ABSTRACT: Interferon and ribavirin treatment in a rhesus macaque model of novel Middle East respiratory syndrome coronavirus (MERS-CoV) infection (hCoV-EMC)
Project description:Background: The recent emergence of a novel coronavirus in the Middle East (designated MERS-CoV) is a reminder of the zoonotic potential of coronaviruses and the severe disease these etiologic agents can cause in humans. Clinical features of Middle East respiratory syndrome (MERS) include severe acute pneumonia and renal failure that is highly reminiscent of severe acute respiratory syndrome (SARS) caused by SARS-CoV. The host response is a key component of highly pathogenic respiratory virus infection. Here, we computationally analyzed gene expression changes in a human airway epithelial cell line infected with two genetically distinct MERS-CoV strains obtained from human patients, MERS-CoV-EMC (designated EMC) and MERS-CoV-London (designated LoCoV). Results: Using topological techniques, such as persistence homology and filtered clustering, we characterized the host response system to the different MERS-CoVs, with LoCoV inducing early kinetic changes, between 3 and 12 hours post infection, compared to EMC. Robust transcriptional changes distinguished the two MERS-CoV strains predominantly at the late time points. Combining statistical analysis of infection and cytokine-stimulated treatment transcriptomics, we identified differential innate and pro-inflammatory responses between the two virus strains, including up-regulation of extracellular remodeling genes following LoCoV infection and differential pro-inflammatory responses between the two strains. Conclusions: These transcriptional differences may be the result of amino acid differences in viral proteins known to modulate innate immunity against MERS infection. Triplicate wells of Calu-3 2B4 cells were infected with Human Coronavirus EMC 2012 (HCoV-EMC) or time-matched mock infected. Cells were harvested at 0, 3, 7, 12, 18 and 24 hours post-infection (hpi), RNA extracted and transcriptomics analyzed by microarray.
Project description:We will use the EMC/2012 strain of the novel beta Coronavirus called Middle East Respiratory Syndrome Coronavirus (MERS-CoV). It was initially passaged on Vero E6 cells in Saudi Arabia before being sequenced at the Erasmus Medical College in Rotterdam, Netherlands by Dr Ron Fouchier. We propose to perform a time course of infection of hCoV-EMC on MRC5 cells (Human Lung origin) and Vero cells (African Green Monkey Kidney cells). Both cell lines readily grow and replicate the virus. Importantly these cell lines show signs of Cytopathic effect (CPE), such as cell rounding and release from the petri dish that coincide with time points high virus replication demonstrating the effects of virus replication on the cells. Transcriptomic analysis will be performed after infection with MERS-CoV and SARS-CoV (Urbani strain) to compare the host gene induction that occurs during infection.
Project description:We will use the EMC/2012 strain of the novel beta Coronavirus called Middle East Respiratory Syndrome Coronavirus (MERS-CoV). It was initially passaged on Vero E6 cells in Saudi Arabia before being sequenced at the Erasmus Medical College in Rotterdam, Netherlands by Dr Ron Fouchier. We propose to perform a time course of infection of hCoV-EMC on MRC5 cells (Human Lung origin) and Vero cells (African Green Monkey Kidney cells). Both cell lines readily grow and replicate the virus. Importantly these cell lines show signs of Cytopathic effect (CPE), such as cell rounding and release from the petri dish that coincide with time points high virus replication demonstrating the effects of virus replication on the cells. Transcriptomic analysis will be performed after infection with MERS-CoV and SARS-CoV (Urbani strain) to compare the host gene induction that occurs during infection.
Project description:We will use the EMC/2012 strain of the novel beta Coronavirus called Middle East Respiratory Syndrome Coronavirus (MERS-CoV). It was initially passaged on Vero E6 cells in Saudi Arabia before being sequenced at the Erasmus Medical College in Rotterdam, Netherlands by Dr Ron Fouchier. We propose to perform a time course of infection of hCoV-EMC on MRC5 cells (Human Lung origin) and Vero cells (African Green Monkey Kidney cells). Both cell lines readily grow and replicate the virus. Importantly these cell lines show signs of Cytopathic effect (CPE), such as cell rounding and release from the petri dish that coincide with time points high virus replication demonstrating the effects of virus replication on the cells. Transcriptomic analysis will be performed after infection with MERS-CoV and SARS-CoV (Urbani strain) to compare the host gene induction that occurs during infection. MRC5 and Vero E6 cells will be infected at an MOI of 0.1 and 3 and RNA harvested from cells at 24 and 48 post infection. RNA will be processed for library creation and sequenced on an Illumina Hiseq. Sequencing reads will be analyzed and compared across the time course and between each virus to identify common response pathways induced during infection as well as unique pathways specific to each virus.
Project description:Differential expression was determined in Calu-3 cells between mock infected and infection with either Human coronavirus EMC and SARS coronavirus at different times post infection. Calu-3 2B4 cells were infected with Human Coronavirus EMC 2012 (HCoV-EMC) or mock infected. Samples were collected 0, 3, 7, 12, 18 and 24 hpi. There are 3 mock and 3 infected replicates for each time point, except for 12 hpi for which there are only 2 infected replicates (one replicate did not pass RNA quality check). There were no mock sampes at 18 hpi, and therefore infected samples at 18 hpi were compared with mocks at 24 hpi. For direct comparison with SARS-CoV infected cells, raw data from HCoV-EMC experiments were quantile normalized together with the SARS-CoV dataset (GEO Series accession number GSE33267).
Project description:The purpose of the experiment was to compare placental transcriptome of rhesus macaque at approximately 80% completed gestation to human placental transcriptomes.
Project description:Microarray analysis of peripheral blood mononuclear cells (PBMCs), lungs, and lung lesions collected over the course of hCoV-EMC infection of 6 rhesus macaques. 6 rhesus macaques were infected intratracheally with hCoV-EMC. PBMCs were collected at days 0, 1, 3, and 6, and lungs were collected from serial sacrifices of 3 animals each at day 3 and day 6. Infection produced a mild-moderate, self-limiting respiratory infection, and was not lethal. We performed microarray analysis (using Agilent Rhesus arrays) on all lungs, lung lesions, and PBMCs collected for the study.