given position consistently revert to the amino acids of the original virulent field strain VR-2332. The evolutionary pressure at this, now called the virulent specific site or simply referred to as the site of the invention or simply the site, is enormous. For two revertant strains of RespPRRS, it was first demonstrated that the amino acid mutation to the original virulent terrain isolate amino acid at this virulent specific site occurred independently of geographical location, in both the US and Europe. Live vaccines with defined mutations as the basis of attenuation according to the invention make it possible to avoid the disadvantages of the current generation of attenuated vaccines. A further advantage of said attenuating mutations lies in their known molecular uniqueness which allows them to be used as distinctive markers for attenuated pestiviruses and to distinguish them from field pestiviruses.

The amino acid and nucleotide sequences in the conventional attenuated RespPRRS virus were compared to the original field isolate VR-2332. To identify virulent specific sites, the two virulent revertants were compared to each other and to VR-2332 as well as RespPRRS.

This allowed the identification of a virulence specific site in an individual viral protein that is involved in the virulence of PRRS viruses.

The present invention provides non-RespPRRS live PRRS viruses that are less virulent than ATCC VR-2332 PRRS virus, and are characterized by containing an open reading frame (ORF) encoded protein selected from the group consisting of ORF 1a as described in Figure 1 for said VR-2332 strain, ORF 1b as described in Figure 2 for said VR-2332 strain and / or aleboRF 2 as described in Figure 3 for said VR-2332 strain, wherein at least one of the amino acids in the identified virulent specific the sites are not identical to at least one of the amino acids of the VR-2332 strain at the corresponding positions.

The numbering of amino acids and nucleotides is consistent with the VR-2332 database entry. Figures 1, 2 and 3 provide information that is representative of all PRRS strains and allow visualization of the invention and identification of preferred amino acids and preferred sites in all PRRS viruses that could be numbered differently. The identification of these positions is achieved by identifying the retained characteristic identical amino acids in the PRRS strain of interest and said reference strain, and then determining the location of the virus site of interest with respect to the site in Figures 1, 2 and / or 3 .

Three of the listed sites for the protein encoded by the viral ORF 1a, ORF 1b and / or ORF 2 have been identified, which are shown for VR-2332 in Figures 1, 2 and / or 3.

Furthermore, the invention relates to a live attenuated PRRS virus comprising ORF 1a, 1b and 2 substantially as in VR 2332, which is not RespPRRS, and characterized in that at least one of the amino acids at positions 321 to 341 of ORF 1a is not identical to the amino acid the amino acid (s) of VR 2332 at the corresponding position (s) as shown in Figure 1 and / or at least one of the amino acids at positions 936 to 956 of ORF 1b is not identical to the amino acid (s) of VR 2332 at the corresponding position (s) as is shown in Figure 2 and / or at least one of the amino acids at positions 1 to 20 of ORF 2 is not identical to the amino acid (s)

-6, VR 2332 at the corresponding position (s) as shown in Figure 3. Thus, at 321-341 ORF 1a, at 936 to 956 ORF 1b, and / or at 1 to 20 ORF 2 (see Figures 4, 5 or 6) is a mutant encoding a nucleotide triplet for one or more than one amino acid, resulting in one or several sequence changes of said sites, either at the nucleic acid level or, preferably, also at the amino acid level; be mutated to all nucleotide or amino acid positions at said site. The present invention encompasses viruses in which either one or two or all three of said sites are mutated. A mutation means replacing the amino acid with another or replacing the coding nucleotide with another (e.g., T for C), a so-called substitution, preferably in such a way that the encoded amino acid is altered, or any other mutation, such as a deletion or insertion. The mutation is usually carried out in the coding nucleotide sequence.

Said mutations may be made by standard methods known in the art, e.g. site-specific mutagenesis (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) of an infectious copy as described, e.g. in Meulenberg et al., Adv. Exp. Med. Biol., 1998, 440: 199-206).

