Accepted Manuscript Integrated profiling of global metabolomic and transcriptomic responses to viral hemorrhagic septicemia virus infection in olive flounder Se-Young Cho, Yong Kook Kwon, Miso Nam, Bipin Vaidya, Seok Ryel Kim, Sunghoon Lee, Joseph Kwon, Duwoon Kim, Geum-Sook Hwang PII:
S1050-4648(17)30607-1
DOI:
10.1016/j.fsi.2017.10.007
Reference:
YFSIM 4878
To appear in:
Fish and Shellfish Immunology
Received Date: 29 May 2017 Revised Date:
30 September 2017
Accepted Date: 6 October 2017
Please cite this article as: Cho S-Y, Kwon YK, Nam M, Vaidya B, Kim SR, Lee S, Kwon J, Kim D, Hwang G-S, Integrated profiling of global metabolomic and transcriptomic responses to viral hemorrhagic septicemia virus infection in olive flounder, Fish and Shellfish Immunology (2017), doi: 10.1016/ j.fsi.2017.10.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Integrated profiling of global metabolomic and transcriptomic responses to
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viral hemorrhagic septicemia virus infection in olive flounder
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Se-Young Choa,†, Yong Kook Kwonb,†, Miso Namb,c,†, Bipin Vaidyad, Seok Ryel Kime, Sunghoon Leef
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Joseph Kwona,*, Duwoon Kimg,*, Geum-Sook Hwangb,h,*
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a
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Korea
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b
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Biological Disaster Analysis Team, Korea Basic Science Institute, Daejeon 169-148, Republic of
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Seoul 03759, Republic of Korea.
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c
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d
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e
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Incheon 400-420, South Korea
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f
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g
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South Korea
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Department of Chemistry, Sungkyunkwan University, Suwon 16419, Republic of Korea.
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EONE-DIAGNOMICS Genome Center Co. Ltd., Korea 406-840
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Department of Food Science and Technology, Chonnam National University, Gwangju 500-757,
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Department of Chemistry and Nano Science, Ewha Womans University, Seoul, Republic of Korea
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Bioenergy Research Center, Chonnam National University, Gwangju 500-757, South Korea
West Sea Fisheries Research Institute, National Fisheries Research and Development Institute,
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Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute,
These authors equally contributed to this work
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*Corresponding authors:
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Joseph Kwon: Korea Basic Science Institute, Daejeon 169-148, Republic of Korea. Email:
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[email protected] 1
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Duwoon Kim: Chonnam National University, Gwangju 500-757, South Korea. Email:
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[email protected]
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Geum-Sook Hwang: Korea Basic Science Institute, Seoul 120-140, Republic of Korea. Phone: +82-2-
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6908-6200. Fax: +82-2-6908-6239. E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Viral hemorrhagic septicemia virus (VHSV) is one of the most serious viral pathogen that
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infects farmed fish. In this study, we measured the replication of VHSV increased steadily at
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9, 24, 72, and 120 hours after infection and progression of necrosis was observed at 72 hpi.
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We performed transcriptomic and metabolomics profiling of kidney tissues collected at each
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infection
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chromatography/quadrupole time-of-flight mass spectroscopy to investigate the mechanisms
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of VHSV infection in the kidneys of olive flounder. A total of 13,862 mRNA molecules and
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72 metabolites were selected to identify the mechanisms of infection and they were integrated
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using KEGG pathway database. Six KEGG metabolic pathways, including carbohydrate
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metabolism, amino acid metabolism, lipid metabolism, transport and catabolism, metabolism
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of cofactors and vitamins, and energy metabolism, were significantly suppressed, whereas the
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immune system was activated by VHSV infection. A decrease in levels of amino acids such
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as valine, leucine, and isoleucine, as well as in their derivative carnitines, was observed after
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VHSV infection. In addition, an increase in arachidonic acid level was noted. Integrated
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analysis of transcriptome and metabolome using KEGG pathway database revealed four
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types of responses in the kidneys of olive flounder to VHSV infection. Among these, the
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mechanisms related to the immune system and protein synthesis were activated, whereas ATP
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synthesis and the antioxidant system activity were suppressed. This is the first study
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describing the mechanisms of metabolic responses to VHSV infection in olive flounder. The
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results suggest that the suppression of ATP synthesis and antioxidant systems, such as
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glutathione and peroxisome signaling, could be the cause of necrosis, whereas the activation
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of the immune system could result in the inflammation of kidney tissue in VHSV-infected
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olive flounder.
using
Illumina
HiSeq2000
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ultra-performance
liquid
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Keywords: olive flounders; viral metabolomics; integrated profiling
hemorrhagic
septicemia
virus;
transcriptomics;
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ACCEPTED MANUSCRIPT 1. Introduction
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Viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus that
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belongs to the family Rhabdoviridae, is one of the most serious virus that affect farmed fish.
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VHSV infection is listed as a notifiable disease by the World Organization for Animal Health
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[1] and has been detected in juvenile olive flounder (Paralichthys olivaceus) at an
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aquaculture farm and in wild marine fish in Korea [2, 3]. Fish infected with VHSV exhibit
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enlargement of internal organs and hemorrhages in the kidneys, skin, and muscle as well as
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high mortality rate of 40–60% [4]. Although an increasing number of studies focus on VHSV
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infection, metabolic changes in fish during VHSV infection have not yet been clearly
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elucidated.
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Several studies on effects of VHSV infection have been published. Takami et al. highlighted
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the role of interferon in the protection of olive flounder against VHSV infection [5]. Aquilino
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et al. studied changes in 19 mRNA molecules in rainbow trout gills upon infection with
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VHSV, by real-time PCR [6]. Verrier et al. reported on quantitative trait loci that were related
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to the resistance to VHSV infection in rainbow trout [7]. However, these studies did not
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clearly explain the biological mechanism of observed responses and resistance to VHSV
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infection [7]. Therefore, a comprehensive multi-omics study is needed to understand the
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biological response upon viral infection.
