proteomic analysis of crucian carp Carassius auratus gibelio in cyprinid herpesvirus 2 infection

Accepted Manuscript Combined transcriptomic/proteomic analysis of crucian carp Carassius auratus gibelio in cyprinid herpesvirus 2 infection Min Liu, Ting Wu, Shuang Li, Panpan Wei, Yuye Yan, Wei Gu, Wen Wang, Qingguo Meng PII:

S1050-4648(18)30465-0

DOI:

10.1016/j.fsi.2018.07.057

Reference:

YFSIM 5457

To appear in:

Fish and Shellfish Immunology

Received Date: 14 April 2018 Revised Date:

10 May 2018

Accepted Date: 28 July 2018

Please cite this article as: Liu M, Wu T, Li S, Wei P, Yan Y, Gu W, Wang W, Meng Q, Combined transcriptomic/proteomic analysis of crucian carp Carassius auratus gibelio in cyprinid herpesvirus 2 infection, Fish and Shellfish Immunology (2018), doi: 10.1016/j.fsi.2018.07.057. 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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ACCEPTED MANUSCRIPT 1

Combined transcriptomic/proteomic analysis of crucian carp Carassius

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auratus gibelio in cyprinid herpesvirus 2 infection

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Min Liu a, Ting Wu a, b, Shuang Lia, Panpan Wei a, Yuye Yan a, Wei Gu a, c, Wen Wang a,

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Qingguo Meng a, c,*

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a

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Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing

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Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key

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Normal University, 1 Wenyuan Road, Nanjing 210023, China

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b

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30# Yeting East Road, Baoying 225800, China

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c

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Lianyungang, Jiangsu 222005, China

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Baoying Center for Control and Prevention of Aquatic Animal Infectious Disease,

Co-Innovation Center for Marine Bio-Industry Technology of Jiangsu Province,

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*

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Tel: +86-25-85891955.

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E-mail address: [email protected]

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Corresponding authors: Qingguo Meng

ACCEPTED MANUSCRIPT Abstract

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Cyprinid herpesvirus 2 (CyHV-2) is a pathogen of herpesviral hematopoietic necrosis disease

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of crucian carp. Our study aimed to investigate the molecular mechanisms and immune

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response at the mRNA and protein levels in head kidney during CyHV-2 infection. Three days

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after infection with CyHV-2, 7,085 differentially expressed genes were identified by

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transcriptome sequencing, of which 3,090 were up-regulated and 3,995 were down-regulated.

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And 338 differentially expressed proteins including 277 up-regulated and 61 down-regulated

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were identified using tandem mass tag labeling followed by liquid chromatography tandem

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mass spectrometry. Notably, 128 differentially co-expressed genes at mRNA and protein

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levels (cDEGs)

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co-down-regulated. In addition, 10 cDGEs in the above pathways were selected for qRT-PCR

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to confirm the validity of the transcriptome and proteome changes by showing that RIG-I,

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MDA5, LGP2, FAS, PKR and PKZ up-regulated and Integrin α, Integrin β2, NCF2 and NCF4

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down-regulated. This indicated that after CyHV-2 infection, the herpes simplex infection

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pathway, RIG-I like receptor signaling pathway, necroptosis pathway and p53 signaling

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pathway were activated and the phagosome pathway was suppressed. Our findings reveal the

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pathogenesis and the host immune mechanism of CyHV-2 infection of crucian carp.

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Keywords: Crucian carp; Carassius auratus gibelio; Cyprinid herpesvirus 2; Transcriptome;

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Proteome

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co-up-regulated

and

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were reliably quantified,

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1. Introduction Crucian carp Carassius auratus gibelio is one of the main freshwater aquaculture species

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in China due to its high commercial value and suitability for use in a variety of culture

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systems [1]. However, with rapid development of intensive culture, viral and bacterial

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infections are causing a serious damage to the aquaculture industry resulting in heavy

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economic damage [2-4]. In particular, the herpesviral hematopoietic necrosis (HVHN) disease

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caused by CyHV-2 has caused enormous economic losses in recent years [5-7]. Epidemics

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caused by CyHV-2 in domestic cyprinid species have been reported in both Asian and

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European countries [3, 8, 9]. CyHV-2 is an emerging pathogen that causes HVHN in fish [10,

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11]. And this disease occurs primarily in the spring and autumn and is affected by water

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temperatures of 15-25 ℃, which is the susceptibility of the fish to CyHV-2 infection. But the

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pathogenesis mechanisms remain unknown, acute CyHV-2 infections generally result in high

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mortality, and the surviving fish become chronic carriers displaying no external clinical signs.

