scholarly journals Bacterial Artificial Chromosome Clones of Viruses Comprising the Towne Cytomegalovirus Vaccine

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaohong Cui ◽  
Stuart P. Adler ◽  
Andrew J. Davison ◽  
Larry Smith ◽  
EL-Sayed E. Habib ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Genetic Manipulation ◽  
Artificial Chromosome ◽  
Phenotypic Properties ◽  
Cultured Fibroblasts ◽  
Starting Point ◽  
Viral Stock ◽  
Bac Cloning ◽  
Cytomegalovirus Vaccine

Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines.

scholarly journals Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Kalpana Dulal ◽  
Benjamin Silver ◽  
Hua Zhu
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Bac Clone ◽  
Dna Viruses ◽  
E Coli ◽  
Recombination System ◽  
Capture Method

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

scholarly journals Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi

2008 ◽  
Vol 82 (10) ◽  
pp. 4955-4964 ◽  
Author(s):  
B. Costes ◽  
G. Fournier ◽  
B. Michel ◽  
C. Delforge ◽  
V. Stalin Raj ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Thymidine Kinase ◽  
Artificial Chromosome ◽  
Wild Type ◽  
Bac Clone ◽  
Recombinant Viruses ◽  
Koi Herpesvirus ◽  
Cyprinus Carpio Koi

ABSTRACT Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

scholarly journals Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

2019 ◽  
Author(s):  
Erin A. Mack ◽  
Yu-Ping Xiao ◽  
David R. Allred
Keyword(s):  
Antigenic Variation ◽  
Genetic Manipulation ◽  
Artificial Chromosome ◽  
Babesia Bovis ◽  
Chromosome 1 ◽  
Gene Encoding ◽  
Persistent Infections ◽  
Essentially Normal ◽  
Regulated Gene Expression

AbstractBabesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

scholarly journals Simulating the evolutionary trajectories of metabolic pathways for insect symbionts in the Sodalis genus

2019 ◽  
Author(s):  
Rebecca J. Hall ◽  
Stephen Thorpe ◽  
Gavin H. Thomas ◽  
A. Jamie Wood
Keyword(s):  
Genetic Manipulation ◽  
Balance Model ◽  
Ecological Change ◽  
Metabolic Modelling ◽  
Flux Balance ◽  
Sodalis Glossinidius ◽  
Starting Point ◽  
Evolutionary Trajectories ◽  
Insect Symbionts

1AbstractInsect-bacterial symbioses are ubiquitous, but there is still much to uncover about how these relationships establish, persist and evolve. The tsetse endosymbiont Sodalis glossinidius displays intriguing metabolic adaptations to its microenvironment, but the process by which this relationship evolved remains to be elucidated. The recent chance discovery of the free-living secies of the Sodalis genus, S. praecaptivus, provides a serendipitous starting point from which to investigate the evolution of this symbiosis. Here, we present a flux balance model for S. praecaptivus. Metabolic modelling is used in combination with a multi-objective evolutionary algorithm to explore the trajectories that S. glossinidius may have undertaken after becoming internalised. The time-dependent loss of key genes is shown to influence the evolved populations, providing possible targets for future in vitro genetic manipulation. This method provides an unusually detailed perspective on possible evolutionary trajectories for S. glossinidius in this fundamental process of evolutionary and ecological change.

scholarly journals Simulating the evolutionary trajectories of metabolic pathways for insect symbionts in the genus Sodalis

2020 ◽  
Vol 6 (7) ◽  
Author(s):  
Rebecca J. Hall ◽  
Stephen Thorpe ◽  
Gavin H. Thomas ◽  
A. Jamie Wood
Keyword(s):  
Type Species ◽  
Genetic Manipulation ◽  
Balance Model ◽  
Ecological Change ◽  
Content Type ◽  
Link Type ◽  
Starting Point ◽  
Evolutionary Trajectories ◽  
Insect Symbionts