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Integrated analyses of omics data, including those of the transcriptome, proteome, and
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metabolome, have been performed to gain a comprehensive understanding of biological
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responses in cells or organs [8-11]. Changes in gene expression levels due to physical or
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biological stress can be visualized using transcriptomics. However, the abundance of
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transcripts does not always correlate with protein abundance and activity [12, 13], because
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the latter can vary dramatically owing to the activity of regulators, abundance of substrates
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advantage of measuring molecules that function directly in the cell, it has a limitation with
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respect to the detection in holistic profiling. This is because proteomic profiling does not
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include compounds of lower molecular weights, such as metabolites [14], which are smaller
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than products reported by transcriptome and proteome analyses in many organisms [15].
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Therefore, metabolomic data can complement proteomic and transcriptomic data, and thereby
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allow researchers to look at the holistic picture of host metabolism. Metabolome is the final
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product of gene expression that is altered by treatment, which is directly connected to the
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biochemical phenotype [10].
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Here, we focus on metabolomic and transcriptomic profiling in the kidneys of olive flounder
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to investigate biological responses to the infection with VHSV 9, 24, 72, and 120 h post-
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infection (hpi).
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2.1. Chemicals and reagents
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HPLC-grade water, acetonitrile, and ethanol were purchased from Honeywell Burdick &
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Jackson. Formic acid (99% purity) was obtained from Sigma-Aldrich.
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2.2. VHSV challenge test in olive flounder
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Olive flounder fish (108 ± 2.4 g) were obtained from a private hatchery in Yeongam, South
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Korea, and acclimatized to the experimental conditions for 24 h in the laboratory under a
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photoperiod cycle of 12 h light/12 h dark. During the experiments, UV-sterilized seawater
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was used with temperature maintained at 12.5 ± 0.2 °C, salinity of 36.2 ± 0.6 ppt. Eighty-
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eight flounder fish were randomly divided into five experimental groups of 11 fish each, and
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subjected to either VHSV infection or mock infection for 9, 24, 72, or 120 h. Each fish
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infected with the virus was intraperitoneally injected with 100 µL of the medium containing
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106.8 TCID50/mL VHSV. Mock-infected groups were treated with infection medium
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(Leibovitz’s L-15 supplemented with 2% FBS, streptomycin 100 µg/mL, and penicillin 100
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U/mL) free from the virus. These experiments were carried out in strict accordance with the
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recommendations of the Guide for Institutional Animal Care and Use issued by the
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Committee of Chonnam National University (Permit Number: CNU IACUC-YS20135).
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2.3. Sample preparation and experimental conditions
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Samples were collected at 9, 24, 72, or 120 hpi. Among the 11 flounder in each group, the
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kidney tissues of five fish were frozen at –80 °C for metabolomic analysis; three were fixed
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in 10% neutral buffered formalin (NBF) for routine histology. Total RNA was isolated
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immediately from pooled tissue of three flounder kidneys, using an RNeasy Mini Extraction
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Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, in order to
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perform quantitative real-time PCR (qRT-PCR) and transcriptomic analysis of kidney tissue
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2.4. Histological examination
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Kidney tissue was processed for routine histology after fixation in 10% NBF for 24 h. Briefly,
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the fixed tissue was dehydrated through a graded series of ethanol dilutions (gradient: 60–
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100%), cleared in xylene, and infiltrated and embedded in paraffin according to the standard
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paraffin-embedded method [16]. The tissues were sliced into 4-µm thick sections, cleared in
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xylene, dewaxed by ethanol (gradient: 100–50%), and finally washed with distilled water.
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The sections were stained with hematoxylin-eosin, rinsed in tap water, dehydrated using
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ethanol (gradient: 80–100%), cleared in xylene, mounted on glass slides, and observed with
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an Olympus BX51 microscope after air drying.
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2.5. VHSV quantification by qRT-PCR
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VHSV N gene mRNA expression level in infected kidney tissues of olive flounder was
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determined by qRT-PCR. To create the standard curve, a fragment of the N gene was
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amplified with a pair of primers, VHSV-N-ORF-F (5'-ATG GAA GGG GGA ATC CGT GC-
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3') and VHSV-N-ORF-R (5'-TTA ATC AGA GTC CCC TGG GTA GTC GT-3'), and cloned
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into the pGEM T-easy vector according to the manufacturer's protocol (Promega, Madison,
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WI, USA). The constructed plasmid DNA was used as a standard for absolute quantification.
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The copy number of the plasmid DNA template was calculated according to the plasmid
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molecular weight and then converted into copy numbers based on Avogadro's number. Ten-
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fold dilutions of the plasmid were used to create the calibration curve for absolute
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quantification of the viral samples [17]. The extracted total RNA was reverse-transcribed into
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cDNA, using a First-Strand cDNA Synthesis Kit (Beams Biotech, Suwon, Korea). For qRT-
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PCR of the specific primer pair, q-VHSV-NF (5'- ATC GAA GCC GGA ATC CTT ATG C-3')
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and q-VHSV-NR (5'- CCT TGA CGA TGT CCA TGA GGT TG-3') were designed based on a
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ACCEPTED MANUSCRIPT highly conserved region of the VHSV N gene. VHSV N-gene titration by real-time PCR was
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carried out in the Thermal Cycler Dice Real-Time System (TP850, Takara) with SYBR
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Premix Ex Tag reagent (Takara), using the following program: denaturation at 95 °C for 10
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sec, followed by 40 cycles of (95 °C for 5 sec, 60 °C for 30 sec, 1 cycle of 95 °C for 15 sec,
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60 °C for 30 sec, and 95 °C for 15 sec). VHSV N-gene copy number was calculated using a
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standard curve [18].
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2.6. RNA isolation and Illumina HiSeq2000 sequencing
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Total RNA from the kidneys was prepared using a miRNeasy Mini Kit according to the
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manufacturer’s instructions (Qiagen, Hiden, Germany). The quality of the RNA was checked
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using an Agilent 2100 Bioanalyzer (Agilent) prior to sequencing. mRNA sequencing libraries
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were generated using an mRNA Seq Sample Prep Kit following the manufacturer’s
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instructions (Illumina, San Diego, CA, USA). Poly(A) mRNA (5 µg) was isolated from total
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RNA, using oligo-DT beads and chemically fragmented. Double-stranded cDNA was
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synthesized using the fragmented mRNA as template and subjected to end-repair, adenylation
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of the 3'-end, and sequencing adapter ligation. This was followed by DNA purification with
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magnetic beads and PCR amplification. Finally, the amplified library was purified, quantified,
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and then applied for template preparation. The Illumina HiSeq2000 platform was utilized to
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generate 99-bp paired-end sequencing reads.
