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A method for the generation of ectromelia virus (ECTV) recombinants: in vivo analysis of ECTV vCD30 deletion mutants.

Alejo A, Saraiva M, Ruiz-Argüello MB, Viejo-Borbolla A, de Marco MF, Salguero FJ, Alcami A - PLoS ONE (2009)

Bottom Line: These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date.The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response.The method developed might be useful for the construction of ECTV mutants for the study of additional genes.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Madrid, Spain.

ABSTRACT

Background: Ectromelia virus (ECTV) is the causative agent of mousepox, a lethal disease of mice with similarities to human smallpox. Mousepox progression involves replication at the initial site of infection, usually the skin, followed by a rapid spread to the secondary replicative organs, spleen and liver, and finally a dissemination to the skin, where the typical rash associated with this and other orthopoxviral induced diseases appears. Case fatality rate is genetically determined and reaches up to 100% in susceptible mice strains. Like other poxviruses, ECTV encodes a number of proteins with immunomodulatory potential, whose role in mousepox progression remains largely undescribed. Amongst these is a secreted homologue of the cellular tumour necrosis factor receptor superfamily member CD30 which has been proposed to modulate a Th1 immune response in vivo.

Methodology/principal findings: To evaluate the contribution of viral CD30 (vCD30) to virus pathogenesis in the infected host, we have adapted a novel transient dominant method for the selection of recombinant ECTVs. Using this method, we have generated an ECTV vCD30 deletion mutant, its corresponding revertant control virus as well as a virus encoding the extracellular domain of murine CD30. These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date.

Conclusions/significance: We show that the vCD30 is expressed as a secreted disulfide linked trimer and that the absence of vCD30 does not impair mousepox induced fatality in vivo. Replacement of vCD30 by a secreted version of mouse CD30 caused limited attenuation of ECTV. The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response. The method developed might be useful for the construction of ECTV mutants for the study of additional genes.

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Transient dominant selection for the generation of ECTV recombinant viruses.A. Schematic representation of the recombination events between the viral genome and the plasmid as well as the phenotypes of intermediate and final viruses obtained during the procedure. B. Representation of the plasmid pMS35 used for the generation of the ECTVΔCD30 virus. The vCD30 locus and the primers used for amplification of the flanking regions are indicated. C. Flowchart of the main steps in the generation of the ECTVΔCD30 virus.
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pone-0005175-g001: Transient dominant selection for the generation of ECTV recombinant viruses.A. Schematic representation of the recombination events between the viral genome and the plasmid as well as the phenotypes of intermediate and final viruses obtained during the procedure. B. Representation of the plasmid pMS35 used for the generation of the ECTVΔCD30 virus. The vCD30 locus and the primers used for amplification of the flanking regions are indicated. C. Flowchart of the main steps in the generation of the ECTVΔCD30 virus.

Mentions: The study of the pathogenesis of orthopoxviruses lacking particular genes involved in the evasion of the immune response provides important information not only on the role of these genes in the virus context but also on the host antiviral defence mechanisms [35]. We have adapted the method described by Sanchez-Puig et al. [34] for the generation of recombinant ECTVs. The principle of the method (Figure 1A) consists on the simultaneous infection of cells with ECTV and transfection with a recombinant plasmid containing approximately 500 bp of the 5′ and 3′ flanking regions of the gene to be deleted and a downstream expression cassette that allows for selection (Figure 1B). Homologous recombination between viral DNA sequences present in the recombinant plasmid and genomic ECTV DNA produces recombinant viruses formed by a single cross-over event that inserts the complete plasmid into the ECTV genome. The intermediate viral species transiently expresses the selectable markers, EGFP, which can be visualized under the fluorescence microscope and pac, which confers resistance to puromycin. This intermediate virus can be isolated by allowing the virus to replicate during several passages and to be amplified in the presence of the antibiotic, which causes cells to detach and does not allow plaque assays. A subsequent plaque assay on cell monolayers in the absence of puromycin selection enables picking of plaques which appear green under UV-light examination (Figure 1C). In a second step, perfomed on cell monolayers in the absence of selection, another recombination event occurs, resulting in the resolution of the intermediate virus and leading to the formation of either a virus lacking the targeted gene or to wild-type virus. Providing that the length of the flanking regions cloned into the recombinant plasmid is similar, the proportion of mutant and wild-type viruses should be similar. At this stage, it is possible to isolate viral plaques that have lost the selectable marker and test them in order to identify a deletion mutant virus and to distinguish it from wild-type viruses. Finally, the desired recombinant virus is purified by successive plaque purification. The production and selection of a revertant virus is achieved exactly in the same way, but in this case the cells are infected with the deletion mutant, instead of the wild-type virus, and transfected with a plasmid containing a full-length wild-type copy of the gene and its flanking regions. In the first step, an intermediate virus, harbouring the fully integrated recombinant plasmid and both the wild type and deleted versions of the gene, is selected by serial passage in the presence of puromycin. In the second step, virus plaques expressing a wild type gene are isolated by plaque assay in the absence of puromycin.


