North American porcine reproductive and respiratory syndrome (PRRS) virus and uses thereof

11351,243

16/809038

March 04, 2020

The invention provides isolated polynucleotide molecules that comprise a DNA sequence encoding an infectious RNA sequence encoding a genetically-modified North American PRRS virus, methods to make it and related polypeptides, polynucleotides, and various components. Vaccines comprising the genetically modified virus and polynucleotides and a diagnostic kit to distinguish between naturally infected and vaccinated animals are also provided.

Zoetis Services LLC (Parsippany, NJ, US)

Zoetis Services LLC (Parsippany, NJ, US)

1. An isolated Porcine Reproductive and Respiratory Syndrome Virus (PRRS) wherein the protein thereof encoded by ORF1a is selected from a group consisting of those amino acid sequences that contain: a) one, two, three or four of: an amino acid N within the amino acid sequence AMANVYD (SEQ ID NO: 9); an amino acid N within the amino acid sequence IGHNAVM (SEQ ID NO: 12); an amino acid Y within the amino acid sequence CWWYLFD (SEQ ID NO: 18); and an amino acid H within the amino acid sequence HGVHGKY (SEQ ID NO: 21); and b) one or both of: an amino acid T within the amino acid sequence PSATDTS (SEQ ID NO: 27); and an amino acid C within the amino acid sequence APMCQDE (SEQ ID NO: 33); and c) one or both of: an amino acid T within the amino acid sequence CAPTGMD (SEQ ID NO: 36); and an amino acid A within the amino acid sequence PKVAKVS (SEQ ID NO: 39); and d) an amino acid N within the amino acid sequence ADFNPEK (SEQ ID NO: 45).

2. A plasmid capable of directly transfecting a suitable host cell and expressing a Porcine Reproductive and Respiratory Syndrome Virus (PRRS) from the suitable host cell so transfected, which plasmid comprises: (a) a DNA sequence encoding an infectious RNA molecule encoding the PRRS virus, and (b) a promoter capable of transcribing said infectious RNA molecule, wherein the protein encoded by ORF1a of said virus has an amino acid sequence that contains: (1) one, two, three, or four of an amino acid N within the amino acid sequence ANV (see SEQ ID NO: 9); an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12); an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18); and an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21), and (2) one or both of an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27); and an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33) and (3) one or both of an amino acid T within the amino acid sequence PTG (see SEQ ID NO: 36); and an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39); and (4) an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45).

3. The plasmid of claim 2 , wherein the promoter is a eukaryotic promoter capable of permitting a DNA launch in targeted eukaryotic cells.

4. The plasmid of claim 2 , wherein the promoter is a prokaryotic or phage promoter capable of directing in vitro transcription of the plasmid.

5. A method of generating a PRRS virus, which method comprises transfecting a suitable host cell with a plasmid according to claim 3 , and obtaining PRRS virus generated by the transfected cell.

6. A method of generating a PRRS virus, which method comprises transcribing the plasmid of claim 4 .

7. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus, wherein said DNA sequence is selected from the group consisting of: (a) a sequence that has at least 85% identity to SEQ ID NO:6, wherein the protein encoded by ORF1a thereof has an amino acid sequence that contains: (1) one, two, three, or four of: an amino acid N within the amino acid sequence ANV (see SEQ ID NO: 9); an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12); an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18); and an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21) and (2) one or both of an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27); and an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33), or any subset thereof; and (3) one or both of an amino acid T within the amino acid sequence PTG (see SEQ ID NO: 36); and an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39), and (4) an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45); and (b) a DNA sequence that hybridizes to the complement of a DNA sequence of (b) under highly stringent conditions which comprise hybridization to filter bound DNA in 0.5 M NaHPo4, 7% SDS, 1 mM EDTA at 65 degrees C., and washing in 0.1 SSC/0/1% SDS at 68 degrees C.

8. A vaccine for protecting a porcine animal against infection by a PRRS virus, which vaccine comprises (a) a genetically modified North American PRRS virus encoded by the polynucleotide molecule according to claim 7 , (b) said infectious molecule, (c) said polynucleotide molecule in the form of a plasmid, or (d) a viral vector comprising said polynucleotide molecule, wherein the PRRS virus is able to elicit an effective immunoprotective response against infection by PRRS virus, in an amount effective to produce immunoprotection against infection, and a carrier suitable for veterinary use.

6500662 December 2002 Calvert
7132106 November 2006 Calvert
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7618797 November 2009 Calvert
7691389 April 2010 Calvert
8481705 July 2013 Calvert
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8609827 December 2013 Calvert
8895025 November 2014 Calvert
9566324 February 2017 Calvert

Alignment of SEQ ID No. 6 with U.S. Pat. No. 6,500,662—SEQ ID Dec. 1, 1999. cited by examiner

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