Equine herpesvirus 1 tk mutants

The equine herpesvirus type 1 (EHV-1) which fails to produce any functional thymidine kinase as a result of an insertion in the EHV-1 thymidine kinase gene produced by the process comprising:

• (1) constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of EHV-1 containing substantially all of the EHV-1 thymidine kinase gene and flanking sequences thereof;
• (2) inserting a detectable foreign DNA sequence within the coding region of the EHV-1 thymidine kinase gene of the hybrid plasmid of step (1);
• (3) co-transfecting semi-permissive, immortalized EHV-1 host cells with the hybrid plasmid of step (2) and genomic DNA isolated from an EHV-1, wherein said genomic DNA comprises the EHV-1 thymidine kinase gene and expresses the EHV-1 thymidine kinase gene upon infection of EHV-1 host cells, wherein said semi-permissive, immortalized EHV-1 host cells are RAB-9 or RAB(BU) cells, and harvesting recombinant EHV-1;
• (4) culturing the resulting recombinant EHV-1 of step (3) in permissive EHV-1 host cells;
• (5) selecting or screening the cells resulting from step (4) or EHV-1 harvested therefrom, for EHV-1 containing the detectable foreign DNA sequence;
• and
• (6) screening the cells or virus from step (5) for EHV-1 which fails to produce any functional thymidine kinase as a result of an insertion in the EHV-1 thymidine kinase gene, and wherein the virus in which the insertion is demonstrated is harvested.

An equine herpesvirus type 1 (EHV-1) which fails to produce any functional thymidine kinase as a result of a deletion in the EHV-1 thymidine kinase gene produced by the process comprising:

• (1) constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of EHV-1 containing substantially all of the EHV-1 thymidine kinase gene and flanking sequences thereof;
• (2) deleting sequences within the coding region of the EHV-1 thymidine kinase gene of the hybrid plasmid of step (1);
• (3) co-transfecting semi-permissive, immortalized EHV-1 host cells with the hybrid plasmid of step (2) and genomic DNA isolated from an EHV-1 which fails to produce any functional thymidine kinase as a result of an insertion of a detectable foreign DNA sequence in the EHV-1 thymidine kinase gene, wherein said semi-permissive, immortalized EHV-1 host cells are RAB-9 or RAB(BU) cells, and harvesting recombinant EHV-1;
• (4) culturing the resulting recombinant EHV-1 of step (3) in permissive EHV-1 host cells;
• (5) selecting or screening the cells resulting from step (4) or EHV-1 harvested therefrom, for EHV-1 which do not contain the detectable foreign DNA sequence insertion;
• and
• (6) screening the cells or virus from step (5) for EHV-1 which fails to produce any functional EHV-1 gene thymidine kinase as a result of a deletion in the EHV-1 thymidine kinase gene, and wherein virus in which the deletion is demonstrated is harvested.