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

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.

Figure 1. 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.

Figure 2. ECTV vCD30 is not essential for virus growth in cell culture.

A. Representation of the genomic structure of the different ECTV generated showing the vCD30 and the adjacent E12 loci. B. Single-step growth curves of the indicated viruses on BSC-1 cells. Shown are means of triplicate samples for each time point.

Figure 3. ECTV vCD30 ligand screening.

Sensorgram showing binding of mTNFSF8 (CD30L) injected at 50 nM to purified vCD30 coupled to a CM5 sensor chip by SPR analysis. TNFSF10 (pink line), TNFSF11 (cyan line), TNFSF12 (red line) and TNFSF14 (green line) injected at 100 nM each did not show interaction.

Figure 4. ECTV vCD30 forms disulfide linked trimers.

Western blot analysis using anti vCD30 (A) or anti CrmD (B) antibodies of supernatants from mock-infected BSC-1 cells (M) or cells infected with ECTV (E), ECTVΔCD30 (Δ) or ECTVRevCD30 (R).

Figure 5. Absence of ECTV vCD30 does not affect viral spread or replication in vivo.

Lung (top row, L) and spleen (S) samples from representative uninfected (NI) or intranasally infected BALB/c mice at day 7 post-infection were analysed by IHC for virus replication and vCD30 expression, as indicated. H&E analysis shows extensive necrosis of spleen tissue of infected animals.

Figure 6. 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.