Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To improve both safety and stability of the vaccines used in the field to vaccinate foxes against rabies by the oral route, a recombinant vaccinia virus, expressing the immunizing G protein of rabies virus (VVTGg RAB-26D3 187 X P strain) has been developed. The c-DNA corresponding to the glycoprotein of the ERA strain of rabies virus has been inserted into the thymidine kinase (TK) gene of the vaccinia virus (Copenhagen strain). The efficacy of this recombinant strain was tested by the oral route, primarily in foxes. The duration of immunity conferred by the VVTGg RAB, a minimum of 12 months in cubs and 18 months in adult animals, corresponds to the length of protection required for fox vaccination in the field. VVTGg RAB innocuity was tested in foxes and in domestic animals as well as in numerous wild animal species that could compete with the red fox in consuming vaccine baits in Europe. During a minimum of 28 days post vaccination, neither clinical signs nor lesions were observed in any of the vaccinated animals. Moreover no transmission of immunizing amounts of the recombinant occurred in the red fox or the other species tested. To study the stability of the vaccine strain, baits containing the vaccine were placed in the field. Despite considerable variations of environmental temperatures, the VVTGg RAB titre remained stable after one month in the field. Since all the baits are taken within one month, it can be assumed that most of the animals taking the baits are effectively vaccinated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Development of a recombinant vaccinia-rabies vaccine for oral vaccination of foxes against rabies. 128 44

The use of an ELISA that can differentiate between swine infected with pseudorabies virus (PRV) and swine vaccinated with a specific PRV vaccine was evaluated on an individual and herd basis, and a system for interpreting ELISA results on a herd basis was developed. In 17 herds, recently introduced replacement gilts, seronegative for PRV, were vaccinated with a thymidine kinase- and glycoprotein X (gpX)-deleted vaccine. After vaccination, blood samples were collected from these gilts approximately every 1 to 2 months for up to 19 months. Serum samples were analyzed for antibodies to gpX antigen, using a commercially available ELISA kit according to the manufacturer's protocol. Herd status was determined as positive, suspect, or negative, according to the serum sample:negative control (S:N) values of the samples collected from the herd. From the 17 herds, 130 evaluations were performed. On 49 (38%) of the 130 herd evaluations, 1 or more gilts had suspect test results. Additional testing was required in 19 (39%) of these 49 herd evaluations to determine the PRV infection status of the herd. Status of herds having gilts with suspect results and no positive results was usually negative after retesting. Herds having gilts with positive results were unlikely to have negative status after retesting.
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PMID:Use of an enzyme-linked immunosorbent assay for detection of infection with pseudorabies virus on a herd basis. 131 89

Bovine herpesvirus 1 (BHV-1) gene expression was examined by RNA blot hybridization using clones representing immediate-early, early, and late genes. An immediate-early protein gene probe hybridized with two transcripts, 3.4 and 5.8 kb, expressed by infected cells in the presence of cycloheximide (CH). During infection of cells without metabolic inhibitors these transcripts were detected as early as 2 hr postinfection (p.i.) and accumulated to 8 hr p.i. The early gene probe, thymidine kinase, hybridized with a 4.3-kb RNA that was detected in the presence of phosphonoacetic acid (PAA), but not in the presence of CH. The late gene probe, glycoprotein III, (gIII) hybridized with a 1.6-kb transcript that was not expressed by infected cells treated with CH and only in very reduced amounts by infected cells treated with PAA. The gIII RNA was not detected until 4 hr p.i. in total cell RNA. Transcripts for the bovine actin and beta-galactosyltransferase genes did not decrease in BHV-1-infected cells until 6 hr p.i., coincident with the increase of BHV-1 DNA and RNA synthesis. Consequently, shutoff of host cell transcription by BHV-1 may be different than what has been described for herpes simplex virus.
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PMID:Relationship of bovine herpesvirus 1 immediate-early, early, and late gene expression to host cellular gene transcription. 131 50