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2.7. Assembly and functional annotation of the transcriptome
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Transcriptome sequencing was conducted using the Illumina Hi-Seq2000 sequencing
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platform. To better assemble the entire transcriptome de novo, a paired-end sequencing
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strategy was used. All raw reads were mapped by Burrows-Wheeler Aligner (BWA) [19]. All
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sequences were examined for possible sequencing errors. Adaptor sequences and low quality
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sequences were trimmed. The raw reads were cleaned by removing adaptor-only reads and
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resulting high-quality sequences were deposited in the NCBI database and de novo assembled
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into contigs with Trinity software, as described for de novo transcriptome assembly without a
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reference genome [22]. The assembled unigenes of the flounder were performed using
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BLASTx programs against sequences in the NCBI non-redundant (nr) protein database (E-
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value ≤ 1E-5) for a sequence homology search [23]. Unigenes were tentatively identified
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according to top hits against known sequences. The transcript expression levels were
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quantified in fragments per kilobase per million mapped reads (FPKM) that measured the
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read density normalized for RNA length and total number of reads in each sample [24]. Due
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to limitations of functional annotation information associated with olive flounder disease
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models, we have employed a database of well-studied zebrafish proteins to illustrate the
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biological function changes to viral infectious disease mechanisms using the BLASTx
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algorithm [25].
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2.8. Extraction of metabolites
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Polar metabolites were extracted from kidney tissues of olive flounder using a 1:1
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methanol/water mixture [26]. From each sample, about 50 mg of kidney tissue was placed in
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a 1.5-mL tube containing 2.8-mm zirconium oxide beads. Each sample was mixed with 1 mL
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of 1:1 methanol/water mixture, and homogenized twice at 5,000 rpm for 20 s using a tissue
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grinder (Precellys 24; Bertin Technologies, Ampere Montigny-le-Bretonneux, France). The
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mixture was vortexed for 1 min, followed by centrifugation at 14,240 × g for 10 min at 4 °C.
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The upper layer was transferred in 120-µL aliquots to new 1.5-mL tubes. The supernatants
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were filtered through a 0.2-µm hydrophilic polytetrafluoroethylene syringe filter (Millex®,
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Millipore, Ireland).
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2.9. Ultra-performance liquid chromatography (UPLC)/quadrupole time-of-flight (QTOF)
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All UPLC/QTOF-MS analyses of the complex chemical components in the kidneys of olive
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flounder were performed on an ACQUITY UPLC system (Waters, Milford, MA, USA) with
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a triple TOFTM 5600 mass spectrometer equipped with an electrospray ionization source (AB
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SCIEX, Concord, ON, Canada). Chromatographic separation of samples was performed on
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an AQUTIC UPLC HSS T3 column (2.1 mm × 100 mm, 1.7 µm) at 45 °C using water
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containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The
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gradient elution program was isocratic at 0.1% B for 2 min, 0.1–25% B for 2–6 min, 25–80%
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B for 6–10 min, 80–90% B for 10–12 min, 90–99.9% B for 12–21 min, 99.9% B for 2 min,
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and then the initial conditions for 2 min. The flow rate was maintained at 0.4 mL/min.
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Data acquisition was performed with a Triple TOF 5600 system with Turbo V sources and a
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Turbo ion spray interface. QTOF-MS data was acquired in positive (negative) using an ion
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spray voltage of 5.5 kV (–4.5 kV), nebulizer gas (Gas 1) of 55 psi, heater gas (Gas 2) of 65
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psi, curtain gas of 30 psi, turbo spray temperature of 500°C, and declustering potential (DP)
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of 90 V (–90 V). MS and MS/MS were performed in positive (negative) mode using the
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UPLC/QTOF-MS equipped with Turbo V sources and a Turbo ion spray interface. For QTOF
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MS/MS data, information-dependent acquisition (IDA) was used with the following
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acquisition conditions: survey scans of 250 ms, product ion scans of 100 ms, high-resolution
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mode, DP of 90 V (−90 V), collision energy of 35 V(−35 V), and collision energy spread of
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15 V. The MS/MS analysis was acquired by automatic fragmentation, in which the five most
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intense mass peaks were fragmented. The QTOF-MS and IDA scans were operated with a
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mass range of m/z 50–1000. TOF MS and product ion calibration were performed every day
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in both high-sensitivity and high-resolution modes using a chromatography data system
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device prior to the analysis.
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All MS data information, including retention times, m/z, and ion intensities, was extracted by
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MarkerView software (AB SCIEX, Concord, Canada) incorporated in the instrument, and the
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resulting MS data were assembled into a matrix. Batch normalization [27] and median fold-
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change normalization [28] were employed to remove systematic variation between
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experimental samples. The normalized data were used for further study as raw data.
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2.11. Identification of metabolites
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The metabolites were tentatively identified by connecting to freely accessible metabolite
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databases, such as Human Metabolome Database (HMDB, (http://www.hmdb.ca/), MassBank
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(http://www.massbank.jp/), and METLIN (http://metlin.scripps.edu/). The neutral mass is the
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primary term for database search, within tolerance. Isotopic pattern similarity was used as the
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second filter to select optimal candidates, by comparing the ratios of the detected isotopes
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and matching isotopes to those with the predicted isotopic pattern in the database compound.
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As the third filter, we compared the fragment pattern of metabolites obtained from our
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experiments, with fragment patterns in databases. The m/z values of precursor ions, isotope
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pattern matching, and fragment pattern similarity were used for identification of metabolites.
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2.12. Statistical analysis
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The chi-squared test (3 ☓ 2) was employed to determine the activation or suppression of
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pathways and classes. mRNA expression patterns in each pathway or class were classified
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into three groups (positive, negative, and no correlation) and inserted into the chi-squared
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table. The correlation between mRNA quantification results and the degree of virus infection
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was assessed using Spearman’s rank correlation coefficient. mRNA results were separated
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into three groups: positive correlation (r ≥ 0.7), negative correlation (r ≤ -0.7), and no
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correlation (-0.7 < r < 0.7).
