Unknown,Transcriptomics,Genomics,Proteomics

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Searching for IPN-specific gene expression profiles in Salmo salar using susceptible and resistant families challenged with the Infectious Pancreatic Necrosis virus (IPNV).

ABSTRACT: Infectious pancreatic necrosis (IPN) is widespread disease with global distribution and highly contagious, which leads to severe economic problems both freshwater and saltwater, particularly in the Atlantic salmon (Salmo salar). In this work we analyzed and evaluated gene expression profiles in resistant and susceptible families associated to S. salar exposed to IPNV in order to understand the defense mechanisms mounted by resistant fish in combating infection. Experimentally, 29 families with known pedigree and variability were infected by immersion with 1x106 PFU/ml of IPNV, and a gamma frailty model analysis based on Kaplan-Meier mortality curves were obtained to identify and classify families as susceptible or resistant. Samples of head kidney tissue were obtained at day 1 and 5 post-infection from susceptible and resistant families and pooled to analyse their expression pattern with realt-ime PCR and one-color salmon-specific oligonucleotide microarray (21K). The microarray results showed clear differences in both families, where resistant fish presented an over-expression of genes associated with endocrine function, and a down-regulation of genes associated principally with tissue differentiation, metabolism protein degradation, cell signaling and immunological process. Moreover qPCR analysis using as target a number of transcripts associated with immunological processes showed the same trend observing a higher expression in susceptible than resistant families, indicating significant differences for CCL-19, CCRM-NM-3c, IL-12, IFN-M-NM-1, and IFN-M-NM-3. The one-color oligonucleotide microarrays included in this study was performed using pooled samples from each families at two different post-infection time condition. In the case of susceptible families were chosen at 1 day post-infection (dpi) the families 5352 (n = 2) and 5469 (n = 2) and at 5 dpi the families 5629 (n = 2) and 5637 (n = 2), whereas in resistant families were selected at 1 dpi the families 5459 (n =3) and 5761 (n =3), and at 5 dpi the families 5463 (n =3) and 5764 (n =3).

ORGANISM(S): Salmo salar

SUBMITTER: Felipe Reyes-Lopez

PROVIDER: E-GEOD-40575 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Infectious Pancreatic Necrosis (IPN) is a highly contagious birnavirus disease of farmed salmonid fish, which often causes high levels of morbidity and mortality. A large genetic component underlying resistance to this disease has been previously described for Atlantic salmon (Salmo salar L.), which mediates high mortality rates in some families and zero mortality in others. A global comparison of the gene expression profiles of resistant and susceptible Atlantic salmon fry following challenge with the IPN virus was undertaken. Full sibling salmon fry from two IPNV-resistant and two IPNV-susceptible families were challenged with the virus and sampled at 1 day, 7 days and 20 days post-challenge. Significant viral titre was observed in both resistant and susceptible fish at all timepoints, although generally at higher levels in susceptible fish. Microarray interrogations were performed using a custom-designed, oligonucleotide microarray platform (Agilent) with 44 K probes per slide (Salar_2; Agilent Design ID:025520). The design is lodged with ArrayExpress ( under accession number A-MEXP-2065. Dual-label hybridisations were undertaken, with each experimental sample (Cy3 labelled) being competitively hybridised against a pooled reference control (Cy5 labelled) comprising equimolar amounts from each experimental RNA sample. The interrogations comprised 144 separate hybridisations; 2 genotypes (susceptible, resistant) Ã 2 families for each genotype Ã 2 challenge states (control, challenged) Ã 3 timepoints (1, 7, 20 dpi) Ã 4 biological replicates for resistant (2 from each of two tanks) and 8 biological replicates for susceptible (4 from each of two tanks). A preliminary analysis suggested evidence for a segregating QTL in both the susceptible families and therefore twice as many offspring were screened. It was later established that the evidence for QTL segregation in one of the families was inconclusive and therefore comparisons were made at the family level only. The analyses took the unbalanced design into account.

Project description:Infectious pancreatic necrosis virus (IPNV) is a fish-derived pathogen and possess a bi-segmented, double-stranded RNA genome. By using zebrafish 14K oligo-microarray and quantitative RT-PCR, we identified differential expression of a defined subset of genes involved in apoptosis and immunity at 6-, 12- and 24- hour after IPNV infection. Transcripts divided into 11 functional categories were significantly modulated by IPNV, including immune response, apoptosis, transcription, signal transduction, lipid and cholesterol metabolism, carbohydrate metabolism, oxidative phosphorylation, cell cycle, protein degradation, protein folding and stress response, protein synthesis, nucleoside metabolism and synthesis. Most of pro-apoptotic bcl-2 family members were up-regulated after IPNV infection. Activation of pro-apoptotic members might disrupt potential of mitochondria and leaded to the mitochondria-mediated apoptosis in the late stage of IPNV infection. After treating the IPNV-infected cells with TNFM-NM-1 inhibitor AP126, expression of two bcl-2 family genes Bad and Bid and activation of caspase-8 and -3 had been inhibited significantly in early stage of IPNV infection. Expression of RIP-1 and Bmf-1 these two necroptosis-related genes and production of ROS were diminished in virus-infected cells which pre-treated with AP126. Our study shows the interactions between host cells and IPNV, the molecular mechanisms involved in IPNV-induced pathogenesis, and the variation of transcriptome through TNFM-NM-1 during infection which shows the important component of host defense. TNFM-NM-1 might lead to apoptosis in early stage and necrosis in late stage in ZF4 cells infected by IPNV. TNFM-NM-1 is crucial to apoptosis and ROS-mediated necrosis caused by IPNV in zebrafish cells. Zebrafish ZF4 cell line was derived from 24h post-fertilization zebrafish embryos. Previous studies have been shown that adult zebrafish and zebrafish cell line could be infected with IPNV. In the present study, ZF4 cells were infected with IPNV, and total RNA was isolated from infected and uninfected control cells at 0 h, 6 h, 12 h, 24 h post-infection. Microarray analysis gene expression between IPNV-infected and uninfected cells relative to internal control on slides. The zebrafish 14K oligo microarray we used comprise 1800 zebrafish gene sequences from the NCBI and a database of 12768 putative open reading frames using NCBI zebrafish EST sequence information. Data files were imported into GeneSpring GX 7.3 for further analysis. Expression data sets must pass all the following quality control categories before used for cluster analysis. Clustering analysis allowed us to observe differences in cellular gene expression. A similar expression pattern was seen when comparing differentially expressed host cell genes between 12 and 24 h post-infection. The expression profiles were significantly different between 6 and 12 h post-infection. Therefore, we conclude that the regulation of host gene expression was changed after 12 h post-infection, and progressed into the late stage. To understand the interactions between host cells and IPNV and the molecular mechanisms involved in IPNV-induced pathogenesis, we used zebrafish oligo microarrays to investigate the gene expression profiles of IPNV-infected zebrafish embryonic cells. We also studied expression of specific genes related to immune response and apoptosis which were not present on the microarray we used by real-time quantitative RT-PCR. We used Pathway StudioM-bM-^@M-^Ys software to analyze the altered genes in cellular pathway for IPNV, TNFM-NM-1 was shown to be crucial to these genes of host response. Our study proved the variation of transcriptome during infection, which shows the activation of important component of host defense, apoptosis and necrosis through TNFM-NM-1 mediate pathway.

Dataset Information

Searching for IPN-specific gene expression profiles in Salmo salar using susceptible and resistant families challenged with the Infectious Pancreatic Necrosis virus (IPNV).

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