North American Porcine Reproductive and Respiratory Syndrome (PRRS) Virus and Uses Thereof

Abstract

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.

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 any of:
 an amino acid N within the amino acid sequence AMA N VYD (SEQ ID NO: 9);   an amino acid N within the amino acid sequence IGH N AVM (SEQ ID NO: 12);   an amino acid D within the amino acid sequence TVP D GNC (SEQ ID NO: 15);   an amino acid Y within the amino acid sequence CWW Y LFD (SEQ ID NO: 18);   an amino acid H within the amino acid sequence HGV H GKY (SEQ ID NO: 21);   an amino acid V within the amino acid sequence AAK V DQY (SEQ ID NO: 24);   an amino acid T within the amino acid sequence PSA T DTS (SEQ ID NO: 27);   an amino acid L within the amino acid sequence LNS L LSK (SEQ ID NO: 30).   an amino acid C within the amino acid sequence APM C QDE (SEQ ID NO: 33);   an amino acid T within the amino acid sequence CAP T GMD (SEQ ID NO: 36);   an amino acid A within the amino acid sequence PKV A KVS (SEQ ID NO: 39);   an amino acid I within the amino acid sequence AGE I VGV (SEQ ID NO: 42);   an amino acid N within the amino acid sequence ADF N PEK (SEQ ID NO: 45); and   an amino acid I within the amino acid sequence QTP I LGR (SEQ ID NO: 48).   
     
     
         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) an amino acid N within the amino acid sequence A N V (see SEQ ID NO: 9);   an amino acid N within the amino acid sequence H N A (see SEQ ID NO: 12);   an amino acid D within the amino acid sequence P D G (see SEQ ID NO: 15),   an amino acid Y within the amino acid sequence W Y L (see SEQ ID NO: 18);   an amino acid H within the amino acid sequence V H G (see SEQ ID NO: 21), or any subset thereof; and/or   (2) an amino acid V within the amino acid sequence K V D (see SEQ ID NO: 24);   an amino acid T within the amino acid sequence A T D (see SEQ ID NO: 27);   an amino acid L within the amino acid sequence S L L (see SEQ ID NO: 30).   an amino acid C within the amino acid sequence M C Q (see SEQ ID NO: 33), or any subset thereof; and/or   (3) an amino acid T within the amino acid sequence P T G (see SEQ ID NO: 36);   an amino acid A within the amino acid sequence V A K (see SEQ ID NO: 39), or any subset thereof; and/or   (4) an amino acid I within the amino acid sequence E I V (see SEQ ID NO: 42);   an amino acid N within the amino acid sequence F N P (see SEQ ID NO: 45);   and amino acid I within the amino acid sequence P I L (see SEQ ID NO: 48), or any subset thereof.   
     
     
         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) SEQ ID NO:6;   (b) a sequence that has at least 85% identity to the DNA sequence of (a) wherein the protein encoded by ORF1a thereof has an amino acid sequence that contains:
 (1) an amino acid N within the amino acid sequence A N V (see SEQ ID NO: 9); 
   an amino acid N within the amino acid sequence H N A (see SEQ ID NO: 12);   an amino acid D within the amino acid sequence P D G (see SEQ ID NO: 15),   an amino acid Y within the amino acid sequence W Y L (see SEQ ID NO: 18);   an amino acid H within the amino acid sequence V H G (see SEQ ID NO: 21), or any subset thereof; or
 (2) an amino acid V within the amino acid sequence K V D (see SEQ ID NO: 24); 
   an amino acid T within the amino acid sequence A T D (see SEQ ID NO: 27);   an amino acid L within the amino acid sequence S L L (see SEQ ID NO: 30).   an amino acid C within the amino acid sequence M C Q (see SEQ ID NO: 33), or any subset thereof; or
 (3) an amino acid T within the amino acid sequence P T G (see SEQ ID NO: 36); 
   an amino acid A within the amino acid sequence V A K (see SEQ ID NO: 39), or any subset thereof; or
 (4) an amino acid I within the amino acid sequence E I V (see SEQ ID NO: 42); 
   an amino acid N within the amino acid sequence F N P (see SEQ ID NO: 45);   and amino acid I within the amino acid sequence P I L (see SEQ ID NO: 48), or any subset thereof; and   (c) a DNA sequence that hybridizes to the complement of a DNA sequence of (a) or (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.