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The numbers in the comparison group were generated using a 12
ACCEPTED MANUSCRIPT random function. The generated random numbers were correlated with the degree of virus
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infection. The numbers in the positive correlation (r ≥ 0.7), negative correlation (r ≤ -0.7),
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and no correlation groups were counted. The correlation between the number of genes in each
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pathway and the degree of virus infection was calculated, and classified into three groups
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(positive correlation: r ≥ 0.7, negative correlation: r ≤ -0.7, no correlation -0.7 < r < 0.7). The
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Jonckheere-Terpstra test was performed to determine an order of metabolites by infection
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time. The correlation between transcripts and metabolites in major pathways was assessed
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using Pearson correlation coefficient. Calculations of the Spearman’s rank correlation
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coefficient, chi-squared test, and Jonckheere-Terpstra test were performed using R program
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(version 2.13.1). Calculation of the Pearson correlation coefficient was performed using
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python (version 2.7.13).
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3.1. Changes in VHSV N gene expression and histology of kidney tissue in VHSV infection
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To validate the infection of flounder by VHSV, we carried out VHSV N gene expression
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analysis by qRT-PCR. The copy number of the VHSV N gene gradually increased from 9 to
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120 hpi in the kidneys of olive flounder (Figure 1A) and reached maximum replication
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capacity at 120 hpi. Flounder fish injected with VHSV exhibited necrosis and inflammation
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of the kidney tissue, as revealed by histopathological investigations (Figure 1B–F). Necrotic
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foci were found at low levels between 9 and 24 hpi. As inflammation increased from 72 to
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120 hpi, necrotic foci became more prominent in the kidney tissue.
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3.2. De novo assembly and functional annotation
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Transcript information for VHSV-infected flounder (P. olivaceus) was characterized by
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constructing a cDNA library prepared from purified mRNA isolated from kidney tissue. The
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Illumina Hi-Seq 2000 sequencing platform generated a total of 215,871,162 raw reads, and
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the raw reads were then filtered to remove adaptor sequences. After trimming the adaptor
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sequences and low-quality reads, 186,446,536 clean reads were generated. The clean reads
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obtained using the Illumina Hi-Seq 2000 transcriptome sequencing platform constituted 86.3%
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of the raw reads. The P. olivaceus transcriptome sequencing and assembly statistics are
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shown in Table 1, Table S1-S3, and Figure S1. Using Trinity software [29], the clean reads
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were assembled into 336,692 contigs with an average length of 1,375 bp. The contigs
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generated in this analysis were further assembled into 278,578 unigenes with a minimum
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length of 200 bp and an average read length of 1,077 bp with lengths ranging from 200 bp to
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over 2,900 bp using Trinity software. The length distribution of the assembled unigenes is
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displayed in Figure S2. Our RNA-Seq analysis of P. olivaceus provided more than 96,627
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previously reported unigenes in comparison with the same species of RNA-Seq analysis [30].
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the nr (non-redundant protein) database. For biological function annotation, the olive
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flounder transcriptomic sequence was compared with the zebrafish (D. rerio) nr database of
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NCBI and 22,517 genes were generated by a similarly mapped zebrafish gene symbol (within
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E-value < 1E-10) (Table S4). It has further been used to determine the VHSV inducible
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disease mechanism studies. Each RNA-seq sample was mapped to the de novo assembled
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contig, the FPKM (Fragments per kilobase per million mapped reads) values of the respective
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contig were summed by their respective gene symbols, and their expression levels were
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quantified by the FPKM value. Information on the 13,862 mRNA that can be used to
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compare gene expression levels tagged with the D. rerio gene symbols were provided in
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Table S5.
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3.3. Changes in metabolomic pathways in VHSV-infected olive flounder by KEGG pathway
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enrichment analysis
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Zebrafish pathway database in KEGG was employed to analyze olive flounder
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transcriptome, which was divided into metabolic classes and pathways, with several pathways
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being representative of a metabolic class. The numbers of mRNA molecules whose
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expression became higher or lower after virus infection were compared to examine the
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activation or suppression of each class and each pathway. The expression levels of
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significantly affected mRNA species in the metabolic classes are displayed in Figure 2A and
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Table S6 showed significantly changed mRNA by virus infection. mRNA expression was
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consistent in the early stage (up to 24 h) of the infection, but was altered during the later
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stages (72 to 120 h) of infection. Metabolic classes, such as carbohydrate metabolism, lipid
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metabolism, amino acid metabolism, metabolism of cofactors and vitamins, transport and
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catabolism, energy metabolism, and metabolism of other amino acids in the class database
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were significantly suppressed, whereas the immune system was activated after VHSV
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infection. The chi-squared test was employed to measure the significance of mRNA changes
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in each class, and the results are summarized in Table 2. The pathways in classes with significantly altered mRNA levels were also analyzed to
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measure the significance of each pathway by the chi-squared test. Glycolysis-
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gluconeogenesis, pentose phosphate metabolism, propanoate metabolism, butanoate
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metabolism, pyruvate metabolism, and fructose and mannose metabolism, which are included
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into carbohydrate metabolism class, were suppressed (p < 0.1). Steroid biosynthesis,
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synthesis and degradation of ketone bodies, and fatty acid degradation were suppressed in
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lipid metabolism class (p < 0.1). Oxidative phosphorylation and nitrogen metabolism were
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suppressed (p < 0.1), and biosynthesis of ATP was decreased owing to the suppression of
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oxidative phosphorylation (p < 0.1). Valine, leucine, and isoleucine degradation, arginine and
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proline metabolism, alanine, aspartate, and glutamate metabolism, tyrosine metabolism,
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phenylalanine metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis were
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significantly suppressed in amino acid metabolism class (p < 0.1). One carbon pool by folate
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pathway was suppressed in metabolism of cofactors and vitamins class after VHSV infection
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(p < 0.1). Glutathione metabolism was suppressed in metabolism of other amino acids class
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(p < 0.1). Peroxisome pathway in transport and catabolism class was suppressed (p < 0.1).
