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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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Expression of murine soluble CD30 attenuates mousepox development.Groups of 15 susceptible BALB/c mice were infected intranasally with 100 pfu each of the indicated viruses and scored daily for signs of illness and weight loss. Survival rates are shown on the bottom panel.
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pone-0005175-g006: Expression of murine soluble CD30 attenuates mousepox development.Groups of 15 susceptible BALB/c mice were infected intranasally with 100 pfu each of the indicated viruses and scored daily for signs of illness and weight loss. Survival rates are shown on the bottom panel.

Mentions: The natural infection route for ECTV is through abrasions in the skin and therefore subcutanous inoculation is possibly the most physiologically relevant model. However, oronasal transmission is also possible. Therefore, we next tested the intranasal inoculation route. The resistant C57BL6 strain shows an enhanced susceptibility to ECTV when infected intranasally. When C57BL6 mice were inoculated intranasally with high doses of parental ECTV virus (104, 105 and 106 pfu per animal), all infected mice died after a 9 to 12 day period. No significant differences were observed among the animals infected with the different recombinant viruses (not shown). Intranasal infection of susceptible BALB/c mice with doses of 1, 10 or 100 pfu per animal showed no significant differences between ECTV or ECTVΔCD30-infected mice, with the LD50 at approximately 100 pfu and mean time to death of 12 days post-infection. As shown in Figure 5, ECTV replication in the bronchiolar epithelia was readily detected by day 7 post-infection in animals infected with either parental, ECTVΔCD30 or ECTVRevCD30 viruses. Concomitant spleen necrosis and replication in this target organ was also detected by immunohistochemistry (IHC). Expression of vCD30 in vivo was similarly confirmed by IHC (Figure 5). Infection with ECTV mCD30 appeared to result in a slightly attenuated disease at these low doses, and to confirm this difference a second experiment using groups of 15 susceptible mice was performed (Figure 6). Indeed, a modest reduction of mortality in the case of ECTV mCD30 infected mice as compared to the parental ECTV, ECTVΔCD30 or control ECTVRevCD30 infected mice was observed. The reason for this slight attenuation is currently unknown and may relate to differences in the specific activity of the vCD30 as compared to the mCD30.


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)

Expression of murine soluble CD30 attenuates mousepox development.Groups of 15 susceptible BALB/c mice were infected intranasally with 100 pfu each of the indicated viruses and scored daily for signs of illness and weight loss. Survival rates are shown on the bottom panel.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005175-g006: Expression of murine soluble CD30 attenuates mousepox development.Groups of 15 susceptible BALB/c mice were infected intranasally with 100 pfu each of the indicated viruses and scored daily for signs of illness and weight loss. Survival rates are shown on the bottom panel.
Mentions: The natural infection route for ECTV is through abrasions in the skin and therefore subcutanous inoculation is possibly the most physiologically relevant model. However, oronasal transmission is also possible. Therefore, we next tested the intranasal inoculation route. The resistant C57BL6 strain shows an enhanced susceptibility to ECTV when infected intranasally. When C57BL6 mice were inoculated intranasally with high doses of parental ECTV virus (104, 105 and 106 pfu per animal), all infected mice died after a 9 to 12 day period. No significant differences were observed among the animals infected with the different recombinant viruses (not shown). Intranasal infection of susceptible BALB/c mice with doses of 1, 10 or 100 pfu per animal showed no significant differences between ECTV or ECTVΔCD30-infected mice, with the LD50 at approximately 100 pfu and mean time to death of 12 days post-infection. As shown in Figure 5, ECTV replication in the bronchiolar epithelia was readily detected by day 7 post-infection in animals infected with either parental, ECTVΔCD30 or ECTVRevCD30 viruses. Concomitant spleen necrosis and replication in this target organ was also detected by immunohistochemistry (IHC). Expression of vCD30 in vivo was similarly confirmed by IHC (Figure 5). Infection with ECTV mCD30 appeared to result in a slightly attenuated disease at these low doses, and to confirm this difference a second experiment using groups of 15 susceptible mice was performed (Figure 6). Indeed, a modest reduction of mortality in the case of ECTV mCD30 infected mice as compared to the parental ECTV, ECTVΔCD30 or control ECTVRevCD30 infected mice was observed. The reason for this slight attenuation is currently unknown and may relate to differences in the specific activity of the vCD30 as compared to the mCD30.

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