MXPA02007198A - Recombinant attenuation of prrsv. - 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

RECOMBINANT ATTENUATION OF PRRSV Field of the invention The present invention relates to live PRRS viruses that are attenuated by mutations of amino acids at specific sites of the viral protein encoded by the open reading frame (ORF), selected from the ORF group, ORF Ib and / or ORF 2. The invention also pertains to nucleotide sequences encoding the viruses, to methods for generating such viruses and to their use for the preparation of a pharmaceutical composition for the prophylaxis and treatment of PRRS infections. BACKGROUND OF THE INVENTION Mysterious swine disease, subsequently renamed porcine reproductive and respiratory syndrome (PRRS), is caused by a virus of positive chain RNA with cover of the family arteviridae (E.J. Snijder, 1998). Approximately 10 to 15 years ago, two strains of different PRRS viruses apparently emerged independently in the USA. and Europe. The disease is r. ** TO *. í *. currently endemic in many countries producing pigs in North America, Europe and Asia. It continues to be a major cause of the disease of reproductive and respiratory loss in pigs. In the USA. It is estimated that the prevalence of the infection is up to 70%. The virus is transmitted by inhalation, ingestion, intercourse, wounds from bites or needles. It replicates in mucosal, pulmonary or regional macrophages. Subclinically, the disease results in resolution or persistent infection. Persistently infested animals conceal the virus in oral / pharyngeal fluids, blood, feces, urine and semen. Clinical symptoms in sows refer to abortion or premature delivery with weak live born pigs, stillborn pigs and autolized fetuses. Infested neonatal pigs have high mortality or suffer from pneumonia. The breeding and subsequent development of pigs are complicated by pneumonia, concurrent bacterial infections and increased mortality. Berracos are prone to fever and morphological changes in semen. As for all arterioviruses, the PRRS virus genome is a single positive strand RNA molecule of approximately 15 kilobases. The ORFs (open reading frame) la and Ib encode replicases, the ORFs 2 to 5 encode putative glycoproteins (gp 1 to 4), ORF 6 encodes a membrane protein (M) and ORF 7 encodes a nucleocapsid protein ( N) The original descriptions of the PRRS infection in the US (ATCC VR-2332 isolated, deposited on July 18, 1991 in American Type Culture Collection in Rockville, Maryland, USA, Genebank U 87392 U00153) and Europe (WO 92/21375, isolated agent Lelystad (CDI-NL- 2.91), deposited on June 5, 1991 at the Pasteur Institute, Paris, accession number 1-1102) identified viruses that had genomic and serological differences. The comparison showed that both had a common ancestor that had diverged before the clinical disease was described in the late 1980s. For a number of PRRS viruses, full length genomic sequences and their protein coding regions have been reported. complete structural (Snijder et al 1998, Meulenberg et al 1993b, Conzelmann et al 1993, Murtaugh et al 1995, Kapur et al 1996). PRRS viruses can be replicated in vitro in pig lung macrophages, monocytes, glia cells and two subpopulations of MA-104 cells (embryonic monkey kidney cell) known as CL-2621 and MARC-145 (KD Rossow, Porcine reproductive and respiratory syndrome, Vet Pathol 35: 1-20, (1998)). Recombinant media are also available to generate infectious PRRS clones (EP 0 839 912 A1). To protect pigs, live attenuated PRRS vaccines are commercially available (RespPRRS / Ingelvac® PRS MLV, Boehringer Ingelheim). Exterminated vaccines (inactivated whole virus) or subunit vaccines (viral proteins conventionally purified or purified in a heterologously expressed manner) are often inferior to live vaccines in their efficacy to produce a complete protective immune response, even in the presence of adjuvants. For the PRRS * ^ * *? * *? * á i íitSm * it has been shown that, compared to the currently available exterminated vaccines, attenuated vaccines induce an immunity against the disease that lasts longer and is more effective (Snijder et al., alluded to above). The present PRRS live vaccines are conventionally attenuated by serially passing the virus into appropriate host cells until their pathogenicity is lost (EP 0529584 Bl). The current live PRRS vaccines are still leaving a wide margin for improvement. On the one hand, they do not prevent reinfection. Second, they do not allow serological discrimination between vaccinated animals and animals infested with the field virus. But the most important of all is that live vaccines from complete microorganisms, although attenuated, can be associated with serious safety problems. This is especially true for RNA viruses such as PRRS virus that are considered to have high mutation rates due to inaccurate replication of the RNA genome resulting from a lack of test reading by the RNA replication enzyme.

