Project description:Three experiments, corresponding to the three figures in the article, are represented in this set. Each experiment was an ex vivo treatment time course as described in the paper. For each of the experiments, mRNA was isolated at the indicated time points, cDNA was directly prepared by reverse transcription in the presence of Cy5-labeled dUTP, and the expression profiled using Cy3-labeled cDNA prepared by reverse transcription of Stratagene Universal Human Reference RNA as a control. One experiment, corresponding to Figure 1 in the paper, is a set of infection, or mock-infection time courses, in which each of three cell types: primary human dermal fibroblasts, primary human macrophages, or HELA cells were respectively infected with Monkeypox virus, Vaccinia virus, or Ebola virus, or mock-infected. Infected cells or mock-infected cells were harvested and lysed at the indicated times after infection and mRNA isolated for analysis following the protocol described above. The second experiment, corresponding to Figure 2 in the paper, is a set of treatments of human dermal fibroblasts, each in 24-well plates with either: PBS only (mock), interferon alpha (IFN-alpha) at 0.6 pM final concentration (Sigma, St. Louis, MO), tumor necrosis factor alpha (TNF-alpha) at 0.6 pM final concentration (Sigma), PMA at 25ng/mL final concentration plus ionomycin at 1micromolar final concentration, polyinosinic-polycytidylic acid as potassium salt (poly(I-C)) at 100 microg/mL final concentration (Sigma), Escherichia coli 055:B5 lipopolysaccharide (LPS) at 1microg/mL final concentration (Sigma), or dexamethasone at 1 micromolar final concentration (Sigma). Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. The third experiment, corresponding to Figure 3 in the paper, is a set of infections or mock-infections of human dermal fibroblasts, or human primary macrophages, with Monkeypox virus or Vaccinia virus, respectively, followed by treatment with either ionomycin + phorbol myristic acid (PMA) or with poly(I-C). The intention of the experiment was to investigate whether prior infection altered the response of the cells to the chemical agents. Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. Description of sample characteristics: Time: Time after infection or Mock infection Infection: Ebola-Zaire/killed Monkeypox Virus/Mock infection/Monkaypox Virus/None/Pre infection/Vaccinia NY/Vaccinia WR Compound Based Treatment: Dexamethasone/Interferon-alpha/Ionomycin + PMA/LPS/PBS/polyinosinic-polycytidylic acid/TNF-alpha Cell Type: Primary Human Dermal Fibroblast/Primary Human Macrophage/HeLa Figure in article: Numbers group slides that are represented in the same figure of the article.

Project description:Three experiments, corresponding to the three figures in the article, are represented in this set. Each experiment was an ex vivo treatment time course as described in the paper. For each of the experiments, mRNA was isolated at the indicated time points, cDNA was directly prepared by reverse transcription in the presence of Cy5-labeled dUTP, and the expression profiled using Cy3-labeled cDNA prepared by reverse transcription of Stratagene Universal Human Reference RNA as a control. One experiment, corresponding to Figure 1 in the paper, is a set of infection, or mock-infection time courses, in which each of three cell types: primary human dermal fibroblasts, primary human macrophages, or HELA cells were respectively infected with Monkeypox virus, Vaccinia virus, or Ebola virus, or mock-infected. Infected cells or mock-infected cells were harvested and lysed at the indicated times after infection and mRNA isolated for analysis following the protocol described above. The second experiment, corresponding to Figure 2 in the paper, is a set of treatments of human dermal fibroblasts, each in 24-well plates with either: PBS only (mock), interferon alpha (IFN-alpha) at 0.6 pM final concentration (Sigma, St. Louis, MO), tumor necrosis factor alpha (TNF-alpha) at 0.6 pM final concentration (Sigma), PMA at 25ng/mL final concentration plus ionomycin at 1micromolar final concentration, polyinosinic-polycytidylic acid as potassium salt (poly(I-C)) at 100 microg/mL final concentration (Sigma), Escherichia coli 055:B5 lipopolysaccharide (LPS) at 1microg/mL final concentration (Sigma), or dexamethasone at 1 micromolar final concentration (Sigma). Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. The third experiment, corresponding to Figure 3 in the paper, is a set of infections or mock-infections of human dermal fibroblasts, or human primary macrophages, with Monkeypox virus or Vaccinia virus, respectively, followed by treatment with either ionomycin + phorbol myristic acid (PMA) or with poly(I-C). The intention of the experiment was to investigate whether prior infection altered the response of the cells to the chemical agents. Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. Description of sample characteristics: Time: Time after infection or Mock infection Infection: Ebola-Zaire/killed Monkeypox Virus/Mock infection/Monkaypox Virus/None/Pre infection/Vaccinia NY/Vaccinia WR Compound Based Treatment: Dexamethasone/Interferon-alpha/Ionomycin + PMA/LPS/PBS/polyinosinic-polycytidylic acid/TNF-alpha Cell Type: Primary Human Dermal Fibroblast/Primary Human Macrophage/HeLa Figure in article: Numbers group slides that are represented in the same figure of the article. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment