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Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, and toll-like
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receptor signaling pathway were activated in immune system class following virus infection
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(p < 0.1). Furthermore, amino acyl-tRNA biosynthesis was activated after virus infection (p
S1050-4648(17)30607-1
DOI:
10.1016/j.fsi.2017.10.007
Reference:
YFSIM 4878
To appear in:
Fish and Shellfish Immunology
Received Date: 29 May 2017 Revised Date:
30 September 2017
Accepted Date: 6 October 2017
Please cite this article as: Cho S-Y, Kwon YK, Nam M, Vaidya B, Kim SR, Lee S, Kwon J, Kim D, Hwang G-S, Integrated profiling of global metabolomic and transcriptomic responses to viral hemorrhagic septicemia virus infection in olive flounder, Fish and Shellfish Immunology (2017), doi: 10.1016/ j.fsi.2017.10.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Integrated profiling of global metabolomic and transcriptomic responses to
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viral hemorrhagic septicemia virus infection in olive flounder
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Se-Young Choa,†, Yong Kook Kwonb,†, Miso Namb,c,†, Bipin Vaidyad, Seok Ryel Kime, Sunghoon Leef
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Joseph Kwona,*, Duwoon Kimg,*, Geum-Sook Hwangb,h,*
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a
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Korea
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b
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Biological Disaster Analysis Team, Korea Basic Science Institute, Daejeon 169-148, Republic of
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Seoul 03759, Republic of Korea.
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c
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d
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e
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Incheon 400-420, South Korea
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f
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g
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South Korea
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Department of Chemistry, Sungkyunkwan University, Suwon 16419, Republic of Korea.
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EONE-DIAGNOMICS Genome Center Co. Ltd., Korea 406-840
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Department of Food Science and Technology, Chonnam National University, Gwangju 500-757,
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Department of Chemistry and Nano Science, Ewha Womans University, Seoul, Republic of Korea
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Bioenergy Research Center, Chonnam National University, Gwangju 500-757, South Korea
West Sea Fisheries Research Institute, National Fisheries Research and Development Institute,
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Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute,
These authors equally contributed to this work
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*Corresponding authors:
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Joseph Kwon: Korea Basic Science Institute, Daejeon 169-148, Republic of Korea. Email:
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[email protected] 1
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Duwoon Kim: Chonnam National University, Gwangju 500-757, South Korea. Email:
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[email protected]
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Geum-Sook Hwang: Korea Basic Science Institute, Seoul 120-140, Republic of Korea. Phone: +82-2-
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6908-6200. Fax: +82-2-6908-6239. E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Viral hemorrhagic septicemia virus (VHSV) is one of the most serious viral pathogen that
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infects farmed fish. In this study, we measured the replication of VHSV increased steadily at
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9, 24, 72, and 120 hours after infection and progression of necrosis was observed at 72 hpi.
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We performed transcriptomic and metabolomics profiling of kidney tissues collected at each
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infection
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chromatography/quadrupole time-of-flight mass spectroscopy to investigate the mechanisms
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of VHSV infection in the kidneys of olive flounder. A total of 13,862 mRNA molecules and
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72 metabolites were selected to identify the mechanisms of infection and they were integrated
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using KEGG pathway database. Six KEGG metabolic pathways, including carbohydrate
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metabolism, amino acid metabolism, lipid metabolism, transport and catabolism, metabolism
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of cofactors and vitamins, and energy metabolism, were significantly suppressed, whereas the
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immune system was activated by VHSV infection. A decrease in levels of amino acids such
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as valine, leucine, and isoleucine, as well as in their derivative carnitines, was observed after
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VHSV infection. In addition, an increase in arachidonic acid level was noted. Integrated
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analysis of transcriptome and metabolome using KEGG pathway database revealed four
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types of responses in the kidneys of olive flounder to VHSV infection. Among these, the
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mechanisms related to the immune system and protein synthesis were activated, whereas ATP
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synthesis and the antioxidant system activity were suppressed. This is the first study
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describing the mechanisms of metabolic responses to VHSV infection in olive flounder. The
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results suggest that the suppression of ATP synthesis and antioxidant systems, such as
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glutathione and peroxisome signaling, could be the cause of necrosis, whereas the activation
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of the immune system could result in the inflammation of kidney tissue in VHSV-infected
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olive flounder.
using
Illumina
HiSeq2000
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ultra-performance
liquid
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Keywords: olive flounders; viral metabolomics; integrated profiling
hemorrhagic
septicemia
virus;
transcriptomics;
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ACCEPTED MANUSCRIPT 1. Introduction
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Viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus that
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belongs to the family Rhabdoviridae, is one of the most serious virus that affect farmed fish.
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VHSV infection is listed as a notifiable disease by the World Organization for Animal Health
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[1] and has been detected in juvenile olive flounder (Paralichthys olivaceus) at an
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aquaculture farm and in wild marine fish in Korea [2, 3]. Fish infected with VHSV exhibit
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enlargement of internal organs and hemorrhages in the kidneys, skin, and muscle as well as
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high mortality rate of 40–60% [4]. Although an increasing number of studies focus on VHSV
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infection, metabolic changes in fish during VHSV infection have not yet been clearly
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elucidated.
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Several studies on effects of VHSV infection have been published. Takami et al. highlighted
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the role of interferon in the protection of olive flounder against VHSV infection [5]. Aquilino
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et al. studied changes in 19 mRNA molecules in rainbow trout gills upon infection with
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VHSV, by real-time PCR [6]. Verrier et al. reported on quantitative trait loci that were related
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to the resistance to VHSV infection in rainbow trout [7]. However, these studies did not
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clearly explain the biological mechanism of observed responses and resistance to VHSV
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infection [7]. Therefore, a comprehensive multi-omics study is needed to understand the
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biological response upon viral infection.
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Integrated analyses of omics data, including those of the transcriptome, proteome, and
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metabolome, have been performed to gain a comprehensive understanding of biological
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responses in cells or organs [8-11]. Changes in gene expression levels due to physical or
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biological stress can be visualized using transcriptomics. However, the abundance of
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transcripts does not always correlate with protein abundance and activity [12, 13], because
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the latter can vary dramatically owing to the activity of regulators, abundance of substrates
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ACCEPTED MANUSCRIPT and products, and reaction conditions. Moreover, although proteomic profiling has the
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advantage of measuring molecules that function directly in the cell, it has a limitation with
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respect to the detection in holistic profiling. This is because proteomic profiling does not
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include compounds of lower molecular weights, such as metabolites [14], which are smaller
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than products reported by transcriptome and proteome analyses in many organisms [15].
