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:We analyzed effects of monkeypox (MPX) and vaccinia (VAC) infection on cultured human cells. Our custom-designed arrays contain >18,000 human genes as well as genes for each open reading frame of MPX and VAC. A reference experiement design type is where all samples are compared to a common reference. Compound Based Treatment: Chemical used for treating infected cells Infection: Virus used for infection Cell Line: Cell line used for infection Time: Time cells were harvested after infection Keywords: reference_design Experiments used primary human monocytes with mock infection, VAC-Western Reserve, VAC-NYBOH, MPX-Zaire, gamma irradiated (killed) MPX-Zaire, and Ebola-Zaire (EBOV) (deposited March 2004). The next set of experiments used primary human monocytes (Mf7.29) and primary human fibroblasts (S100) with mock infection, VAC-Western Reserve, MPX-Zaire, and gamma irradiated MPX-Zaire (g-MPX) (deposited August 2004). The final set used primary human monocytes (Mph), primary human fibroblasts (NHDF), and HeLa cells with mock infection, VAC-Western Reserve, MPX-Zaire, and gamma irradiated MPX-Zaire (g-MPX). Some cells were treated with ionomycin + PMA (I+P), cycloheximide (CHX) or hydroxyurea (HU). (deposited November 2005). Total RNA from cells was harvested at each timepoint, preserved in Trizol, Trizol-extracted total RNA, 1 round amplified (Ambion MessageAmp I), directly labeled by Cy5 incorporation during reverse transcription of amplified RNA. Each sample was hybridized versus a common reference (Stratagene Universal Human Reference RNA plus a mix of poxvirus transcripts from all timepoints). The reference was1 round of amplification (Ambion MessageAmp I), directly labeled by Cy3 incorporation during reverse transcription of amplified reference RNA. Cy3 and Cy5 samples were hybridized to a custom human-poxvirus array. Fluorescent images of hybridized microarrays were acquired using the GenePix 4000B and GenePix 4200AL microarray scanners.

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Monkeypox virus induces the synthesis of less dsRNA than vaccinia virus, and is more resistant to the anti-poxvirus drug, IBT, than vaccinia virus.

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Monkeypox virus induces the synthesis of less dsRNA than vaccinia virus, and is more resistant to the anti-poxvirus drug, IBT, than vaccinia virus.

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