A potential reversion of live attenuated viruses can impose a serious threat to vaccinated animals. For conventionally attenuated attenuated viruses, where attenuation is achieved by a conventional multiple passage, the molecular origin, as well as the genetic stability remain unknown, and the outbreak of revertants is unpredictable. Therefore, the technical problem on which this invention is based was to provide PRRS viruses that are less likely to reverse than wild type viruses. The invention The present invention relates to live PRRS viruses that are attenuated by amino acid mutations at specific sites of the viral protein encoded by the open reading frame (ORF), selected from the group ORF, ORF Ib and / or ORF 2. The invention also pertains to nucleotide sequences encoding the viruses, to methods for generating such viruses and to their use for the preparation of a pharmaceutical composition for the prophylaxis and treatment of PRRS infections.

Legends of the figures Fig. 1: ORF amino acid sequence of ATTC VR-2332 with marked attenuation sites according to the invention. Fig. 2: amino acid sequence of ORF Ib of ATTC VR-2332 with marked attenuation sites according to the invention. Fig. 3: amino acid sequence of ATTC ORF 2 VR-2332 with labeled preferred attenuation sites according to the invention. Fig. 4: ORT nucleotide sequence ATTC VR-2332 with preferred attenuation sites according to the invention in bold type and most preferred underlined sites. Fig. 5: Nucleotide sequence of ORF Ib of ATTC VR-2332 with preferred attenuation sites according to the invention in bold type and the most preferred underlined sites. Fig. 6: nucleotide sequence of ATTC ORF 2 VR-2332 with preferred attenuation sites according to the invention in bold and the most preferred sites underlined.

Description of the invention The solution to the above technical problem is achieved by the description and the embodiments characterized in the claims. Prior to the embodiments of the present invention it should be noted that, as used herein and in the appended claims, the singular forms of "a (a) "and" the (the) "include the plural reference, unless the context clearly dictates otherwise.

Thus, for example, the reference to "a PRRS virus" includes a plurality of such PRRS viruses, the reference to "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art. , etc. Unless defined otherwise, all technical and scientific terms and expressions used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Although in practice or in the test of the present invention, any methods and materials similar or equivalent to those described herein can be used., the preferred methods, devices and materials are now described. All publications mentioned herein are incorporated herein by reference in order to describe and explain the cell lines, vectors and methodologies outlined in the publications, which could be used in connection with the invention. Nothing in this specification is to be construed as an admission that the invention is not authorized to anticipate such a description by virtue of the prior invention. It has been found, surprisingly, that PRRSs comprise specific genomic sites in some open reading frames that consistently revert to the amino acids of the original virulent field strain VR-2332 at that position. The evolutionary pressure on this, from now called "specific site of virulence" or simply called "site of invention" or simply "site" is immense. For two RESPPRRS revertant strains, it was possible to demonstrate for the first time that the mutation of amino acids to the amino acid of the original virulent field isolate at this virulence-specific site 5 occurred geographically independently in both the USA and the USA. as in Europe. Live vaccines with mutations defined as the basis of the attenuation according to the invention make it possible to avoid the disadvantages of the present generation of vaccines 10 attenuated. An additional advantage of attenuating mutations lies in their known molecular singularity which allows them to be used as distinctive markers for attenuated pestiviruses and to distinguish them from pestiviruses from the field. 15 The amino acid and nucleotide sequence of the virus The conventionally attenuated RespPRRS was compared to the original field VR-2332 isolate. To identify the specific sites of virulence, the two mentioned virulent revertants were compared with each other and with VR-20 2332 as well as RespPRRS, respectively. This allowed the identification of the specific site of virulence in an individual viral protein that is involved in the virulence of PRRS virus. Accordingly, one aspect of the invention relates to live PRRS viruses that are not RespPRRS and that are * *. *** ** .... * - ^ * ..... less virulent than the PRRS virus ATCC VR-2332, which are characterized in that they comprise a protein encoded by the open reading frame (ORF), selected from the ORF group as described in figure 1 for strain VR-2332, ORF Ib as described in figure 2 for strain VR-2332 and / or ORF 2 as described in figure 3 for strain VR-2332, where at least one of the amino acids at the specific virulence sites identified does not is identical to at least one of the amino acids of strain VR-2332 in the corresponding positions. The numbering of amino acids and nucleotides is in accordance with the database entry of VR-2332. Figures 1, 2 and 3 provide information that is representative of all PRRS strains and allows to visualize the invention and identify the preferred amino acids and the preferred site in all PRRS viruses that may be numbered differently. The identification of these positions is achieved by identifying characteristic identical amino acids conserved in a PRRS strain of interest and the listed reference strain and subsequently determining the position of the site of the virus of interest relative to the site in Figure 1, 2 and / or 3 , respectively. Three of the sites have been identified for the protein encoded by the ORF, ORF Ib and / or viral ORF 2 that are described for VR-2332 in Figure 1, 2 and / or 3.