A method for the generation of ectromelia virus (ECTV) recombinants: in vivo analysis of ECTV vCD30 deletion mutants.

Alejo A, Saraiva M, Ruiz-Argüello MB, Viejo-Borbolla A, de Marco MF, Salguero FJ, Alcami A - PLoS ONE (2009)

Transient dominant selection for the generation of ECTV recombinant viruses.A. Schematic representation of the recombination events between the viral genome and the plasmid as well as the phenotypes of intermediate and final viruses obtained during the procedure. B. Representation of the plasmid pMS35 used for the generation of the ECTVΔCD30 virus. The vCD30 locus and the primers used for amplification of the flanking regions are indicated. C. Flowchart of the main steps in the generation of the ECTVΔCD30 virus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664468&req=5

pone-0005175-g001: Transient dominant selection for the generation of ECTV recombinant viruses.A. Schematic representation of the recombination events between the viral genome and the plasmid as well as the phenotypes of intermediate and final viruses obtained during the procedure. B. Representation of the plasmid pMS35 used for the generation of the ECTVΔCD30 virus. The vCD30 locus and the primers used for amplification of the flanking regions are indicated. C. Flowchart of the main steps in the generation of the ECTVΔCD30 virus.
Mentions: The study of the pathogenesis of orthopoxviruses lacking particular genes involved in the evasion of the immune response provides important information not only on the role of these genes in the virus context but also on the host antiviral defence mechanisms [35]. We have adapted the method described by Sanchez-Puig et al. [34] for the generation of recombinant ECTVs. The principle of the method (Figure 1A) consists on the simultaneous infection of cells with ECTV and transfection with a recombinant plasmid containing approximately 500 bp of the 5′ and 3′ flanking regions of the gene to be deleted and a downstream expression cassette that allows for selection (Figure 1B). Homologous recombination between viral DNA sequences present in the recombinant plasmid and genomic ECTV DNA produces recombinant viruses formed by a single cross-over event that inserts the complete plasmid into the ECTV genome. The intermediate viral species transiently expresses the selectable markers, EGFP, which can be visualized under the fluorescence microscope and pac, which confers resistance to puromycin. This intermediate virus can be isolated by allowing the virus to replicate during several passages and to be amplified in the presence of the antibiotic, which causes cells to detach and does not allow plaque assays. A subsequent plaque assay on cell monolayers in the absence of puromycin selection enables picking of plaques which appear green under UV-light examination (Figure 1C). In a second step, perfomed on cell monolayers in the absence of selection, another recombination event occurs, resulting in the resolution of the intermediate virus and leading to the formation of either a virus lacking the targeted gene or to wild-type virus. Providing that the length of the flanking regions cloned into the recombinant plasmid is similar, the proportion of mutant and wild-type viruses should be similar. At this stage, it is possible to isolate viral plaques that have lost the selectable marker and test them in order to identify a deletion mutant virus and to distinguish it from wild-type viruses. Finally, the desired recombinant virus is purified by successive plaque purification. The production and selection of a revertant virus is achieved exactly in the same way, but in this case the cells are infected with the deletion mutant, instead of the wild-type virus, and transfected with a plasmid containing a full-length wild-type copy of the gene and its flanking regions. In the first step, an intermediate virus, harbouring the fully integrated recombinant plasmid and both the wild type and deleted versions of the gene, is selected by serial passage in the presence of puromycin. In the second step, virus plaques expressing a wild type gene are isolated by plaque assay in the absence of puromycin.

Bottom Line: These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date.The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response.The method developed might be useful for the construction of ECTV mutants for the study of additional genes.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Madrid, Spain.

ABSTRACT

Background: Ectromelia virus (ECTV) is the causative agent of mousepox, a lethal disease of mice with similarities to human smallpox. Mousepox progression involves replication at the initial site of infection, usually the skin, followed by a rapid spread to the secondary replicative organs, spleen and liver, and finally a dissemination to the skin, where the typical rash associated with this and other orthopoxviral induced diseases appears. Case fatality rate is genetically determined and reaches up to 100% in susceptible mice strains. Like other poxviruses, ECTV encodes a number of proteins with immunomodulatory potential, whose role in mousepox progression remains largely undescribed. Amongst these is a secreted homologue of the cellular tumour necrosis factor receptor superfamily member CD30 which has been proposed to modulate a Th1 immune response in vivo.

Methodology/principal findings: To evaluate the contribution of viral CD30 (vCD30) to virus pathogenesis in the infected host, we have adapted a novel transient dominant method for the selection of recombinant ECTVs. Using this method, we have generated an ECTV vCD30 deletion mutant, its corresponding revertant control virus as well as a virus encoding the extracellular domain of murine CD30. These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date.

Conclusions/significance: We show that the vCD30 is expressed as a secreted disulfide linked trimer and that the absence of vCD30 does not impair mousepox induced fatality in vivo. Replacement of vCD30 by a secreted version of mouse CD30 caused limited attenuation of ECTV. The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response. The method developed might be useful for the construction of ECTV mutants for the study of additional genes.

Show MeSH
Related in: MedlinePlus