In November 2011 Sockeye Salmon (600 fry, average weight = 5.5 g) were immersed for one hour in a 85 L static, aerated, 5°C freshwater bath containing IHNV (Okanagan 2000 isolate, U genogroup; K. Garver, unpublished) at a dose of 1.98 x 103 pfu ml-1. After the one hour exposure to virus, the fish along with bath water were transferred into a 700 L tank. Sockeye Salmon from a separate tank that were not handled or exposed to IHNV served as control fish (naïve fish). Challenged and naïve fish were maintained in freshwater in one tank each, and monitored daily for signs of disease and morbidity for a period of nine months. To mimic temperature changes that would be normally experienced in freshwater, fish were held for four months at 5 °C (± 1 °C) followed by a slow transition over several days to 9 °C (± 1 °C) for the remaining five months. At the end of the study (nine months post challenge), naïve and surviving Sockeye Salmon had similar average weights of 23 g and 20 g, respectively. At 271 dpc, 240 surviving fish from each of the challenge and naïve groups were reassigned into smaller 400 L tanks (120 fish per tank; total of four tanks) supplied with the same source of water. Shortly after the transfer, one group each of the challenged and naïve fish received an intraperitoneal (ip) injection of poly(I:C) (Sigma-Aldrich) dissolved in saline solution at a dose of 10 µg per g fish. For controls, a group each of challenged and naïve fish were transferred without injection. A sample of 39 fish were removed from each tank at 3 (274 dpc), 7 (278 dpc) and 10 (281 dpc) days after poly(I:C) injection/transfer. At sampling fish were euthanized in an overdose of buffered tricaine methanesulfonate (TMS) and brain tissues were aseptically removed, individually flash-frozen in liquid N2 and transferred to -80 °C until RNA extraction.
Growth protocol
In June 2011 Sockeye Salmon fry (1000 fish from Sakinaw Lake stock, brood year 2010) were transferred from Rosewall Creek hatchery, Fanny Bay, British Columbia (B.C.) to the Pacific Biological Station, Nanaimo, B.C.. Fish were reared in 2.7 m3 tanks and 5 °C (± 1 °C) freshwater under a natural photoperiod.
Extracted molecule
total RNA
Extraction protocol
Samples were randomized and total RNA was extracted from brain samples using TRIzol (Invitrogen) by physical disruption with a TissueLyser II (Retsch). The remainders of each sample were then purified by treatment with DNase I (Qiagen) and subsequently column-purified using the RNeasy® MinElute® Cleanup kit (Qiagen) following manufacturer instructions. Total RNA was quantified and quality checked by spectrophotometry (NanoDrop-1000) and 1% agarose gel electrophoresis, then stored at -80 °C.
Label
Cy5-CTP
Label protocol
cDNA synthesis and cRNA amplification was performed on 200 ng input total RNA using the Low Input Quick Amp system (Agilent Technologies; v6.5) with Cy5- or Cy3-CTP (Perkin Elmer). Experimental groups were labeled with Cy5-CTP (Perkin Elmer) and these consisted of naïve (n = 7), survivors (n = 7), and carriers (n = 5), as well as poly(I:C)-injected naïve (n = 7), poly(I:C)-injected survivors (n = 7), and poly(I:C)-injected carriers (n = 2). A reference pool was generated by synthesizing Cy3-labeled cRNA from two individuals from each of the six conditions (total in reference = 12 samples). Equimolar concentrations of each synthesized Cy3-cRNA sample (n = 12) was pooled to create a common reference used to hybridize to each array alongside sample Cy5-cRNA. All cRNA samples were quality checked for specific activity as per manufacturers’ protocol (Agilent) and kept at -80 °C in the dark until hybridization.
Channel 2
Source name
pool of 2 samples from each experimental conditions (total = 12 samples), containing equimolar sample(s) per condition
In November 2011 Sockeye Salmon (600 fry, average weight = 5.5 g) were immersed for one hour in a 85 L static, aerated, 5°C freshwater bath containing IHNV (Okanagan 2000 isolate, U genogroup; K. Garver, unpublished) at a dose of 1.98 x 103 pfu ml-1. After the one hour exposure to virus, the fish along with bath water were transferred into a 700 L tank. Sockeye Salmon from a separate tank that were not handled or exposed to IHNV served as control fish (naïve fish). Challenged and naïve fish were maintained in freshwater in one tank each, and monitored daily for signs of disease and morbidity for a period of nine months. To mimic temperature changes that would be normally experienced in freshwater, fish were held for four months at 5 °C (± 1 °C) followed by a slow transition over several days to 9 °C (± 1 °C) for the remaining five months. At the end of the study (nine months post challenge), naïve and surviving Sockeye Salmon had similar average weights of 23 g and 20 g, respectively. At 271 dpc, 240 surviving fish from each of the challenge and naïve groups were reassigned into smaller 400 L tanks (120 fish per tank; total of four tanks) supplied with the same source of water. Shortly after the transfer, one group each of the challenged and naïve fish received an intraperitoneal (ip) injection of poly(I:C) (Sigma-Aldrich) dissolved in saline solution at a dose of 10 µg per g fish. For controls, a group each of challenged and naïve fish were transferred without injection. A sample of 39 fish were removed from each tank at 3 (274 dpc), 7 (278 dpc) and 10 (281 dpc) days after poly(I:C) injection/transfer. At sampling fish were euthanized in an overdose of buffered tricaine methanesulfonate (TMS) and brain tissues were aseptically removed, individually flash-frozen in liquid N2 and transferred to -80 °C until RNA extraction.
