The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromo... more The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, includi...
The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictalur... more The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 6...
The Edwardsiella piscicida-like sp. is a Gram-negative facultative anaerobe that causes disease i... more The Edwardsiella piscicida-like sp. is a Gram-negative facultative anaerobe that causes disease in some fish species. We report here the complete genome sequence of a virulent isolate from a diseased white grouper (Epinephelus aeneus) raised on the Red Sea in Israel, which contains a chromosome of 3,934,167 bp and no plasmids.
EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri ... more EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase (PTL) activity, including OspF from Shigella and SpvC from Salmonella. A yeast-2-hybrid system was used to identify the major vault protein (MVP) as a specific host-cell binding partner for EseN, and the proximity ligation assay (PLA) confirmed the interaction. Similar to other pathogens, E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Structurally, EseN contains a highly conserved docking motif that is required for specific binding to mitogen-activated protein kinases, such as ERK1/2, and a motif that is essential for PTL activity. Immunoblotting and immunofluorescence analyses indicate that EseN inactivates ERK1/2 by dephosphorylation in vivo in the head kidney of infected fish and ex vi...
Motile aeromonads are ubiquitous aquatic bacteria that can cause motile aeromonad septicemia (MAS... more Motile aeromonads are ubiquitous aquatic bacteria that can cause motile aeromonad septicemia (MAS), a disease which affects channel catfish and can produce significant economic loss. Motile aeromonads isolated from commercially-raised channel catfish were screened for production of S-layer protein in order to evaluate its potential role in natural epizootics. The S-layer protein was produced by 14 of 24 (58%) isolates from epizootics evaluated in this study. Concomitant infections with other internal pathogens were detected in 10 of the 24 cases used in this study, and only one of those 10 isolates (10%) produced the S-layer protein. When Aeromonas sp. was the only internal pathogen diagnosed, 13 of 14 (93%) isolates produced the S-layer protein.
Page 1. Effect of Delivery Method and Antigen Preparation on the Production of Antibodies Against... more Page 1. Effect of Delivery Method and Antigen Preparation on the Production of Antibodies Against A eromonas hydrophila in Channel Catfish Immunization by hyperosmotic infiltration (HI) is an important tool in controlling and ...
An Edwardsiella sp. was isolated from the kidney of diseased groupers (Epinephelus aeneus and E. ... more An Edwardsiella sp. was isolated from the kidney of diseased groupers (Epinephelus aeneus and E. marginatus) cultured in Eilat (Israel, Red Sea). Affected fish presented a severe suppurative nephritis with large abscesses occasionally spreading into the surrounding musculature. Biochemical profiles and phenotypic comparisons failed to provide a clear identification to the species level, and genetic analysis of the 16S subunit failed to discriminate between Edwardsiella piscicida, E. tarda and E. ictaluri. Analysis of the gyrB gene, however, placed the grouper isolates into the E. piscicida-like group, a newly recognized taxon which also encompasses the non-motile strains previously classified as atypical E. tarda. Initial genomic analysis revealed the presence of the Edwardsiella type 3 secretion system (T3SS) but also revealed a pathogenicity island encoding a second T3SS with homology to the locus of enterocyte effacement of Escherichia coli. Further analysis revealed 3 different type 6 secretion systems that were also present in all sequenced isolates of Edwardsiella piscicida-like strains. Based on estimated DNA-DNA hybridization values and the average nucleotide index, the grouper strain fits into the E. piscicida-like phylogroup described as E. anguillarum sp. nov. The peculiarities associated with this isolate and the association of other conspecific piscine isolates from multiple marine and brackish water species suggest a link of the entire E. piscicida-like phylogroup to the marine environment.