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Therefore, metabolomic data can complement proteomic and transcriptomic data, and thereby
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allow researchers to look at the holistic picture of host metabolism. Metabolome is the final
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product of gene expression that is altered by treatment, which is directly connected to the
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biochemical phenotype [10].
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Here, we focus on metabolomic and transcriptomic profiling in the kidneys of olive flounder
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to investigate biological responses to the infection with VHSV 9, 24, 72, and 120 h post-
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infection (hpi).
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ACCEPTED MANUSCRIPT 2. Materials and Methods
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2.1. Chemicals and reagents
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HPLC-grade water, acetonitrile, and ethanol were purchased from Honeywell Burdick &
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Jackson. Formic acid (99% purity) was obtained from Sigma-Aldrich.
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2.2. VHSV challenge test in olive flounder
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Olive flounder fish (108 ± 2.4 g) were obtained from a private hatchery in Yeongam, South
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Korea, and acclimatized to the experimental conditions for 24 h in the laboratory under a
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photoperiod cycle of 12 h light/12 h dark. During the experiments, UV-sterilized seawater
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was used with temperature maintained at 12.5 ± 0.2 °C, salinity of 36.2 ± 0.6 ppt. Eighty-
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eight flounder fish were randomly divided into five experimental groups of 11 fish each, and
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subjected to either VHSV infection or mock infection for 9, 24, 72, or 120 h. Each fish
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infected with the virus was intraperitoneally injected with 100 µL of the medium containing
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106.8 TCID50/mL VHSV. Mock-infected groups were treated with infection medium
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(Leibovitz’s L-15 supplemented with 2% FBS, streptomycin 100 µg/mL, and penicillin 100
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U/mL) free from the virus. These experiments were carried out in strict accordance with the
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recommendations of the Guide for Institutional Animal Care and Use issued by the
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Committee of Chonnam National University (Permit Number: CNU IACUC-YS20135).
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2.3. Sample preparation and experimental conditions
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Samples were collected at 9, 24, 72, or 120 hpi. Among the 11 flounder in each group, the
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kidney tissues of five fish were frozen at –80 °C for metabolomic analysis; three were fixed
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in 10% neutral buffered formalin (NBF) for routine histology. Total RNA was isolated
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immediately from pooled tissue of three flounder kidneys, using an RNeasy Mini Extraction
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Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, in order to
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perform quantitative real-time PCR (qRT-PCR) and transcriptomic analysis of kidney tissue
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2.4. Histological examination
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Kidney tissue was processed for routine histology after fixation in 10% NBF for 24 h. Briefly,
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the fixed tissue was dehydrated through a graded series of ethanol dilutions (gradient: 60–
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100%), cleared in xylene, and infiltrated and embedded in paraffin according to the standard
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paraffin-embedded method [16]. The tissues were sliced into 4-µm thick sections, cleared in
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xylene, dewaxed by ethanol (gradient: 100–50%), and finally washed with distilled water.
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The sections were stained with hematoxylin-eosin, rinsed in tap water, dehydrated using
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ethanol (gradient: 80–100%), cleared in xylene, mounted on glass slides, and observed with
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an Olympus BX51 microscope after air drying.
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2.5. VHSV quantification by qRT-PCR
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VHSV N gene mRNA expression level in infected kidney tissues of olive flounder was
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determined by qRT-PCR. To create the standard curve, a fragment of the N gene was
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amplified with a pair of primers, VHSV-N-ORF-F (5'-ATG GAA GGG GGA ATC CGT GC-
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3') and VHSV-N-ORF-R (5'-TTA ATC AGA GTC CCC TGG GTA GTC GT-3'), and cloned
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into the pGEM T-easy vector according to the manufacturer's protocol (Promega, Madison,
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WI, USA). The constructed plasmid DNA was used as a standard for absolute quantification.
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The copy number of the plasmid DNA template was calculated according to the plasmid
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molecular weight and then converted into copy numbers based on Avogadro's number. Ten-
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fold dilutions of the plasmid were used to create the calibration curve for absolute
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quantification of the viral samples [17]. The extracted total RNA was reverse-transcribed into
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cDNA, using a First-Strand cDNA Synthesis Kit (Beams Biotech, Suwon, Korea). For qRT-
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PCR of the specific primer pair, q-VHSV-NF (5'- ATC GAA GCC GGA ATC CTT ATG C-3')
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and q-VHSV-NR (5'- CCT TGA CGA TGT CCA TGA GGT TG-3') were designed based on a
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ACCEPTED MANUSCRIPT highly conserved region of the VHSV N gene. VHSV N-gene titration by real-time PCR was
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carried out in the Thermal Cycler Dice Real-Time System (TP850, Takara) with SYBR
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Premix Ex Tag reagent (Takara), using the following program: denaturation at 95 °C for 10
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sec, followed by 40 cycles of (95 °C for 5 sec, 60 °C for 30 sec, 1 cycle of 95 °C for 15 sec,
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60 °C for 30 sec, and 95 °C for 15 sec). VHSV N-gene copy number was calculated using a
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standard curve [18].
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2.6. RNA isolation and Illumina HiSeq2000 sequencing
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Total RNA from the kidneys was prepared using a miRNeasy Mini Kit according to the
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manufacturer’s instructions (Qiagen, Hiden, Germany). The quality of the RNA was checked
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using an Agilent 2100 Bioanalyzer (Agilent) prior to sequencing. mRNA sequencing libraries
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were generated using an mRNA Seq Sample Prep Kit following the manufacturer’s
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instructions (Illumina, San Diego, CA, USA). Poly(A) mRNA (5 µg) was isolated from total
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RNA, using oligo-DT beads and chemically fragmented. Double-stranded cDNA was
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synthesized using the fragmented mRNA as template and subjected to end-repair, adenylation
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of the 3'-end, and sequencing adapter ligation. This was followed by DNA purification with
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magnetic beads and PCR amplification. Finally, the amplified library was purified, quantified,
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and then applied for template preparation. The Illumina HiSeq2000 platform was utilized to
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generate 99-bp paired-end sequencing reads.