Essentially, at least 75% of the sequence, preferably 85%, most preferably the entire sequence except the sites of the invention is identical to VR 2332. However, other nucleic acids encoding amino acids outside the sites of the invention may also be mutated. Said virus according to the invention must meet the criteria according to the invention, ie. it must be less susceptible to wild type reversion and less virulent than VR 2332.

The live PRRS virus of the invention refers to the PRRS virus as defined in E. J. Snijder et al. (Cited above), which is able to infect pigs and is able to replicate in pigs. RespPRRS virus attenuated in the usual manner is specifically exempted from the claims. It is not a virus according to the invention and is specifically excluded from the scope of the claims. RespPRRS virus is commercially available from Boehringer Ingelheim Vetmedica (Ingelvac® PRS MLV). Most of its sequence is publicly available (Genebank AJ 223082).

The site and amino acids of particular importance according to the invention are in no way limited to the exact position as defined for the VR-2332 strain, but are simply used as an example to indicate preferred amino acids that are at a given position or position, which corresponds to it in other PRRS strains. For different PRRS viruses, the numbering of preferred amino acid positions may vary, but one skilled in the art of molecular biology of the arteriviridae will be able to easily identify these preferred amino acids by determining their position relative to other conserved amino acids of said proteins.

The term less virulent than PRRS virus VR-2332 is to be understood in relation to the comparison of clinical symptoms of the virus of interest with VR-2332. Preferred methods for determining whether the PRRS virus is less virulent than the PRRS virus VR2332 are set forth in Example 1. Not all possible preferred amino acid mutations at a virulent specific site can contribute to reducing virulence. The procedure of Example 1 provides accurate and direct experimental guidance for determining whether a live PRRS virus containing a protein of the invention is less virulent than a virulent field isolate VR-2332.

The virulent specific site was identified by reverting at least one amino acid from the attenuated virus to the amino acid from VR-2332. This particular amino acid is part of a larger secondary peptide structure, such as an alpha helix or a β-fold or a hairpin β motif or others. It is therefore highly probable that neighboring amino acids are also involved in the regulation of virulence of this protein. A person skilled in the art of protein chemistry would therefore expect a high probability that additional amino acids with properties contributing to virulence within 10 amino acids to the left and right of the originally identified amino acid position would be identified. 10 to 20 amino acids is a typical range of peptide motifs in proteins. Therefore, preferred viruses of the invention include the protein shown by way of example in Figure 1 or its corresponding protein in other strains, wherein the virulent specific site comprises 10 upstream and 10 downstream amino acids from the originally identified amino acid position. More preferred are those viruses as mentioned above in which the virulent specific site comprises 5 amino acids

-8 upstream and 5 amino acids downstream of the originally identified amino acid position. Most preferred are those viruses as mentioned above, in which the virulent specific site comprises 3 upstream amino acids and 3 downstream amino acids from the originally identified amino acid position.

With the guidance of the present invention, it is possible to generate attenuated PRRS strains from virulent field strains by mutating amino acids at a virulent specific site. There is still a safety problem associated with the high frequency of mutations in RNA viruses. This problem can be greatly reduced by deleting specific amino acids at the virulent specific site of the viral protein. The invention therefore relates to PRRS viruses as described above, characterized in that at least one of the amino acids at the virulence specific site of the viral protein is deleted. The term deleted should be understood to be missing when compared to the amino acids shown in the figure at or at these positions. Thus, in accordance with the invention, deletion means the removal of one or more nucleotides or amino acids.

Therefore, in a more preferred embodiment, the invention relates to a live attenuated PRRS virus according to the invention, characterized in that at least one of the amino acids at positions 321 to 341 of ORF 1b is deleted and / or at least one of the amino acids at positions 936 to 956 of ORF1b is deleted and / or at least one of the amino acids at positions 1-20 of ORF2 is deleted. So either at 321 to

341 ORF 1a, or at 936 to 956 ORF 1b, or at 1 to 20 ORF 2, or at all three sites, is a nucleotide triplet encoding an amino acid or several triplets deleted, resulting in one or more deletions at said sites. The present invention encompasses viruses in which only one nucleotide or only one amino acid is deleted, as well as viruses in which one or all of said sites are completely deleted (see Figures 4, 5 or 6).