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As a consequence, understanding the host immune response against viruses in fish is critical

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to develop prophylactic and preventive control measures [12].

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Crucian carp Carassius auratus gibelio, belonging to the family Cyprinidae, is the

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lowest equivalent vertebrate with both innate and adaptive immune systems [13,14]. However,

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the innate immunity is stronger than adaptive immunity and constitutes the first line of the

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host defense after pathogen invasion [15-18]. And the innate immune response represents an

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important part of the host defense mechanism preventing viral replication after infection

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because fish possess evolutionary conserved pattern recognition receptors (PRRs) that are

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responsible for sensing the presence of pathogen-associated molecular patterns (PAMPs)

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ACCEPTED MANUSCRIPT which are structurally conserved among many microorganisms and playing an essential role

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in the innate immune response by sensing pathogens and activating appropriate immune

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signaling [12, 19-21]. These PRRs mainly contain RIG-I-like receptors (RLRs), toll-like

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receptors (TLRs), and NOD-like receptors (NLRs) which are directly involved in the

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activation of the interferon (IFN) system. Among those, cytosolic RLRs can recognize viral

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nucleic acids and trigger the innate immune responses. The current research evidence shows

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that fish and mammals are fairly close in molecular composition and function of immunity,

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however, there are significant morphological changes [22]. The thymus, kidney and spleen are

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the major lymphoid organs of teleost fish [23]. Studies have shown that head kidney is an

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important immune tissue in teleost fish, functionally equivalent to the bone narrow in

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mammals [24]. In teleost fish, head kidney contains a large number of T and B lymphocytes,

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macrophages and granulocytes that is an important organ involved in innate immunity, and is

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also the major source of antibody production [25-28]. Therefore, the head kidneys have

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important significances for the research of the immune mechanism of fish.

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With the development of modern biological technology, there are many methods that can

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be used to study the pathogenic mechanism. Transcriptome sequencing of cDNA using

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Illumina technology has become an efficient strategy for generating enormous sequences that

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represent expressed genes [29-31]. Researchers have applied transcriptomic analysis to

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research the pathogenic mechanism of fish viral diseases to cope with infections by different

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pathogens, such as the transcripts in Sarmo sala infected with salmonid alphavirus (SAV-1)

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and salmon alphavirus subtype 3 (SAV-3) [32, 33], in Sarmo sala head-kidney, liver , spleen

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and gills upon infectious salmon anaemia virus (ISAV) infection [34, 35]，in the Cyprinus

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ACCEPTED MANUSCRIPT carpio head kidney response to cyprinid herpesvirus 3 (CyHV-3) infection [36], in the Asian

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seabass epithelial cells response to nervous necrosis virus (NNV) infection [37], in the

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rainbow trout gills and zebrafish fin upon channel catfish virus (CCV) infection [38,39] and

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in the rainbow trout gills response to response to viral hemorrhagic septicemia virus (VHSV)

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infection [40], ect. Meanwhile, the executor of animal life activities is protein, so the study of

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proteome can better define the pathogenesis. Mass spectrometry (MS)-based protein

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quantification has emerged as a powerful technology for proteome wide quantitative profiling

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of differentially proteins and stable isotope labeling has been applied to increase the accuracy

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of quantitative results [41, 42]. The isotopes can be incorporated chemically as in isobaric

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labeling methods include family of reagents referred to as ‘‘isobaric mass tags’’ such as

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isobaric tag for relative and absolute quantification (iTRAQ) and tandem mass tag (TMT) or

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stable isotope [43, 44]. Previous study found that TMT labeling was a powerful tool for

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proteomics to find differences in protein [42]. As proteomic is a newly developed technology,

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only a small amount of diseases are currently reported in fish, such as Danio rerio gills upon

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Aeromonas hydrophila infection, Paralichthys olivaceus liver’s response to Edwardsiella

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tarda infection, epithelioma papulosum cyprini cells of spring viremia of carp virus (SVCV)

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infection and the VHSV infected zebrafish [39,45-47] , ect.

