N Protein Muants of Porcine Reproductive and Respiratory Syndrome Virus
The present invention provides a genetically modified PRRS virus, methods to make it and related polypeptides, polynucleotides and various components. Vaccines comprising the genetically modified virus and polynucleotides are also provided.
1 . A composition comprising a PRRS infectious agent selected from the group consisting of:
a.) a genetically modified PRRS virus comprising an N protein which has been modified in at least one conserved region selected from the group consisting of the NLS-2 region, NoLS region and the NES region such that the conserved region has been eliminated, and wherein the genetically modified PRRS virus is attenuated;
b.) an infectious RNA molecule encoding the genetically modified PRRS virus of a.); and
c.) an isolated polynucleotide molecule comprising a DNA sequence encoding the infectious RNA molecule of b.).
2 . The composition of claim 1 which has been further modified to result in the elimination of the conserved NLS-1 region.
3 . The composition of claim 1 wherein the conserved region has been eliminated by the introduction of a non-conservative amino acid substitution.
4 . The composition of claim 1 or 2 wherein the conserved region has been at least partially deleted.
5 . The composition of claim 1 wherein the conserved region is the NoLS region.
6 . The composition of claim 1 wherein the conserved region is the NLS-2 region.
7 . The composition of claim 1 wherein the conserved region is the NES region.
8 . The composition of claim 1 wherein the PRRS virus is a North American PRRS virus.
9 . The composition of claim 1 wherein the PRRS virus is a European PRRS virus.
10 . The composition of claim 8 wherein the conserved region is the NLS-2 region.
11 . The composition of claim 10 wherein residues 42 and 43 of the N protein are glycines.
12 . The composition of claim 10 wherein residues 42 and 43 of the N protein are glycine and residue 44 is an asparagine.
13 . The composition of claim 10 wherein the NLS-2 region has been at least partially deleted.
14 . The composition of claim 13 wherein at least one of residues 43 through 48 of the N protein have been deleted.
15 . The composition of claim 14 wherein both residues 43 and 44 of the N protein have been deleted.
16 . The composition of claim 14 wherein residues 43, 44, and 46 of the N protein have been deleted. The composition of claim 14 wherein residues 44, 46, and 47 of the N protein have been deleted.
17 . The composition of claim 14 wherein residues 46, 47, and 48 of the N protein have been deleted.
18 . The composition of claim 1 that contains additional nucleotide mutation, substitutions and/or deletions, designed to minimize the probability of reversion.
19 . A vaccine for protecting a porcine animal from infection by a PRRS virus comprising the composition of claims 1 in an amount effective to produce immunoprotection against infection by a PRRS virus; and a carrier acceptable for veterinary use.
20 . A method for protecting a porcine animal from infection by a PRRS virus, which comprises vaccinating the animal with an amount of the vaccine of claim 19 that is effective to produce immunoprotection against infection by a PRRS virus.
21 . A transfected host cell comprising a composition according to claim 1 .
22 . A method for making a genetically modified and attenuated PRRS virus, which method comprises:
a.) mutating a DNA sequence encoding an infectious RNA molecule which encodes a PRRS virus, to produce a genetically modified PRRS virus comprising an N protein which has been modified in an at least one conserved region selected from the group consisting of the NLS-2 region, NoLS region and the NES region such that the conserved region has been eliminated;
b.) introducing the genetically modified PRRS virus into a host cell capable of supporting PRRS replication.
23 . A method of claim 22 wherein the genetically modified PRRS virus is a North American PRRS virus.
24 . A method of claim 22 wherein the genetically modified PRRS virus is a European PRRS virus.
25 . The method of claim 22 wherein the host cell capable of supporting PRRS replication is a MARC-145 cell.
26 . The method of claim 22 wherein the host cell capable of supporting PRRS replication is comprised within a live porcine animal.