Another aspect of the invention relates to a live attenuated PRRS virus comprising ORF la, Ib and 2 essentially as in VR-2332, which is not RespPRRS, characterized in that at least one of the amino acids at position 321 to 341 of ORF is not or are identical to the amino acid (s) of strain VR-2332 in the corresponding position (s) as described in Figure 1, and / or at least one of the amino acids in position 936 to 956 of ORF Ib is not or are identical to the amino acid (s) of strain VR-2332 in the corresponding position or positions as described in Figure 2, and / or at least one of the amino acids in position 1 to 20 of ORF 2 is not or are identical or the amino acids of strain VR-2332 in the corresponding position (s) as described in figure 3. Thus, at site 321-341 of ORF la, site 936-956 of ORF Ib and / or site 1 -20 of ORF 2 (see figures 4, 5 or 6, respectively), the nucleotide triplet encoding an amino acid, or more than one amino acid, is mutated, resulting in one or more changes in the sequence at the sites, either at the nucleic acid level or , additionally and preferably, also at the amino acid level, after which all the nucleotide or amino acid positions at the site may be mutated. Viruses in which one, two or all three of the sites are mutated are encompassed by the present invention. "Mutation" means the replacement of one amino acid by another or the replacement of the coding nucleotide by another (for example C by a T), the so-called "substitution", preferably in a manner that changes the encoded amino acid, or any other mutation such as " deletion "or" insertion ". The mutation is always carried out in the coding nucleotide sequence. Mutations can be carried out by conventional methods known in the art, for example site-directed mutagenesis (see, for example, 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, for example, by Meulenberg et al., Adv. Exp. Med. Biol., 1998, 440: 199-206). "Essentially" means that at least 75% of the sequence, preferably 85%, most preferably the entire sequence, except the "sites" according to the invention is identical to VR-2332. However, additional nucleic acids encoding amino acids outside the sites according to the invention may also be mutated. The virus according to the invention continues to meet the criteria according to the invention, ie it is less likely to revert to the wild type and is also less virulent than VR-2332. A live PRRS virus according to the invention refers to a PRRS virus as defined in E.J. Snijder et al. (referenced above), which is capable of infesting pigs and is capable of replicating in pigs. The conventionally attenuated RespPRRS virus is specifically dereclaimed. It is not a virus according to the invention and is specifically excluded from the scope of the claims. The RespPRRS virus is commercially available from the company Boehringer Ingelheim Vetmedica (Ingelvac® PRS MLV). Most of its sequence is publicly available (Genebank AJ 223082). The site and the amino acids of particular importance of the invention are by no means limited to the exact position as defined for strain VR-2332, but are simply used in an exemplary manner to designate the preferred amino acids found therein. position or correspond to that position in other strains of PRRS. For different PRRS viruses, the numbering of the positions of the preferred amino acids could be different, but an expert in the Molecular Biology sector of the family of arteriviridae viruses will easily identify these preferred amino acids by their position in relation to the other conserved amino acids. of proteins. The expression "less virulent than the PRRS virus VR-2332" is to be understood in terms of a comparison of clinical symptoms of the virus of interest with VR-2332. A preferred method for determining whether a PRRS virus is less virulent than the PRRS virus VR-2332 is listed in Example 1. Not all possible mutations of preferred amino acids at the virulence specific site could be involved to reduce virulence. The procedure of Example 1 provides a precise and direct experimental arrangement for determining whether a live PRRS virus, comprising the protein according to the teaching of the invention, is less virulent than the virulent field isolated VR-2332.

The specific virulence site was identified by reverting at least one amino acid from the attenuated virus to the amino acid of VR-2332.

