Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Classification of three temporal kinetic transcripts of SGIV

ABSTRACT: Singapore grouper iridovirus (SGIV) is the major agent that causes severe iridovirus diseases in grouper maricluture. Based on the genomic information, a DNA microarray, containing probes corresponding to 162 putative SGIV open reading frames (ORFs) was constucted. The viral microarrays wereused to classify the majority of SGIV transcripts into three temporal kinetic classes (immediate-early, IE; early, E; late, L) during an in vitro infection by their dependence on de novo protein synthesis inhibitor and viral DNA replication. Keywords: drug response To map the SGIV transcripts into temporal kinetic classes during the infection in vitro, GS cells were treated with drug inhibitors as previously described with some slight modifications. Briefly, GS monolayers were treated for 1 h prior to, during and throughout the viral infection with either CHX or PAA, which inhibited de novo protein synthesis and viral DNA replication mechanisms, respectively. To distinguish between viral IE transcripts and other transcripts, cells infected with SGIV (MOI of 5) or mock infected were treated with CHX (100 Âµg/ml), and then harvested at 8 h p.i. Whereas, viral E and L transcripts were categorized when cells infected with SGIV (MOI of 5) in the presence and absence of PAA (200 Âµg/ml), respectively. Until at 36 h p.i., cells were collected for RNA extraction. In a comparison analysis, we used Significance Analysis of Microarrays (SAM) software to identify the groups of different expression genes between the drug-treated samples and untreated samples. SAM identifies genes with statistically significant changes in expression by assimilating a set of gene-specific t tests. Significance is based on an estimated of FDRâ¤5% and a 2 fold-change cutoff. Only the genes that met the following three criteria could be categorized into various temporal kinetic classes: (i) a viral gene was considered to be a IE transcript if its expression level increased or weakly affected under CHX treatment, (ii) a viral gene was considered to be a L transcript if its median intensity ratio in the PAA-treated samples was at least two-fold lower than that in the untreated samples and (iii) a viral gene was considered to be a E transcript if it expressed higher or remained within the two- fold range after PAA treatment but was inhibited distinctly by CHX treatment. To identify the significant differences in gene expression, a criterion with at least two-fold change combined with studentâs one sample t-test p value <0.05 was adopted.

ORGANISM(S): Epinephelus coioides

SUBMITTER: Qiwei Qin

PROVIDER: E-GEOD-7154 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Whole-genome transcriptional profiles of a novel marine fish iridovirus, Singapore grouper iridovirus (SGIV) in virus-infected grouper spleen cell cultures and in orange-spotted grouper, Epinephulus coioides.

Teng Yong Y Hou Zhiwei Z Gong Jie J Liu Hong H Xie Xiayang X Zhang Liang L Chen Xiaohong X Qin Qi Wei QW

Virology 20080701 1

A DNA microarray containing all Singapore grouper iridovirus (SGIV) open reading frames (ORFs) was constructed to map the viral gene transcriptional profiles in virus-infected grouper spleen (GS) cells and in spleen tissues of virus-infected grouper. The results showed that viral genes started to be transcribed as early as 1 h postinfection (p.i.), and followed by a rapid increasing gene expression along with virus infection in cell cultures. The three temporal kinetic classes (15 immediate-earl ...[more]

PMID: 18555886

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Project description:Singapore grouper iridovirus (SGIV) is the major agent that causes severe iridovirus diseases in grouper maricluture. Based on the genomic information, a DNA microarray, containing probes corresponding to 162 putative SGIV open reading frames (ORFs), was constructed to map the viral gene transcriptional profiles over the time course by establishing the models of SGIV-infected GS cells and SGIV-infected grouper. All the data from real-time RT-PCR, RT-PCR and dilution RT-PCR assays were confirmed with the findings of microarrays, which were clustered into groups with the similarity expression profiles by the Self-Organizing Maps (SOMs) approach. The microarray analysis showed that SGIV had big differential expression profiles in the special infected cells and organ and the viral DNA replication mechanisms were firstly prevented as an important strategy of the host defense during the natural course infection.Our studies firstly uncover the relative of a marine viral gene expression patterns between in vitro and in vivo infection, which provides a better understanding of SGIV transcription regulation and a greater degree shared with other iridoviruses on their repliaction and pathogenesis. Keywords: time course To further characterize SGIV gene expression patterns and to monitor the gene temporal kinetic transcription program on a genome-wide scale, we monitored viral gene expression profiles in vitro and in vivo infection.For the experiment of infection in vitro, GS cell monolayers cultured in 75cm2 flask were inoculated with 1ml of SGIV (5.0 Ã105.5 TCID50/ml) at 25oC. The mock-infected cells (as reference samples) were treated in the same manner as SGIV-infected cells but with fresh culture medium. Total RNA was isolated from the cells at 1, 2, 4, 6, 8, 10, 12, 16, 24, 36, 48, 72, and 96 hours post-infection (h p.i.), respectively. For the experiment of infection in vivo, Grouper Epinephelus tauvina juveniles with approximately 40-50 g were experimentally infected with 150 Âµl of the SGIV inoculum (5.0Ã105.5 TCID50/ml) and held in tanks supplied with running seawater at 25oC. Control fishes were injected with the same volume of EMEM. Total RNA was harvested from the spleens of 5 fishes randomly selected from the experimental population at 1, 2, 3, 4, 5, 7, 9, 11, and 15 days post-infection (d p.i.). Total RNA from the spleens of 35 mock-infected fishes was used as reference samples. After visual inspection for the presence of image artifacts, such as scratches, dirt, contamination, high region, or overall background on the array, the scanned images were saved as TIF files and further analyzed to generate raw data using SpotDataTM software (CapitalBio). After filtering the low-intensity spots and background-noise value, a global scaling procedure was performed to normalize among the different arrays and the different channels of same arrays using housekeeping gene of piscine 18S RNA which was also spotted in triplicate. To estimate the variance result from dye-bias, a swap-dye strategy was used for the virus-infected and mock-infected samples at 48 h p.i.

