Orthopoxvirus Zoonoses-Do We Still Remember and Are Ready to Fight?

The eradication of smallpox was an enormous achievement due to the global vaccination program launched by World Health Organization. The cessation of the vaccination program led to steadily declining herd immunity against smallpox, causing a health emergency of global concern. The smallpox vaccines induced strong, humoral, and cell-mediated immune responses, protecting for decades after immunization, not only against smallpox but also against other zoonotic orthopoxviruses that now represent a significant threat to public health. Here we review the major aspects regarding orthopoxviruses' zoonotic infections, factors responsible for viral transmissions, as well as the emerging problem of the increased number of monkeypox cases recently reported. The development of prophylactic measures against poxvirus infections, especially the current threat caused by the monkeypox virus, requires a profound understanding of poxvirus immunobiology. The utilization of animal and cell line models has provided good insight into host antiviral defenses as well as orthopoxvirus evasion mechanisms. To survive within a host, orthopoxviruses encode a large number of proteins that subvert inflammatory and immune pathways. The circumvention of viral evasion strategies and the enhancement of major host defenses are key in designing novel, safer vaccines, and should become the targets of antiviral therapies in treating poxvirus infections.

Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

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.

Differential Activation of Splenic cDC1 and cDC2 Cell Subsets following Poxvirus Infection of BALB/c and C57BL/6 Mice.

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α+ in mice and CD141+ in human) and cDC2 (CD11b+ in mice and CD1c+ in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4+ T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge.

Integrity of the Intestinal Barrier: The Involvement of Epithelial Cells and Microbiota-A Mutual Relationship.

The gastrointestinal tract, which is constantly exposed to a multitude of stimuli, is considered responsible for maintaining the homeostasis of the host. It is inhabited by billions of microorganisms, the gut microbiota, which form a mutualistic relationship with the host. Although the microbiota is generally recognized as beneficial, at the same time, together with pathogens, they are a permanent threat to the host. Various populations of epithelial cells provide the first line of chemical and physical defense against external factors acting as the interface between luminal microorganisms and immunocompetent cells in lamina propria. In this review, we focus on some essential, innate mechanisms protecting mucosal integrity, thus responsible for maintaining intestine homeostasis. The characteristics of decisive cell populations involved in maintaining the barrier arrangement, based on mucus secretion, formation of intercellular junctions as well as production of antimicrobial peptides, responsible for shaping the gut microbiota, are presented. We emphasize the importance of cross-talk between gut microbiota and epithelial cells as a factor vital for the maintenance of the homeostasis of the GI tract. Finally, we discuss how the imbalance of these regulations leads to the compromised barrier integrity and dysbiosis considered to contribute to inflammatory disorders and metabolic diseases.

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

Macrophages are the first encounters of invading bacteria and are responsible for engulfing and digesting pathogens through phagocytosis leading to initiation of the innate inflammatory response. Intracellular digestion occurs through a close relationship between phagocytic/endocytic and lysosomal pathways, in which proteolytic enzymes, such as cathepsins, are involved. The presence of cathepsins in the endo-lysosomal compartment permits direct interaction with and killing of bacteria, and may contribute to processing of bacterial antigens for presentation, an event necessary for the induction of antibacterial adaptive immune response. Therefore, it is not surprising that bacteria can control the expression and proteolytic activity of cathepsins, including their inhibitors - cystatins, to favor their own intracellular survival in macrophages. In this review, we summarize recent developments in defining the role of cathepsins in bacteria-macrophage interaction and describe important strategies engaged by bacteria to manipulate cathepsin expression and activity in macrophages. Particularly, we focus on specific bacterial species due to their clinical relevance to humans and animal health, i.e., Mycobacterium, Mycoplasma, Staphylococcus, Streptococcus, Salmonella, Shigella, Francisella, Chlamydia, Listeria, Brucella, Helicobacter, Neisseria, and other genera.

NF-κB as an Important Factor in Optimizing Poxvirus-Based Vaccines against Viral Infections.

Poxviruses are large dsDNA viruses that are regarded as good candidates for vaccine vectors. Because the members of the Poxviridae family encode numerous immunomodulatory proteins in their genomes, it is necessary to carry out certain modifications in poxviral candidates for vaccine vectors to improve the vaccine. Currently, several poxvirus-based vaccines targeted at viral infections are under development. One of the important aspects of the influence of poxviruses on the immune system is that they encode a large array of inhibitors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is the key element of both innate and adaptive immunity. Importantly, the NF-κB transcription factor induces the mechanisms associated with adaptive immunological memory involving the activation of effector and memory T cells upon vaccination. Since poxviruses encode various NF-κB inhibitor proteins, before the use of poxviral vaccine vectors, modifications that influence NF-κB activation and consequently affect the immunogenicity of the vaccine should be carried out. This review focuses on NF-κB as an essential factor in the optimization of poxviral vaccines against viral infections.

