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Salmonid host response to Hematopoietic Necrosis virus: cellular receptors, viral control and novel pathways of defence

ABSTRACT: The transcriptional response of Atlantic salmon (Salmo salar) to infectious hematopoietic necrosis (IHN) virus was elucidated using 16,008 gene GRASP cDNA microarrays. S. salar were exposed to the IHN virus in a waterborne challenge, and kidney samples from five fish sampled on each of days 0, 1, 5 and 9 were analysed in the microarray study. Validation of nine of the significant genes was conducted on fish sampled on alternate days from 1 to 13 and compared to the unchallenged control sampled on day 0. The key pathways up-regulated in response to the virus included endosomal transport, type I and II interferon responses, the alternative complement pathway, apoptosis, phagocytosis and phagocytic cell oxidation, natural killer cell activity, antiviral replication via iron sequestering, virally-induced disruption of the cell cycle, retroviral DNA integration, T-cell activation and cellular immunity. Fish that did not contain viral titer on days 5 and 9 responded in a similar manner to those with titer, but further induced a number of genes involved in anti-viral replication (Glutathione peroxidase 1 and ferritin H), anti-proliferation (PTEN and B-cell receptor associated protein 32), lysosomal response (CD63 antigen), pro-inflammatory response (lipopolysaccharide binding protein, stress activated JNK1, thiol peroxidase), and T-cell activation (T-cell activation Rho-GTPase activating protein) that may increase resistance to the virus. Three genes that were potentially co-opted by the virus to enhance infectivity were also identified, including uPAR (angiogenesis), CypA (viral replication and infectivity), and BAF1 (viral protein biosynthesis). Perhaps the most notable finding was the up-regulation of uPAR which can function to increase the density of fibronectin, the receptor of the IHN virus, on the cell surface, hence facilitate viral entry into the cell. Keywords: Immunlogical response to IHN virus challenge

ORGANISM(S): Salmo salar Oncorhynchus mykiss

PROVIDER: GSE6924 | GEO | 2007/03/31

SECONDARY ACCESSION(S): PRJNA99347

REPOSITORIES: GEO

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Project description:The transcriptional response of Atlantic salmon (Salmo salar) to infectious hematopoietic necrosis (IHN) virus was elucidated using 16,008 gene GRASP cDNA microarrays. S. salar were exposed to the IHN virus in a waterborne challenge, and kidney samples from five fish sampled on each of days 0, 1, 5 and 9 were analysed in the microarray study. Validation of nine of the significant genes was conducted on fish sampled on alternate days from 1 to 13 and compared to the unchallenged control sampled on day 0. The key pathways up-regulated in response to the virus included endosomal transport, type I and II interferon responses, the alternative complement pathway, apoptosis, phagocytosis and phagocytic cell oxidation, natural killer cell activity, antiviral replication via iron sequestering, virally-induced disruption of the cell cycle, retroviral DNA integration, T-cell activation and cellular immunity. Fish that did not contain viral titer on days 5 and 9 responded in a similar manner to those with titer, but further induced a number of genes involved in anti-viral replication (Glutathione peroxidase 1 and ferritin H), anti-proliferation (PTEN and B-cell receptor associated protein 32), lysosomal response (CD63 antigen), pro-inflammatory response (lipopolysaccharide binding protein, stress activated JNK1, thiol peroxidase), and T-cell activation (T-cell activation Rho-GTPase activating protein) that may increase resistance to the virus. Three genes that were potentially co-opted by the virus to enhance infectivity were also identified, including uPAR (angiogenesis), CypA (viral replication and infectivity), and BAF1 (viral protein biosynthesis). Perhaps the most notable finding was the up-regulation of uPAR which can function to increase the density of fibronectin, the receptor of the IHN virus, on the cell surface, hence facilitate viral entry into the cell. Keywords: Immunlogical response to IHN virus challenge A preliminary microarray experiment was run on the waterborne-challenged S. salar whereby the five individuals sampled on a given day were combined and run on a single slide, and we compared the expression profiles from each day (days 0 (unchallenged control) through 11). From this experiment, we noted that the most profound changes were occurring around days 1, 5, and 9, so we performed subsequent experiments at these time points, with five fish per day and 5 control fish analysed in total.

Project description:Cardiomyopathy syndrome (CMS) is a disease associated with severe myocarditis in adult Atlantic salmon (Salmo salar L.), which is presumably caused with a double-stranded RNA virus (piscine myocarditis virus; PMCV). This study addressed the host immune response in Atlantic salmon during experimentally induced CMS as assessed by microarray transcriptome profiling. Post-smolts were infected by intraperitoneal injection of PMCV and developed cardiac pathology consistent with CMS. From analysis of heart samples at several time points and different tissues at early and clinical stages by oligonucleotide microarrays, six gene sets representing a broad range of immune responses were identified. Histopathological examination of cardiac tissue showed myocardial lesions from 6 weeks post infection (wpi) that peaked at 8-9 wpi and was followed by a recovery. Viral RNA was detected in all organs from 2 wpi. The highest and equal viral load was observed in heart, spleen and kidney, peaking at 8-9 wpi. Strong and systemic induction of antiviral and IFN-dependent genes from 2 wpi that leveled off during infection, was followed by a biphasic activation of pathways for B cells and MHC antigen presentation, both peaking at clinical pathology. This was preceded by a distinct cardiac activation of complement at 6 wpi. Most severe cardiac pathology and peak viraemia coincided with a cardiac-specific activation of T cell responses dominated by genes involved in effector CD8 cell responses and splenic induction of complement. These changes preceded the reduction in viral load and pathology, suggesting an important role of cytotoxic T cells for viral elimination in infected heart. Atlantic salmon post-smolts (average weight 50g) were challenged with the putative CMS-causing pathogen (PMCV). Hearts were sampled from challenged and control fish at 2, 4, 6, 8, 9 and 10 wpi. Pools of equal amounts of total RNA from three fish hearts each were combined and hybridized against control pools of 8 to 10 fish hearts per time point. Spleen, liver, mid-kidney, red blood cells (RBC) and peripheral blood leukocytes (PBL) were analyzed at 4 wpi (latest time point before clinical stage) and 8 wpi (peak pathology). The samples of infected fish were labeled with Cy5 and hybridized to control samples from the same time-points labeled with Cy3. Competitive hybridization to the arrays was followed by washing, scanning, image analysis, and data analysis. Selected genes were analyzed with RT-qPCR.

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.

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Salmonid host response to Hematopoietic Necrosis virus: cellular receptors, viral control and novel pathways of defence

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