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Human interleukin-18 (hIL-18) is a cytokine that plays an important role in inflammation and host defense against microbes. Its activity is regulated in vivo by a naturally occurring antagonist, the human IL-18-binding protein (IL-18BP). Functional homologs of human IL-18BP are encoded by all orthopoxviruses, including variola virus, the causative agent of smallpox. They contribute to virulence by suppressing IL-18-mediated immune responses. Here, we describe the 2.0-Å resolution crystal structure of an orthopoxvirus IL-18BP, ectromelia virus IL-18BP (ectvIL-18BP), in complex with hIL-18. The hIL-18 structure in the complex shows significant conformational change at the binding interface compared with the structure of ligand-free hIL-18, indicating that the binding is mediated by an induced-fit mechanism. EctvIL-18BP adopts a canonical Ig fold and interacts via one edge of its β-sandwich with 3 cavities on the hIL-18 surface through extensive hydrophobic and hydrogen bonding interactions. Most of the ectvIL-18BP residues that participate in these interactions are conserved in both human and viral homologs, explaining their functional equivalence despite limited sequence homology. EctvIL-18BP blocks a putative receptor-binding site on IL-18, thus preventing IL-18 from engaging its receptor. Our structure provides insights into how IL-18BPs modulate hIL-18 activity. The revealed binding interface provides the basis for rational design of inhibitors against orthopoxvirus IL-18BP (for treating orthopoxvirus infection) or hIL-18 (for treating certain inflammatory and autoimmune diseases).

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs’ immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs’ organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8 + T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8 + T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8 + T cells were not recognized by VACV-reactive CD8 + T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R 70–78 variant of the dominant VACV B8R 70–78 epitope. C4R 70–78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

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. 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. 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.

The heightened concern about the intentional release of variola virus has led to the need to develop safer smallpox vaccines. While subunit vaccine strategies are safer than live virus vaccines, subunit vaccines have been hampered by the need for multiple boosts to confer optimal protection. Here we developed a protein-based subunit vaccine strategy that provides rapid protection in mouse models of orthopoxvirus infections after a prime and single boost. Mice vaccinated with vaccinia virus envelope proteins from the mature virus (MV) and extracellular virus (EV) adjuvanted with CpG ODN and alum were protected from lethal intranasal challenge with vaccinia virus and the mouse-specific ectromelia virus. Organs from mice vaccinated with three proteins (A33, B5 and L1) and then sacrificed after challenge contained significantly lower titers of virus when compared to control groups of mice that were not vaccinated or that received sub-optimal formulations of the vaccine. Sera from groups of mice obtained prior to challenge had neutralizing activity against the MV and also inhibited comet formation indicating anti-EV activity. Long-term partial protection was also seen in mice challenged with vaccinia virus 6 months after initial vaccinations. Thus, this work represents a step toward the development of a practical subunit smallpox vaccine.

The recent spread of the monkeypox virus among humans has heightened concerns regarding orthopoxvirus infections. Consequently, conducting a comprehensive study on the immunobiology of the monkeypox virus is imperative for the development of effective therapeutics. Ectromelia virus (ECTV) closely resembles the genetic and disease characteristics of monkeypox virus, making it a valuable research tool for studying orthopoxvirus–host interactions. Guanylate-binding proteins (GBPs), highly expressed interferon-stimulated genes (ISGs), have antagonistic effects against various intracellular pathogenic microorganisms. Our previous research has shown that GBP2 has a mild but statistically significant inhibitory effect on ECTV infection. The presence of a significant number of molecules in the poxvirus genome that encode the host immune response raises questions about whether it also includes proteins that counteract the antiviral activity of GBP2. Using IP/MS and co-IP technology, we discovered that the poly(A) polymerase catalytic subunit (PAPL) protein of ECTV is a viral regulatory molecule that interacts with GBP2. Further studies have shown that PAPL antagonizes the antiviral activity of GBP2 by reducing its protein levels. Knocking out the PAPL gene of ECTV with the CRISPR/Cas9 system significantly diminishes the replication ability of the virus, indicating the indispensable role of PAPL in the replication process of ECTV. In conclusion, our study presents preliminary evidence supporting the significance of PAPL as a virulence factor that can interact with GBP2.

