RECOMBINANT NORTH AMERICAN TYPE 1 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND METHODS OF USE

• US 20100136047A1
• Filed: 06/24/2008
• Published: 06/03/2010
• Est. Priority Date: 06/25/2007
• Status: Active Grant

• Current Assignee: South Dakota State University (Board of Regents South Dakota)
• Original Assignee: South Dakota State University (Board of Regents South Dakota)
• Inventors: Fang, Ying, Nelson, Eric A., Hennings, Jane
• Granted Patent: US 8,182,984 B2
• Time in Patent Office: Days
• US Class Current: 424/199.100
• CPC Class Codes: A61K 2039/5254 avirulent or attenuated
• CPC Class Codes: A61K 2039/5256 expressing foreign proteins
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/06 Antiabortive agents; Labour repressants
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2770/10021 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2770/10022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2770/10034 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/10043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2770/10061 Methods of inactivation or attenuation
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major problem in the pork industry worldwide. The inclusion of markers in vaccines will allow for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. Using a cDNA infectious clone of North American Type 1 PRRSV, a recombinant green fluorescent protein (GFP) tagged PRRSV has been made, containing deletion of an immunogenic epitope, ES4, in the nsp2 region. GFP and ES4 epitope-based ELISAs compliment the marker identification.

Claims

1. A recombinant North American Type 1 porcine reproductive and respiratory syndrome virus (PRRSV), comprising one or more mutations in open reading frame (ORF) 1a, wherein said mutations are such that the recombinant PRRSV fails to produce at least one functional polypeptide corresponding to ORF1a.

2. The recombinant PRRSV of claim 1, wherein the mutation comprises a deletion.

3. The recombinant PRRSV of claim 2, wherein the deletion is in the nsp2 region.

4. The recombinant PRRSV of claim 3, wherein the deletion includes epitope ES4.

5. The recombinant PRRSV of claim 4, wherein the deletion includes amino acids 736-790 of ORF1a.

6. The recombinant PRRSV of claim 3, wherein the mutation comprises an insertion of a heterologous DNA sequence.

7. The recombinant PRRSV of claim 6, wherein the insertion is between amino acids 733 and 734 of ORF1a.

8. The recombinant PRRSV of claim 6, wherein the insertion is green fluorescent protein (GFP).

9. A recombinant North American PRRS virus encoded by an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus, wherein said DNA sequence is SEQ ID NO:

43.

10. A North American Type 1 PRRSV vaccine comprising a recombinant PRRSV having a mutation in ORF1a, said mutation including a deletion, an insertion, or both, in ORF1a, and a pharmaceutically acceptable carrier.

11. The vaccine of claim 10, wherein said mutation comprises a deletion in the nsp2 region.

12. The vaccine of claim 11, wherein said deletion is of amino acids 736-790 in the nsp2 region.

13. The vaccine of claim 11, wherein said mutation further comprises an insertion of a heterologous DNA sequence.

14. The vaccine of claim 13, wherein said insertion is between amino acids 733 and 734 in the nsp2 region.

15. A kit comprising:

a vaccine comprising a PRRSV mutant having a mutation in ORF1a, said mutation being such that said PRRSV mutant fails to produce a functional ORF1a polypeptide, said mutation including an insertion of a heterologous DNA sequence, and a pharmaceutically acceptable carrier;

one or more first polypeptides encoded by the heterologous DNA sequence; and

one or more second polypeptides encoded by the functional ORF1a.

16. The kit of claim 15, wherein the mutation in the PRRSV vaccine is a deletion in the nsp2 region.

17. The kit of claim 16, wherein the deletion is an ES4 epitope, wherein the one or more first polypeptides include GFP and the one or more second polypeptides include the ES4 epitope.

18. A method of differentiating an animal vaccinated with a PRRSV marker vaccine from an animal naturally infected with PRRSV, wherein the PRRSV marker vaccine includes an insertion mutation and a deletion mutation, the method comprising:

providing a first recombinant PRRSV protein including the insertion mutation;

providing a second recombinant PRRSV protein including the deletion mutation;

incubating a serum sample from the animal with the first and second recombinant PRRSV proteins; and

detecting binding of antibodies in the sample with the first and second recombinant PRRSV proteins;

wherein binding of antibodies in the sample with the first recombinant PRRSV protein is indicative of a vaccinated animal and binding of antibodies in the sample with the second recombinant PRRSV protein is indicative of a naturally infected animal.

19. The method of claim 18, wherein the first recombinant PRRSV protein includes a GFP insertion.

20. The method of claim 18, wherein the second recombinant PRRSV protein includes an ES4 deletion.