Recombinant DNA techniques were used to insert foreign genes into bovine herpesvirus-1 [infectious bovine rhinotracheitis virus (IBRV)] vectors which were attenuated by deletion and/or insertion mutations in the IBRV thymidine kinase (tk) gene. In one recombinant, the regulatory and coding sequences of the late pseudorabies virus (PRV) glycoprotein gIII gene, were inserted into the early IBRV tk gene. This recombinant efficiently expressed the PRV gIII gene indicating that immediate early IBRV proteins were competent to transactivate the late PRV gIII gene. IBRV vector viruses were also prepared in which the coding sequences of the early PRV tk gene, the late PRV gIII gene, and the E. coli beta-galactosidase gene were ligated to the late IBRV gIII promoter. Genotypes and phenotypes of the recombinant viruses were verified by restriction endonuclease and molecular hybridization experiments, thymidine plaque autoradiography, beta-gal plaque assays, and by immunoprecipitation experiments on extracts from 3H-mannose-labelled cells. The recombinant IBRV expressing beta-gal from the IBRV gIII promoter has been useful as an intermediate in the construction of IBRV vectors harboring foreign DNA sequences. The infectivity of the IBRV recombinant that expressed PRV gIII from the IBRV gIII promoter, was neutralized by polyclonal PRV antisera and by monoclonal antibodies to PRV gIII. The PRV gIII glycoprotein synthesized by the preceding recombinant has been used to coat microtiter test plate wells in a PRV gIII differential diagnostic test kit.
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PMID:Expression of porcine pseudorabies virus genes by a bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) vector. 131 33

Pigs with low levels of maternally derived antibodies were vaccinated twice intramuscularly with 10(5), 10(5.5), or 10(6) plaque forming units (p.f.u.) of the genetically engineered Aujeszky's disease virus (ADV) vaccine strain 783. Strain 783 has deletions in genes encoding glycoprotein gI and thymidine kinase. All vaccinated pigs showed a high level of protection against clinical disease after challenge infection with virulent ADV. Vaccination also reduced virus excretion. The daily mean virus excretion and the mean number of days with virus excretion, fever, mild clinical signs, and growth retardation were higher in pigs vaccinated with 10(5) than in pigs vaccinated with 10(5.5) or 10(6) p.f.u. of strain 783.
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PMID:Comparative efficacy of three doses of the genetically engineered Aujeszky's disease virus vaccine strain 783 in pigs with maternal antibodies. 131 82

Genetic recombination between field strains and vaccine strains of pseudorabies virus (PRV) has been suggested as a scenario that might arise from use of deletion-mutant modified-live vaccine strains, particularly those strains attenuated by deletions within the thymidine kinase (TK-) gene locus. To address this hypothesis experimentally, it is necessary to screen large numbers of PRV isolates for their TK genotype. Techniques to detect the native TK genotype are routinely used in molecular virology laboratories, but are time-consuming. We adapted the polymerase chain reaction to define the genotypic status of PRV isolates with regard to the presence or absence of deletions in the TK gene locus. Used in tandem with the existing glycoprotein-specific ELISA that discriminate between PRV-vaccinated and field strain-infected swine populations, the described technique may help to clarify whether vaccine-derived recombinants are generated under natural conditions and after normal vaccine usage.
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PMID:Genotypic screening of pseudorabies virus strains for thymidine kinase deletions by use of the polymerase chain reaction. 132 Aug 13

Primary ocular herpes is usually seen as a follicular conjunctivitis and blepharitis, with or without involvement of the cornea. It is unknown, however, to what extent asymptomatic and/or subclinical primary disease occurs, and whether primary ocular herpes follows direct droplet spread to the eye. Previous models of murine ocular herpes have used trauma (scarification) to introduce virus into the cornea, producing disease which results in significant corneal scarring. To mimic a likely route of infection in humans, a droplet containing virus was placed on the mouse eye and clinical disease recorded. At least 1 month after inoculation, serum was assayed for neutralising antibodies and the cornea, iris, and trigeminal ganglion were investigated for evidence of herpes simplex virus type 1, by cocultivation and the polymerase chain reaction. Some animals showed a severe ulcerative blepharitis with little to no involvement of the cornea, while disease was undetectable in others. The development of disease depended on the dose and strain of virus and age of the animal, with older mice appearing more resistant. Virus was isolated from the trigeminal ganglion of younger animals inoculated with higher doses of virus, after 21 days in culture, suggesting that latency had been established. Neutralising antibodies were present in most mice irrespective of the presence of recognisable clinical disease. Using primers for the thymidine kinase and glycoprotein C regions of the viral genome, herpes simplex virus type 1 DNA was found in the cornea, iris, and trigeminal ganglion of most animals and showed a good correlation with the presence of neutralising antibodies. It would thus appear that herpes simplex virus type 1 is able to accede into the cornea, iris, and trigeminal ganglion following nontraumatic application of virus onto the mouse eye. This model mimics primary ocular disease in humans and may be useful for studies on recurrent disease and the spread of ocular herpes.
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PMID:Non-traumatic acquisition of herpes simplex virus infection through the eye. 132 Sep 25

