Mitochondria-related gene expression profiles in murine fibroblasts and macrophages during later stages of ectromelia virus infection in vitro.

Mitochondria are multitasking organelles that play a central role in energy production, survival and primary host defense against viral infections. Therefore, viruses target mitochondria dynamics and functions to benefit their replication and morphogenetic processes. We endeavor to understand the role of mitochondria during infection of ectromelia virus (ECTV), hence our investigations on mitochondria-related genes in non-immune (L929 fibroblasts) and immune (RAW 264.7 macrophages) cells. Our results show that during later stages of infection, ECTV significantly decreases the expression of mitochondria-related genes regulating many aspects of mitochondrial physiology and functions, including mitochondrial transport, small molecule transport, membrane polarization and potential, targeting proteins to mitochondria, inner membrane translocation, and apoptosis. Such down-regulation is cell-specific, since macrophages exhibited a more profound down-regulation of mitochondria-related genes compared to infected L929 fibroblasts. Only L929 cells exhibited up-regulation of two important genes responsible for oxidative phosphorylation and subsequent ATP production: Slc25a23 and Slc25a31. Changes in the expression of mitochondria-related genes are accompanied by altered mitochondria morphology and distribution in both types of cells. In depth Ingenuity Pathway Analysis (IPA) identified the "Sirtuin Signaling Pathway" as the most significant top canonical pathway associated with ECTV infection in both analyzed cell types. Taken together, down-regulation of mitochondria-related genes observed especially in macrophages indicates dysfunctional mitochondria, possibly contributing to energy collapse and induction of intrinsic pathway of apoptosis. Meanwhile, alteration of the expression of several mitochondria-related genes in fibroblasts without apoptosis induction may represent poxviral strategy to control cellular energy metabolism for efficient replication. Keywords: ectromelia virus; mitochondria; fibroblasts; macrophages.

Pathogenesis of mousepox in H-2(d) mice: evidence for MHC class I-restricted CD8(+) and MHC class II-restricted CD4(+) CTL antiviral activity in the lymph nodes, spleen and skin, but not in the conjunctivae.

Genetically sensitive mice (i.e. H-2(d) haplotype) infected with a natural mouse pathogen named ectromelia virus (EV) can develop a mousepox. Virus replicates well in the skin, next in the draining lymph nodes (DLNs) and then in the spleen and liver, where it may induce extensive necrosis with strong inflammatory reaction. It is well known from the studies defined on some other viruses that a correlation, functional link and powerful help exist between MHC class I-restricted CD8(+) and MHC class II-restricted CD4(+) virus-specific cytotoxic T lymphocytes (CTLs). However, in the case of mousepox the role of CD4(+) CTLs is still controversial and some reports support the notion that induction of EV-specific CD4(+) CTLs is nonessential for the generation of virus-specific immune response. Consequently, this study was designed to evaluate EV-specific CD8(+) and CD4(+) CTL activity in the DLNs, spleen, skin and conjunctivae of BALB/c (H-2(d)) mice at 7 and 14 days p.i. with Moscow strain of EV. By using bulk cytotoxicity assay and immunosurgery of effector T cells with mAb specific for CD4(+) and/or CD8(+) T cells our data show that EV-specific CD8(+) CTLs predominated in DLNs and spleen at 7 days (67 and 66% of total CTLs, respectively) and 14 days p.i. (63 and 69% of total CTLs, respectively). In contrast, we found that EV clearance from the cutaneous lesions during mousepox is CD4(+) CTL-dependent at 7 days p.i. (59% of total CTLs), whereas at 14 days p.i. CD8(+) CTLs predominated in the epidermis, accounting for 72% of the total EV-specific CTLs. Our studies showed that the population of EV-specific CTLs is heterogeneous and contains cells of both phenotypes: CD8(+) and CD4(+). However, these effector cells did not express a similar tendency in cytotoxic activity in the DLNs, spleen and skin in comparison to the conjunctivae where EV-specific CD8(+) and CD4(+) CTLs were not detected at 7 days p.i. and at peak of mousepox conjunctivitis (14 days p.i.). Our results are discussed in terms of the value of EV to study antiviral CTL responses in the genetically susceptible host.

Homing studies on distribution of ectromelia (mousepox) virus-specific T cells adoptively transferred into syngeneic H-2d mice: paradigm of lymphocyte migration.

Mousepox (infectious ectromelia) may be used as a model for studies on the cellular immune response and pathogenesis of generalized viral infections. Ectromelia virus (EV) initially replicates in the footpad (f.p.) skin at the site of infection, next in draining lymph nodes, and then in the spleen and liver where the virus may induce extensive necrotic process with inflammatory reaction. We show in this study that after recipient BALB/c mice (H-2d) f.p. infection with EV prior to the adoptive transfer of syngeneic donor EV-specific cytotoxic T lymphocytes interferon-gamma-positive (IFN-gamma-+), interleukin-2-positive (IL-2+), and IL-4+ of both phenotypes, CD8+ approximately 70%, and CD4+ approximately 30%) preferentially migrated to the inguinal and auxiliary lymph nodes, spleen, liver, and skin at the site of infection (f.p.). Many particles of EV with the morphology characteristic for orthopoxviruses and virus-specific immunofluorescence within the cells of inguinal and auxiliary lymph nodes, liver, spleen, and skin have been observed using high-resolution transmission electron microscopy and fluorescence antibody technique, respectively. Results presented in this article support the concept that immune T cells adoptively transferred into infected recipient mice are able not only to specific migration in the host and homing in the sites of virus replication, but also to develop immunoprotection in the transferred animals.