Insect–bacterial symbioses are ubiquitous, but there is still much to uncover about how these relationships establish, persist and evolve. The tsetse endosymbiont Sodalis glossinidius displays intriguing metabolic adaptations to its microenvironment, but the process by which this relationship evolved remains to be elucidated. The recent chance discovery of the free-living species of the genus Sodalis , Sodalis praecaptivus , provides a serendipitous starting point from which to investigate the evolution of this symbiosis. Here, we present a flux balance model for S. praecaptivus and empirically verify its predictions. Metabolic modelling is used in combination with a multi-objective evolutionary algorithm to explore the trajectories that S. glossinidius may have undertaken from this starting point after becoming internalized. The order in which key genes are lost is shown to influence the evolved populations, providing possible targets for future in vitro genetic manipulation. This method provides a detailed perspective on possible evolutionary trajectories for S. glossinidius in this fundamental process of evolutionary and ecological change.

scholarly journals Patient-Derived Cytomegaloviruses with Different Ganciclovir Sensitivities from UL97 Mutation Retain Their Replication Efficiency and Some Kinase Activity In Vitro

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Diana D. Wong ◽  
Wendy J. van Zuylen ◽  
Stuart T. Hamilton ◽  
Mirjam Steingruber ◽  
Eric Sonntag ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Kinase Activity ◽  
Artificial Chromosome ◽  
Antiviral Resistance ◽  
Wild Type ◽  
Resistance Mutations ◽  
Kinase Gene ◽  
Replication Efficiency ◽  
Resistant Phenotype

ABSTRACT Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro.

scholarly journals Genotypic characterization of two bacterial artificial chromosome clones derived from a single DNA source of the very virulent gallid herpesvirus-2 strain C12/130

2011 ◽  
Vol 92 (7) ◽  
pp. 1500-1507 ◽  
Author(s):  
Stephen J. Spatz ◽  
Lorraine P. Smith ◽  
Susan J. Baigent ◽  
Lawrence Petherbridge ◽  
Venugopal Nair
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Genetic Changes ◽  
Mixed Genotype ◽  
Bac Clones ◽  
Gallid Herpesvirus 2 ◽  
The Individual

The identification of specific genetic changes associated with differences in the pathogenicity of Marek's disease virus strains (GaHV-2) has been a formidable task due to the large number of mutations in mixed-genotype populations within DNA preparations. Very virulent UK isolate C12/130 induces extensive lymphoid atrophy, neurological manifestations and early mortality in young birds. We have recently reported the construction of several independent full-length bacterial artificial chromosome (BAC) clones of C12/130 capable of generating fully infectious viruses with significant differences in their pathogenicity profiles. Two of these clones (vC12/130-10 and vC12/130-15), which showed differences in virulence relative to each other and to the parental strain, had similar replication kinetics both in vitro and in vivo in spite of the fact that vC12/130-15 was attenuated. To investigate the possible reasons for this, the nucleotide sequences of both clones were determined. Sequence analysis of the two genomes identified mutations within eight genes. A single 494 bp insertion was identified within the genome of the virulent vC12/130-10 clone. Seven non-synonymous substitutions distinguished virulent vC12/130-10 from that of attenuated vC12/130-15. By sequencing regions of parental DNA that differed between the two BAC clones, we confirmed that C12/130 does contain these mutations in varying proportions. Since the individual reconstituted BAC clones were functionally attenuated in vivo and derived from a single DNA source of phenotypically very virulent C12/130, this suggests that the C12/130 virus population exists as a collection of mixed genotypes.

scholarly journals Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System

2007 ◽  
Vol 81 (17) ◽  
pp. 9024-9033 ◽  
Author(s):  
Zhen Zhang ◽  
Jenny Rowe ◽  
Weijia Wang ◽  
Marvin Sommer ◽  
Ann Arvin ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Varicella Zoster Virus ◽  
Artificial Chromosome ◽  
Wild Type Virus ◽  
Growth Curve Analysis ◽  
Type Virus ◽  
Wild Type ◽  
Varicella Zoster

ABSTRACT To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZVBAC) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZVLuc). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZVLuc genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.

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