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In order to further study the immunity and related components in the defense system of

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teleost fish, we used the crosstalk between transcriptome sequencing and TMT labeling

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followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine the

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changes in transcripts and proteins expression in the head kidneys of crucian carp at 3 d after

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infection with CyHV-2. In this study, we first reported the combined transcriptomic and

ACCEPTED MANUSCRIPT proteomic analysis of head kidneys of crucian carp to elucidate the transcriptional and

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translational levels of teleost fish immune response in CyHV-2 infection. These results

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provide some new and important information about the immune response of the teleost fish

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against viral infections.

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2. Materials and methods

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2.1. Experimental animals CyHV-2 infection and head kidney tissue sampling

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CyHV-2 was isolated from crucian carp with typical "gill bleeding" symptoms. Briefly,

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head kidneys obtained from moribund specimens of crucian carp were homogenized in a

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mortar with sterile PBS. Tissue extracts were centrifuged at 4,000 rpm for 15 min and the

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supernatant was taken. And the supernatant (filtered with 0.22 µM filter) was inoculated at

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tenfold dilutions (1:10) onto healthy crucian carp. Notably, the CyHV-2 in the experiment

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needed to be detected by PCR. Tissue sample homogenate was diluted 1:1 with PBS, and then,

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DNA was extracted from the sample using the EasyPure Genomic DNA Kit (TransGen

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Biotech, China) according to the manufacturer’s instructions. A conserved fragment of gene

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sequence was amplified by PCR using oligonucleotide primers CyHVpol [5].

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Healthy crucian carp (about 300 g) were taken from an aquaculture farm in Baoying,

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Jiangsu Province, China and cultivated for 7 d in an ultraviolet radiation sterilization

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circulating water temperature controlled aquaculture system before the experiment. The water

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temperature was controlled at 24 ℃. Healthy crucian carp were randomly divided into two

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groups, experimental group and control group, with 10 in each group. Ten fish (with 75%

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alcohol disinfection) in the experimental group received an injection of 100 µL CyHV-2 crude

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extract, individually. Ten fish (with 75% alcohol disinfection) in the control group received an

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ACCEPTED MANUSCRIPT injection of 100 µL PBS (filtered with 0.22 µM filter). Three fishes from the control group

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and 3 fishes from the experimental group at 3 d post-injection were randomly collected to

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prepare head kidney samples (0.1 g each fish). Samples of the control and experimental

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groups were mixed separately for RNA and protein extraction. Samples were immediately

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stored at -80 ℃.

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2.2. RNA extraction, cDNA library construction and transcriptome sequencing

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Total RNA was extracted from head kidney of crucian carp in the experimental group

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and the control group using standard protocols (Trizol; Invitrogen, USA). mRNA selection,

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library preparation and sequencing was performed by the TruseqTM RNA sample prep Kit

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(Illumina, USA) on an Illumina Hiseq sequencer according to manufacturer specifications.

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Briefly, the mRNA was enriched using oligo (dT) magnetic beads and fragmented into short

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fragments (about 200 bp) by mixing with the fragmentation buffer. Thereafter, a random

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hexamer-primer was used to synthesized the first strand of cDNA. The second strand was

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synthesized using a buffer containing dNTPs, RNase H and DNA polymerase I. The double

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strand cDNA was purified with AMPure XP beads. End reparation (by End Repair Mix) and

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3’-end single nucleotide A (adenine) addition was then performed. Finally, sequencing

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adaptors were ligated to the fragments and the fragments were enriched by PCR (PTC-100,

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Bio-Rad) amplification. During quality check, TBS380 Picogreen (Invitrogen, USA) and

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bridge PCR were used to qualify and quantify the sample library. Thereafter, used Illumina

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Hiseq (Illumina, USA) for sequencing. Prior to assembly and mapping (described below), we

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applied filters to remove suspect reads from all the data to get clean data. First, the base of the

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sequence (3’ end) of low quality (mass less than 20) is trimmed off. Next, we removed the

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reads containing N ratios over 10%, the adapters and the mass-pruned sequences less than 20

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bp.

