Modified live pseudorabies viruses

A temperature-resistant pseudorabies virus which fails to produce any functional TK as a result of mutagen-induced mutation produced by a process comprising:

• (1) plaque-purifying a PRV tk.sup.+ strain in tk.sup.- host cells at a permissive temperature for a temperature-sensitive virus;
• (2) propagating the resulting virus of step (1) in tk.sup.- host cells 2 to 5 times in the presence of a mutagen at a permissive temperature for a temperature-sensitive virus;
• (3) propagating the resulting virus of step (2) in tk.sup.+ host cells in the presence of a mutagen at a non-permissive temperature for a temperature-sensitive virus;
• (4) propagating the resulting virus of step (3) in tk.sup.+ host cells in the presence of a mutagen at a permissive temperature for a temperature-sensitive virus;
• (5) propagating the resulting virus of step (4) in tk.sup.+ host cells in the presence of a selective agent at a permissive temperature for a temperature-sensitive virus;
• and
• (6) propagating the resulting virus of step (5) in tk.sup.+ host cells in the presence of a mutagen at a permissive temperature for a temperature-sensitive virus to produce a temperature-resistant PRV tk.sup.- mutagen-induced mutant.

A temperature-resistant pseudorabies virus which fails to produce any functional TK as a result of a deletion in the tk gene produced by a process comprising:

• (1) constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of PRV containing substantially all of the PRV tk gene;
• (2) co-transfecting, in tk.sup.+ host cells, the hydrid plasmid of step (1) with DNA from a temperature-resistant PRV tk.sup.- mutagen-induced mutant;
• (3) selecting, in tk.sup.- host cells, for PRV tk.sup.+ from the virus produced in step (2);
• (4) deleting DNA sequences from the hydrid plasmid of step (1) such that less than substantially all of the PRV tk gene is present, while retaining PRV DNA sequences adjacent to each side of the deletion;
• (5) co-transfecting, in tk.sup.+ host cells, PRV tk.sup.+ DNA derived from the PRV tk.sup.+ obtained in step (3) with the resulting hybrid plasmid of step (4);
• and
• (6) selecting, in tk.sup.- host cells, for PRV tk.sup.- from the virus produced in step (5) to produce temperature-resistant PRV tk.sup.- deletion mutants.