Cytotoxic T lymphocyte control during ectromelia (mousepox) virus infection: interaction between MHC-restricted cells analyzed by non-radioactive fluorometry.

Cytotoxic T lymphocyte (CTL) activity of draining lymph node (DLN) cells isolated from BALB/c mice infected with ectromelia virus (EV) was examined using a fluorometric cell-mediated cytotoxicity (CMC) assay. Specific lysis of target cells A20 and EMT-6 primed with EV was demonstrated. The classical CD8+ cytolytic pathway dominated (72.7%) as compared to that of CD4+ (27.3%) in the cellular response during acute EV infection. Also an alternative method for determining CMC, employing a bisbenzamide dye for labelling target cells, is described. Coefficient variations of relative fluorescence were below 6%, that makes the method sensitive and reliable.

Comparison of different methods of maintenance of Malassezia pachydermatis (s. Pityrosporum pachydermatis) strains.

The maintenance of Malassezia pachydermatis isolated from canine otitis and dermatitis was compared on Sabouraud Dextrose Agar slants under liquid paraffin, an broth and in water, and by paper replica method, silica gel method, freeze-drying and deep freezing at-80 degrees C. Hydrolase production before and after storage was determined with API-ZYM Bio-Merieux kits. Deep freezing and freeze-drying were the best methods of maintaining M. pachydermatis. Hydrolase production was stable throughout the maintenance period.

Present status of classification of viruses of vertebrates.

The VIth Report of the International Committee on Taxonomy of Viruses (ICTV) was published in 1995. We have briefly characterized its contents and discussed the most important changes that have been made in the classification of viruses of vertebrates. The present line up of families and genera of viruses of vertebrates, and subviral agents and unassigned viruses is also provided. We propose an allocation of families of viruses of vertebrates according to a conjectural evolutional connection between the type and class of genomic nucleic acid. Finally the directions and intentions of the ICTV have been reviewed from the Virology Division News in Archives of Virology (1994/1995). These will be considered in the VIIth Report of the ICTV scheduled for publication after the XIth International Congress of Virology in Sydney in 1999.

Controlling orthopoxvirus infections--200 years after Jenner's revolutionary immunization.

An 11-year global WHO campaign for eradication of smallpox finished in October 1977 as the result of Edward Jenner's primary success in 1796, who for the first time applied human vaccination against variola virus (VARV). The 200th anniversary of this happening is a good occasion to summarize the current status of the knowledge about the role of B and T lymphocytes in the control of orthopoxvirus infections. This short review concentrates on general characteristics of orthopoxviruses and the immune response to infection, mainly by vaccinia virus (VV) and ectromelia virus (EV).

The cytosolic free Ca2+ in ectromelia (mousepox) virus stimulated cytotoxic T lymphocytes.

The objective of these studies was to assess the intracellular calcium (Ca2+) signal involved in the activation of ectromelia virus (EV)-specific cytotoxic T lymphocytes (CTL) upon stimulation with EV-sensitized (A20) target cells or concanavalin A (Con A). The CTL originated from mice previously infected with EV. The level of cytosolic Ca2+ in EV-specific CTL was measured using Fluo-3 AM. In both cases transient [Ca2+]i rise and a sustained plateau (1723 nM) were observed in buffer with 1 mM extracellular calcium. The [Ca2+]i response of EV-specific CTL to EV-sensitized target cells or Con A in extracellular calcium free buffer consisted of only one peak at 852 nm. The cytotoxic activity EV-specific CTL assessed in normal medium was 53.0%, but significantly reduced to 15% when verapamil was added to the buffer. No enhancement of [Ca2+]i response in EV-specific CTL was observed upon costimulation with PMA. Association of the level of intracellular [Ca2+]i in EV-specific CTL and the percentage of their cytotoxicity was found. These results show partial dependence of EV-specific CTL killing on extracellular calcium.

The inflammatory and immune response to mousepox (infectious ectromelia) virus.

The ectromelia virus (EV) has been recognized as the etiological agent of a relatively common infection in laboratory mouse colonies around the world, i.e., Europe (including Poland), USA and Asia. Due to widespread use of mice in biomedical research, it is important to study the biology of strains characteristic for a given country. This is particularly significant for the diagnosis, prevention and control ectromelia. In severe epizootics, approximately 90% morbidity is observed within colonies and mortality rate exceeding 70% is observed within 4 to 20 days from the appearance of clinical symptoms. The resistance to lethal infection is mouse strain-dependent. Several inbred strains of mice, including C57BL/6 and AKR are resistant to the lethal effects of EV infection, while others, such as A and BALB/c are susceptible. Recent studies indicate that (1) T lymphocytes, NK cells and interferon (IFN)-dependent host defenses must operate for the expression of resistance, (2) virus-specific T-cell precursors appear earlier in regional lymph nodes of resistant than susceptible mice, and (3) resistance mechanisms are expressed during early stages of infection. Over the past several years, (1) induction of anti-EV cytotoxic CD8+ T lymphocytes (CTL) responses in vivo in the absence of CD4+ (T helper) cells, (2) importance of some cytokines e.g., IFN-gamma in EV clearance at all stages of infection, and (3) induction of nitric oxide (NO) synthase, which is necessary for a substantial antiviral activity of IFN-gamma, have been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)