This particular amino acid is part of a larger secondary peptide structure such as an alpha helix or a β-sheet or a hairpin motif β or others. Therefore, it is very likely that neighboring amino acids are also involved in the regulation of the virulence of that protein. An expert in the field of protein chemistry would therefore expect a high probability of identifying additional amino acids with properties involved in virulence within the vicinity of 10 amino acids to the left and right of the position of the originally identified amino acid. 10 to 20 amino acids is the typical interval for peptide motifs in proteins. Therefore, preferred viruses according to the invention comprise the protein described in Figure 1 in an exemplary manner or correspondingly thereto in other strains, wherein the virulence-specific site comprises 10 amino acids located above and 10 amino acids located below the amino acid position originally identified. More preferred are the viruses as mentioned above, wherein the specific virulence site comprises 5 amino acids located above and 5 amino acids located below the position of the originally identified amino acid. Most preferred are viruses as mentioned above, wherein the virulence-specific site comprises 3 amino acids located above and 3 amino acids located below the position of the originally identified amino acid. With the teaching of the present invention it is possible to generate attenuated PRRS strains from virulent field strains by mutating the amino acids at the specific site of virulence. The safety problem associated with the high frequency of mutation in RNA viruses still remains. This problem can be greatly reduced by suppressing specific amino acids at the specific virulence site of the virus protein. Therefore, the invention relates to PRRS viruses as mentioned above, which are characterized in that at least one of the amino acids at the specific site of virulence of the viral protein is deleted. The term "suppressed" is to be understood as absent in comparison with the amino acids referenced in the figure in that or in those positions. Thus, according to the invention, "deletion" means the separation of one or more nucleotides or amino acids. In a more preferred embodiment, the invention therefore relates to a live attenuated PRRS virus according to the invention, characterized in that at least one of the amino acids at position 321 to 341 of ORF is deleted or deleted. / or at least one of the amino acids at position 936 to 956 of ORF Ib is deleted or and / or at least one of the amino acids at position 1-20 is deleted or deleted. Thus, at any of the sites 321- 341 of ORF la, of sites 936-956 of ORF Ib or of sites 1-20 of ORF 2, or in all three sites the encoding nucleotides for one triplet of amino acid are deleted or are deleted, for several triplets, giving as result one or more deletions in the sites. The present invention encompasses viruses in which only one nucleotide or one amino acid is deleted, as well as viruses in which one of the sites or even all sites are completely deleted (see Figure 4, 5 or 6, respectively). For the specific site of virulence identified in the particular PRRS virus protein it has been shown, in a preferred example, that at least one amino acid is under a high mutational pressure and is involved in the virulent properties of revertants of the VR-strain. 2332 of field. This individual position of the amino acid is a more preferred embodiment of the invention. Thus, in a more preferred embodiment, the present invention relates to a live attenuated PRRS virus according to the invention that is non-RespPRRS and that is less virulent than ATCC VR-2332, characterized in that the amino acid at position 331 of ORF and / or the amino acid at position 946 of ORF Ib and / or the amino acid at position 10 of ORF 2 is not or are identical to the amino acid of strain ATCC VR-2332 in the corresponding position. Thus, at either the ORF la site 331, site 946 of ORF Ib or site 10 of ORF 2, or at all three sites the coding nucleotide is mutated for an amino acid triplet or amino acid, resulting in a three mutations in the sites (see Figure 4, 5 or 6, respectively). For the reasons presented above, it is safer and preferable to avoid the reversal of altered amino acids in the virulent type, simply by suppressing the amino acid that is prone to revert. Therefore, the present invention relates, in this most preferred embodiment, to a live attenuated PRRS virus according to the invention, characterized in that the amino acid at position 331 of the ORF and / or the amino acid in the position is deleted. 946 of ORF Ib and / or the amino acid at position 10 of ORF 2, in other words, is absent when compared at that position in VR-2332. A further, more preferred embodiment is a live attenuated PRRS virus according to the invention, characterized in that the amino acid at position 331 of the ORF la, ie the coding triplet, is deleted. A further, more preferred embodiment is a live attenuated PRRS virus according to the invention, characterized in that the amino acid at position 946 of ORF Ib, ie the coding triplet, is deleted. A further, more preferred embodiment is a live attenuated PRRS virus according to the invention, characterized in that the amino acid at position 10 of ORF 2, ie the coding triplet (see Figure 4, 5 or 6, respectively, is deleted). ). The most preferred virus is identical to VR-2332 in all other positions. The teaching 