Applied and Environmental Microbiology, Mar 1, 1996
In this report, a complete defined medium and a minimally defined medium are described for Edward... more In this report, a complete defined medium and a minimally defined medium are described for Edwardsiella ictaluri. The complete defined medium consists of 46 individual components, including a basal salt solution, glucose, magnesium sulfate, iron sulfate, six trace metals, four nucleotides, 10 vitamins, and 19 amino acids. This medium supports growth in broth and on solid media. Optimal growth at 30؇C was obtained at pH 7.0, and at an osmolality of 390 mosmol/kg of H 2 O, with a glucose concentration of 4 g/liter. The defined minimal medium reduces the 46 components of the complete medium to eight essential components, including the basal salt solution, glucose, magnesium sulfate, pantothenic acid, and niacinamide. In addition, specific amino acids that depend on the specific requirements of the individual strains of E. ictaluri are added.
We evaluated DNA vaccination against channel catfish virus (CCV) by comparing 11 encoded genes, m... more We evaluated DNA vaccination against channel catfish virus (CCV) by comparing 11 encoded genes, multiple doses, codelivery of DNA vaccines, and the resultant immune responses, as well as making a direct comparison with previously published DNA vaccines for CCV. The genes selected included immediate early genes, membrane genes, the major capsid gene, and the putative major envelope glycoprotein gene. The
Proteins in whole-cell lysates (WCLs) of two Edwardsiella ictaluri isolates grown in brain–heart ... more Proteins in whole-cell lysates (WCLs) of two Edwardsiella ictaluri isolates grown in brain–heart infusion broth (BHI) and defined minimal medium broth (MM19) were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In denatured 2D-PAGE, there were no differences in the expression of proteins between virulent isolates 92-266 and 93-146 grown in BHI or between growth temperatures of 25°C and 30°C for
Ronald L. Thune1,2 *, Lidiya P. Dubytska1 1Department of Pathobiological Sciences, School of Vete... more Ronald L. Thune1,2 *, Lidiya P. Dubytska1 1Department of Pathobiological Sciences, School of Veterinary Medicine thune@vetmed.lsu.edu and ldubyt1@lsu.edu 2 School of Animal Sciences, Louisiana State Agricultural Center, Louisiana State University, Baton Rouge, LA 70803 We previously identified a Type III secretion system (T3SS) in Edwardsiella ictaluri that is essential for intracellular replication and virulence. We also identified seven T3SS effector proteins encoded in the E. ictaluri genome, and demonstrated active translocation to the host cell cytoplasm. In this work, the translocated effectors of the T3SS were evaluated for their role in pathogenesis. Individual effector mutants were constructed by over-lap extension PCR and allelic replacement. Briefly, the amino and carboxyl termini of each effector were amplified with linkers, then combined and used as a template for PCR that amplified Δeffector constructs. The deleted amplicon was cloned into pRE107, transformed into wild-type E. ictaluri, and selected on ampicillin for a single crossover integration of the plasmid into the chromosome. Resulting colonies were replica-plated to BHI-sucrose, inducing expression of sacB on pRE107, which is lethal in Gram negative bacteria. This resulted in selection of colonies in which a double crossover event excised the plasmid, which produced both wild-type and deleted genotypes. Mutant constructs were identified by PCR and confirmed by DNA sequencing. One mutant was significantly more virulent than the wild-type, while one was statistically the same as the wild-type. Four mutants were moderately attenuated and not significantly different from each other. The one most attenuated mutant was not significantly different from the media only controls or the T3SS knock-out. There was no significant difference in initial uptake of the mutants into HKDM or invasion into catfish. Only three mutants were significantly reduced in their ability to replicate in HKDM. All of the most attenuated mutants persisted at least nine days in the head-kidney, and some for at least 13 days.
Vaccination with bacterial auxotrophs, particularly those with an interruption in the common path... more Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This ∆aroA::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.
The ability of Edwardsiella ictaluri to enter, survive, and replicate in head kidney-derived macr... more The ability of Edwardsiella ictaluri to enter, survive, and replicate in head kidney-derived macrophages from channel catfish Ictalurus punctatus was investigated in vitro. Primary macrophage cultures were exposed to E. ictaluri and were sampled after 0 (90 min postinfection), 4, 8, and 12 ...
