Modified live pseudorabies viruses

Claim 2

A temperature-resistant pseudorabies virus which fails to produce any functional TK as a result of mutagen-induced mutation.

Claim 3

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 1, wherein said virus has the identifying characteristics of PRV(BUK-5A) (ATCC No. VR-2078).

Claim 4

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 1, wherein said virus is lypholized.

Claim 5

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 + strain in tk - host cells at a permissive temperature for a temperature-sensitive virus; (2) propagating the resulting virus of step (1) in tk - 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 + 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 + 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 + 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 + host cells in the presence of a mutagen at a permissive temperature for a temperature-sensitive virus to produce a temperature-resistant PRV tk - mutagen-induced mutant.

Claim 6

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 + strain in tk - host cells at a permissive temperature for a temperature-sensitive virus; (2) propagating the resulting virus of step (1) in tk - 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 + 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 + 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 + 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 + host cells in the presence of a mutagen at a permissive temperature for a temperature-sensitive virus to produce a temperature-resistant PRV tk - mutagen-induced mutant.

Claim 7

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said virus has the characteristics of PRV(BUK-5A) (ATCC No. VR-2078).

Claim 8

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said virus is lypholized.

Claim 9

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said PRV tk + strain is selected from the group consisting of PRV(BUK) strain, SUCH-1 strain, K strain, Norden strain, PRV(Auj) strain, P-2208 strain, KC-152D strain, S62/26 Iowa strain, IND-FH strain, IND-S strain, IND-R strain and Shope strain.

Claim 10

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 7, wherein said PRV tk + strain is PRV(BUK) strain.

Claim 11

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said permissive temperature for a temperature-sensitive virus is about 33° to 37.5° C.

Claim 12

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 9, wherein said permissible temperature for a temperature-sensitive virus is 34.5° C.

Claim 13

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said non-permissive temperature for a temperature-sensitive virus is about 38.5° to 40° C.

Claim 14

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 11, wherein said non-permissive temperature for a temperature-sensitive virus is 39.1° C.

Claim 15

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said tk - host cells are selected from the group consisting of rabbit Rab(BU), mouse LM(TK - ), human HeLa(BU25), syrian hamster BHK 21 (TK - ) and human line 143.

Claim 16

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 13, wherein said tk - host cells are rabbit Rab(BU).

Claim 17

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said tk + host cells are selected from the group consisting of Rab-9, primary rabbit kidney cells, secondary rabbit kidney cells, monkey cells, human cells, human embryonic kidney cells and chick embryo fibroblast cells.

Claim 18

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 15, wherein said tk + host cells are Rab-9.

Claim 19

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said mutagen is selected from the group consisting of BrdUrd, NH 2 OH, HONO, nitrosoguanidine and UV light.

Claim 20

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 17, wherein said mutagen is BrdUrd.

Claim 21

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 4, wherein said selective agent is selected from the group consisting of BrdUrd, araT, BVDU, FMAU and AIdUrd.

Claim 22

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 19, wherein said selective agent is araT.

Claim 23

A temperature-resistant pseudorabies virus which fails to produce any functional TK as a result of a deletion in the tk gene.

Claim 24

A temperature-resistant pseudorabies virus which fails to produce any functional TK as a result of a deletion in the tk gene.

Claim 25

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 21, wherein said deletion is about 10 to 1500 bp in size.

Claim 26

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 22, wherein said deletion is about 75 to 750 bp in size.

Claim 27

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 21, wherein said virus has the identifying characteristics of PRV(BUK-dl 3) (ATCC No. VR-2074).

Claim 28

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 102, wherein said virus is lypholized.

Claim 29

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 + host cells, the hydrid plasmid of step (1) with DNA from a temperature-resistant PRV tk - mutagen-induced mutant; (3) selecting, in tk - host cells, for PRV tk + 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 + host cells, PRV tk + DNA derived from the PRV tk + obtained in step (3) with the resulting hybrid plasmid of step (4); and (6) selecting, in tk - host cells, for PRV tk - from the virus produced in step (5) to produce temperature-resistant PRV tk - deletion mutants.

Claim 30

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 + host cells, the hydrid plasmid of step (1) with DNA from a temperature-resistant PRV tk - mutagen-induced mutant; (3) selecting, in tk - host cells, for PRV tk + 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 + host cells, PRV tk + DNA derived from the PRV tk + obtained in step (3) with the resulting hybrid plasmid of step (4); and (6) selecting, in tk - host cells, for PRV tk - from the virus produced in step (5) to produce temperature-resistant PRV tk - deletion mutants.

Claim 31

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein said deletion is about 10 to 1500 bp in size.

Claim 32

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 27, wherein said deletion is about 75 to 750 bp in size.

Claim 33

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein said virus is PRV (BUK-dl 3) (ATCC No. VR-2074).

Claim 34

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein said virus is lypholized.

Claim 35

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, 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 36

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 31, wherein said cloning vector is pBR322.

Claim 37

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein said hybrid plasmid of step (1) is pBB-11.

Claim 38

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein the PRV DNA sequences adjacent to each side of the deletion are at least about 400 bp in size.

Claim 39

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein the resulting hybrid plasmid of step (4) is pBB-11 dl SacA26.

Claim 40

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein said temperature-resistant PRV tk - mutagen-induced mutant is PRV(BUK-5A) (ATCC No. VR-2078).

Claim 41

The temperature-resistant pseudorabies virus which fails to produce any functional TK as in claim 26, wherein said PRV tk + DNA of step (5) is derived from PRV(BUK-5A-R1).

Claim 42

The temperature-resistant pseudorabies virus as in claim 26, wherein said tk + host cells are selected from the group consisting of Rab-9, primary rabbit kidney cells, secondary kidney cells, monkey cells, human cells, human embrynoic kidney cells and chick embryo fibroblast cells.

Claim 43

The temperature-resistant pseudorabies virus as in claim 38, wherein said tk + host cells are Rab-9.