Growth protocol
In June 2011 Sockeye Salmon fry (1000 fish from Sakinaw Lake stock, brood year 2010) were transferred from Rosewall Creek hatchery, Fanny Bay, British Columbia (B.C.) to the Pacific Biological Station, Nanaimo, B.C.. Fish were reared in 2.7 m3 tanks and 5 °C (± 1 °C) freshwater under a natural photoperiod.
Extracted molecule
total RNA
Extraction protocol
Samples were randomized and total RNA was extracted from brain samples using TRIzol (Invitrogen) by physical disruption with a TissueLyser II (Retsch). The remainders of each sample were then purified by treatment with DNase I (Qiagen) and subsequently column-purified using the RNeasy® MinElute® Cleanup kit (Qiagen) following manufacturer instructions. Total RNA was quantified and quality checked by spectrophotometry (NanoDrop-1000) and 1% agarose gel electrophoresis, then stored at -80 °C.
Label
Cy3-CTP
Label protocol
cDNA synthesis and cRNA amplification was performed on 200 ng input total RNA using the Low Input Quick Amp system (Agilent Technologies; v6.5) with Cy5- or Cy3-CTP (Perkin Elmer). Experimental groups were labeled with Cy5-CTP (Perkin Elmer) and these consisted of naïve (n = 7), survivors (n = 7), and carriers (n = 5), as well as poly(I:C)-injected naïve (n = 7), poly(I:C)-injected survivors (n = 7), and poly(I:C)-injected carriers (n = 2). A reference pool was generated by synthesizing Cy3-labeled cRNA from two individuals from each of the six conditions (total in reference = 12 samples). Equimolar concentrations of each synthesized Cy3-cRNA sample (n = 12) was pooled to create a common reference used to hybridize to each array alongside sample Cy5-cRNA. All cRNA samples were quality checked for specific activity as per manufacturers’ protocol (Agilent) and kept at -80 °C in the dark until hybridization.
Hybridization protocol
Samples and reference were combined, fragmented, then hybridized to randomized-order cGRASP 4x44k salmonid arrays (Agilent eArray AMADID: 025055) as per manufacturers’ instructions with the added stabilization solution step to prevent ozone-related problems (Agilent). Slides were kept in the dark in a low ozone atmosphere until scanned.
Scan protocol
Slides were scanned on a ScanArray® Express (Perkin Elmer) at 5 µm resolution with constant PMT settings (Cy5:75; Cy3:80) that had been optimized to obtain 1% of all spots saturated.
Description
Brain_Survivor-polyIC_3dpi_rep4
Data processing
Images were quantified in Imagene 8.1 (Biodiscovery). Poor and control spots were flagged for downstream filtering, and the median background intensity was subtracted from the median signal value for each probe. Samples were loaded into GeneSpring 11.5.1 (Agilent). Negative raw values were set at 1.0, each array was normalized by an intensity-dependent Lowess, and a baseline to median transformation of normalized expression values was performed per gene. Control spots, and any probes not passing the following were removed: raw values ≥ 500 in both channels, and no poor quality flags in at least 65% of the samples in any one condition. A two-way ANOVA (p ≤ 0.01; no multiple test correction) was performed in GeneSpring (Agilent) to identify differentially expressed genes influenced by IHNV status (naïve, survivor, or carrier), poly(I:C)-injection (control or injected), or an interaction of both factors. Probes with a significant interaction effect were removed from the main effect lists. Probes with a significant effect of IHNV status were additionally filtered by fold change (FC), finding those probes with a 1.5-fold difference between survivor and control groups, or carrier and control groups concurrently in both poly(I:C) injected and non-injected groups. Probes with a significant effect of poly(I:C) injection were additionally filtered by fold change to identify probes different between the poly(I:C)-injected and the non-injected control groups (FC ≥ 1.5) consistently in all of the following three comparisons: poly(I:C)-naïve vs. non-injected-naïve; poly(I:C)-survivor vs. non-injected-survivor; and poly(I:C)-carrier vs. non-injected-carrier. Probes with a significant interaction between IHNV status and poly(I:C)-injection were filtered by fold change to retain probes that were differentially expressed by 1.5-fold between any two groups.
