Project description:Orthopoxviruses are large DNA viruses which can cause disease in numerous host species. Even though the eradication of variola virus - the causative agent of human smallpox M-bM-^@M-^S succeeded, with the end of vaccinations several other orthopoxviruses emerged as potential threat to human health. For instance, animal-borne monkeypox virus, cowpox virus and closely related vaccinia virus are all capable of establishing zoonotic infections in humans. The disease caused by each virus differs in terms of expression and severity, but we still know little about the reasons for these different phenotypes. They may be explained by the unique repertoire of host cell modulating factors encoded by each virus. In this study, we aimed at characterizing the specific modulation of the host cells gene expression profile by orthopoxvirus infection. In our study we analyzed changes in host cell gene expression of HeLa cells after infection with cowpox virus, monkeypox virus or vaccinia virus and compared these to each other and to the gene expression profile of non-infected cells using Agilent Whole Genome Microarray technology. We could identify major differences in viral modulation of host cell immune response genes, especially an induction of genes involved in leukocyte migration and Toll-like receptor signalling in cowpox and monkeypox virus infected cells. This was not observed following vaccinia virus infection. If these differences contribute to the different clinical manifestation of cowpox, monkeypox and vaccinia virus infections in certain host species remains to be elucidated. We analyzed the gene expression profile of HeLa cells wich were either mock-infected or infected with Vaccinia virus strain IHD-W, Cowpox virus strain Brighton Red or Monkeypox virus strain MSF#6 at a multiplicity of infection of 5. Experiments were performed in duplicate. At 6 h post infection total RNA was isolated from infected cells and used for microarray analysis.
Project description:Orthopoxviruses are large DNA viruses which can cause disease in numerous host species. Even though the eradication of variola virus - the causative agent of human smallpox – succeeded, with the end of vaccinations several other orthopoxviruses emerged as potential threat to human health. For instance, animal-borne monkeypox virus, cowpox virus and closely related vaccinia virus are all capable of establishing zoonotic infections in humans. The disease caused by each virus differs in terms of expression and severity, but we still know little about the reasons for these different phenotypes. They may be explained by the unique repertoire of host cell modulating factors encoded by each virus. In this study, we aimed at characterizing the specific modulation of the host cells gene expression profile by orthopoxvirus infection. In our study we analyzed changes in host cell gene expression of HeLa cells after infection with cowpox virus, monkeypox virus or vaccinia virus and compared these to each other and to the gene expression profile of non-infected cells using Agilent Whole Genome Microarray technology. We could identify major differences in viral modulation of host cell immune response genes, especially an induction of genes involved in leukocyte migration and Toll-like receptor signalling in cowpox and monkeypox virus infected cells. This was not observed following vaccinia virus infection. If these differences contribute to the different clinical manifestation of cowpox, monkeypox and vaccinia virus infections in certain host species remains to be elucidated.
Project description:Human melanoma tumor cells (HS294T) and monocytes (THP-1) were infected with a double deleted (-VGF, -TK) oncolytic vaccinia virus expressing human DAI (DNA-dependent activator of interferon-regulatory factors). Total RNA was collected and gene expresson profiles were determined with Agilent microarray. An oncolytic vaccinia virus that does not express DAI was used to control the effect of DAI and uninfected cells (PBS treated) were used to control the effect of virus infection. In oncolytic virotherapy the ability of the virus to activate the immune system against tumors is nowadays generally understood to be a key mechanism in full eradication of cancer and for long-term anti-tumor effects. We armed an oncolytic vaccinia virus with DAI to increase the immunogenicity and the vaccine potency of the virus. The aim of this study was to study if the expression of DAI by a replicating vaccinia virus would alter the gene expression profile of infected cells and to study what are the differentially expressed genes. Three-condition experiment: vvdd-tdTomato-hDAI vs. vvdd-tdTomato vs. PBS treated cells. 2 cell lines: HS294T tumor cells and THP-1 monocytes. 3 biological replicates of virus infected cells per cell line and 2 uninfected replicates per cell line. HS294T and THP-1 cells were treated with vvdd-tdTomato-hDAI or vvdd-tdTomato control virus, or with PBS only to have an uninfected control. 16 hours after infection total RNA was extracted and whole genome gene pfofiles were analyzed and differentially expressed genes determined.
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: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:Human melanoma tumor cells (HS294T) and monocytes (THP-1) were infected with a double deleted (-VGF, -TK) oncolytic vaccinia virus expressing human DAI (DNA-dependent activator of interferon-regulatory factors). Total RNA was collected and gene expresson profiles were determined with Agilent microarray. An oncolytic vaccinia virus that does not express DAI was used to control the effect of DAI and uninfected cells (PBS treated) were used to control the effect of virus infection. In oncolytic virotherapy the ability of the virus to activate the immune system against tumors is nowadays generally understood to be a key mechanism in full eradication of cancer and for long-term anti-tumor effects. We armed an oncolytic vaccinia virus with DAI to increase the immunogenicity and the vaccine potency of the virus. The aim of this study was to study if the expression of DAI by a replicating vaccinia virus would alter the gene expression profile of infected cells and to study what are the differentially expressed genes.
Project description:We infected bone marrow derived macrophages (BMDM) with wild type vaccinia virus Western Reserve (WT-WR) and mutant virus deleting A26 protein (WRΔA26) and analyzed transcriptional profiling of cellular genes at 1, 2, 4 and 8 hours post-infection times (p.i.).
Project description:Primary human astrocytes were infected with either monkeypox virus (MPXV clade IIb lineage), vaccinia virus (VACV: Acambis 2000), or controls (MC=monkeypox control, AC = Vaccinia control) at an MOI of 10 for 6 h. Samples (n=4) were analyzed by LC-MS/MS with label-free quantification where the data was acquired by data-dependent acquisition (DDA).
Project description:Vaccinia virus (VACV) is a large cytoplasmic dsDNA virus that causes dramatic alterations to many cellular pathways including microRNA biogenesis. Here we use small RNA sequencing to comprehensively quantify the impact of VACV infection on the expression of endogenous small non-coding RNAs (sncRNAs) at both early (6 h) and late (24 h) times post infection. Non-coding RNAs (sncRNAs) were assessed at 6 hours and 24 hours, in naive and VACV-infected HeLa cells, in the presence or absence of the inhibitor AraC. There were three replicates for each of the eight groups.