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 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.

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.

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Classification of three temporal kinetic transcripts of SGIV

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