# US 5,352,596 A · Pseudorabies virus deletion mutants involving the EPO and LLT genes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,352,596 A - Document ID: US5352596A - Status: Granted - Kind: A - Application number: 07/945,283 - Filed: 1992-09-11 - Granted: 1994-10-04 - Earliest claimed: 1992-09-11 - Assignees: The United States of America as represented by the Secretary of Agriculture - Inventors: Andrew K. Cheung; Ronald D. Wesley - Web page: https://patentgrep.com/patent/US5352596A ## CPC - C07K 14/005: from viruses - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12N 2710/16721: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2710/16722: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - Y10S 424/815: Viral vaccine for porcine species, e.g. swine - Y10S 435/948: using viruses or cell lines - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract An attenuated pseudorabies virus (PRV) having a reduced ability to reactivate from latency is produced by introducing (1) a genomic modification in the early protein 0 (EP0) gene whereby said virus is characterized by the inability to express the early protein 0; or (2) a genomic modification in the large latency transcript (LLT) gene whereby said virus is characterized by disruption of the synthesis of said large latency transcript; or (3) the genomic modifications described in both (1) and (2). The attenuated virus is useful in a vaccine for psuedorabies-susceptible animals, particularly swine. Swine vaccinated with a deletion mutant in the EP0/LLT overlap region displayed reduced virus shedding and fewer clinical signs than animals inoculated with a wild type virus. The deletion mutant-vaccinated swine also harbored less PRV DNA in the nervous tissue and showed reduced ability to reactivate the virus. ## Claims ### Claim 1 A pseudorabies virus having a genomic modification selected from the group consisting of a deletion, and insertion or both in: (1) the early protein 0 gene whereby said virus is characterized by the inability to express the early protein 0; or (2) the large latency transcript gene whereby said virus is characterized by the inability to express said large latency transcript, with the provision that the modification to the large latency transcript is in conjunction with another modification which serves to attenuate the virus. ### Claim 2 (depends on 1) A pseudorabies virus as described in claim 1 wherein said modification is in an overlapping region of the early protein 0 gene and the large latency transcript gene. ### Claim 3 (depends on 1) A pseudorabies virus as described in claim 1 wherein said modification is in an overlapping region of the large latency transcript gene and the immediately-early 180 gene. ### Claim 4 (depends on 1) A pseudorabies virus as described in claim 1 wherein said modification comprises a deletion. ### Claim 5 (depends on 1) A pseudorabies virus as described in claim 1 wherein said modification comprises a deletion in the overlapping region of the early protein 0 gene and the large latency transcript gene. ### Claim 6 (depends on 5) A pseudorabies virus as described in claim 5 wherein said virus is EL.beta.-001. ### Claim 7 (depends on 1) A vaccine comprising the virus of claim 1 in an effective immunization dosage with a pharmaceutically acceptable carrier or diluent. ### Claim 8 (depends on 2) A vaccine comprising the virus of claim 2 in an effective immunization dosage with a pharmaceutically acceptable carrier or diluent. ### Claim 9 (depends on 5) A vaccine comprising the virus of claim 5 in an effective immunization dosage with a pharmaceutically acceptable carrier or diluent. ### Claim 10 (depends on 6) A vaccine comprising the virus of claim 6 in an effective immunization dosage with a pharmaceutically acceptable carrier or diluent. ### Claim 11 A method of immunizing an animal against psuedorabies comprising administering to said animal a vaccine comprising a pseudorabies virus having a genomic modification selected from the group consisting of a deletion, and insertion or both in: (1) the early protein 0 gene whereby said virus is characterized by the inability to express the early protein 0; or (2) the large latency transcript gene whereby said virus is characterized by the inability to express said large latency transcript with the provision that the modification to the large latency transcript is in conjunction with another modification which serves to attenuate the virus. ### Claim 12 (depends on 11) A method as described in claim 11 wherein said modification is in an overlapping region of the early protein 0 gene and the large latency transcript gene. ### Claim 13 (depends on 11) A method as described in claim 11 wherein said modification is in an overlapping region of the large latency transcript gene and the immediately-early 180 gene. ### Claim 14 (depends on 11) A method as described in claim 11 wherein said modification is a deletion. ### Claim 15 (depends on 11) A method as described in claim 11 wherein said modification is a deletion in the overlapping region of the early protein 0 gene and the large latency transcript gene. ### Claim 16 (depends on 15) A method as described in claim 15 wherein said virus is EL.beta.-001. ### Claim 17 (depends on 11) A method as described in claim 11, wherein said animal is a swine. ### Claim 18 (depends on 11) A method as described in claim 11 wherein said virus is present in said vaccine at level of 10.sup.3 -10.sup.6 PFU per dose. ### Claim 19 A method for producing a pseudorabies virus deletion mutant comprising the steps: (a) constructing a hybrid plasmid comprising a cloning vector, a selectable marker, and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus EP0 gene and flanking sequences thereof; (b) deleting DNA sequences from the hybrid plasmid such that the early protein 0 is not expressed, while retaining pseudorabies virus DNA sequences adjacent to each side of the deletion; (c) replacing the deleted sequences from step (b) with the selectable marker; (d) co-transfecting in pseudorabies virus host cells the resulting plasmid from step (b) with infectious psuedorabies virus DNA; and (e) selecting by means of the selectable marker psuedorabies virus deletion mutants which are characterized by the inability to express the early protein 0.