The channel catfish herpesvirus (CCV) thymidine kinase (TK) gene was mapped on the CCV genome by ... more The channel catfish herpesvirus (CCV) thymidine kinase (TK) gene was mapped on the CCV genome by marker rescue analysis using a TK-deficient channel catfish ovary cell line (CCO), a TK-negative CCV mutant, and a panel of cloned CCV genomic DNA fragments. The TK-deficient cell line (CCOBr) was isolated after repeated propagation of CCO cells in increasing concentrations of 5-bromo-2'-deoxyuridine. Infection of CCOBr cells with CCV produced high levels of TK activity. The TK- virus (CCVAr) was isolated after repeated propagation in the presence of the TK-activated antiherpetic agent, 1-beta-D-arabinofuranosylthymine (Ara-T). A CCV genomic DNA library was constructed into cosmid pHC 79. Marker rescue analysis mapped the mutation within a 3.1-kb fragment located internal to the 18-kb repeat ends of the CCV genome. These genomic coordinates contained a putative TK gene identified by homology to other herpesvirus TK and cellular deoxycytidine kinase genes. DNA sequencing of the mapped coordinates identified the presence of a single mutation in the CCVAr mutant virus which resulted in a stop codon at amino acid position 97. These results functionally confirm that ORF 5 identified by Davison (Virology 186, 9-14, 1992) is the TK gene and show that CCV is amenable to marker rescue and marker transfer genetic analyses extensively used for investigations of the molecular biology of other herpesviruses.
... In general the use of the optimized conjugation procedures described here could, with minor m... more ... In general the use of the optimized conjugation procedures described here could, with minor modifications, be used to perform a variety ... the protein that is required to bind to the R6K ori before replication can be initiated, is also integrated into the ... 2001) Phenotype and Virulence ...
The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromo... more The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, includi...
The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictalur... more The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 6...
The Edwardsiella piscicida-like sp. is a Gram-negative facultative anaerobe that causes disease i... more The Edwardsiella piscicida-like sp. is a Gram-negative facultative anaerobe that causes disease in some fish species. We report here the complete genome sequence of a virulent isolate from a diseased white grouper (Epinephelus aeneus) raised on the Red Sea in Israel, which contains a chromosome of 3,934,167 bp and no plasmids.
EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri ... more EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase (PTL) activity, including OspF from Shigella and SpvC from Salmonella. A yeast-2-hybrid system was used to identify the major vault protein (MVP) as a specific host-cell binding partner for EseN, and the proximity ligation assay (PLA) confirmed the interaction. Similar to other pathogens, E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Structurally, EseN contains a highly conserved docking motif that is required for specific binding to mitogen-activated protein kinases, such as ERK1/2, and a motif that is essential for PTL activity. Immunoblotting and immunofluorescence analyses indicate that EseN inactivates ERK1/2 by dephosphorylation in vivo in the head kidney of infected fish and ex vi...
Motile aeromonads are ubiquitous aquatic bacteria that can cause motile aeromonad septicemia (MAS... more Motile aeromonads are ubiquitous aquatic bacteria that can cause motile aeromonad septicemia (MAS), a disease which affects channel catfish and can produce significant economic loss. Motile aeromonads isolated from commercially-raised channel catfish were screened for production of S-layer protein in order to evaluate its potential role in natural epizootics. The S-layer protein was produced by 14 of 24 (58%) isolates from epizootics evaluated in this study. Concomitant infections with other internal pathogens were detected in 10 of the 24 cases used in this study, and only one of those 10 isolates (10%) produced the S-layer protein. When Aeromonas sp. was the only internal pathogen diagnosed, 13 of 14 (93%) isolates produced the S-layer protein.