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2.3. De Novo assembly and gene annotation After obtaining clean data, all sequencing reads need to be made de novo assembly to

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generate contig and singleton. Trinit program (http://trinityrnaseq.sourceforge.net/) was used

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for de novo assembly and ORF prediction. Thereafter, assembled unigenes were used for

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annotation so that they could be classified for gene functioning by searching different protein

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databases. To do this, we used BlastX (version 2.2.23) alignment against four public protein

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databases. The four public protein databases used include: (1) NCBI non-redundant (NR); (2)

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String; (3) Swiss-Prot; (4) Kyoto Encyclopedia of Genes and Genomes (KEGG) at e-value <

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0.00001. The direction of the identified unigenes was determined using the best alignments

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obtained from the four databases. Data obtained from BlastX was used to extract the coding

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regions (CDS) from unigene sequences and translate them into peptide sequences. Unigenes

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with NR annotation were further analyzed with Blast2go to obtain their gene ontology (GO)

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annotations, and were then further classified according to GO functions using the Web Gene

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Ontology (WEGO) annotation software. Finally, the gene function and expression levels were

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analyzed

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(http://www.biomedsearch.com/nih/RSEM-accurate-transcript-quantification-from/21816040.

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html) program.

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2.4. Analysis of differentially expressed transcripts

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using

KEGG

database

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RSEM

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Gene expression pattern was clustered using h-cluster (complete algorithm) to analysis

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the different transcripts. Statistical comparison between CyHV-2-infected (experimental) and

ACCEPTED MANUSCRIPT non-infected (control) groups was conducted using edgeR. Genes with fold change above 1

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and corrected p < 0.05 were deemed significantly differentially expressed. Differently

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expressed genes were analyzed with GO and KEGG enrichment.

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2.5. Protein preparation and TMT labeling

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Head kidney samples from CyHV-2-infected and control groups were respectively

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homogenized to powder in liquid nitrogen. The powder was separately extracted with 4-fold

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volume of lysis buffer (8 M Urea, 1% protease inhibitor, 3 µM TSA, 50 mM NAM and 2 mM

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EDTA) and lysed by sonication. Centrifuged at 20,000 g for 10 min at 4 ℃ to remove cell

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debris. Protein concentration was then measured using the BCA Protein Assay Kit (Beyotime,

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China). Dithiothreitol (Promega, USA) was added to the protein solution to a final

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concentration of 5 mM and was reduced at 56 ℃ for 30 min. Then iodoacetamide (Promega,

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USA) was added to a final concentration of 11 mM and incubated in the dark for 15 min at

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room temperature. Finally, the urea concentration of the sample was diluted to less than 2 M.

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Trypsin (Promega, USA) and protein samples were mixed at a 1:50 mass ratio and digested

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overnight at 37 ℃. Then, trypsin was added to the trypsin and protein sample at a mass ratio

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of 1:100 for 4 h.

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The tryptic digested peptides were desalted with Strata X C18 (Phenomenex, USA) and

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vacuum freeze-dried and the peptides were solubilized with 0.5 M TEAB (Applied

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Biosystems, USA). The TMT labeling was performed according to the manufacturer’s

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instructions (Thermo, USA). The experimental and control groups were labeled as 130 and

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131, respectively. Briefly, the labeled reagents were thawed and then dissolved with

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acetonitrile (Fisher Chemical), mixed with the peptides and incubated at room temperature for

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2 h. The labeled peptides were mixed and then desalted and vacuum freeze-dried.

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2.6. LC-MS/MS analysis The peptides were fractionated by high pH reversed-phase High Performance Liquid

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Chromatography (HPLC) (Thermo, USA) with an Agilent 300 Extend C18 (5 µm particle size,

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4.6 mm ID, 250 mm length). The peptide gradient was 8%-32% acetonitrile (pH 9) and 60

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components were separated within 60 min. The peptides were then combined into 18

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components and the combined components were freeze-dried in vacuum. Then the peptides

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were separated using the EASY-nLC 1000 Ultra Performance Liquid Chromatography (UPLC)

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system after dissolved in the mobile phase A phase. Mobile phase A is an aqueous solution

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containing 0.1% formic acid (Fluka, USA) and 2% acetonitrile and mobile phase B contains

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0.1% formic acid and 90 % acetonitrile. The liquid phase gradient is: 0-26 min, 7%-25% B;

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26-34 min, 25%-40% B; 34-37 min, 40%-80% B; 37-40 min, 80% B. The flow rate was

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maintained at 350 nL/min.

