JP2003520601A - Recombinant attenuation of PRRSV - Google Patents

Abstract

  (57) [Summary] The present invention relates to a live PRRS virus attenuated by site-specific amino acid mutations in a viral protein encoded by an open reading frame (ORF) selected from the group of ORF 1a, ORD 1b and / or ORF 2 About. The invention also relates to nucleotide sequences encoding said viruses, methods for producing such viruses and their use for the preparation of pharmaceutical compositions for the prevention and treatment of PRRS infection.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention is attenuated by amino acid mutations at specific sites in a viral protein encoded by an open reading frame (ORF) selected from the group consisting of ORF 1a, ORF 1b and / or ORF 2. Concerning the live PRRS virus. The present invention also relates to the nucleotide sequences encoding said viruses, methods for producing such viruses and their use for preparing pharmaceutical compositions for the prevention and treatment of PRRS infections.

[0002] named the mystery of pig disease and porcine reproductive and respiratory syndrome (PRRS) after the background of the invention, positive-strand RNA virus having an envelope of arterial virus family (arteriviridae) is causing (EJSnijder, 1998). About 10 to 15 years ago, two different PRRS virus strains emerged independently in the United States and Europe. The disease is endemic to many pigs currently produced in North America, Europe and Asian countries. It is subsequently the leading cause of hyporeproductive and respiratory disease in pigs. In the United States, infection rates are expected to be up to 70%. The virus is transmitted by inhalation, ingestion, intercourse, bites or needles. It replicates in mucosal macrophages, pulmonary macrophages or regional macrophages.

Asymptomatically, the disease results in remission or persistent infection. Persistently infected animals shed the virus in oral / pharyngeal fluids, blood, feces, urine and semen. The clinical manifestations of sows include weakly producing pigs, stilbone pigs and autolyzed fetu.
ses) abortion or premature birth. Infected newborn pigs have a high mortality rate or develop pneumonia. Subsequent breeding and pig growth are exacerbated by pneumonia, concurrent bacterial infections and increased mortality. Boars tend to develop fever and morphological changes in semen. For all arteriviruses, the PRRS viral genome is approximately
It is a single plus-strand RNA molecule of 15 kilobases. ORFs (open reading frames) 1a and 1b encode replicase, and ORFs 2-5 are putative glycoproteins (
ORF 6 encodes a membrane protein (M) and ORF 7 encodes a nucleocapsid protein (N).

[0004] USA (Isolated ATCC VR-2332, Genebank U 87392 U00153) inserted July 18, 1991 in the American Type Culture Collection in Rockville, Maryland, USA and Europe (WO First PRRS infection in an isolated Lelystad Agent (CDI-NL-2.91), accession number I-1102, inserted on June 5, 1991, at the Pasteur Institute in Paris, 92/21375. Characterization identified viruses with genomic and serological differences. Comparisons showed that both had common ancestors that diverged before the clinical disease was described in the late 1980s. A full-length genomic sequence was reported for the PRRS virus number and its region encoding the complete structural protein (Snijder et al., 1998; Meu).
lenberg et al., 1993b; Conzelmann et al., 1993; Murtaugh et al., 1995; Kapur et al., 1996).
. PRRS virus is associated with porcine lung macrophages, monocytes, glial cells and CL-2621.
And in two MA-104 cell subpopulations known as MARC-145 (monkey embryonic kidney cells) that can be replicated in vitro (KD Rossow, Porcine reproductive and re
spiratory syndrome, Vet Pathol 35: 1-20, 1998). Recombinant means for producing infectious PRRS clones are also available (EP 0 839 912 A1).