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2.7. Assembly and functional annotation of the transcriptome
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Transcriptome sequencing was conducted using the Illumina Hi-Seq2000 sequencing
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platform. To better assemble the entire transcriptome de novo, a paired-end sequencing
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strategy was used. All raw reads were mapped by Burrows-Wheeler Aligner (BWA) [19]. All
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sequences were examined for possible sequencing errors. Adaptor sequences and low quality
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sequences were trimmed. The raw reads were cleaned by removing adaptor-only reads and
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ACCEPTED MANUSCRIPT low-quality reads (Phred qualiy score < 20) using the the Fastq_filter software [20, 21]. The
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resulting high-quality sequences were deposited in the NCBI database and de novo assembled
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into contigs with Trinity software, as described for de novo transcriptome assembly without a
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reference genome [22]. The assembled unigenes of the flounder were performed using
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BLASTx programs against sequences in the NCBI non-redundant (nr) protein database (E-
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value ≤ 1E-5) for a sequence homology search [23]. Unigenes were tentatively identified
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according to top hits against known sequences. The transcript expression levels were
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quantified in fragments per kilobase per million mapped reads (FPKM) that measured the
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read density normalized for RNA length and total number of reads in each sample [24]. Due
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to limitations of functional annotation information associated with olive flounder disease
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models, we have employed a database of well-studied zebrafish proteins to illustrate the
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biological function changes to viral infectious disease mechanisms using the BLASTx
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algorithm [25].
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2.8. Extraction of metabolites
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Polar metabolites were extracted from kidney tissues of olive flounder using a 1:1
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methanol/water mixture [26]. From each sample, about 50 mg of kidney tissue was placed in
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a 1.5-mL tube containing 2.8-mm zirconium oxide beads. Each sample was mixed with 1 mL
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of 1:1 methanol/water mixture, and homogenized twice at 5,000 rpm for 20 s using a tissue
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grinder (Precellys 24; Bertin Technologies, Ampere Montigny-le-Bretonneux, France). The
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mixture was vortexed for 1 min, followed by centrifugation at 14,240 × g for 10 min at 4 °C.
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The upper layer was transferred in 120-µL aliquots to new 1.5-mL tubes. The supernatants
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were filtered through a 0.2-µm hydrophilic polytetrafluoroethylene syringe filter (Millex®,
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Millipore, Ireland).
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2.9. Ultra-performance liquid chromatography (UPLC)/quadrupole time-of-flight (QTOF)
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All UPLC/QTOF-MS analyses of the complex chemical components in the kidneys of olive
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flounder were performed on an ACQUITY UPLC system (Waters, Milford, MA, USA) with
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a triple TOFTM 5600 mass spectrometer equipped with an electrospray ionization source (AB
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SCIEX, Concord, ON, Canada). Chromatographic separation of samples was performed on
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an AQUTIC UPLC HSS T3 column (2.1 mm × 100 mm, 1.7 µm) at 45 °C using water
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containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The
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gradient elution program was isocratic at 0.1% B for 2 min, 0.1–25% B for 2–6 min, 25–80%
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B for 6–10 min, 80–90% B for 10–12 min, 90–99.9% B for 12–21 min, 99.9% B for 2 min,
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and then the initial conditions for 2 min. The flow rate was maintained at 0.4 mL/min.
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Data acquisition was performed with a Triple TOF 5600 system with Turbo V sources and a
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Turbo ion spray interface. QTOF-MS data was acquired in positive (negative) using an ion
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spray voltage of 5.5 kV (–4.5 kV), nebulizer gas (Gas 1) of 55 psi, heater gas (Gas 2) of 65
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psi, curtain gas of 30 psi, turbo spray temperature of 500°C, and declustering potential (DP)
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of 90 V (–90 V). MS and MS/MS were performed in positive (negative) mode using the
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UPLC/QTOF-MS equipped with Turbo V sources and a Turbo ion spray interface. For QTOF
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MS/MS data, information-dependent acquisition (IDA) was used with the following
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acquisition conditions: survey scans of 250 ms, product ion scans of 100 ms, high-resolution
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mode, DP of 90 V (−90 V), collision energy of 35 V(−35 V), and collision energy spread of
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15 V. The MS/MS analysis was acquired by automatic fragmentation, in which the five most
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intense mass peaks were fragmented. The QTOF-MS and IDA scans were operated with a
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mass range of m/z 50–1000. TOF MS and product ion calibration were performed every day
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in both high-sensitivity and high-resolution modes using a chromatography data system
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device prior to the analysis.
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All MS data information, including retention times, m/z, and ion intensities, was extracted by
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MarkerView software (AB SCIEX, Concord, Canada) incorporated in the instrument, and the
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resulting MS data were assembled into a matrix. Batch normalization [27] and median fold-
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change normalization [28] were employed to remove systematic variation between
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experimental samples. The normalized data were used for further study as raw data.
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2.11. Identification of metabolites
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The metabolites were tentatively identified by connecting to freely accessible metabolite
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databases, such as Human Metabolome Database (HMDB, (http://www.hmdb.ca/), MassBank
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(http://www.massbank.jp/), and METLIN (http://metlin.scripps.edu/). The neutral mass is the
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primary term for database search, within tolerance. Isotopic pattern similarity was used as the
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second filter to select optimal candidates, by comparing the ratios of the detected isotopes
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and matching isotopes to those with the predicted isotopic pattern in the database compound.
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As the third filter, we compared the fragment pattern of metabolites obtained from our
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experiments, with fragment patterns in databases. The m/z values of precursor ions, isotope
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pattern matching, and fragment pattern similarity were used for identification of metabolites.
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2.12. Statistical analysis
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The chi-squared test (3 ☓ 2) was employed to determine the activation or suppression of
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pathways and classes. mRNA expression patterns in each pathway or class were classified
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into three groups (positive, negative, and no correlation) and inserted into the chi-squared
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table. The correlation between mRNA quantification results and the degree of virus infection
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was assessed using Spearman’s rank correlation coefficient. mRNA results were separated
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into three groups: positive correlation (r ≥ 0.7), negative correlation (r ≤ -0.7), and no
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correlation (-0.7 < r < 0.7).