First Insight into the Modulation of Noncanonical NF-κB Signaling Components by Poxviruses in Established Immune-Derived Cell Lines: An In Vitro Model of Ectromelia Virus Infection.

Dendritic cells (DCs) and macrophages are the first line of antiviral immunity. Viral pathogens exploit these cell populations for their efficient replication and dissemination via the modulation of intracellular signaling pathways. Disruption of the noncanonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling has frequently been observed in lymphoid cells upon infection with oncogenic viruses. However, several nononcogenic viruses have been shown to manipulate the noncanonical NF-κB signaling in different cell types. This study demonstrates the modulating effect of ectromelia virus (ECTV) on the components of the noncanonical NF-κB signaling pathway in established murine cell lines: JAWS II DCs and RAW 264.7 macrophages. ECTV affected the activation of TRAF2, cIAP1, RelB, and p100 upon cell treatment with both canonical and noncanonical NF-κB stimuli and thus impeded DNA binding by RelB and p52. ECTV also inhibited the expression of numerous genes related to the noncanonical NF-κB pathway and RelB-dependent gene expression in the cells treated with canonical and noncanonical NF-κB activators. Thus, our data strongly suggest that ECTV influenced the noncanonical NF-κB signaling components in the in vitro models. These findings provide new insights into the noncanonical NF-κB signaling components and their manipulation by poxviruses in vitro.

Deficiency of Selected Cathepsins Does Not Affect the Inhibitory Action of ECTV on Immune Properties of Dendritic Cells.

Ectromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins - cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4+ T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.

Mitochondrial Heat Shock Response Induced by Ectromelia Virus is Accompanied by Reduced Apoptotic Potential in Murine L929 Fibroblasts.

Poxviruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxviruses on mtHsps has never been studied. We used highly infectious Moscow strain of ectromelia virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.

Ectromelia virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process.

BACKGROUND: Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors - cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxvirus infection on host cells, we investigated the influence of ectromelia virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxvirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. RESULTS: Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. CONCLUSIONS: Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the virus to replicate effectively in infected cells.

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner.

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4+ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4+ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4+ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.

Ectromelia virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α-tubulin acetylation.

Ectromelia virus (ECTV) is an orthopoxvirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.

Manipulation of Non-canonical NF-κB Signaling by Non-oncogenic Viruses.

Nuclear factor (NF)-κB is a major regulator of antiviral response. Viral pathogens exploit NF-κB activation pathways to avoid cellular mechanisms that eliminate the infection. Canonical (classical) NF-κB signaling, which regulates innate immune response, cell survival and inflammation, is often manipulated by viral pathogens that can counteract antiviral response. Oncogenic viruses can modulate not only canonical, but also non-canonical (alternative) NF-κB activation pathways. The non-canonical NF-κB signaling is responsible for adaptive immunity and plays a role in lymphoid organogenesis, B cell development, as well as bone metabolism. Thus, non-canonical NF-κB activation has been linked to lymphoid malignancies. However, some data strongly suggest that the non-canonical NF-κB activation pathway may also function in innate immunity and is modulated by certain non-oncogenic viruses. Collectively, these findings show the importance of studying the impact of different groups of viral pathogens on alternative NF-κB activation. This mini-review focuses on the influence of non-oncogenic viruses on the components of non-canonical NF-κB signaling.

NF-κB Signaling in Targeting Tumor Cells by Oncolytic Viruses-Therapeutic Perspectives.

In recent years, oncolytic virotherapy became a promising therapeutic approach, leading to the introduction of a novel generation of anticancer drugs. However, despite evoking an antitumor response, introducing an oncolytic virus (OV) to the patient is still inefficient to overcome both tumor protective mechanisms and the limitation of viral replication by the host. In cancer treatment, nuclear factor (NF)-κB has been extensively studied among important therapeutic targets. The pleiotropic nature of NF-κB transcription factor includes its involvement in immunity and tumorigenesis. Therefore, in many types of cancer, aberrant activation of NF-κB can be observed. At the same time, the activity of NF-κB can be modified by OVs, which trigger an immune response and modulate NF-κB signaling. Due to the limitation of a monotherapy exploiting OVs only, the antitumor effect can be enhanced by combining OV with NF-κB-modulating drugs. This review describes the influence of OVs on NF-κB activation in tumor cells showing NF-κB signaling as an important aspect, which should be taken into consideration when targeting tumor cells by OVs.

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line.

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.

Long actin-based cellular protrusions as novel evidence of the cytopathic effect induced in immune cells infected by the ectromelia virus.

