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
Overall design: 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 ×105.5 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 µl of the SGIV inoculum (5.0×105.5 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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