Live, attenuated PRRS vaccines are commercially available to protect pigs (Re
spPRRS / Ingelvac® PRS MLV, Boehringer Ingelheim). Killed vaccines (whole inactivated virus) or subunit vaccines (conventionally purified or heterologously expressed purified viral proteins) are used to generate a systemic protective immune response, even in the presence of adjuvants. In most cases, it is inferior to the live vaccine in its effect. Compared to currently available killed vaccines, attenuated vaccines for PRRS
Produces immunity to diseases that are longer lasting and more effective (Snijder
Et al., Supra). This live PRRS vaccine is conventionally attenuated until it is no longer pathogenic by continuously culturing the virus in a host cell that seems appropriate (EP 0529584 B1). Still, this live PRRS vaccine leaves us with insufficient capacity for improvement. First, they do not prevent reinfection. Secondly, they do not allow serological discrimination between vaccinated and field virus infected animals. But most importantly, live vaccines from whole organisms, although attenuated, can be associated with serious safety concerns. This is like the PRRS virus
This is especially true for RNA viruses, which are believed to have a high probability of mutation due to incorrect copying of the RNA genome resulting from lack of proofreading by RNA copy enzymes. The potential for live virus backmutations to be attenuated can pose a serious threat to vaccinated animals. For conventional attenuated viruses, attenuation is achieved by normal multiple passages, molecular origin and genetic stability remain unknown, and backmutation occurrence is unpredictable. Therefore, the technical problem of the present invention was to provide a PRRS virus that is unlikely to revert to a wild-type virus.

SUMMARY OF THE INVENTION The present invention is attenuated by amino acid mutations at specific sites in a viral protein encoded by an open reading frame (ORF) selected from the group ORF 1a, ORF 1b and / or ORF 2. , About the live PRRS virus.
The present invention also relates to the nucleotide sequences encoding said viruses, methods for producing such viruses and their use for preparing pharmaceutical compositions for the prevention and treatment of PRRS infections.

[0007] Resolution of the disclosed above technical problem of the invention is achieved by the description and embodiments characterized in the claims. Prior to the embodiments of the present invention, as used in the specification and claims,
It should be noted that the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "PRRS virus" includes more than one such PRRS virus, and reference to "cell" is 1
References to one or more cells and their equivalents known to those of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although the same or equivalent methods and materials described herein can be used in the methods or tests of the invention, the preferred methods, devices, and materials are described herein. All publications mentioned herein are incorporated herein by reference for the purpose of describing or disclosing cell lines, vectors, and methodologies reported in publications that may be used in connection with the present invention. Incorporated into. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Surprisingly, it was found that the PRRS virus always contains a specific genomic site in some open reading frame returning in its position to amino acids of the original pathogenic region of the VR-2332 strain. The evolutionary pressure of this form, referred to herein as the "pathogenic specific site," or of this form, referred to simply as the "site of the invention" or simply as the "site," is enormous. For the two revertants of RespPRRS, we were able to demonstrate for the first time that amino acid mutations to isolated amino acids of the original pathogenic region at this pathogenic specific site occur geographically independently in the United States and Europe. . Live vaccines with mutations defined as the basis for the attenuation of the invention can avoid the drawbacks of the current generation of attenuated vaccines. Another advantage of said attenuating mutations is their known molecular specificity which allows them to be used as a unique marker for attenuated pestiviruses and to distinguish them from pestiviruses from that region. is there.

The amino acid and nucleotide sequences of the virus RespPRRS, which were attenuated by conventional methods, were compared to the isolated VR-2332 of the original region. In order to identify pathogenic specific sites, the two described virulence revertants were isolated from each other, and VR-2332, and Resp.
Compared to PRRS. This allowed the identification of virulence specific sites of individual viral proteins involved in PRRS virus virulence. As a result, one aspect of the invention is the PRRS virus ATCC VR-2, but not RespPRRS.
ORF 1a which is less pathogenic than 332 and is described in Figure 1 for the VR-2332 strain,
A protein encoded by an open reading frame (ORF) selected from the group of ORF 1b described in FIG. 2 for the VR-2332 strain and / or ORF 2 described in FIG. 3 for the early VR-2332k region is selected. A live PRRS virus, characterized in that it comprises at least one of the amino acids at the identified pathogenic site
Is not identical to at least one of the amino acids of the VR-2332 strain at the corresponding position above).