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Peptides were separated by UPLC system and injected into an NSI ion source (2.0 kV)

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for ionization and then analyzed by Q ExactiveTM Plus mass spectrometry. Both the peptide

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precursor ions and its secondary fragments were detected and analyzed using high-resolution

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Orbitrap (Thermo, USA). The MS scan range was set to 350-1,800 m/z with the resolution at

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7,000 and the secondary MS scanning fixed origin was 100 m/z with the Orbitrap scan

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resolution at 17,500. The data acquisition mode used a Data Dependent Acquisition (DDA)

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procedure, which selected the first 20 peptide precursor ions with the highest signal intensity

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into the HCD collision cell after a first-pass scan and used 31% of the fragmentation energy

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for fragmentation. Secondary MS analysis was also performed sequentially. In order to

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ACCEPTED MANUSCRIPT increase the effective utilization of MS, the automatic gain control (AGC) was set to 5E4, the

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signal threshold was set to 10,000 ions/s with the maximum injection time at 200 ms, and the

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dynamic exclusion time of the MS/MS scan was set to 30 s to avoid the repeated scanning of

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peptide parent ions.

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2.7. Proteome data analysis

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Protein identification was performed by using Maxquant (v1.5.2.8). To reduce the

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probability of false peptide identification, an anti-library was added to calculate the false

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discovery rate (FDR) caused by random matches and a common pollution database was added

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to the database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing

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cleavages. And the minimum length of the peptide was set to 7 amino acid residues with a

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maximum modification number of 5; Mass error was set to 20 peptide mass error (ppm) of

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First search and 5 ppm of Main search for precursor ions and 0.02 Da for fragment ions.

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Alkylation on cysteine, was specified as fixed modification and oxidation on methionine and

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acetylation on protein N-term were specified as variable modifications. For protein

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quantification method, TMT 6-plex was selected in Mascot (Matrix Science, UK). FDR rate

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was adjusted to < 0.01 at protein, peptide and propensity score matching level. And each

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confident protein identification involved at least one unique peptide. The quantitative protein

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ratios were weighted and normalized by the median ratio in Mascot. Proteins with

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quantitative ratio above 2 or below 0.5 and p < 0.05 were deemed significantly differentially

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expressed. Protein identification was then performed using the transcriptome of Carassius

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auratus gibelio (61369 sequence) database. Functional annotations of the proteins were

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conducted using Blast2GO program against the NR protein database.

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2.8. Transcriptome and proteomic crosstalk analysis The gene expression profiling of the transcriptome was correlated with the protein

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expression profiling of the proteomic to compare and categorize the differential expression of

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genes at mRNA and protein levels. Meanwhile the cDEGs were screened out with p < 0.05.

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Then the cDEGs were analyzed with GO and KEGG enrichment.

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2.9. Real-time PCR

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The head kidney tissues were collected as described above. After extraction, total RNA

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was reverse-transcribed into cDNA with a PrimeScript RT reagent Kit (TAKARA, Japan).

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The partial sequences of immune genes were amplified by primers listed in Supplemental

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Table S1. β-actin was used as a housekeeping gene. Real-time PCR was carried out with a

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Mastercycler ep realplex (Eppendorf, Germany) to study the expression of immune genes in

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head kidneys. The PCR reaction was performed in a 25 µL volume with a SYBR Premix Ex

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Taq™ Kit (Takara, Japan), 2 µL of each specific primer and 1 µL of cDNA using the

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following procedure: initial denaturation at 95 ℃ for 30 s; followed by 40 cycles of

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amplification (95 ℃ for 5 s, 60 ℃ for 30 s). The relative expression levels of different

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genes in head kidneys were calculated according to the 2−∆∆CT method. Statistical analysis was

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performed using SPSS software (Ver11.0). Statistical analysis was performed using SPSS

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software (Ver11.0). The data are presented as the mean ± SD (n=3). Statistical significance

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was determined by one-way ANOVA, and posthoc Duncan multiple range tests. Significance

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was set at p < 0.05.

