Thymidine kinase deletion mutants of bovine herpesvirus-1

Claim 2

An infectious bovine rhinotracheitis virus which fails to produce any functional TK as a result of a deletion in the tk gene.

Claim 3

The infectious bovine rhinotracheitis virus as claimed in claim 1, wherein said deletion is about 10 to 1500 bp in size.

Claim 4

The infectious bovine rhinotracheitis virus as claimed in claim 2, wherein said deletion is about 75 to 750 bp in size.

Claim 5

The infectious bovine rhinotracheitis virus as claimed in claim 1, wherein said virus is temperature resistant.

Claim 6

The infectious bovine rhinotracheitis virus as claimed in claim 1, wherein an oligonucleotide linker is present in the deletion site of the tk gene.

Claim 7

The infectious bovine rhinotracheitis virus as claimed in claim 5, wherein said linker is about 7 to 50 nucleotides in length.

Claim 8

The infectious bovine rhinotracheitis virus as claimed in claim 1, wherein said virus has the identifying characteristics of IBRV(NG) dl TK clone 1 (ATCC No. VR-2112).

Claim 9

The infectious bovine rhinotracheitis virus as claimed in claim 1, wherein said virus is lyophilized.

Claim 10

An infectious bovine rhinotracheitis virus which fails to produce any functional TK as a result of a deletion in the tk gene produced by the process comprising: (1) constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of IBRV containing substantially all of the IBRV tk gene; (2) co-transfecting, in tk + host cells, the hybrid plasmid of step (1) with infectious DNA from a tk - IBRV mutagen-induced mutant; (3) selecting, in tk - host cells, for tk + IBRB from the virus produced in step (2); (4) deleting DNA sequences from the hybrid plasmid of step (1) such that less than substantially all of the IBRV tk gene is present; (5) co-transfecting, in tk + host cells, IBRV tk + DNA derived from the tk + IBRV obtained in step (3) with the resulting tk - hybrid plasmid of step (4); and (6) selecting, in tk - host cells, for tk - IBRV from the virus produced in step (5) so as to produce tk - IBRV mutants which fail to produce any functional TK as a result of a deletion in the tk gene.

Claim 11

An infectious bovine rhinotracheitis virus which fails to produce any functional TK as a result of a deletion in the tk gene produced by the process comprising: (1) constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of IBRV containing substantially all of the IBRV tk gene; (2) co-transfecting, in tk + host cells, the hybrid plasmid of step (1) with infectious DNA from a tk - IBRV mutagen-induced mutant; (3) selecting, in tk - host cells, for tk + IBRB from the virus produced in step (2); (4) deleting DNA sequences from the hybrid plasmid of step (1) such that less than substantially all of the IBRV tk gene is present; (5) co-transfecting, in tk + host cells, IBRV tk + DNA derived from the tk + IBRV obtained in step (3) with the resulting tk - hybrid plasmid of step (4); and (6) selecting, in tk - host cells, for tk - IBRV from the virus produced in step (5) so as to produce tk - IBRV mutants which fail to produce any functional TK as a result of a deletion in the tk gene.

Claim 12

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein said deletion is about 10 to 1500 bp in size.

Claim 13

The infectious bovine rhinotracheitis virus as claimed in claim 10, wherein said deletion is about 75 to 750 bp in size.

Claim 14

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein the tk - IBRV mutagen-induced mutant in step (2) is a temperature-resistant mutant such that the resulting mutant in step (6) is both temperature resistant and a tk - deletion mutant.

Claim 15

The infectious bovine rhinotracheitis virus as claimed in claim 9, additionally comprising step (7): (7) propagating the resulting IBRV which fails to produce any functional TK as a result of a deletion in the tk gene of step (6) at a non-permissive temperature for a temperature-sensitive virus so as to select for and produce a temperature-resistant IBRV which fails to produce any functional TK as a result of a deletion in the tk gene.

Claim 16

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein an oligonucleotide linker is present in the deletion site of the IBRV DNA of step (4) while retaining IBRV DNA sequences adjacent to each side of the deletion site.

Claim 17

The infectious bovine rhinotracheitis virus as claimed in claim 14, wherein said linker is about 7 to 50 nucleotides in length.

Claim 18

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein said virus is lyophilized.

Claim 19

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein said cloning vector is selected from the group consisting of pBR322, pMB9, pBR325, pKH47, pBR328, pHC79, phage Charon 28, pKB11, pKSV-10, pMAR420 and oligo (dG)-tailed pBR322.

Claim 20

The infectious bovine rhinotracheitis virus as claimed in claim 17, wherein said cloning vector is pBR322.

Claim 21

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein said hybrid plasmid of step (1) is pLATK dl NdeI.

Claim 22

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein in step (4) the IBRV DNA sequences adjacent to each side of the deletion are at least 400 bp in size.

Claim 23

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein the resulting hybrid plasmid of step (4) is pLATK dl NdeI dl BgII/NG/SstI.

Claim 24

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein said tk + host cells are selected from the group consisting of RAB-9 (ATCC No. CRL-1414); primary rabbit kidney cells, secondary rabbit kidney cells; rabbit cornea (SIRC) cells (ATCC No. CCL-60), rabbit kidney (LLC-RK1) cells (ATCC No. CCL-106), embryo bovine trachea (EBTR) cells (ATCC No. CCL-44), bovine turbinate (BT) cells (ATCC No. CRL-1390), and bovine kidney (MDBK) cells (ATCC No. CCL-22).

Claim 25

The infectious bovine rhinotracheitis virus as claimed in claim 22, wherein said tk + host cells are RAB-9.

Claim 26

The infectious bovine rhinotracheitis virus as claimed in claim 9, wherein the tk - IBRV mutagen-induced mutant is selected from the group consisting of the non-temperature-resistant bromovinyldeoxyuridine-resistant IBRV mutant of IBRV(P8-2) and the temperature-resistant IBRV(B8-D53) (ATCC No. VR-2066).

Claim 27

The infectious bovine rhinotracheitis virus as claimed in claim 24, wherein the tk - IBRV mutagen-induced mutant is the temperature-resistant IBRV(B8-D53) (ATCC No. VR-2066).