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The numbers in the comparison group were generated using a 12
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infection. The numbers in the positive correlation (r ≥ 0.7), negative correlation (r ≤ -0.7),
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and no correlation groups were counted. The correlation between the number of genes in each
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pathway and the degree of virus infection was calculated, and classified into three groups
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(positive correlation: r ≥ 0.7, negative correlation: r ≤ -0.7, no correlation -0.7 < r < 0.7). The
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Jonckheere-Terpstra test was performed to determine an order of metabolites by infection
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time. The correlation between transcripts and metabolites in major pathways was assessed
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using Pearson correlation coefficient. Calculations of the Spearman’s rank correlation
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coefficient, chi-squared test, and Jonckheere-Terpstra test were performed using R program
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(version 2.13.1). Calculation of the Pearson correlation coefficient was performed using
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python (version 2.7.13).
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3.1. Changes in VHSV N gene expression and histology of kidney tissue in VHSV infection
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To validate the infection of flounder by VHSV, we carried out VHSV N gene expression
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analysis by qRT-PCR. The copy number of the VHSV N gene gradually increased from 9 to
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120 hpi in the kidneys of olive flounder (Figure 1A) and reached maximum replication
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capacity at 120 hpi. Flounder fish injected with VHSV exhibited necrosis and inflammation
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of the kidney tissue, as revealed by histopathological investigations (Figure 1B–F). Necrotic
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foci were found at low levels between 9 and 24 hpi. As inflammation increased from 72 to
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120 hpi, necrotic foci became more prominent in the kidney tissue.
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3.2. De novo assembly and functional annotation
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Transcript information for VHSV-infected flounder (P. olivaceus) was characterized by
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constructing a cDNA library prepared from purified mRNA isolated from kidney tissue. The
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Illumina Hi-Seq 2000 sequencing platform generated a total of 215,871,162 raw reads, and
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the raw reads were then filtered to remove adaptor sequences. After trimming the adaptor
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sequences and low-quality reads, 186,446,536 clean reads were generated. The clean reads
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obtained using the Illumina Hi-Seq 2000 transcriptome sequencing platform constituted 86.3%
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of the raw reads. The P. olivaceus transcriptome sequencing and assembly statistics are
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shown in Table 1, Table S1-S3, and Figure S1. Using Trinity software [29], the clean reads
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were assembled into 336,692 contigs with an average length of 1,375 bp. The contigs
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generated in this analysis were further assembled into 278,578 unigenes with a minimum
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length of 200 bp and an average read length of 1,077 bp with lengths ranging from 200 bp to
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over 2,900 bp using Trinity software. The length distribution of the assembled unigenes is
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displayed in Figure S2. Our RNA-Seq analysis of P. olivaceus provided more than 96,627
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previously reported unigenes in comparison with the same species of RNA-Seq analysis [30].
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the nr (non-redundant protein) database. For biological function annotation, the olive
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flounder transcriptomic sequence was compared with the zebrafish (D. rerio) nr database of
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NCBI and 22,517 genes were generated by a similarly mapped zebrafish gene symbol (within
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E-value < 1E-10) (Table S4). It has further been used to determine the VHSV inducible
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disease mechanism studies. Each RNA-seq sample was mapped to the de novo assembled
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contig, the FPKM (Fragments per kilobase per million mapped reads) values of the respective
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contig were summed by their respective gene symbols, and their expression levels were
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quantified by the FPKM value. Information on the 13,862 mRNA that can be used to
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compare gene expression levels tagged with the D. rerio gene symbols were provided in
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Table S5.
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3.3. Changes in metabolomic pathways in VHSV-infected olive flounder by KEGG pathway
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enrichment analysis
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Zebrafish pathway database in KEGG was employed to analyze olive flounder
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transcriptome, which was divided into metabolic classes and pathways, with several pathways
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being representative of a metabolic class. The numbers of mRNA molecules whose
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expression became higher or lower after virus infection were compared to examine the
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activation or suppression of each class and each pathway. The expression levels of
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significantly affected mRNA species in the metabolic classes are displayed in Figure 2A and
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Table S6 showed significantly changed mRNA by virus infection. mRNA expression was
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consistent in the early stage (up to 24 h) of the infection, but was altered during the later
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stages (72 to 120 h) of infection. Metabolic classes, such as carbohydrate metabolism, lipid
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metabolism, amino acid metabolism, metabolism of cofactors and vitamins, transport and
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catabolism, energy metabolism, and metabolism of other amino acids in the class database
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were significantly suppressed, whereas the immune system was activated after VHSV
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infection. The chi-squared test was employed to measure the significance of mRNA changes
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in each class, and the results are summarized in Table 2. The pathways in classes with significantly altered mRNA levels were also analyzed to
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measure the significance of each pathway by the chi-squared test. Glycolysis-
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gluconeogenesis, pentose phosphate metabolism, propanoate metabolism, butanoate
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metabolism, pyruvate metabolism, and fructose and mannose metabolism, which are included
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into carbohydrate metabolism class, were suppressed (p < 0.1). Steroid biosynthesis,
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synthesis and degradation of ketone bodies, and fatty acid degradation were suppressed in
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lipid metabolism class (p < 0.1). Oxidative phosphorylation and nitrogen metabolism were
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suppressed (p < 0.1), and biosynthesis of ATP was decreased owing to the suppression of
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oxidative phosphorylation (p < 0.1). Valine, leucine, and isoleucine degradation, arginine and
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proline metabolism, alanine, aspartate, and glutamate metabolism, tyrosine metabolism,
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phenylalanine metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis were
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significantly suppressed in amino acid metabolism class (p < 0.1). One carbon pool by folate
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pathway was suppressed in metabolism of cofactors and vitamins class after VHSV infection
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(p < 0.1). Glutathione metabolism was suppressed in metabolism of other amino acids class
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(p < 0.1). Peroxisome pathway in transport and catabolism class was suppressed (p < 0.1).
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Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, and toll-like
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receptor signaling pathway were activated in immune system class following virus infection
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(p < 0.1). Furthermore, amino acyl-tRNA biosynthesis was activated after virus infection (p