Amino acid and nucleotide numbering follows VR-2332 database entry. Figures 1, 2 and 3 give representative information for all PRRS strains, visualize the invention and identify preferred amino acids and preferred sites of all potentially differently numbered PRRS viruses. Identification of these positions is based on the desired PRRS.
Identifying a characteristic identical amino acid conserved in the strain and the reference strains listed,
This is accomplished by subsequently locating the site of the virus of interest relative to the site of Figures 1, 2 and / or 3. The three said sites are the viral ORFs described in Figures 1, 2 and 3 for VR-2332.
Identified for proteins encoded by 1a, ORF 1b and / or ORF 2.

Another aspect of the invention is in VR-2332, which is not RespPRRS, at positions 321-34 of ORF 1a.
At least one amino acid of 1 is not identical to the amino acid of the VR 2332 strain at the corresponding position described in FIG. 1 and / or at least one amino acid of positions 936-956 of ORF 1b is the corresponding amino acid described in FIG. Essentially not identical to the amino acid of the VR 2332 strain at position and / or at least one amino acid at positions 1 to 20 of ORF 2 is not identical to the amino acid of the VR 2332 strain at the corresponding position depicted in FIG. Characteristic ORF
It relates to live attenuated PRRS viruses including 1a, 1b and 2. Thus, ORF
Nucleotides coding for one or more amino acids at sites 321-341 of 1a, sites 936-956 of ORF 1b, and / or sites 1-20 of ORF 2 (see FIGS. 4, 5 or 6, respectively). The triplet may be mutated, resulting in one or more alterations in the sequence at the site, at the nucleic acid level, or more preferentially at the amino acid level, thus mutating to all nucleotide or amino acid positions at the site. is there.
Viruses in which one, two or all three of said sites are mutated are included in the invention. A "mutation" is the replacement of an amino acid with another, called a "substitution", or the replacement of a nucleotide encoding with another (eg, C with T), preferably replacing the encoded amino acid, Or other mutations such as "deletion" or "addition". Mutations are always made in the encoding nucleotide sequence. Said mutations are standard methods known in the art, for example Meulenberg et al., Adv.
Exp. Med. Biol, 1998, 440: 199-206) site-directed mutagenesis of infectious copies (see, eg, Sambrook et al., (1989) Molecular Cloning: A Laboratory Man.
ual, 2 ^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N
ew York).

By “essentially” is meant that at least 75% of the sequences, preferably 85%, and most preferably all of the sequences except the “sites” of the present invention are identical to VR 2332.
However, additional nucleic acids encoding amino acids outside of said sites of the invention may also be mutated. The virus of the invention still meets the criteria of the invention (ie unlikely to revert to wild type and less pathogenic than VR 2332). Live PRRS virus of the present invention refers to the PRRS virus defined in EJ Snijder et al. (See above), which is capable of infecting pigs and capable of replicating in the body of pigs. RespPRRS virus, which is conventionally attenuated, is clearly denied. It is not a virus of the invention and is specifically excluded from the scope of the claims. RespPRRS virus is Boehringer Ingelheim Fetmedica (Ingelvac®)
From PRS MLV). Most of the sequences are publicly available (G
enebank AJ 223082).

Sites and amino acids of particular interest to the present invention are not limited to the exact positions defined for VR-2332, but point to preferred amino acids at that position or corresponding to that position in other PRRS strains. Simply used in a typical way for. different
For the PRRS virus, the numbering of the preferred amino acid positions may vary, but experts in the field of molecular biology of viruses of the arterial viridae will be dependent on their position relative to other conserved amino acids of the protein. These preferred amino acids will be easily identified. The term "PRRS virus less pathogenic than VR-2332" should be understood in terms of a comparison of clinical manifestations of the virus of interest by VR-2332. A preferred method for determining whether a PRRS virus is less pathogenic than PRRS virus VR-2332 is described in Example 1. Preferred amino acid mutations at a pathogenic site are not always associated with reduced pathogenicity. The method of Example 1 is accurate for determining whether a live PRRS virus containing a protein according to the teachings of the present invention is less virulent than that of isolated field VR-2332, Provide a simple experimental device.

