Infectious cDNA clone of North American porcine reproductive and respiratory syndrome (PRRS) virus and uses thereof

An isolated polynucleocide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that when it infects a porcine animal it is unable to produce PRRS in said animal, wherein said DNA sequence is SEQ ID NO:

• 1 or a sequence homologous to SEQ ID NO:
• 1, except that said DNA sequence contains one or more deletions of at least a portion of an open reading frame (ORF) selected from the group consisting of ORF 2, and ORF 4, corresponding to, SEQ ID NO:
• 4, and SEQ ID NO:
• 6, respectively, wherein said one or more deletions genetically disable the encoded PRRS virus in its ability to produce PRRS disease in said animal, wherein said PRRS virus is only capable of growing on a complementing cell line, and is not capable of growing on an unalter cell line, wherein said sequence homologous to SEQ ID NO:
• 1 has at least 70% identity to SEQ ID NO:
• 1 or hybridizes to the complement of SEQ ID NO:
• 1 under highly stringent conditions which comprise hybridization to filter bound DNA in 0.5M NaHPO4, 7% SDS, 1mM EDTA at 65° C., and washing in 0.1xSSC/0.1%SDS at 68° C.

A method for preparing a genetically modified PRRS virus that is capable of eliciting an immunoprotective response in a mammal or a bird vaccinated therewith, which method comprises obtaining an isolated DNA encoding an infectious RNA molecule which encodes a wild-type PRRS virus;

• genetically mutating the DNA so as to obtain a mutated DNA encoding an infectious RNA molecule which encodes a genetically modified PRRS virus which virus is replication-defective and remains able to elicit an effective immunoprotective response against infection by the wild-type PRRS virus in a mammal or a bird;
• placing said mutated DNA in a plasmid in an operable linkage to a promoter;
• transfecting a host cell with said plasmid;
• and expressing the genetically modified PRRS virus.

An isolated polynucleotide molecule comprising a nucleotide sequence that encodes a PRRS virus, wherein said nucleotide sequence is an RNA molecule corresponding to a DNA sequence that is SEQ ID NO:

• 1 or a sequence homologous to SEQ ID NO:
• 1, except for a deletion of at least a portion of ORF 2 that corresponds to SEQ ID NO:
• 4, wherein said sequence homologous to SEQ ID NO:
• 1has at least 70% identity to SEQ ID NO:
• 1 or hybridizes to the complement of SEQ II) NO:
• 1 under highly stringent conditions which comprise hybridization to filter bound DNA in 0.5M NaHPO4, 7% SDS, 1mM EDTA at 65° C., and washing in 0.1xSSC/0.1%SDS at 68° C. and wherein said deletion renders the PRRS virus replication defective.

An isolated polynucleotide molecule comprising a nucleotide sequence that encodes a PRRS virus, wherein said nucleotide sequence is an RNA molecule corresponding to a DNA sequence that is SEQ ID NO:

• 1 or a sequence homologous to SEQ ID NO:
• 1, except for a deletion of at least a portion of ORF 4 that corresponds to SEQ ID NO:
• 6, wherein said sequence homologous to SEQ ID NO:
• 1 has at least 70% identity to SEQ ID NO:
• 1 or hybridizes to the complement of SEQ ID NO:
• 1 under highly stringent conditions which comprise hybridization to filter bound DNA in 0.5M NaHPO4, 7% SDS, 1mM EDTA at 65° C., and washing in 0.1xSSC/0.1%SDS at 68° C. and wherein said deletion renders the PRRS virus replication defective.

An isolated polynucleotide molecule comprising a nucleotide sequence that encodes a PRRS virus, wherein said nucleotide sequence is an RNA molecule corresponding to a DNA sequence that is SEQ ID NO:

• 1 or a sequence homologous to SEQ ID NO:
• 1, except for one or more deletions of at least a portion of an open reading frame (ORF) selected from the group consisting of ORF 2and ORF 4, corresponding to SEQ ID NO. 4, and SEQ ID NO. 6, respectively, wherein said sequence homologous to SEQ ID NO:
• 1 has at least 70% identity to SEQ ID NO:
• 1 or hybridizes to the complement of SEQ ID NO:
• 1 under highly stringent conditions which comprise hybridization to filter bound DNA in 0.5M NaHPO4, 7% SDS, 1mM EDTA at 65°C, and washing in 0.1 xSSC/0.1% SDS at 68° C. and wherein said deletion renders the PRRS virus replication defective.