In order to identify a virulent specific site in a particular PRRS viral protein, in a preferred example, it has been demonstrated that at least one amino acid is under strong mutation pressure and is involved in the virulent properties of revertants of the VR-2332 field strain. This individual amino acid position is the most preferred embodiment of the invention.

Therefore, the most preferred embodiment of the present invention relates to a live attenuated PRRS virus of the invention which is not RespPRRS and which is less virulent than ATCC VR 2332, characterized in that the amino acid at position 331 of ORF 1a and / or amino acid at position 946 of ORF 1b and / or the amino acid at position 10 of ORF 2 is not identical to the amino acid of ATCC VR 2332 at the corresponding position. Thus, either at 331 of ORF 1a, or at 946 of ORF 1b, or at 10 of ORF 2, or at all three sites, the nucleotide triplet encoding the amino acid or amino acid is mutated, resulting in one to three mutations at those sites (see Figures 4 , 5 or 6).

For the above reasons, it is safer and advantageous to prevent the reversion of altered amino acids to the virulent type by simply deleting an amino acid that is prone to reverse.

Therefore, the most preferred embodiment of the present invention relates to the live attenuated PRRS virus of the invention, characterized in that the amino acid at position 331. ORF 1a and / or the amino acid at position 946 of ORF 1b and / or the amino acid at position 10 ORF2 is deleted. when compared to the corresponding position in VR-2332.

Another most preferred embodiment is the live attenuated PRRS virus of the invention, characterized in that the amino acid at position 331 of ORF 1a, i. encoding the triplet is deleted. Another most preferred embodiment is the live attenuated PRRS virus of the invention, characterized in that the amino acid at position 946 of ORF 1b, i. encoding the triplet is deleted. Another most preferred embodiment is the live attenuated PRRS virus of the invention, characterized in that the amino acid at position 10 of the ORF 2, i. encoding the triplet is deleted (see Figures 4, 5 or 6). Said most preferred virus is identical to VR-2332 at all other positions.

The disclosure of the present invention now allows the skilled artisan to recombinantly produce infectious clones of PRRS viruses that are less virulent than VR-2332 and are useful for preparing a live pharmaceutical composition. All the information necessary for the production of recombinant infectious positive-strand RNA virus clones is readily available in the art, in particular for PRRSV. For example, European patent application EP 0839912 on behalf of Meulenberg et al.

Which is fully incorporated herein, provides clear guidance on the preparation of recombinant live PRRS viruses. Therefore, another aspect of the present invention relates to nucleotide sequences encoding a virus of the invention. Due to the degeneracy of the genetic code, multiple nucleotide variants will result in identical amino acid translation. The invention also encompasses these degenerate variants.

As mentioned in the introductory part, the ability to distinguish between a less virulent live vaccine strain of a pharmaceutical composition and virulent wild-type viral infections is important for the organization of pig health care. This is often difficult, especially when the clinical symptoms of a field infection are not as specific or overlapped by other infections, or when there is a short time to observe and evaluate. Recombinant generation of the viruses of interest allows for the incorporation of modifications in the genetic code that introduce a serological marker and / or a virulent marker. A serological marker refers to an antigen-detectable molecule such as a peptide, 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 virulent 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, a preferred embodiment of the present invention relates to a nucleotide sequence of the invention that has been modified to encode a virulent marker and / or a serological marker. Mutations or deletions introduced to attenuate virulence are particularly useful as virulent and serological markers. By monitoring these mutations at the described virulent specific sites, it is possible to predict the appearance of possible virulent revertants at an early stage.

More preferably, the nucleotide sequences of the invention are such that the nucleotide sequence encoding said marker is located within any of the open reading frames encoding structural viral proteins.

Furthermore, the invention relates to a method for generating an infectious live attenuated PRRS virus of the invention, which comprises producing recombinant

A nucleic acid as described above comprising at least one complete DNA copy or an in vitro transcribed RNA copy or derivative of one or the other.