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3. Results

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3.1. Identification of differential expression genes and proteins

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ACCEPTED MANUSCRIPT The differentially expressed genes were analyzed by edgeR in the Trinity platform with

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FDR < 0.05. The results revealed that 7,085 genes were differently expressed between

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CyHV-2-infected and non-infected groups, including 3,090 up-regulated genes and 3,995

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down-regulated genes. And the differentially expressed genes were identified and classified

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by GO enrichment. The biological process ontology analysis was as follows: metabolic

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process (22%)，single-organism process (19%) and cellular process (18%) of up-regulated

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genes (Fig.1A); metabolic process (22%)，cellular process (17%) and catalytic activity (16%)

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of down-regulated genes (Fig.1D). The cellular component ontology analysis: cell part (19%),

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cell (19%), membrane (16%) and organelle (15%) of up-regulated genes (Fig.1B); cell (29%),

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membrane (25%) and organelle (22%) of down-regulated genes (Fig.1E). The molecular

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function ontology analysis: catalytic activity (49%), binding (28%) and transporter activity

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(11%) of up-regulated genes (Fig.1C); metabolic process (32%), cellular process (26%) and

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establishment of localization (13%) of down-regulated genes (Fig.1F).

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Using fold change >2 and p < 0.05 as standards, 338 differentially expressed proteins

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were reliably quantified by TMT and LC-MS/MS analysis, including 277 up-regulated

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proteins and 61 down-regulated proteins subsequent to CyHV-2 infection. GO analysis of

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differentially expressed proteins in head kidneys was as follows. The biological process

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ontology analysis: metabolic process (21%)，cellular process (20%) and single-organism

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process (16%) of up-regulated proteins (Fig.1G); metabolic process (20%)，single-organism

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process (20%) and cellular process (18%) of down-regulated proteins (Fig.1J). However, the

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immune system process (5%) was only found in up-regulated proteins. The cellular

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component ontology analysis: the extracellular region (19%) of up-regulated proteins was

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ACCEPTED MANUSCRIPT significantly higher than that of down-regulated proteins (4%) and the membrane (17%) was

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significantly lower than that of down-regulated proteins (26%) (Fig.1H, K).The molecular

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function ontology analysis: the binding (64%) proteins of up-regulated proteins was

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significantly higher than that of down-regulated proteins (52%) and the molecular function

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regulator (4%) of up-regulated proteins was significantly lower than that of down-regulated

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protein (9%) (Fig.1I, L).

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3.2. Functional annotation based on GO and KEGG analysis

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In order to detect whether the differentially expressed genes and proteins are

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significantly enriched in certain functional types, GO and KEGG enrichment and other

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functional annotation types were analyzed. A negative logarithm (-log10) conversion was

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performed on the p-value obtained by the enrichment test, and the larger the converted value,

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the more significant of this functional type.

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Up-regulated genes in the molecular function was mainly enriched in oxidoreductase

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activity, argininosuccinate synthase activity and triose-phosphate isomerase activity, etc; the

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biological process was mainly enriched in respiratory electron transport chain, electron

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transport chain and aerobic respiration, etc (Fig. 2A). Down-regulated genes in the molecular

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function was mainly enriched in nitronate monooxygenase activity, structural constituent of

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peritrophic membrance and oxidoreductase activity, etc; the biological process was mainly

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enriched in chitin metabolic process, mesodermal cell differentiation and hemangioblast cell

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differentiation, etc (Fig. 2B).

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GO functional enrichment analysis of differential proteins was as follows: up-regulated

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proteins in the cell component were mainly enriched in extracellular region, while

ACCEPTED MANUSCRIPT down-regulated proteins were mainly enriched in intracellular organelle part; differential

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proteins were mainly enriched in peptidase regulator or inhibitor activity in molecular

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function; up-regulated proteins in the biological process were mainly enriched in blood

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coagulation, hemostasis, and injury response and injury healing, while down-regulated

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proteins were mainly enriched in response to oxidative stress and adhesion (Fig. 2C, D).

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Differently expressed proteins from CyHV-2-infected and control groups were further

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annotated by KEGG pathway with p<0.05. Up-regulated of TMT quantitative proteins after

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CyHV-2 infection was mainly enriched in herpes simple infection pathway, p53 signaling

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pathway and pyrimidine metabolism pathway (Fig. 3A). The up-regulated proteins with flod

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change

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differentiation-associated protein 5 (MDA5), tumor necrosis factor receptor superfamily

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member 6-like isoform X1 (FAS), laboratory of genetics and physiology 2 (LGP2),

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dsRNA-dependent protein kinase (PKR) and Z-DNA binding protein kinase (PKZ) were

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mainly enriched in the herpes simple infection pathway and E3 ubiquitin-protein ligase

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(MDM2) , FAS and so on were mainly enriched in p53 signaling pathway. CyHV-2 is a kind

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of herpes virus, so herpes simple infection pathway plays an important role in CyHV-2

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infection and p53 signaling pathway may fight the CyHV-2 by causing apoptosis.

