PRRS virus variant, european PRRS virus cDNA clone, and uses thereof

Abstract

The present invention belongs to the field of animal health and provides means to study Porcine Reproductive and Respiratory Syndrome (PRRS), a viral disease affecting swine, and for the development of vaccines, therapeutics and diagnostics for the prophylaxis, treatment and diagnosis of PRRS. In a first consideration, the invention relates to a new PRRS virus variant. and, in a second consideration, to a nucleic acid sequence which comprises the genome of an infectious genotype I (EU) PRRS virus clone. Based on this, new PRRS vaccine candidates with improved properties are provided.

Claims

1. A genotype I Porcine Reproductive and Respiratory Syndrome (PRRS) virus whose genome is encoded by a nucleic acid molecule comprising a sequence selected from SEQ ID NOS: 49, 56, 57, and 58.

2. The virus of claim 1, wherein the nucleic acid comprises the sequence of SEQ ID NO: 49.

3. The virus of claim 1, wherein the nucleic acid comprises the sequence of SEQ ID NO: 56.

4. The virus of claim 1, wherein the nucleic acid comprises the sequence of SEQ ID NO: 57.

5. The virus of claim 1, wherein the nucleic acid comprises the sequence of SEQ ID NO: 58.

6. A vaccine comprising the virus according to claim 1.

7. The vaccine of claim 6, further comprising a pharmaceutically acceptable excipient.

8. The vaccine of claim 7, wherein the pharmaceutically acceptable excipient is selected from a solvent, a dispersion media, an adjuvant, a stabilizing agent, a diluent, a preservative, an antibacterial agent, an antifungal agent, and an isotonic agent.

9. The vaccine of claim 6, wherein the vaccine comprises 10.sup.4 to 10.sup.6 particles of the virus per dose.

10. The vaccine of claim 6, wherein the nucleic acid comprises the sequence of SEQ ID NO: 49.

11. The vaccine of claim 6, wherein the nucleic acid comprises the sequence of SEQ ID NO: 56.

12. The vaccine of claim 6, wherein the nucleic acid comprises the sequence of SEQ ID NO: 57.

13. The vaccine of claim 6, wherein the nucleic acid comprises the sequence of SEQ ID NO: 58.

14. A method for treating and/or prophylaxis of PRRS in swine, comprising administering to the swine the vaccine according to claim 6.

15. The method of claim 14, wherein the vaccine is administered by an intranasal, an intramuscular, an oral, or an intrauterine route.

16. The method of claim 14, wherein the administration induces a protective immune response against PRRS in the swine that reduces and/or prevents PRRS symptoms.

17. The method of claim 14, wherein the nucleic acid comprises the sequence of SEQ ID NO: 56.

18. The method of claim 14, wherein the nucleic acid comprises the sequence of SEQ ID NO: 58.

19. A method for differentiating between infection and vaccination of a swine, comprising detecting for a vaccine marker in the swine that differentiates between native infection with PRRS and the vaccine according to claim 1.

20. The method of claim 19, wherein the vaccine marker is a deletion or insertion marker that permits serological and/or sequence differentiation.

Description

LIST OF FIGURES

(1) FIG. 1: A. Infectious virus recovered from the BI EU cDNA clone induced a strong CPE on MA104 cells as shown by bright field microscopy. B. PRRSV capsid protein-specific immunofluorescence (IF) staining of BI EU-infected MA104 cells.

(2) FIG. 2: Growth of virus recovered from the infectious cDNA clone BI EU on MA104 cells.

(3) FIG. 3: nsp1 N-terminal domain (NTD) amino acid sequence alignment of several US (type II, top) and EU (type I, bottom) PRRSV strains. The NTD aminoacid sequence of BI EU is given at the very bottom. Amino acids R22, PR24, E32, SFP and H52 are indicated above the alignment and have been shown to be crucial for nsp1 homodimerization (Xue et al., 2010). Target regions for nsp1 mutagenesis are framed in red. The SDGRSR (SEQ ID NO: 64) motif corresponds to the region described in WO 2013017570 A1 using PRRSV EU cDNA clone LoN94-13. FIG. 3 discloses SEQ ID NOS: 65-88, respectively, in order of appearance.

(4) FIG. 4: Amino acid sequence alignment of BI EU-nsp1 deletion mutants. FIG. 4 discloses SEQ ID NOS: 89-99, respectively, in order of appearance.

(5) FIG. 5: Growth of BI EU-nsp1 deletion mutants on IFN-competent MA104 cells. FIG. 5 references SEQ ID NOS: 99, 89, 93, 90, 95, 92, 97, 94, 98, and 96, respectively, in order of appearance.

(6) FIG. 6: IFN- levels measured at different timepoints in the cell culture supernatant of MA104 cells infected with the BI EU-nsp1 deletion mutants or with parent BI EU virus. FIG. 6 references SEQ ID NOS: 89, 98, 96, 94, 92, 99, 95, 90, 93, and 97, respectively, in order of appearance.