Another preferred embodiment of the invention relates to a method according to the invention in which specific mutations are introduced by molecular biology methods in which the nucleic acid corresponding to amino acid positions 321 to 341 of ORF 1a and / or nucleic acid corresponding to amino acid positions 936 to 956 of ORF 1b and / or a nucleic acid corresponding to amino acid positions 1 to 20, mutated in such a way that at least one nucleotide is substituted or deleted at said positions.

Another important aspect of the invention is a pharmaceutical composition comprising the PRRS virus of the invention and a pharmaceutically acceptable carrier.

The pharmaceutical composition essentially consists of one or more components which are capable of modifying physiological, e.g. the immunological, functions of the organism to which they are administered, or the organisms that live in or on it, such as, but not limited to, antibiotics or antiparasitics, as well as other ingredients that are added to the composition to achieve certain other objectives, such as, but not limited to, processing properties, sterility, stability, the ability to simply administer the composition in an enteral or parenteral manner such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal but other appropriate means, post-administration tolerance, controlled release properties.

The pharmaceutically acceptable carrier may contain physiologically acceptable compounds that serve, for example, to stabilize or enhance absorption, or form part of a slow secreted PRRS virus formulation of the invention. Such physiologically acceptable compounds include, for example, hydrocarbons 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, e.g., Remington's Pharmaceutical Sciences (1990). 18). (Mack Publ., Easton). It will be apparent to one of ordinary skill in the art that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the mode of administration of the compound.

Another object of the invention relates to the use of the viruses according to the invention. By making available live attenuated PRRS viruses of the invention, they can now be used to produce a vaccine for the prophylaxis and treatment of PRRS infections. Defining the molecular basis of their attenuation makes them excellent compared to currently attenuated viruses. Particularly preferred is the use of viruses according to the invention which contain deletions at virulent specific sites, since the deletions are less susceptible to reversion.

The following example serves to illustrate the present invention in more detail, but is not to be construed as limiting the scope of the invention described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Amino acid sequence of ORF 1a ATTC VR-2332 with preferred attenuation sites designated according to the invention.

Figure 2: Amino acid sequence of ORF 1b ATTC VR-2332 with preferred attenuation sites designated according to the invention.

Figure 3: Amino acid sequence of ORF 2 of ATTC VR-2332 with preferred weakening sites of the invention indicated.

Figure 4: Nucleotide sequence of ORF 1a ATTC VR-2332 with bold outlined preferred attenuation sites of the invention and the most preferred sites are underlined.

Figure 5: Nucleotide sequence of ORF 1b ATTC VR-2332 with bold outlined preferred attenuation sites of the invention and the most preferred sites are underlined.

Figure 6: Nucleotide sequence of ORF 2 ATTC VR-2332 with bold outlined preferred attenuation sites of the invention and the most preferred sites are underlined.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Introduction of weakness

This example provides a clear guide to comparing the virulence of two different strains of PRRS viruses.

At least 10 young sows per group that are from a PRRS-free farm are included in each experiment.

I

The animals are tested to be free of PRRS virus specific serum antibodies and are PRRSV negative. All animals included in the study are from the same source and breed. The animals are divided into groups at random.

Infection is performed on day 90 of pregnancy by intranasal administration of 1 ml PRRSV with 10 ⁵ TDCID 50 (third passage) per nostril. There are at least three groups for each test set: One group per VR-2332 infection; one test group for infection with a possible attenuated virus; and a strict control group.

The value of the study is confirmed when strict controls remain negative during the PRRS study and when at least 25% fewer live healthy piglets are born in the VR 2332-infected group compared to strict controls.

Attenuation, in other words, lower virulence, is defined as a statistically significant change in one or more parameters determining reproductive performance:

A significant reduction of at least one of the following parameters in the test group is preferred:

- frequency of stillbirths

- Abortion for 112 days or before 112 days of pregnancy

- number of shriveled piglets

- number of live and weak piglets

- premature weaning mortality or, in addition, a significant increase in one of the following parameters in the test group is advantageous:

- number of suckling piglets per litter

- number of live healthy piglets born per litter

- compared to VR2332 infected group.