Project description:E2 exposure significantly decreased peak viral titer in hNECs from female donors. We used microarray analyses to identify global gene expression patterns between E2 and vehicle exposed hNECs from female donors Influenza causes an acute infection characterized by virus replication in respiratory epithelial cells. The severity of influenza and other respiratory diseases changes over the life course and during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult male and female donors, we exposed cultures to the endogenous 17Î²-estradiol (E2) or select estrogen receptor modulators (SERMs), then infected cultures with a seasonal influenza A virus (IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and whether these effects where sex-dependent. Estradiol, raloxifene, and bisphenol A decreased IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was dependent on the genomic estrogen receptor- 2 (ESR2) as neither genomic ESR1 nor non- genomic GPR30 were expressed in hNEC cultures and addition of the genomic ER antagonist ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on interferon or chemokine secretion, but significantly downregulated cell metabolic processes, including genes that encode for zinc finger proteins, many of which contain estrogen response elements in their promoters. These data provide novel insights into the cellular and molecular mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial cells derived from humans. Primary human nasal epithelial cells from females were exposed to E2 for 24h prior to infection, then infected with an H3N2 strain of influenza a virus for 2 hours. At 24 and 48h post infection, hNECs were collected in Trizol for RNA extraction and hybridization on Affymetrix Human Gene ST 2.0 microarrays.

Project description:Small non-coding RNAs have emerged as key players in modulation of viral infection. A unique example is the critical dependence of hepatitis C virus (HCV) on the liver-specific microRNA (miRNA), miR-122, which has surfaced as therapeutic target. Here, we used crosslinking immunoprecipitation (CLIP) of the Argonaute (AGO) protein to characterize strengths and specificities of miRNA interactions across 15 viral genomes. Intriguingly, replication of pestiviruses, which are major threats to milk and meat industry, critically depends on cellular miR-17 and let-7 interactions with the viral 3âUTR. Like HCV, miRNA binding enhanced translation and prevented viral RNA degradation. On the cellular transcriptome, pestiviral miR-17 sequestration in vitro and ex vivo conferred reduced AGO binding and functional mRNA de-repression for miR-17 targets. These findings generalize the concept of RNA virus dependence on cellular miRNAs, highlight such interactions as therapeutic targets, and connect functional regulation of the transcriptome in primary cells to miRNA sequestration. Several subseries of analyses were performed. For each sample, subseries to which it belongs are indicated. In the âVirus AGO-CLIPâ subseries, AGO-CLIP was performed on cells infected with virus as indicated. Processed reads were aligned to the host genome (hg18 or BosTau7) and to the respective viral genome. The primary data of interest from this subseries concerns AGO binding to viral RNA, and is given as processed data file âTable S1â. In the âVirus AGO-CLIP: BVDV vs. Mockâ subseries, AGO-CLIP was performed on four replicates each of BVDV and mock infected MDBK cells. CLIP reads aligned to BosTau7 were clustered, and differential analysis was performed on binding to each cluster. In addition, differential analysis of AGO bound miRNAs was performed. The associated data is given as processed data file âTable S3 and S4â. In the âAGO-CLIP: tinyLNA-17 vs. Mockâ AGO-CLIP was performed on four replicates each of tinyLNA-17 and mock treated MDBK cells. CLIP reads aligned to BosTau7 were clustered, and differential analysis was performed on binding to each cluster. The associated data is given as processed data file âTable S5â. In the âRNA-seq: BVDV vs. Mockâ subseries, mRNA-seq was performed on two replicates each of MDBK cells infected with different biotypes of BVDV or with miR-17 seed site mutant BVDV. The latter (incl. mock controls) were trans-complemented with the corresponding mutated miR-17. Differential gene expression analysis was performed between selected conditions. The associated data is given as processed data file âTable S6â.

Dataset Information

Classification of three temporal kinetic transcripts of SGIV

Publications

Whole-genome transcriptional profiles of a novel marine fish iridovirus, Singapore grouper iridovirus (SGIV) in virus-infected grouper spleen cell cultures and in orange-spotted grouper, Epinephulus coioides.

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