The pathogenic site is identified by a backmutation of at least one amino acid from the attenuated virus to the amino acid of VR-2332. This particular amino acid is a large secondary peptide structure, such as an alpha helix or β sheet or hairpin β motif or other part. Therefore, adjacent amino acids are likely to be involved in the regulation of protein pathogenicity. Therefore, experts in the field of protein chemistry also expect high probability of identifying amino acids by virtue of their pathogenicity-related properties within 10 amino acids to the left of the originally identified amino acid position and to the right. To do. Ten~
The 20 amino acids are a typical region of the peptide motif of proteins. Accordingly, preferred viruses of the invention include proteins corresponding to those of FIG. 1 in an exemplary manner, or of other strains, wherein the virulence site is upstream of the originally identified amino acid position. Contains 10 amino acids and 10 amino acids downstream.
More preferably, those viruses described above, the pathogenicity-specific site of which comprises 5 amino acids upstream and 5 amino acids downstream of the originally specified amino acid position. Most preferred are those viruses mentioned above, whose pathogenicity-specific site comprises 3 amino acids upstream and 3 amino acids downstream of the originally specified amino acid position.

In accordance with the teachings of the present invention, it is possible to produce an attenuated PRRS strain from a pathogenic field strain by mutating amino acids at the pathogenic site. The safety issues associated with the high probability of mutations in RNA viruses remain. This problem can be greatly reduced by deleting certain amino acids at the pathogenic site of viral proteins. Therefore, the present invention relates to the above-mentioned PRRS virus, which is characterized in that at least one amino acid at the pathogenic specific site of the viral protein is deleted. The term "deletion" should be understood to be deleted at that position or relative to the amino acids referenced in the figures at those positions. Thus, according to the present invention, "deletion" means the removal of one or more nucleotides or amino acids.

Thus, in a more preferred embodiment, the invention provides that at least one amino acid at positions 321-341 of ORF 1a is deleted and / or at least one amino acid at positions 936-956 of ORF 1b is deleted. It relates to a live attenuated PRRS virus according to the invention, characterized in that it is deleted and / or has at least one amino acid in position 1 to 20 deleted. Thus, ORF 1a sites 321-341, ORF 1b sites 936-956, or ORF 2
Nucleotides encoding one amino acid triplet, multiple triplets at sites 1-20, or at all three sites, are deleted, resulting in one or more deletions at said sites. Viruses in which only one nucleotide or one amino acid is deleted and viruses in which one or all of said sites are completely deleted are encompassed by the invention (FIG. 4, respectively). 5 or 6).

At least one amino acid is under high mutagenic pressure for the identified pathogenic site of the particular PRRS viral protein, implicated in the pathogenicity of revertants of the field strain VR-2332. , Shown in the preferred embodiment. This individual amino acid position is the most preferred embodiment of the invention. Therefore, in a most preferred embodiment, the invention relates to a live attenuated PRRS virus of the invention that is less pathogenic than ATCC VR 2332, but not RespPRRS, with the amino acid at position 331 of ORF 1a and / or ORF 1b. At position 946 and / or at position 10 of ORF 2 is not identical to the amino acid of ATCC VR 2332 strain at the corresponding position. Thus, site 331 of ORF 1a and site 946 of ORF 1b.
, Or ORF 2 at site 10 or all three sites, the nucleotide or amino acid encoding a single amino acid triplet is mutated resulting in 1-3 mutations at said site (FIGS. 4, 5 or respectively, respectively). See 6).

