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 DNA molecule which encodes a genotype I PRRS virus and which is capable of producing live virus when transfected into cells, wherein said molecule comprises a nucleic acid sequence having at least 99.1% sequence identity with the nucleic acid sequence of SEQ ID NO:48, wherein maximally 0.8% of the differences in sequence identity are located within the first 110 nucleotides of the 5 end of SEQ ID NO:48 and within the the last 97 nucleotides of the 3 end of SEQ ID NO:48 and wherein maximally 0.1% of the differences in sequence identity are located in the remaining nucleotides of SEQ ID NO:48.

2. The DNA molecule of claim 1, wherein said virus is attenuated and/or wherein said virus is able to induce a protective immune response against respiratory and/or reproductive signs of disease after infection with Porcine Reproductive and Respiratory Syndrome (PRRS) virus in swine; and/or wherein said virus is able to reach titers of at least 5?10.sup.5 to 1?10.sup.6 tissue culture infectious dose 50 (TCID.sub.50) per milliliter (ml) within 24 hours post infection of MA104 cells, preferably at an MOI (multiplicity of infection) of 0.001 to 0.1, and/or wherein said virus is able to reach titers of at least 5?10.sup.6 to 1?10.sup.7 tissue culture infectious dose 50 (TCID.sub.50) per milliliter (ml) within 48 hours post infection of MA104 cells, at an MOI (multiplicity of infection) of 0.001 to 0.1.

3. An RNA transcript of the DNA construct of claim 1.

4. A cell transfected with the DNA construct of claim 1 or with the RNA transcript of claim 3.

5. A genotype I PRRS virus produced by the cell of claim 4.

6. A genotype I PRRS virus whose genome comprises a nucleic acid molecule according to claim 1.

7. A method for producing a genotype I PRRS virus comprising transfecting a cell with the DNA construct of claim 1.

8. A composition comprising a nucleic acid molecule of claim 1 suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.

9. A method of prophylaxis or treatment of Porcine Reproductive and Respiratory Syndrome comprising administering the virus according to claim 5 to an animal.

10. A kit comprising the PRRS virus or a fragment thereof according to claim 5 as a detection marker for the differentiation between infected and vaccinated animals (DIVA).

11. A nucleic acid molecule of claim 1, wherein said nucleic acid sequence of SEQ ID NO: 48 comprises an nsp2 region from 1380 to 3808 and an ORF4 region encoding GP4 from 12805 to 13356.

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? 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 motif (SEQ ID NO: 64), 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 SED 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.

IN THE SEQUENCE LISTING

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

(10) 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;

(11) 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;

(12) 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;

(13) 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;

(14) 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;

(15) 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;

(16) 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;

(17) 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;

(18) 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);

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

(20) 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;

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

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

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

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

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

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

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

(28) 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;

(29) 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;

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

(31) 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);

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

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

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

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

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

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

(38) 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;

(39) 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;

(40) 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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