We studied toxicity and antiviral activity of bioactive substances extracted from the roots (ethylacetate extracts) and aerial parts (ethanol extracts) of lady’s mantle ( Alchemilla vilgaris L.). Plant extracts are characterized by low toxicity for continuous Vero cell culture, but inhibit the reproduction of orthopoxviruses (vaccinia virus and ectromelia virus) in these cells. Of all studied extracts, ethylacetate extract from lady’s mantle roots characterized by the highest content of catechins in comparison with other samples demonstrated the highest activity in vitro towards the studied viruses (neutralization index for vaccinia and ectromelia viruses were 4.0 and 3.5 lg, respectively). The antiviral effect of Alchemilla vulgaris L. extracts was shown to be dose dependent.

We studied toxicity and antiviral activity of aqueous and ethanol extracts of bioactive substances from the biomass of nematophagous fungus Duddingtonia flagrans prepared by submerged culturing of the mycelium. It is found that both extracts were characterized by low toxicity for cultured Vero cells and inhibited reproduction of DNA-viruses in this cell line. Ethanol extract of the fungus exhibited higher in vitro antiviral activity against Herpes simplex virus type 2, ectromelia virus, and vaccinia virus than water extract, which can be due to higher content of proteins, polysaccharides, flavonols, catechins, or carotenes or more effective their combination. The extracts of cultured mycelium of Duddingtonia flagrans fungus containing a complex of bioactive substances can be used for creation of broad-spectrum antiviral drugs against DNA-viruses.

Fas receptor-Fas ligand (FasL) signalling is involved in apoptosis of immune cells as well as of the virus infected target cells but increasing evidence accumulates on Fas as a mediator of apoptosis-independent processes such as induction of activating and proinflammatory signals. In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6. MRL-Faslpr/J, and B6Smn.C3-Faslgld/J mice. Ectromelia virus (ECTV) infection of Fas- and FasL-deficient mice led to increased virus titers in lungs and decreased migration of IFN-γ expressing NK cells, CD4+ T cells, CD8+ T cells, and decreased IL-15 expression. The lungs of ECTV-infected Fas- and FasL-deficient mice showed significant inflammation during later phases of infection accompanied by decreased expression of anti-inflammatory IL-10 and TGF-β1 cytokines and disturbances in CXCL1 and CXCL9 expression. Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis. Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

Abstract During mousepox in resistant (C57BL/6) or susceptible (BALB/c) strains of mice, stimulation of Th1 or Th2 cytokine immune response, respectively, is observed. Because mechanisms of different polarization of T cells remain elusive, in this study, we quantitatively assessed the phenotype of antigen-presenting cells (APCs) involved in ectromelia virus (ECTV) antigen presentation and cluster formation with effector cells in secondary lymphoid organs of BALB/c and C57BL/6 mice. We showed that both strains of mice display similar dynamics and kinetics of viral antigen presentation by CD11c + , CD11b + , and CD19 + cells. CD11c + and CD11b + cells highly participated in viral antigen presentation during all stages of mousepox, whereas CD19 + cells presented viral peptides later in infection. The main population of dendritic cells (DCs) engaged in ECTV antigen presentation and cell junction formation with effector cells was a population of myeloid CD11b + DCs (mDCs). We suggest that, on the one hand, ECTV may differentially affect the functions of APCs depending on the strain of mice. On the other hand, we suggest that some types of APCs, such as mDCs or other DCs subsets, have different abilities to direct the shape of immune response depending on the host resistance to mousepox. This paper analyzes antigen presentation in ectromelia infected susceptible and resistent mice, and explains the differences in T cell polarization between the strains. Presence of infected CD11c+, CD11b+ and CD19+ cells in draining lymph nodes and spleens of infected mice is analyzed over time and interactions between pAPC and ‘effector’ cells are analyzed.