For the above reasons, it is safe and preferred to avoid the reversion of the modified amino acid to the highly pathogenic type by easily deleting the reversible amino acid. Therefore, in this most preferred embodiment, the invention relates to the live attenuated PRRS virus of the invention, when compared to its position in VR-2332, the amino acid at position 331 of ORF 1a and / or the ORF. The amino acid at position 946 of 1b and / or the amino acid at position 10 of ORF 2 is deleted, ie is absent. Furthermore, the most preferred embodiment is a live attenuated PRRS virus of the invention, characterized in that the amino acid at position 331 of ORF 1a, ie the coding triplet, is deleted. Furthermore, the most preferred embodiment is the live attenuated PRRS virus of the invention, characterized in that the amino acid at position 946 of ORF 1b, ie the coding triplet, is deleted. Furthermore, the most preferred embodiment is a live attenuated PRRS virus of the invention, characterized by a deletion of the amino acid at position 10 of ORF 2, ie the coding triplet (FIG. 4, respectively). 5 or 6). The most preferred virus is identical to VR-2332 in all other positions.

Now, in accordance with the teachings of the present invention, one skilled in the art will genetically engineer a PRRS virus infectious clone that is less pathogenic than VR-2332 and that is useful in preparing raw pharmaceutical compositions. To be able to produce. All the information required to produce recombinant infectious clones of positive-strand RNA viruses can be found in particular PRRSV.
Regarding, it has been easily available. For example, the European patent application EP 0 839 912 of Meulenberg et al. (Which is hereby incorporated by reference in its entirety)
Clearly teach how to prepare PRRS virus. Therefore, in another aspect, the invention relates to nucleotide sequences encoding the viruses of the invention. Due to the degeneracy of the multiple nucleotides in the genetic code, variants produce identical amino acid translations. Also, degenerate variants thereof are included in the present invention.

As described on the first page, it is important that porcine health care can distinguish between less pathogenic live vaccine strains of pharmaceutical compositions and pathogenic wild-type virus infections. This is often difficult, especially if the clinical manifestations of field infections are not multi-infections with specific or other infections, or if the observation and evaluation period is short. Recombinant production of the virus of interest allows the introduction of alterations in the genetic code that establish serum and / or pathogenic markers. Serum markers refer to antigenically detectable molecules such as peptides, proteins, glycoproteins that can be isolated from infected cells or body fluids such as, but not limited to, pharyngeal fluid or nasal fluid or urine. Pathogenic markers should be understood as markers of the genetic code that can be identified by recombinant analysis methods such as, but not limited to, PCR and conventional sequencing. Therefore, in a preferred embodiment, the invention relates to the nucleotide sequences of the invention, which nucleotide sequences are modified to encode a pathogenic marker and / or a serum marker. In particular, mutations or deletions introduced for the purpose of attenuating pathogenicity are useful as pathogenicity and serum markers. By monitoring these mutations of the disclosed virulence site, the occurrence of pathogenic revertants can be predicted as early as possible.

More preferably, the nucleotide sequence of the present invention is such that the nucleotide sequence encoding the marker is located in any open reading frame encoding a structural viral protein. Another aspect of the invention relates to a method of producing an infectious live attenuated PRRS virus of the invention, wherein said method comprises at least one full length DNA copy or an in vitro transcribed RNA copy or derivative thereof. Producing a recombinant nucleic acid as described above. Another preferred embodiment of the present invention relates to the method of the present invention, wherein the particular mutation is inserted in a molecular biology method, the nucleic acid corresponding to amino acid positions 321-341 of ORF 1a and / or amino acid position 936 of the ORF. Nucleic acid and / or amino acid positions 1-20 corresponding to 956
Is characterized in that the nucleic acid corresponding to is mutated in such a way that at least one nucleotide is replaced or deleted at said position.

Another important aspect of the present invention is a pharmaceutical composition comprising the PRRS virus of the present invention and a pharmaceutically acceptable carrier. A "pharmaceutical composition" is a component that is not limited to one or more antibiotics or anthelmintic drugs that can modify the physiological, e. Purpose of, for example, traits to be treated, infertility, stability,
Possibility of administering the composition by the enteral or parenteral route, for example oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal or other suitable routes, tolerability after administration, controlled release It consists essentially of the other ingredients that are added to achieve the properties, including but not limited to. Pharmaceutically acceptable carriers can include, for example, physiologically acceptable compounds that act on stability or increase absorption or form part of a slow release formulation of the PRRS virus of the present invention. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. Agents (eg Remington's Pharmaceutical
Sciences (1990). 18 ^th ed. Mack Publ., Easton). Those skilled in the art will know that the choice of pharmaceutically acceptable carrier, including the physiologically acceptable compound, will depend, for example, on the route of administration of the composition.