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Down-regulated proteins were mainly enriched in two pathways of phagosome pathway and

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arginine and proline metabolism pathway (Fig. 3B). And the down-regulated proteins

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including integrin beta2-chain (Integrin β2), complement C3 (iC3b), immunoglobulin heavy

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chain (IG) and so on were mainly enriched in phagosome pathway. So the phagosome

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pathway may play an important role in CyHV-2 infection.

including

retinoic

acid-inducible

protein

I

(RIG-I),

melanoma

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3.3. Correlations between transcriptome and proteome data analysis. Gene expression can be described at both transcriptional (mRNA) and translational

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(protein) level. And the immune response is associated with highly regulated processes that

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need to be precisely controlled at both the mRNA and protein levels. So we investigated

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whether changes in protein levels correlated with changes in the corresponding transcriptions.

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The analysis result shown that the expression of protein and transcription had positive

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relationship (Fig. 4A). Meanwhile we identified 8 groups based on their abundances as

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follows: unchanged protein and mRNA, unchanged protein and down-regulated mRNA,

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unchanged protein and up-regulated mRNA, down-regulated protein and unchanged mRNA,

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up-regulated protein and unchanged mRNA, up-regulated protein and down-regulated mRNA

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(Supplemental

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Table S2), co-down-regulated protein and mRNA and co-up-regulated protein and mRNA

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(Fig. 4B).

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Using a 2-fold increase or decrease in cDEGs as a benchmark for physiologically

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significant change, 128 cDEGs were reliably quantified, including 86 co-up-regulated and 42

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co-down-regulated subsequent to CyHV-2 infection. The specific results were as follows. Of

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the up-regulated cDEGs, 16 were involved with immunologic proteins, 4 were cytoskeleton/

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extracellular proteins, 3 were transport proteins, 33 were physiologic proteins, 10 were

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intracellular proteins and 20 were listed as uncharacterized/hypothetical proteins. The

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up-regulated immunity genes in head kidneys included RIG-I with 5.78-fold change of

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expression and 19.03-fold change of transcription, MDA5 with 2.96-fold change of

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expression and 5.54-fold change of transcription and FAS with 2.25-fold change of

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expression and 3.51-fold change of transcription. In addition, it also included LGP2, PKR and

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PKZ, etc. The up-regulated cDEGs identified with known physiologic or immunologic roles

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and the corresponding change ratios are presented in Table 1. Of the down-regulated cDEGs, 11 were grouped within the immune system proteins, 4

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were cytoskeleton/extracellular proteins, 1 were transport proteins, 21 were physiologic

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proteins, 2 were intracellular proteins and 3 were in uncharacterized/hypothetical protein

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category. The down-regulated proteins that were involved in head kidneys immunity included

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integrin alpha (Integrin α) with 0.477-fold change of expression and 0.0292-fold change of

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transcription, Integrin β2 with 0.41-fold change of expression and 0.0439-fold change of

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transcription, neutrophil cytosol factor 2 (NCF2) with 0.486-fold change of expression and

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0.1571-fold change of transcription and neutrophil cytosol factor 4 (NCF4) with 0.43-fold

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change of expression and 0.1161-fold change of transcription, etc. The down-regulated

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cDEGs with notable physiologic or immunologic roles and the corresponding change ratios

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are presented in Table 2.

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The result of GO enrichment show up-regulated cDEGs in the cell component were

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mainly enriched in fibrinogen complex and extracellular region; up-regulated cDEGs in the

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molecular function were mainly enriched in protein or receptor binding, while

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down-regulated cDEGs were mainly enriched in peptidase regulator or inhibitor activity and

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peroxidase activity; up-regulated cDEGs in the biological process (similar to the results of

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proteomic) were mainly enriched in platelet activation, agglutination, hemostasis, and injury

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response and wound healing, while down-regulated cDEGs were mainly enriched in response

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to oxidative stress and adhesion (Fig. 5).

