Thymidine kinase negative insertion mutants of pseudorabies virus and methods for the production of same

Claim 2

A pseudorabies virus which fails to produce any functional thymidine kinase as a result of an insertion in the tk gene.

Claim 3

The pseudorabies virus as claimed in claim 1, wherein said insertion is about 8 to 5000 bp in size.

Claim 4

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus also fails to produce any glycoprotein gI as a result of a mutation in the gI gene.

Claim 5

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus is also temperature-resistant.

Claim 6

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus is lyopholized.

Claim 7

A pseudorabies virus which fails to produce any functional thymidine kinase as a result of an insertion in the tk gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus tk gene and flanking sequence thereof; (2) Inserting a foreign DNA sequence within the pseudorabies virus tk gene of the resulting hybrid plasmid of step (1); (3) Co-transfecting into pseudorabies virus host cells, the hybrid plasmid of step (2) with infectious DNA from a tk + pseudorabies virus; and (4) Selecting in tk - pseudorabies virus host cells, for tk - pseudorabies virus from the virus produced in step (3) so as to produce a pseudorabies virus mutant which fails to produce any functional thymidine kinase as a result of an insertion in the tk gene.

Claim 8

A pseudorabies virus which fails to produce any functional thymidine kinase as a result of an insertion in the tk gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus tk gene and flanking sequence thereof; (2) Inserting a foreign DNA sequence within the pseudorabies virus tk gene of the resulting hybrid plasmid of step (1); (3) Co-transfecting into pseudorabies virus host cells, the hybrid plasmid of step (2) with infectious DNA from a tk + pseudorabies virus; and (4) Selecting in tk - pseudorabies virus host cells, for tk - pseudorabies virus from the virus produced in step (3) so as to produce a pseudorabies virus mutant which fails to produce any functional thymidine kinase as a result of an insertion in the tk gene.

Claim 9

The pseudorabies virus as claimed in claim 6, wherein said insertion is about 8 to 5000 bp in size.

Claim 10

The pseudorabies virus as claimed in claim 6, wherein said pseudorabies virus mutant also fails to produce any gI glycoprotein as a result of a mutation in the gI gene.

Claim 11

The pseudorabies virus as claimed in claim 6, wherein the infectious DNA of step (3) is derived from temperature-resistant pseudorabies virus such that the resulting mutants of step (3) are temperature-resistant pseudorabies virus mutants which fail to produce any functional thymidine kinase as a result of an insertion in the tk gene.

Claim 12

The pseudorabies virus as claimed in claim 6, additionally comprising step (5): (5) Propagating the resulting pseudorabies virus of step (4) at a non-permissive temperature for a temperature-sensitive virus so as to select for and produce temperature-resistant pseudorabies virus mutants which fail to produce any functional thymidine kinase as a result of an insertion in the tk gene.

Claim 13

The pseudorabies virus as claimed in claim 6, wherein said cloning vector is selected from the group consisting of pBR322, pBR325, pKH47, pBR328, pHC79, phage Charon 28, pKB11, pKSV-10 and pMAR420.

Claim 14

The pseudorabies virus as claimed in claim 11, wherein said cloning vector is pBR322.

Claim 15

The pseudorabies virus as claimed in claim 6, wherein said pseudorabies virus host cells in step (3) are tk + pseudorabies virus host cells.

Claim 16

The pseudorabies virus as claimed in claim 6, wherein said pseudorabies virus as lyopholized.