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 that are useful for preparing a living pharmaceutical composition. All the information required to produce recombinant infectious clones of positive strand RNA viruses, particularly for PRRSV, is readily available in the art. For example, European patent application EP 0 839 912 of Meulenberg et al, hereinabove referred to in its entirety, provides clear teaching for the preparation of recombinant live PRRS viruses. Therefore, in a further aspect, the present invention relates to nucleotide sequences encoding a virus according to the invention. Due to the degeneracy of the genetic code, multiple nucleotide variants will result in the translation of identical amino acids. These degenerate variants are also included in the invention. As mentioned in the introduction 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 viral infections. This is often difficult, especially when the clinical symptoms of a field infection are not specific or overlapping with other infections or the period of time for observation and evaluation is short. The recombinant generation of the viruses of interest allows the introduction of modifications in the genetic code that establishes a serological marker and / or a marker of 10 virulence. A serological marker refers to an antigenically detectable molecule such as a peptide, a protein, glycoprotein that can be isolated from infested cells or bodily fluids such as, but not limited to pharyngeal or nasal fluids or urine. A The 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 preferred embodiment, the present invention is 20 refers 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. In particular, mutations or mutations are useful as virulence and serological markers 25 deletions introduced in order to attenuate the - - '- "-" virulence. By monitoring these mutations at the specific sites of virulence described, it is possible to predict the outbreak of potentially virulent revertants at an early stage. It is more preferred that the nucleotide sequences of the invention be such that the nucleotide sequence encoding the marker is located within any of the open reading frames that encode structural viral proteins. A further aspect of the present invention relates to a method for the generation of an infectious live attenuated PRRS virus according to the invention, the method comprising producing a recombinant nucleic acid as described above comprising at least one DNA copy of full length or a copy of RNA transcribed in vitro or a derivative of any of them. Another preferred embodiment according to the invention relates to a method according to the invention, in which specific mutations are inserted with molecular biology methods, characterized in that the nucleic acid corresponding to the amino acid positions 321 to 341 of ORF and / or the nucleic acid corresponding to amino acid positions 936 to 956 of ORF and / or the nucleic acid corresponding to amino acid positions 1 to 20 is mutated so that at least one nucleotide at the positions is substituted or deleted. Another important aspect of the invention is a pharmaceutical composition comprising a PRRS virus according to the invention and a pharmaceutically acceptable carrier. A "pharmaceutical composition" consists essentially of one or more ingredients capable of modifying physiological functions, for example immunological, of the organism to which it is administered or of organisms living in or on its surface such as, but not limited to antibiotics or antiparasitics, as well as other constituents added thereto in order to achieve certain other objectives such as, but not limited to, features of treatment, sterility, stability, ability to administer the compositions enterally or parenterally such as orally, intranasally, intravenous, intramuscular, subcutaneous, intradermal or other suitable route, tolerance after administration, controlled release properties. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds which act, for example, to stabilize or increase absorption or form part of a slow release formulation of the PRRS virus according to the invention. Physiologically acceptable compounds of this type include, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (see also, for example, Remington's Pharmaceutical Sciences (1990). 18th ed. Mack Publ., Easton). One skilled in the art will know that the choice of a pharmaceutically acceptable vehicle, which includes a physiologically acceptable compound, depends, for example, on the route of administration of the composition. A further aspect of the invention relates to the use of the viruses according to the invention. With the availability of the live attenuated PRRS viruses of the invention it is now possible to use these in the manufacture of a vaccine for the prophylaxis and treatment of PRRS infections. Their defined molecular attenuation base makes them superior to conventionally attenuated current viruses. Especially, the use of viruses according to the invention comprising deletions at the specific sites of virulence is preferred, since the deletions are less prone to revert. The following example serves to further illustrate the present invention, but it should not be considered as limiting the scope of the invention described herein. References l.Snijder E.J., Meulenberg J.J.M., 1998. The 20 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. 