(7) FIG. 7: Growth kinetics of recombinant BI EU viruses harboring deletions or insertions within the ORF4 protein.

(8) FIG. 8: Serum neutralization tests for the recombinant virus BI EU-GP5-36-46-AtoC and the parental virus BI EU.

(9) In the sequence listing:

(10) SEQ ID NOs:1-24 correspond to sequences of the ectodomain of PRRSV ORF4 protein with a deletion;

(11) SEQ ID NO:25 and SEQ ID NO:26 correspond to sequences of the first two predicted N-terminal -sheets of PRRSV (genotype I) ORF4 protein;

(12) SEQ ID NO:27 and SEQ ID NO:28 correspond to sequences of the first two predicted N-terminal -sheets of PRRSV (genotype II) ORF4 protein;

(13) SEQ ID NO:29 and SEQ ID NO:30 correspond to sequences of the first two predicted N-terminal -sheets of PRRSV (genotype I) ORF4 protein;

(14) SEQ ID NO:31 and SEQ ID NO:32 correspond to sequences of the first two predicted N-terminal -sheets of PRRSV (genotype II) ORF4 protein;

(15) SEQ ID NO:32 corresponds to a (partial) sequence of a PRRSV (genotype I) ORF4 protein having a deletion of 11 amino acid residues in the region between the first two predicted N-terminal -sheets;

(16) SEQ ID NO:33 corresponds to a (partial) sequence of a PRRSV (genotype II) ORF4 protein having a deletion of 7 amino acid residues in the region between the first two predicted N-terminal -sheets;

(17) SEQ ID NO:34 corresponds to the sequence of the ectodomain of a PRRSV (genotype I) ORF4 protein having a deletion of 11 amino acid residues;

(18) SEQ ID NO:35 corresponds to the sequence of the ectodomain of a PRRSV (genotype II) ORF4 protein having a deletion of 7 amino acid residues;

(19) SEQ ID NO:36 corresponds to the sequence of a PRRSV (genotype I) ORF4 protein having a deletion of 11 amino acid residues (and including the sequence of SEQ ID NO:34, respectively);

(20) SEQ ID NO:37 corresponds to a nucleotide sequence encoding the sequence of SEQ ID NO:36;

(21) SEQ ID NO:38 corresponds to a nucleotide sequence encoding a genotype I PRRSV whose genome comprises a nucleic acid molecule which codes for the sequence of SEQ ID NO:36;

(22) SEQ ID NO:39 corresponds to the sequence of a peptide encoded by the ORF5 gene of PRRS virus;

(23) SEQ ID NO:40 corresponds to the sequence of a peptide encoded by the ORF5 gene of PRRS virus;

(24) SEQ ID NO:41 corresponds to Lelystad virus complete genome;

(25) SEQ ID NO:42 corresponds to VR2332 virus complete genome;

(26) SEQ ID NO:43 corresponds to the sequence of ORF4 protein of the Lelystad virus;

(27) SEQ ID NO:44 corresponds to the sequence of ORF4 protein of the VR2332 virus;

(28) SEQ ID NO:45 corresponds to a first nucleic acid sequence as described herein;

(29) SEQ ID NO:46 corresponds to a second nucleic acid sequence as described herein, which flanks the 5 end of the first nucleic acid sequence;

(30) SEQ ID NO:47 corresponds to a third nucleic acid sequence as described herein, which flanks the 3 end of the first nucleic acid sequence;

(31) SEQ ID NO:48 corresponds to BI EU complete viral cDNA insert;

(32) SEQ ID NO:49 corresponds to the sequence of SEQ ID NO:48 with a deletion, thereby encoding an ORF4 protein having a deletion of 13aa (aa 57-69);

(33) SEQ ID NO:50 corresponds to the sequence of SEQ ID NO:39 with the substitution N>Q at position 9;

(34) SEQ ID NO:51 corresponds to the sequence of aa 1-11 of SEQ ID NO:39;

(35) SEQ ID NO:52 corresponds to the sequence of SEQ ID NO:51 with the substitution N>Q at position 9;

(36) SEQ ID NO:53 corresponds to the sequence of SEQ ID NO:51 with a Gly-Gly linker;

(37) SEQ ID NO:54 corresponds to the sequence of SEQ ID NO:52 with a Gly-Gly linker;

(38) SEQ ID NO:55 corresponds to the sequence of SEQ ID NO:53 with an N-terminal proline residue;

(39) SEQ ID NO:56 corresponds to the sequence of SEQ ID NO:49 with an insert, thereby encoding the sequence of SEQ ID NO:53;

(40) SEQ ID NO:57 corresponds to the sequence of SEQ ID NO:49 with an insert, thereby encoding the sequence of SEQ ID NO:54;

(41) SEQ ID NO:58 corresponds to the sequence of SEQ ID NO:48 with a deletion, thereby encoding an ORF4 protein having a deletion of 14aa (aa 56-69), wherein an insert coding for the sequence of SEQ ID NO: 55 is included.

REFERENCE LIST

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