Project description:BACKGROUND: Animal-borne orthopoxviruses, like monkeypox, vaccinia and the closely related cowpox virus, are all capable of causing zoonotic infections in humans, representing a potential threat to human health. The disease caused by each virus differs in terms of symptoms and severity, but little is yet know about the reasons for these varying phenotypes. They may be explained by the unique repertoire of immune and host cell modulating factors encoded by each virus. In this study, we analysed the specific modulation of the host cell's gene expression profile by cowpox, monkeypox and vaccinia virus infection. We aimed to identify mechanisms that are either common to orthopoxvirus infection or specific to certain orthopoxvirus species, allowing a more detailed description of differences in virus-host cell interactions between individual orthopoxviruses. To this end, we analysed changes in host cell gene expression of HeLa cells in response to infection with cowpox, monkeypox and vaccinia virus, using whole-genome gene expression microarrays, and compared these to each other and to non-infected cells. RESULTS: Despite a dominating non-responsiveness of cellular transcription towards orthopoxvirus infection, we could identify several clusters of infection-modulated genes. These clusters are either commonly regulated by orthopoxvirus infection or are uniquely regulated by infection with a specific orthopoxvirus, with major differences being observed in immune response genes. Most noticeable was an induction of genes involved in leukocyte migration and activation in cowpox and monkeypox virus-infected cells, which was not observed following vaccinia virus infection. CONCLUSION: Despite their close genetic relationship, the expression profiles induced by infection with different orthopoxviruses vary significantly. It may be speculated that these differences at the cellular level contribute to the individual characteristics of cowpox, monkeypox and vaccinia virus infections in certain host species.

Project description:Monkeypox virus (MPV) is a zoonotic Orthopoxvirus and a potential biothreat agent that causes human disease with varying morbidity and mortality. Members of the Orthopoxvirus genus have been shown to suppress antiviral cell defenses, exploit host cell machinery, and delay infection-induced cell death. However, a comprehensive study of all host genes and virus-targeted host networks during infection is lacking. To better understand viral strategies adopted in manipulating routine host biology on global scale, we investigated the effect of MPV infection on Macaca mulatta kidney epithelial cells (MK2) using GeneChip rhesus macaque genome microarrays. Functional analysis of genes differentially expressed at 3 and 7 hours post infection showed distinctive regulation of canonical pathways and networks. While the majority of modulated histone-encoding genes exhibited sharp copy number increases, many of its transcription regulators were substantially suppressed; suggesting involvement of unknown viral factors in host histone expression. In agreement with known viral dependence on actin in motility, egress, and infection of adjacent cells, our results showed extensive regulation of genes usually involved in controlling actin expression dynamics. Similarly, a substantial ratio of genes contributing to cell cycle checkpoints exhibited concerted regulation that favors cell cycle progression in G1, S, G2 phases, but arrest cells in G2 phase and inhibits entry into mitosis. Moreover, the data showed that large number of infection-regulated genes is involved in molecular mechanisms characteristic of cancer canonical pathways. Interestingly, ten ion channels and transporters showed progressive suppression during the course of infection. Although the outcome of this unusual channel expression on cell osmotic homeostasis remains unknown, instability of cell osmotic balance and membrane potential has been implicated in intracellular pathogens egress. Our results highlight the role of histones, actin, cell cycle regulators, and ion channels in MPV infection, and propose these host functions as attractive research focal points in identifying novel drug intervention sites.

Dataset Information

Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

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Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

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