25 3. Conzelmann K.K. , Visser N., Van Oensel P., Thiel * n? ufre? * i * H.J., 1993. Molecular characterization of reproductive reproductive and respiratory syndrome virus, a member of the arterivirus group, Virology 103, 329-339. 4. Murtaugh M.P., Elam M.R. , Ka ach 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. 5. Kapur V., Elam M.R. , Pa tovich T.M. Murtaugh M.P., 1996. Genetic variation in reproductive and respiratory syndrome virus isolates in the midwestern United States, Journal of General Virology, 77, 1271-1276. ß.Rossow K.D., 1998. Porcine reproductive and respiratory syndrome, Vet Pathol 35: 1-20. Example 1 Establishment of attenuation This example provides a clear guide for the comparison of the virulent character of two different strains of PRRS virus. Each test includes at least 10 young sows per group, which come from a farm free of PRRS. The animals are tested free of serum PRRSV-specific antibodies and 5 negative for PRRSV. All the animals included in the trial are from the same source and breed. The allocation of animals to the groups is random. The confrontation is carried out on the 90th day of pregnancy with intranasal application of 1 ml of PRRSV with 105 TCID50 (third step) through the nostril. For each test set there are at least three groups: a group for the confrontation with VR-2332; a test group for the confrontation with the possibly attenuated virus; and a strictly witnessed group. 15 The validity of the study is given when the strict witnesses remain negative PRRS throughout the course of the study and at least 25% fewer live healthy piglets are born compared to the strict controls in the group confronted with VR-2332. 20 Attenuation, in other words less virulence, is defined as the statistically significant change of one or more parameters that determine reproductive behavior: A significant reduction is preferred in at least 25 one of the following parameters for the test group: ^^^^ jg ^ s. 5 frequency of stillbirths abortion on or before day 112 of pregnancy number of mummified piglets number of live and weak piglets pre-weaning mortality or, in addition, a significant increase in one of the following parameters is preferred for the test group: number of piglets weaned per sow number of live healthy piglets born by 15 sow compared to the group infested with VR-2332. twenty 25 »Ihtih? 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Claims (1)

1. CLAIMS 1. - A live attenuated PRRS virus comprising ORF la, Ib and 2, essentially as in VR-2332, which is non-RespPRRS, characterized in that at least one of the amino acids at positions 321 to 341 of ORF is not or is not identical to the amino acid (s) of strain VR-2332 at the corresponding position (s), and / or at least one of the amino acids at positions 936 to 956 of ORF Ib is not or is not identical to the amino acid (s) of the VR strain -2332 in the corresponding position (s) and / or at least one of the amino acids in positions 1 to 20 of ORF 2 is not or is not identical to the amino acid (s) of strain VR-2332 in the corresponding position (s). 2. - A live attenuated PRRS virus according to claim 1, characterized in that said at least one of the amino acids at positions 321 to 341 of ORF is or are deleted, and / or at least one of the amino acids in the positions 936 to 956 of ORF Ib are or are deleted and / or at least one of the amino acids at positions 1 to 20 is or are deleted. 3. - A live attenuated PRRS virus according to any one of claims 1 or 2, characterized in that the amino acid at position 331 of the ORF la and / or the amino acid at position 936 of ORF Ib and / or the amino acid in the position 10 of ORF 2 is not or is not identical to the amino acid of strain ATCC VR 2332 in the corresponding position. 4. A live attenuated PRRS virus according to any one of claims 1 or 3, characterized in that the amino acid at position 331 of ORF la and / or the amino acid at position 946 of ORF Ib and / or the amino acid in position 10 of ORF 2 is or are deleted. 5. A nucleotide sequence encoding a virus according to any one of claims 1 to 4. 6. A nucleotide sequence according to claim 5, wherein the nucleotide sequence has been modified to encode a nucleotide sequence. virulence marker and / or a serological marker. 1 . - A nucleotide sequence according to claim 6, wherein the nucleic acid encoding said marker is located within any of the open reading frames that encode structural viral proteins. 8. A method for the generation of an infectious live attenuated PRRS virus, the method comprising producing a recombinant nucleic acid comprising at least one full-length DNA copy or an RNA copy transcribed in vitro or a derivative of any one of them, characterized in that the nucleotide sequence is a nucleotide sequence according to any one of claims 5 to 7. 9. A method according to claim 8, wherein specific mutations are inserted with molecular biology methods. , characterized in that the nucleic acid corresponding to the amino acid positions 321 to 341 of ORF la and / or the nucleic acid corresponding to amino acid positions 936 to 956 of ORF and / or the nucleic acid corresponding to amino acid positions 1 to 20 it is mutated so that at least one nucleotide is substituted or deleted at the positions. 10. - Pharmaceutical composition comprising a PRRS virus according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. 11. Use of a PRRS virus according to any one of claims 1 to 4 in the manufacture of a vaccine for the prophylaxis and treatment of PRRS infections. i .. ** i. - - * - ^^ ga