Page 1. Effect of Delivery Method and Antigen Preparation on the Production of Antibodies Against... more Page 1. Effect of Delivery Method and Antigen Preparation on the Production of Antibodies Against A eromonas hydrophila in Channel Catfish Immunization by hyperosmotic infiltration (HI) is an important tool in controlling and ...
An Edwardsiella sp. was isolated from the kidney of diseased groupers (Epinephelus aeneus and E. ... more An Edwardsiella sp. was isolated from the kidney of diseased groupers (Epinephelus aeneus and E. marginatus) cultured in Eilat (Israel, Red Sea). Affected fish presented a severe suppurative nephritis with large abscesses occasionally spreading into the surrounding musculature. Biochemical profiles and phenotypic comparisons failed to provide a clear identification to the species level, and genetic analysis of the 16S subunit failed to discriminate between Edwardsiella piscicida, E. tarda and E. ictaluri. Analysis of the gyrB gene, however, placed the grouper isolates into the E. piscicida-like group, a newly recognized taxon which also encompasses the non-motile strains previously classified as atypical E. tarda. Initial genomic analysis revealed the presence of the Edwardsiella type 3 secretion system (T3SS) but also revealed a pathogenicity island encoding a second T3SS with homology to the locus of enterocyte effacement of Escherichia coli. Further analysis revealed 3 different type 6 secretion systems that were also present in all sequenced isolates of Edwardsiella piscicida-like strains. Based on estimated DNA-DNA hybridization values and the average nucleotide index, the grouper strain fits into the E. piscicida-like phylogroup described as E. anguillarum sp. nov. The peculiarities associated with this isolate and the association of other conspecific piscine isolates from multiple marine and brackish water species suggest a link of the entire E. piscicida-like phylogroup to the marine environment.
Applied and Environmental Microbiology, Mar 1, 1996
In this report, a complete defined medium and a minimally defined medium are described for Edward... more In this report, a complete defined medium and a minimally defined medium are described for Edwardsiella ictaluri. The complete defined medium consists of 46 individual components, including a basal salt solution, glucose, magnesium sulfate, iron sulfate, six trace metals, four nucleotides, 10 vitamins, and 19 amino acids. This medium supports growth in broth and on solid media. Optimal growth at 30؇C was obtained at pH 7.0, and at an osmolality of 390 mosmol/kg of H 2 O, with a glucose concentration of 4 g/liter. The defined minimal medium reduces the 46 components of the complete medium to eight essential components, including the basal salt solution, glucose, magnesium sulfate, pantothenic acid, and niacinamide. In addition, specific amino acids that depend on the specific requirements of the individual strains of E. ictaluri are added.
We evaluated DNA vaccination against channel catfish virus (CCV) by comparing 11 encoded genes, m... more We evaluated DNA vaccination against channel catfish virus (CCV) by comparing 11 encoded genes, multiple doses, codelivery of DNA vaccines, and the resultant immune responses, as well as making a direct comparison with previously published DNA vaccines for CCV. The genes selected included immediate early genes, membrane genes, the major capsid gene, and the putative major envelope glycoprotein gene. The
Proteins in whole-cell lysates (WCLs) of two Edwardsiella ictaluri isolates grown in brain–heart ... more Proteins in whole-cell lysates (WCLs) of two Edwardsiella ictaluri isolates grown in brain–heart infusion broth (BHI) and defined minimal medium broth (MM19) were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In denatured 2D-PAGE, there were no differences in the expression of proteins between virulent isolates 92-266 and 93-146 grown in BHI or between growth temperatures of 25°C and 30°C for