Another aspect of the invention relates to the use of the viruses of the invention. The utility of the live attenuated PRRS viruses of the present invention allows their use in the manufacture of vaccines for the prevention and treatment of PRRS infections. The defined molecule, which is the center of attenuation, produces better than this conventionally attenuated virus. In particular,
The use of the virus of the present invention containing a deletion of the pathogenic specific site is preferred because the deletion is less likely to revert. The following examples are intended to illustrate the invention in more detail, but this should not be construed as limiting the scope of the invention disclosed herein.

References 1. Snijder EJ, Meulenberg JJM, 1998. The molecular biology of ar
teriviruses, Journal of General Virology May 79 (5): 961-971. 2. Meulenberg JJM, Hulst, MM et al., 1993b. Lelystad virus, the
causative agent of porcine epidemic abortion and respiratory syndrome, V
irology 192,62-72. 3. Conzelmann KK, Visser N., Van Woensel P., Thiel HJ, 1993. Molec
ular characterization of porcine reproductive and respiratory syndrome v
irus, a member of the arterivirus group, Virology 103,329-339. 4. Murtaugh MP, Elam MR, Kakach LT, 1995. Comparison of the str
uctural protein coding sequences of the VR-2332 and Lelystad virus strai
ns of the PRRS virus, Archives of Virology 140,1451-1460. 5. Kapur V., Elam MR, Pawtovich TM Murtaugh MP, 1996. Genetic v
ariation in porcine reproductive and respiratory sydrome virus isolates
in the midwestem United States, Journal of General Virology 77,1271-1276
. 6. Rossow KD, 1998. Porcine reproductive and respiratory syndrome, Ve
t Pathol 35: 1-20.

Example 1 Formation of Attenuation This example provides clear guidance for the comparison of pathogenicity of two different strains of PRRS virus. At least 10 heifers per group are included in each trial and are derived from farms without PRRS. Animals will be tested for no PRRS virus specific serum antibody or negative for PRRSV. All animals included in the study are of the same source and strain. Animals assigned to groups are chaotic. Validation is performed at day 90 of gestation with an intranasal application of 1 ml PRRSV with 10 ⁵ TDCID ₅₀ per nostril (3rd passage). There are at least 3 groups for each test setting: a group for VR-2332 validation; a test group for validation with as attenuated viruses as possible; a strict control group. Study relevance is given when strict controls are PRRS negative for the time course of the study, resulting in healthy piglets with at least 25% shorter lifespans compared to the VR2332 validation group strict controls. . Attenuation, or weak pathogenicity, is defined as a systematically significant change in one or more parameters that determine fertility: A large reduction in at least one of the following parameters of the test group is preferred: Frequency, abortion before 112 days of pregnancy, number of mummified piglets, number of life and weak piglets, pre-weaning mortality or one apparent increase in one of the following parameters of the test group: More preferred: -Number of weaned piglets per sow-Number of life-threatening piglets born per sow-Comparison with VR2332 infected group

[Brief description of drawings]

FIG. 1 OR of ATTC VR-2332 with a preferred attenuated site of the invention labeled.
This is the amino acid sequence of F 1a. It is an alignment of the amino acid sequences of the PRRS virus ATCC VR-2332 strain.

FIG. 2 OR of ATTC VR-2332 with preferred attenuated sites of the invention labeled.
This is the amino acid sequence of F 1b. It is an alignment of the amino acid sequences of the PRRS virus ATCC VR-2332 strain.

FIG. 3: OR of ATTC VR-2332 with preferred attenuated sites of the invention labeled.
This is the amino acid sequence of F 2. It is an alignment of the amino acid sequences of the PRRS virus ATCC VR-2332 strain.

FIG. 4 is the nucleotide sequence of ORF 1a of ATTC VR-2332 with preferred attenuated sites of the invention in bold and most preferred underlined sites.

