ABSTRACT: HT29 cells was infected with EV71 at MOI 1 or nil respectively and harvested at 36hpi We use miRNA microarray to profile and identify miRNA which are up or down regulated due to EV71 infection 3 biological replicate (6 samples)
Project description:Enterovirus 71 (EV71), a member of Picornaviridae, causes severe neurological and systemic illness in children. To better understand the virus-host cell interactions, we performed a triple-SILAC-based quantitative proteomics study monitoring host cell proteome changes after EV71 infection. Based on the quantitative data for more than 4100 proteins, ~17% of the proteins were found as significantly changed (p<0.01) at either 8 or 20 hours post infection (h.p.i.). Five biological processes and seven protein classes showed significant differences. Functional screening of 9 regulated proteins discovered the regulatory role of CHCH2, a mitochondrial protein known as a transcriptional activator for cytochrome c oxidase (COX), in EV71 replication. Further studies showed that CHCH2 served as a negative regulator of innate immune responses.
Project description:To characterize the primary and recall responses to EV71 vaccines, PBMC from 19 recipients before and after vaccination with EV71 vaccine are collected and their gene expression signatures after stimulation with EV71 antigen were compared. Four-condition experiment,pre-vaccination PBMCs (stimulation vs. no stimulation with EV71 antigen) vs. post-vaccination PBMCs (stimulation vs. no stimulation with EV71 antigen)
Project description:We attempted to identify potential miRNAs that can regulate viral replication by screening host miRNAs in EV71-infected RD cells. To compare the differential expression of miRNAs in EV71-infected cells at different time points, miRNA expression profiles were analyzed using Agilent Human MicroRNA Array chips. At 5 h p.i., the expression levels of most miRNAs in EV71-infected cells were not different from those of mock-infected cells. However, five miRNAs (miR-574-3p, miR-574-5p, miR-181d, miR-197, and miR-939) showed a miRNAs (miR-193a-3p and miR-324-5p) exhibited a increase in expression level in response to EV71 infection at 10 h p.i. RD cells were seeded (2*106 cells) in 10 cm dishes and incubated overnight. The cells were infected with EV71 (strain 2231) on ice at an M.O.I. of 10. After adsorption for 1 h, the virus suspension was replaced with DMEM containing 2% FBS, and the cells were harvested at the indicated times. Total cellular RNA extraction and miRNA microarray analyses were performed using the Agilent Human MicroRNA Array V2 chip, which contains 723 human and 76 viral miRNAs, each with 16 duplicates. Total gene signals were detected and analyzed using the GeneSpring 7.3.1 software and were normalized to the 75th percentile.
Project description:To compare MicroRNA expression in 16HBE infected with EV71 and CA16 we defined the following experimental groups: EV71-0h, EV71-6h, EV71-12h, CA16-0h, CA16-6h and CA16-12h
Project description:Enterovirus 71 (EV71) is one of the leading causes of hand, foot and mouth disease with neurological complications in some cases. To study the pathogenesis of EV71 infection, large scale analyses of EV71 infected cells have been performed. However, most of these studies employed rhabdomyosarcoma (RD) cells or used transcriptomic strategy. Here, we performed SILAC-based quantitative proteomic analysis of EV71-infected U251 cells, a human glioma cell line. A total of 3,125 host proteins were quantified, 451 of which were differentially regulated as a result of EV71 infection at 8 hpi or 20 hpi or both.
Project description:We have previously shown that Heparin (Hep) significantly inhibited Enterovirus 71 (EV71) infection and binding in both Vero and a human neural cell line, SK-N-SH, in vitro. Therefore, in this study we intended to gain insight into the cellular and molecular mechanisms of action of Hep against clinical EV71 infection in SK-N-SH cells. Instead of stating a long list of gene functions and pathways, we tried to select for EV71-induced genes that were exclusively affected by antiviral activity of Hep through a multi-level comparison and characterization. Overall, our microarray analysis may suggest that Hep might exert its anti-EV71 activity in SK-N-SH cells, at least in part, through the following mechanisms: i. Reduction of the down-regulation effect of EV71 on TOX that would lead to an increased population of effective, mature T-cells and NK-cells; ii. Reduction of the up-regulation effect of EV71 on GIP, that in turn, would reduce recruiting different GPCRs, leading to a reduced viral infection in host cells; iii. Partially neutralization of the EV71-mediated apoptosis through expression of CARP2 that acts as an anti-apoptosis ubiquitin protein ligase (E3). EV71 is a neurovirological virus that can sometimes cause severe and fatal CNS complications in infected patients. Since still there is no approved drug for prophylaxis of EV71-casued disease, discovering a molecular drug target(s) for EV71 infection would be crucial. This was the first study to report direct assessment of mechanisms of action of antivirals against EV71 infection. SK-N-SH cells were infected with a clinical EV71 isolate followed by treatment with 125 µg/mL of Hep. At 72 hours post infection, antiviral activity and cytotoxicity of Hep at 125 µg/mL in 12-well plates were carried out at the same time as RNA extraction. This way, we could ensure that we would assess transcript profiles of the host cells under the same condition and time as assessment of antiviral activity and cytotoxicity for the same replicates. Changes in expression profiles of the host cells were comparatively assessed under four conditions: cell control (neither infection nor treatment, designated CC), treated only with Hep (compound control, designated Cyto), EV71-infected cells treated with Hep (treatment, designated H), and infected with EV71 without treatment with Hep (virus control, designated V). All experiments were applied in triplicate, and totally twelve GeneChip® Human Gene 1.0 ST arrays were purchased from Affymetrix and processed. Then, the following five contrasts were made: Hep vs. CC; VC vs. CC; Cyto vs CC; Hep vs. VC; and Hep vs. Cyto. For each contrast, only samples from the two target groups were included. The statistical parameters of ANOVAs, p values, multiple test corrections, and fold changes were calculated within each contrast. Then, a multi-level selection and analysis procedure was employed in order to attribute changes in the gene transcription level to antiviral activity of Hep.
Project description:Ribosome profiling (Ribo-Seq) (maps positions of translating ribosomes on the transcriptome) analysis of human (RD) cells infected with enterovirus strains EV7, EV71, and PV1.
Project description:We attempted to identify potential miRNAs that can regulate viral replication by screening host miRNAs in EV71-infected RD cells. To compare the differential expression of miRNAs in EV71-infected cells at different time points, miRNA expression profiles were analyzed using Agilent Human MicroRNA Array chips. At 5 h p.i., the expression levels of most miRNAs in EV71-infected cells were not different from those of mock-infected cells. However, five miRNAs (miR-574-3p, miR-574-5p, miR-181d, miR-197, and miR-939) showed a miRNAs (miR-193a-3p and miR-324-5p) exhibited a increase in expression level in response to EV71 infection at 10 h p.i.
Project description:To characterize the primary and recall responses to EV71 vaccines, PBMCs from 19 recipients before and after vaccination with EV71 vaccine were collected. 14 samples pre-vaccination and 16 samples post-vaccination were detected by microarray and their gene expression signatures after stimulation with EV71 antigen were compared.
Project description:HT29 cells was infected with EV71 at MOI 1 or nil respectively and harvested at 36hpi We use miRNA microarray to profile and identify miRNA which are up or down regulated due to EV71 infection