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


RESULTS
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
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.
Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Madrid, Spain.
Ali
A
Alejo
Margarida
M
Saraiva
Maria Begoña
MB
Ruiz-Argüello
Abel
A
Viejo-Borbolla
Mar Fernández
MF
de Marco
Francisco Javier
FJ
Salguero
Antonio
A
Alcami
D000818:Animals; D002460:Cell Line; D018450:Disease Progression; D004481:Ectromelia virus / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D004482:Ectromelia, Infectious / Q000276:immunology*; D005260:Female; D006801:Humans; D017730:Ki-1 Antigen / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D055503:Protein Multimerization; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D014779:Virus Replication
D017730:Ki-1 Antigen; D008024:Ligands; D014764:Viral Proteins