FIG. 5 is the nucleotide sequence of ORF 1b of ATTC VR-2332 with preferred attenuated sites of the invention in bold and most preferred underlined sites.

FIG. 6 is the nucleotide sequence of ORF 2 of ATTC VR-2332 with the preferred attenuated site of the invention in bold and the most preferred underlined site.

─────────────────────────────────────────────────── ─── Continued front page    (31) Priority claim number 100 03 372.5 (32) Priority date January 26, 2000 (January 26, 2000) (33) Priority country Germany (DE) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), EA (AM , AZ, BY, KG, KZ, MD, RU, TJ, TM) , AE, AU, BG, BR, CA, CN, CZ, EE, HR, HU, ID, IL, IN, JP, KR, LT, L V, MX, NO, NZ, PL, RO, SG, SI, SK , UA, US, UZ, VN, YU, ZA (72) Inventor Leng Bells Haike             Federal Republic of Germany 49545 Techrembe             Luk Niederdolfelstraße             Two F-term (reference) 4B024 AA01 BA32 CA04 CA06 DA02                       EA04 GA11                 4B065 AA97X AA99Y AB01 AC20                       BA02 BA14 CA43                 4C085 AA03 BA51 DD01 EE01 EE07                       GG02 GG03 GG04 GG05 GG08

Claims (11)

[Claims] 1. An essentially O as found in VR-2332 that is not RespPRRS.
A live attenuated PRRS virus comprising RF 1a, 1b and 2 and the location of ORF 1a
At least one of the amino acids 321 to 341 is not identical to the amino acid of the VR 2332 strain at the corresponding position and / or is identical to an amino acid of the VR 2332 strain at the corresponding position to at least one of the amino acids at positions 936 to 956 of ORF 1b. And / or positions 1-2 of ORF 2
Said live attenuated PRRS virus, characterized in that at least one of the 0 amino acids is not identical to the amino acid of the VR 2332 strain at the corresponding position. 2. At least one of the amino acids at positions 321-341 of ORF 1a is deleted and / or at least one of the amino acids at positions 936-956 of ORF 1b is deleted and / or at positions 1-20. The live attenuated PRRS virus of claim 1, wherein at least one of the amino acids is deleted. 3. An amino acid at position 331 of ORF 1a and / or an amino acid at position 946 of ORF 1b and / or an amino acid at position 10 of ORF 2 are not identical to the amino acid of the ATCC VR 2332 strain at the corresponding position. Live attenuated PR according to paragraph 1 or 2
RS virus. 4. The method according to any one of claims 1 to 3, wherein 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 deleted. A live attenuated PRRS virus according to claim 1. 5. A nucleotide sequence encoding the virus according to any one of claims 1 to 4. 6. The nucleotide sequence according to claim 5, which has been modified to encode a toxicity marker and / or a serum marker. 7. The nucleotide sequence according to claim 6, wherein the nucleic acid encoding the marker is present in any sequence of the open reading frame encoding a structural viral protein. 8. A method of producing an infectious, live attenuated PRRS virus, the method comprising producing a recombinant nucleic acid comprising at least one full length DNA copy or an in vitro transcribed RNA copy, or a derivative thereof. 8. The method, comprising, wherein the nucleotide sequence is the nucleotide sequence according to any one of claims 5 to 7. 9. A nucleic acid and / or amino acid position corresponding to amino acid position 321 to 341 of ORF 1a and / or amino acid position 936 to 956 of ORF 1a, wherein the specific mutation is inserted by a molecular biological method. 9. A method according to claim 8 wherein the nucleic acid corresponding to 1 to 20 is mutated in such a way that at least one nucleotide at said position is replaced or deleted. 10. The PRRS according to any one of claims 1 to 4.
A pharmaceutical composition comprising a virus and a pharmaceutically acceptable carrier. 11. Use of the PRRS virus according to any one of claims 1 to 4 in the manufacture of a vaccine for the prevention and treatment of PRRS infection.