Project description:This study demonstrates the ability of an Ebolavirus resequencing microarray to determine the sequence of the Zaire ebolavirus glycoprotein that has been engineered into the place of the surface protein of a recombinant vesicular stomatitis virus expressing green fluorescent protein (rVSV-EBOVgp-GFP). The rVSV-EBOVgp-GFP was cultured in VERO-E6 cells for three passages either in the presence of a monoclonal antibody that blocks infection (KZ52) or in control cultures with no antibody. Culture supernatant and cell lysate was collected before passaging and after each passage. RNA was extracted from each sample and the sequence of the Ebola glycoprotein in each sample was determined by the Ebola resequencing microarray. Illumina Next Generation Sequencing was performed on the initial virus stock, before passaging and on the third passage of the KZ52 antibody selected virus stock to validate the microarray sequence results.
Project description:This study demonstrates the ability of a Ebolavirus resequencing microarray to determine the sequence of the Zaire ebolavirus glycoprotein that has been engineered into the place of the surface protein of a recombinant vesicular stomatitis virus expressing green fluorescent protein (rVSV-EBOVgp-GFP). The rVSV-EBOVgp-GFP was cultured in VERO-E6 cells for three passages either in the presence of a monoclonal antibody that blocks infection (KZ52) or in control cultures with no antibody. Culture supernatant and cell lysate was collected before passaging and after each passage. RNA was extracted from each sample and the sequence of the Ebola glycoprotein in each sample was determined by the Ebola resequencing microarray.
Project description:The purpose of this experiment was to obtain samples for mRNA analysis in IHH cells infected with Zaire Ebola virus and mutants: Zaire Ebola virus: This wild-type Ebola virus - strain Mayinga - was isolated from a fatal human case in Zaire (now known as the Democratic Republic of Congo) in 1976 Zaire Ebola virus, VP35 R312A possesses a R312A mutation in the VP35 protein. Zaire Ebola virus, delta sGP. Lacks the ability to produce non-structural protein, the secreted glycoprotein (sGP). Zaire Ebola virus, delta mucin. Lacks the mucin-like domain (MLD), which contains both N-linked and O-linked glycosylation sites, for the glycoproteins.
Project description:The purpose of this experiment was to obtain samples for mRNA analysis in IHH cells infected with Zaire Ebola virus and mutants: Zaire Ebola virus: This wild-type Ebola virus - strain Mayinga - was isolated from a fatal human case in Zaire (now known as the Democratic Republic of Congo) in 1976 Zaire Ebola virus, VP35 R312A possesses a R312A mutation in the VP35 protein. Zaire Ebola virus, delta sGP. Lacks the ability to produce non-structural protein, the secreted glycoprotein (sGP). Zaire Ebola virus, delta mucin. Lacks the mucin-like domain (MLD), which contains both N-linked and O-linked glycosylation sites, for the glycoproteins.
Project description:The purpose of this experiment was to obtain samples for mRNA analysis in IHH cells infected with Zaire Ebola virus and mutants: Zaire Ebola virus: This wild-type Ebola virus - strain Mayinga - was isolated from a fatal human case in Zaire (now known as the Democratic Republic of Congo) in 1976 Zaire Ebola virus, VP35 R312A possesses a R312A mutation in the VP35 protein. Zaire Ebola virus, delta sGP. Lacks the ability to produce non-structural protein, the secreted glycoprotein (sGP). Zaire Ebola virus, delta mucin. Lacks the mucin-like domain (MLD), which contains both N-linked and O-linked glycosylation sites, for the glycoproteins. Overview of Experiment: Cells: Immortalized Human Hepatocytes (IHH); seed 60,000 cells per well in a 24-well plate. Infected with a multiplicity of infection (MOI) of 0.5. After infection, 3x wash with PBS and replace with 5% FCS DMEM without NaPyr or NEAA. Time matched mocks done in triplicate from same cell stock as rest of samples. Time Points = 0, 6, 12, 24, 48, and 72 hrs post infection in triplicate.
Project description:The purpose of this experiment was to obtain samples for mRNA analysis in IHH cells infected with Zaire Ebola virus and mutants: Zaire Ebola virus: This wild-type Ebola virus - strain Mayinga - was isolated from a fatal human case in Zaire (now known as the Democratic Republic of Congo) in 1976 Zaire Ebola virus, VP35 R312A possesses a R312A mutation in the VP35 protein. Zaire Ebola virus, delta sGP. Lacks the ability to produce non-structural protein, the secreted glycoprotein (sGP). Zaire Ebola virus, delta mucin. Lacks the mucin-like domain (MLD), which contains both N-linked and O-linked glycosylation sites, for the glycoproteins. Overview of Experiment: Cells: Immortalized Human Hepatocytes (IHH); seed 60,000 cells per well in a 24-well plate. Infected with a multiplicity of infection (MOI) of 0.5. After infection, 3x wash with PBS and replace with 5% FCS DMEM without NaPyr or NEAA. Time matched mocks done in triplicate from same cell stock as rest of samples. Time Points = 0, 6, 12, 24, 48, and 72 hrs post infection in triplicate.
Project description:Highly pathogenic Zaire ebolavirus (EBOV) infection is associated with a dysregulated immune response and high levels of cytokines and chemokines are observed in fatal human cases. . In stark contrast Reston ebolavirus (RESTV) might be non-pathogenic for humans yet the underlying mechanisms determining pathogenicity for different Ebola viruses are not understood. In this study we investigate antiviral immune responses in EBOV- and RESTV- infected primary human monocyte-derived macrophages (MDM). We provide evidence that increased pathogenicity of the highly pathogenic EBOV is associated with a strong activation of host responses from infected MDM. The observed cytokine response after EBOV infection is strikingly similar to LPS-mediated immune signatures however EBOV caused significant induction of the interferon response in addition. In contrast we show that the low pathogenic RESTV fails to elicit significant immune responses in infected MDM. These results demonstrate a correlation of pathogenicity and excessive MDM activation for different Ebola virus species. Interaction of the viral glycoprotein (GP) with Toll-like receptor 4 (TLR4) leading to activation of NF_B signaling is responsible for this effect rather than differences in replication or blocking of immune signaling. We demonstrate that inhibition of TLR4 is able to abolish EBOV-GP mediated NF_B activation which might offer the possibility to develop targeted treatments for EBOV limiting the extreme immune response that seems to be detrimental to the host.
Project description:Since the discovery of filoviruses, comprised of Marburg and Ebola species, seemingly random, sporadic fatal outbreaks of disease in humans and non-human primates have evoked interest in delineation of host tropisms and potential reservoirs. We now describe identification of Reston ebolavirus (REBOV) by microarray, in domestic swine from the Philippines experiencing unusually severe outbreaks of porcine reproductive and respiratory disease syndrome. While REBOV is the only member of Filoviridae that has not been associated with disease in humans, its emergence in the human food chain heightens concerns for public health, swine agriculture and international trade. REBOV isolates were found to be significantly more divergent from each other than from the original virus isolate from 1989, indicating polyphyletic origins in swine. This further suggests that REBOV has been circulating since, and possibly before, the initial discovery of REBOV in monkeys. These results identify swine as the first non-primate susceptible host for REBOV.