Project description:We conducted a high-throughput drug repositioning screen using the LOPAC®1280 and the ReFRAME drug libraries to identify existing drugs that harbor antiviral activity against SARS-CoV-2, in a Vero E6 cell-based assay. We additionally performed RNA sequencing on control and SARS-CoV-2 infected Vero E6 cells to study the biological changes after SARS-CoV-2 infection and to elucidate the potential mechanisms underlying the positive hits identified from our high-throughput screen. Vero E6 cells were either mock-infected or infected with SARS-CoV-2 USA-WA1/2020 (MOI = 0.3) with three replicates. Cells were harvested 24 hours after infection, and total RNA was extracted using the Qiagen® RNeasy® Plus Mini Kit. The quality of the extracted RNA was assessed with the Agilent® 2100 Bioanalyzer. Libraries were prepared from total RNA following ribosome RNA depletion using standard protocol according to Illumina®. Total RNA sequencing was then performed on the Illumina® NextSeq system; 150bp paired-end runs were performed and 100 million raw reads per sample were generated.
Project description:Purpose: The goals of this study are to monitor the evolution pattern of SARS-CoV2 in depending host cells by viral transcriptome sequencing analysis of Vero, A549, Caco2, and HRT18 cells infected with SARS-CoV2. Methods: SARS-CoV-2 isolate was passaged 4 time on Vero cells and used to extract RNA for the high-throughput sequencing. The 8×104 PFU of SARS-CoV2 stocks passaged on vero cells were inoculated to the monolayer of A549, CaCO2, and HRT-18 cell lines in 75T flask for 1hour at 37℃ in a 5% CO2 incubator with gentle shaking of 15 minutes interval. After that, the infected cells were washed two times with DPBS and incubated with the fresh maintenance medium for 3 days. The virus inoculation was performed in triplicate for each cell lines. In case of the first passage, the infected cell pellets were resuspended to 250µl with fresh medium, to extract RNA for the high-throughput sequencing. The cultured cell supernatant of the virus-infected A549, CaCO2, and HRT18 cells was centrifuged at 3,000g for 10min to use for the next passage, and stored at -80℃. The serial passage of SARS-CoV-2 on A549, CaCO2, and HRT18 cell lines were continued to passage 12 and the cultured cell supernatant of the infected cells in passage 12 was centrifuged at 3,000g for 10 min, and used to extract RNA for the high-throughput sequencing. The RNA samples were sequenced with illumine TruSeq Strand Total RNA LT kit and illumine NovaSeq6000 plaform form Macrogen, Inc (Seoul, Korea) for high throughput sequencing. The raw reads were trimmed with BBDuk and mapped the isolate SARS-CoV-2/human/KOR/KCDC03-NCCP43326/2020 (Genebank accession number. MW466791) with Bowtie 2 using Geneious program 2021.2.2 Result: Using SNP analysis workflow, our result showed the sequence variations pattern of SARS-CoV2 depending on host cell (A549, CaCO2, and HRT18 cell lines) and it was confirmed that a relatively large number of SNPs were commonly observed in spike protein. Some SNPs affect amino acid changes, and a common pattern of amino acid changes was observed the genomic sequence of SARS-CoV2 passaged in A549, CaCO2 and HRT18 cells. Conclusion: In this study, we tried to monitor the SARS-CoV-2 (GenBank accession No. MW466791 in 2020, Korea) evolution pattern in different host cells using high throughput sequencing analysis, and compare the selected mutations by each host cells with natural mutations found in currently circulating SARS-CoV-2 variants.
Project description:RNA sequencing was performed on control and SARS-CoV-2 infected Vero E6 cells, with or without remdesivir treatment to study the biological changes after SARS-CoV-2 infection and to evaluate the effectiveness of remdesivir on the gene expression level. 500,000 Vero E6 cells were seeded in 6-well plates. The following day, the cell medium was replaced with fresh medium supplemented with either DMSO or 1 µM remdesivir, and cells were either mock-infected or infected with SARS-CoV-2 USA-WA1/2020 (MOI=0.3), with three replicates per experimental condition. Cells were harvested 24 hours after infection, and total RNA was extracted using the Qiagen® RNeasy® Plus Mini Kit. The quality of the extracted RNA was assessed with the Agilent® 2100 Bioanalyzer. Libraries were prepared from total RNA following ribosome RNA depletion using standard protocol according to Illumina®. Total RNA sequencing was then performed on the Illumina® NextSeq system; 150bp paired-end runs were performed and 100 million raw reads per sample were generated.
Project description:To compare MicroRNA expression in Vero cells infected with DENV-2 adapted strain of Vero cells and its source srain derived from C6/36 cells
Project description:We report a novel mechanism of gene origination in the segmented negative sense viruses (sNSVs), including IAV and LASV. During replication, these viruses use "snatched" host caps to prime their own transcription (cap-snatching). We show that a proportion of cap-snatched host sequences contain translational start codons (AUGs), that are recognized by the host ribosome. This allows for the translation of host and viral “untranslated regions” (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting.
Project description:PEDV wildtype strain (HNXX) infected Vero cells could form typical syncytial lesions, resulting in cell membrane rearrangement. In order to study which proteins are involved in the rearrangement of cell membrane, we infected Vero cells with PEDV. To capture the intact cell membrane at the same time as the occurrence of lesions caused by PEDV, membrane protein was collected at 18 hours post infection. iTRAQ-based quantitative membrane proteomics was used in analysing host proteins differentially expressed in PEDV-infected Vero cells and the control group. In this quantitative proteomics experiments results, a total of 4579 proteins were identified. Significantly upregulated or downregulated proteins were determined with a p-value of <0.05 and FC of <0.83 or >1.20. After bioinformatics data analysis, Ten proteins were found significantly upregulated, and eleven proteins were markedly downregulated after virus infection. Approximately 47% of these proteins were membrane proteins or membrane-associated proteins.
Project description:Two rounds of TMT relative quantitative proteomics were performed to detect cellular factors involved in p-eIF4E regulation of the synthesis of viral proteins.our first round of screening identified differentially expressed proteins in PEDV-infected cells and mock-infected cells; the cellular pathways involved were mainly the estrogen, cAMP, and calcium signaling pathways. Second round screening identified differentially expressed proteins in the PEDV-infected S209A-Vero cells vs. the PEDV-infected WT-Vero cells; the regulated cellular pathways were found to be mainly in the PI3K-Akt, focal adhesion, and mTOR signaling pathways, and the biological processes and molecular functions in which p-eIF4E played a role were related mainly to metabolism and biogenesis, catalytic activity, and stimuli response.4006 host factors were detected, of which 193 (in brown) were significantly upregulated (ratio ≥1.2, P<0.05) and 191 (in green) were down-regulated upon PEDV infection (ratio ≤0.83, P<0.05). 29 of the 191 down-regulated proteins were susceptible to a low level of p-eIF4E . Notably, among the 193 upregulated cellular proteins, 77 were upregulated in the WT-Vero over the S209A-Vero cells , suggesting that the WT-Vero cells are more susceptible to a high level of p-eIF4E.
Project description:Proteomic analysis of Monkeypox virus produced with experimentally infected Vero E6 cells using Data-Dependent Detection for a comprehensive coverage. The analysis was performed to prioritize the most responsive peptides amenable to mass spectrometry for the development of mass spectrometry-based detection methods.
Project description:Next-generation proteomics of Vero E6 cells infected by Italy-INMI1 SARS-CoV-2 virus for defining the kinetics of whole viral particle antigen production for vaccines. Cells from Day1, Day2, Day3, Day4, Day7 post-infection at two multiplicities of infection.