Ronald L. Thune1,2 *, Lidiya P. Dubytska1 1Department of Pathobiological Sciences, School of Vete... more Ronald L. Thune1,2 *, Lidiya P. Dubytska1 1Department of Pathobiological Sciences, School of Veterinary Medicine thune@vetmed.lsu.edu and ldubyt1@lsu.edu 2 School of Animal Sciences, Louisiana State Agricultural Center, Louisiana State University, Baton Rouge, LA 70803 We previously identified a Type III secretion system (T3SS) in Edwardsiella ictaluri that is essential for intracellular replication and virulence. We also identified seven T3SS effector proteins encoded in the E. ictaluri genome, and demonstrated active translocation to the host cell cytoplasm. In this work, the translocated effectors of the T3SS were evaluated for their role in pathogenesis. Individual effector mutants were constructed by over-lap extension PCR and allelic replacement. Briefly, the amino and carboxyl termini of each effector were amplified with linkers, then combined and used as a template for PCR that amplified Δeffector constructs. The deleted amplicon was cloned into pRE107, transformed into wild-type E. ictaluri, and selected on ampicillin for a single crossover integration of the plasmid into the chromosome. Resulting colonies were replica-plated to BHI-sucrose, inducing expression of sacB on pRE107, which is lethal in Gram negative bacteria. This resulted in selection of colonies in which a double crossover event excised the plasmid, which produced both wild-type and deleted genotypes. Mutant constructs were identified by PCR and confirmed by DNA sequencing. One mutant was significantly more virulent than the wild-type, while one was statistically the same as the wild-type. Four mutants were moderately attenuated and not significantly different from each other. The one most attenuated mutant was not significantly different from the media only controls or the T3SS knock-out. There was no significant difference in initial uptake of the mutants into HKDM or invasion into catfish. Only three mutants were significantly reduced in their ability to replicate in HKDM. All of the most attenuated mutants persisted at least nine days in the head-kidney, and some for at least 13 days.
Vaccination with bacterial auxotrophs, particularly those with an interruption in the common path... more Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This ∆aroA::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.
The ability of Edwardsiella ictaluri to enter, survive, and replicate in head kidney-derived macr... more The ability of Edwardsiella ictaluri to enter, survive, and replicate in head kidney-derived macrophages from channel catfish Ictalurus punctatus was investigated in vitro. Primary macrophage cultures were exposed to E. ictaluri and were sampled after 0 (90 min postinfection), 4, 8, and 12 ...
The channel catfish herpesvirus (CCV) thymidine kinase (TK) gene was mapped on the CCV genome by ... more The channel catfish herpesvirus (CCV) thymidine kinase (TK) gene was mapped on the CCV genome by marker rescue analysis using a TK-deficient channel catfish ovary cell line (CCO), a TK-negative CCV mutant, and a panel of cloned CCV genomic DNA fragments. The TK-deficient cell line (CCOBr) was isolated after repeated propagation of CCO cells in increasing concentrations of 5-bromo-2'-deoxyuridine. Infection of CCOBr cells with CCV produced high levels of TK activity. The TK- virus (CCVAr) was isolated after repeated propagation in the presence of the TK-activated antiherpetic agent, 1-beta-D-arabinofuranosylthymine (Ara-T). A CCV genomic DNA library was constructed into cosmid pHC 79. Marker rescue analysis mapped the mutation within a 3.1-kb fragment located internal to the 18-kb repeat ends of the CCV genome. These genomic coordinates contained a putative TK gene identified by homology to other herpesvirus TK and cellular deoxycytidine kinase genes. DNA sequencing of the mapped coordinates identified the presence of a single mutation in the CCVAr mutant virus which resulted in a stop codon at amino acid position 97. These results functionally confirm that ORF 5 identified by Davison (Virology 186, 9-14, 1992) is the TK gene and show that CCV is amenable to marker rescue and marker transfer genetic analyses extensively used for investigations of the molecular biology of other herpesviruses.
... In general the use of the optimized conjugation procedures described here could, with minor m... more ... In general the use of the optimized conjugation procedures described here could, with minor modifications, be used to perform a variety ... the protein that is required to bind to the R6K ori before replication can be initiated, is also integrated into the ... 2001) Phenotype and Virulence ...
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