Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF–derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc.

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission–fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis.

Newborn higher vertebrates are largely immuno-incompetent and generally survive infections -including poxviruses -by maternal antibody protection. Here, we show that mice survived epidemics as adults only if exposed to lethal orthopoxvirus infections during infancy under the umbrella of maternal protective antibodies. This implies that both the absence of exposure to infection during early infancy or of effective vaccination renders the population highly susceptible to new or old re-emerging pathogens.Key words: Ectromelia . Maternal antibody . Protection . Vaccination . Vaccinia IntroductionIn the last 50 years, several viral diseases have been largely confined or even eradicated, as in the case of smallpox (variola virus) for which routine vaccinations with vaccinia virus (VACV) have been subsequently stopped in the 1970s. Due to loss of regular exposure to immunizing viral antigens, herd immunity might wane and leave the population susceptible to pathogen reemergence. Therefore, the consequences of interruption of the natural re-exposure cycles in the absence of infectious antigen contact need to be better understood.For these studies, we used orthopoxvirus infections because in both humans and mice the species-specific pox virus has devastating consequences with mortalities reaching 50-80% in the 14th century in Europe or up to 100% for certain mouse colonies in research establishments. Newborn and infant mice are highly susceptible to WT of ectromelia (mousepox) virus (ECTV) and the empirically selected VACV [1][2][3]. Resistance to ECTV varies between mouse strains; C57BL/6 are one of the genetically more resistant strains. Orthopox viruses such as variola virus, VACV, and ECTV share the cross-reactive neutralizing protective epitope [4] in addition to many T-cell specificities. Accordingly, vaccination with VACV provides solid protection to smallpox or mousepox (named sometimes ''smallpox of mice'') [5,6]. In this paper, we describe that long-lasting immunological protection results after immuno-incompetent pups of immune mice encounter an otherwise lethal pox-virus infection. Results and discussionWe compared survival of Moscow strain of Ectromelia virus (ECTV-MOS) infection at the age of 12-42 wk when specific pathogen-free infant C57BL/6 (H-2 b ) mice from immune mothers had been exposed to a lethal orthopox virus infection within the first days of life or not. C57BL/6 dams were immunized with 2 Â 10 6 pfu of VACV WR (LD50$10 8 pfu). Four weeks later they were mated with normal male mice. The newborn pups were infected intranasally at day 1 of age with 3-10 LD 50 corresponding to about 10 3 pfu of VACV WR ( We do not define maternal antibodies as the sole effector mechanism in this study, although they are the only ones transferred from the mother to the offspring. The theoretical possibility that viral antigen persisting in the mother could have been transferred to immunize newborns is unlikely because first, mothers were vaccinated 4 wk before pregnancy and second, without challenge infection early a...

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