A detailed sequence and transcript analysis of the thymidine kinase (tk) gene of bovine herpesvirus 1 (BHV1, Cooper strain) was carried out. A tk open reading frame (ORF) of 1077 bp was identified and compared with tk ORFs previously published for other strains of BHV1. The Cooper sequence was in good agreement with that of strain Q3932 but differed significantly from strains 6660 and LA. Reanalysis of the LA tk sequence, however, failed to confirm this difference. Except for five single base substitutions, our results indicate that the Cooper, Q3932, and LA strains share the same tk sequence. The size of the tk mRNA was determined by Northern blot analysis. In contrast to the size of other herpesvirus tk mRNAs (approximately 1.5 kb), the BHV1 tk transcript was 4.3 kb. Northern blot analysis indicated that the tk transcript was 3' coterminal with the downstream 3.1-kb transcript which encodes a BHV1 homologue of the HSV1 H glycoprotein (gH). The 5' ends of the tk and gH mRNAs were mapped by S1 analysis to positions 135 and 91 bp upstream of their respective translation start sites. The 5' end of the tk transcript was found to overlap a 5.2-kb transcript with opposite polarity to the tk mRNA.
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PMID:Sequence and transcript analysis of the bovine herpesvirus 1 thymidine kinase locus. 132 82

The role of cis-acting promoter elements associated with herpes simplex virus type 1 (HSV-1) early and late genes was evaluated during productive infection with regard to activation of gene expression by the HSV-1 transactivator ICP4 and control of temporal regulation. A set of recombinant viruses was constructed such that expression of an HSV-1 early gene, thymidine kinase (tk), was placed under the control of either the tk TATA box or the TATA box from the late gene, glycoprotein C (gC), in the presence or absence of the upstream Sp1 and CCAAT sites normally found in the tk promoter. The presence of Sp1 sites in the promoter or replacement of the tk TATA box with the gC TATA box resulted in a decreased activation of tk mRNA expression by ICP4. Substitution of the A + T-rich region from the gC TATA box in the context of the remainder of the surrounding tk sequences resulted in a promoter that bound recombinant TATA-binding protein (TBP) better at lower concentrations than the wild-type tk promoter did. These results indicate that tk promoters that are better able to utilize TBP are less responsive to ICP4 activation and suggest that activation by ICP4 involves the general transcription factors that interact with TBP or TBP itself. Additionally, all of the viruses expressed tk at early times postinfection, indicating that cis-acting promoter elements that control the level of expression of HSV-1 early and late genes do not determine temporal regulation.
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PMID:Substitution of a TATA box from a herpes simplex virus late gene in the viral thymidine kinase promoter alters ICP4 inducibility but not temporal expression. 132 6

This report describes a novel method for complementation studies of defective herpes simplex virus (HSV) genes. Viral test gene and nonviral reporter gene cassettes were rapidly integrated into the HSV genome in a site-specific and reversible manner by using the P1 phage-based Cre-lox recombination system. Shuttle plasmids contained a functional loxP recombination site, an expressible form of the bacterial lacZ gene, and a copy of the wild-type glycoprotein B (gB) gene or double mutant gB allele containing both a temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viral genome, K delta T::lox1, was constructed from the HSV type 1 (syn) gB-deficient mutant virus, K delta T, by marker transfer of the loxP recombination site into the viral thymidine kinase locus. Shuttle plasmids of up to 12.9 kb in length were recombined with high efficiency (11 to 20%) into the K delta T::lox1 genome in cell-free, Cre-mediated recombination reactions. Expression of a functional wild-type or double mutant gB polypeptide complemented the nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production of either wild-type or Syn- plaques on Vero cells. The latter recombinant virus was also ts for growth. The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.
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PMID:A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome. 132 9


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