References

1. Snijder E.J., Meulenberg J.J.M., 1998. The molecular biology of arteriviruses, Journal of General Virology, May 79 (5): 961-971.

2. Meulenberg J.J.M., Hulst, M.M. et al., 1993b. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome, Virology 192, 62-72.

3. Conzelmann K.K., Visser N., Van Woensel P., Thiel H. J., 1993. Molecular characterization of porcine reproductive and respiratory syndrome virus, and member of the arterivirus group, Virology 103, 329-339.

4. Murtaugh M. P., Elam M. R., Kakach L. T., 1995. Comparison of the structural protein coding sequences of VR-2332 and Lelystad virus strains of the PRRS virus, Archives of Virology 140, 1451-1460.

5. Kapur V., Elam M. R., Pawtovich T. M. Murtaugh M. P., 1996. Genetic Variation in Porcine Reproductive and Respiratory Virus Isolates Syndrome in the Midwestern United States, Journal of General Virology 77,1271-1276.

Rossow K. D., 1998. Porcine reproductive and respiratory syndrome, Vet Pathol 35: 1-20.

Claims (11)

PATENT CLAIMS A live attenuated PRRS virus comprising ORF 1a, 1b and 2, essentially as in VR-2332, which is not RespPRRS, and characterized in that at least one of the amino acids at positions 321 to 341 of ORF 1a is not identical to the amino acid the amino acid (s) of VR-2332 at the corresponding position (s) and / or at least one of the amino acids at positions 936 to 956 of ORF 1b is not identical to the amino acid (s) of VR-2332 at the corresponding position (s) and / or of the amino acids at positions 1 to 20 of ORF 2 is not identical to the amino acid (s) of the VR-2332 strain at the corresponding position (s). The live attenuated PRRS virus according to claim 1, characterized in that at least one of the amino acids at position 321 to 341 of ORF 1a is deleted and / or at least one of the amino acids at position 936 to 956 of ORF 1b is deleted and / or of the amino acids at positions 1-20 of ORF2 is deleted. The live attenuated PRRS virus according to claim 1 or 2, characterized in that the amino acid at position 331 of ORF 1a and / or the amino acid at position 946 of ORF 1b and / or the amino acid at position 10 of ORF 2 is not identical to the amino acid of ATTC VR. 2332 in the corresponding position. The live attenuated PRRS virus according to any one of claims 1 to 3, characterized in that the amino acid at position 331 of ORF 1 'a and / or the amino acid at position 946 of ORF 1b and / or the amino acid at position 10 of ORF 2 is deleted. The nucleotide sequence encoding the virus of any one of claims 1 to 4. Nucleotide sequence according to claim 5, which has been modified to encode a virulent marker and / or a serological marker. The nucleotide sequence of claim 6, wherein the nucleic acid encoding the marker is located within any of the open reading frames encoding structural viral proteins. A method for generating an infectious live attenuated virus, comprising producing a recombinant nucleic acid comprising at least one full DNA copy or an in vitro transcribed RNA copy or derivative thereof, wherein said nucleotide sequence is a nucleotide sequence according to any one of claims 5 to 7. The method of claim 8, wherein specific mutations are introduced by molecular biology methods, characterized in that a nucleic acid corresponding to amino acid positions 321 to 341 of ORF 1a and / or a nucleic acid corresponding to amino acid positions 936 to 956 of ORF 1b and / or nucleic acid the acid corresponding to amino acid positions 1 to 20 of ORF 2 is mutated in such a way that at least one nucleotide is substituted or deleted at said positions. A pharmaceutical composition comprising a PRRS virus according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. Use of a PRRS virus according to any one of claims 1 to 4 for the manufacture of a vaccine for the prophylaxis and treatment of PRRS infections.