The aim of the study was to evaluate the influence of ectromelia virus (ECTV) infection on actin cytoskeleton rearrangement in immune cells, such as macrophages and dendritic cells (DCs). Using scanning electron and fluorescence microscopy analysis we observed the presence of long actin-based cellular extensions, formed by both types of immune cells at later stages of infection with ECTV. Such extensions contained straight tubulin filaments and numerous punctuate mitochondria. Moreover, these long cellular projections extended to a certain length and formed convex structures termed "cytoplasmic packets". These structures contained numerous viral particles and presumably were sites of progeny virions' release via budding. Further, discrete mitochondria and separated tubulin filaments that formed a scaffold for accumulated mitochondria were visible within cytoplasmic packets. ECTV-induced long actin-based protrusions resemble "cytoplasmic corridors" and probably participate in virus dissemination. Our data demonstrate the incredible capacity for adaptation of ECTV to its natural host immune cells, in which it can survive, replicate and induce effective mechanisms for viral spread and dissemination.

Functional paralysis of GM-CSF-derived bone marrow cells productively infected with ectromelia virus.

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.

The in Vitro Inhibitory Effect of Ectromelia Virus Infection on Innate and Adaptive Immune Properties of GM-CSF-Derived Bone Marrow Cells Is Mouse Strain-Independent.

Ectromelia virus (ECTV) belongs to the Orthopoxvirus genus of the Poxviridae family and is a natural pathogen of mice. Certain strains of mice are highly susceptible to ECTV infection and develop mousepox, a lethal disease similar to smallpox of humans caused by variola virus. Currently, the mousepox model is one of the available small animal models for investigating pathogenesis of generalized viral infections. Resistance and susceptibility to ECTV infection in mice are controlled by many genetic factors and are associated with multiple mechanisms of immune response, including preferential polarization of T helper (Th) immune response toward Th1 (protective) or Th2 (non-protective) profile. We hypothesized that viral-induced inhibitory effects on immune properties of conventional dendritic cells (cDCs) are more pronounced in ECTV-susceptible than in resistant mouse strains. To this extent, we confronted the cDCs from resistant (C57BL/6) and susceptible (BALB/c) mice with ECTV, regarding their reactivity and potential to drive T cell responses following infection. Our results showed that in vitro infection of granulocyte-macrophage colony-stimulating factor-derived bone marrow cells (GM-BM-comprised of cDCs and macrophages) from C57BL/6 and BALB/c mice similarly down-regulated multiple genes engaged in DC innate and adaptive immune functions, including antigen uptake, processing and presentation, chemokines and cytokines synthesis, and signal transduction. On the contrary, ECTV infection up-regulated Il10 in GM-BM derived from both strains of mice. Moreover, ECTV similarly inhibited surface expression of major histocompatibility complex and costimulatory molecules on GM-BM, explaining the inability of the cells to attain full maturation after Toll-like receptor (TLR)4 agonist treatment. Additionally, cells from both strains of mice failed to produce cytokines and chemokines engaged in T cell priming and Th1/Th2 polarization after TLR4 stimulation. These data strongly suggest that in vitro modulation of GM-BM innate and adaptive immune functions by ECTV occurs irrespective of whether the mouse strain is susceptible or resistant to infection. Moreover, ECTV limits the GM-BM (including cDCs) capacity to stimulate protective Th1 immune response. We cannot exclude that this may be an important factor in the generation of non-protective Th2 immune response in susceptible BALB/c mice in vivo.

Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

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.

[MAVS protein and its interactions with hepatitis A, B and C viruses]. / Bialko MAVS i jego interakcje z wirusami zapalenia watroby typu A, B i C.

Mitochondrial antiviral signaling protein (MAVS) transmits activation signal of type I interferon (IFN) gene transcription in the molecular intracellular pathway, which depends on the protein encoded by retinoic acid inducible gene I (RIG-I) or melanoma differentiation-associated protein-5 (MDA-5). MAVS, as a signal molecule, performs an essential function in the development of an antiviral immune response. The molecule of MAVS consists of two domains: the N-terminal domain and the C-terminal domain. The N-terminal end of MAVS contains the caspase activation and recruitment domain (CARD). CARD is responsible for MAVS interaction with RIG-I and MDA-5, which act as cytosolic sensors detecting foreign viral genetic material in the host cell. After binding to viral RNA, RIG-I or MDA-5 activates MAVS and transmits the signal of IFN type I gene expression. The C-terminal transmembrane domain (TM) of MAVS anchors the protein to the outer mitochondrial membrane. In this paper interactions between MAVS and hepatitis virus type A (HAV), type B (HBV) and type C (HCV) are presented. Mechanisms of indirect activation of MAVS by viral DNA and RNA, as well as the strategies of HAV, HBV and HCV for blocking of the intracellular signaling pathway at the level of MAVS, are described.