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ACCEPTED MANUSCRIPT The transcriptome and proteome of the head kidney after CyHV-2 infected was

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combined to analysis by KEGG pathway with p<0.05. Up-regulated cDEGs were mainly

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enriched in 6 signaling pathways of herpes simplex infection pathway, RIG-I like receptor

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signaling pathway, necroptosis pathway, p53 signaling pathway, nicotinate and nicotinamide

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metabolism and pyrimidine metabolism (Fig. 6A). And down-regulated cDEGs were mainly

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enriched in 2 signaling pathways of phagosome pathway and arginine and proline metabolism

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(Fig. 6B). The results of the joint analysis are basically consistent with the results of proteome

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analysis. Therefore, the two signaling pathways of the herpes simplex infection and

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phagosome played an important role in the up-regulation and down-regulation of cDGEs,

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respectively, and were closely related to CyHV-2 infection.

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In order to found the more biological information, we analyzed protein function of

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co-regulated proteins or transcriptions. GO and KEGG pathway enrichment clustering

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analysis were performed. The clustering result was as follows: up-regulated cDEGs in the

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biological process were mainly enriched in hemostasis, blood coagulation and wound healing

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while down-regulated cDEGs were mainly enriched in proteolysis (Fig.7 A); up-regulated

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cDEGs in the cell component were mainly enriched in fibrinogen complex (Fig.7 B);

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up-regulated cDEGs in the molecular function were mainly enriched in dsRNA adenosine

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deaminase activity, protein binding, bridging and receptor binding, while down-regulated

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cDEGs were mainly enriched in endopeptidase regulator and inhibitor activity (Fig.7 C).

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Up-regulated cDEGs were mainly enriched in 6 signaling pathways of pyrimidine metabolism,

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nicotinate and nicotinamide metabolism, necroptosis pathway, p53 signaling pathway, herpes

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simplex infection pathway and RIG-I like receptor signaling pathway while down-regulated

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cDEGs were mainly enriched in phagosome pathway and arginine and proline metabolism

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(Fig.7 D). The information in Fig. 7 was basically the same as the results obtained above.

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3.4. RT-PCR analysis of the mRNA To investigate the results of the transcriptome and proteome after CyHV-2 infected, we

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performed qRT-PCR on some selected targets in both the experimental group and control

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group at 0 d and 3 d post infection. We measured the mRNA transcription levels of 8 proteins,

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including 5 genes of up-regulated proteins and 4 genes of down-regulated proteins, among

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which the up-regulated proteins include: RIG-I, PKR, PKZ, FAS, MDA5 and LGP2 involved

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in herpes simplex infection pathway, RIG-I like receptor signaling pathway necroptosis

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pathway and p53 signaling pathway; the down-regulated proteins include: Integrin α, Integrin

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β2, NCF2 and NCF4 involved in phagosome pathway.

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As shown in Fig.8, the mRNA expression of RIG-I, PKR, PKZ, FAS, MDA5 and LGP2

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in head kidneys after the infection with CyHV-2 at 3 d were significantly higher than those of

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the control group. This further suggested that the herpes simplex infection pathway, p53

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signaling pathway, necroptosis pathway and RIG1-I like receptor signaling pathway were

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activated after CyHV-2 infection. And the mRNA expression of Integrin α, Integrin β2, NCF2

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and NCF4 were significantly reduced after CyHV-2 infection compared to the control group.

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This indicated that the phagosome was inhibited in head kidneys after infected with CyHV-2.

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To sum up, all the results by RT-PCR analysis were consistent with the proteome and

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transcriptome analysis.

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4. Discussion

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HVHN caused by CyHV-2 is a very serious disease of crucian carp in aquaculture.

ACCEPTED MANUSCRIPT However, the molecular pathogenesis of HVHN induced by CyHV-2 is still unknown.

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Therefore, the relationship between crucian carp and CyHV-2 is worthy of in-depth study,

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which is of great significance for us to control the disease. Transcriptomic and proteomic have

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been increasingly applied to examine differentially expressed genes and proteins in cells,

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organs or tissues to infer the corresponding biological functions. In this study, we first

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reported the transcriptomics and proteomics crosstalk analysis of fish viral diseases.

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We used high-throughput sequencing and TMT labeling to screen out 128 cDGEs. With

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the exception of genes related to cellular structure and metabolism, abundant sequences were

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found to be homologous to known immune-relevant genes in other species, based on the GO

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and KEGG annotation. Among 86 up-regulated cDGEs, multiple genes involved in the herpes

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simplex infection, RIG-I like receptor signaling pathway, necroptosis pathway and p53

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signaling pathway, such as RIG-I, MDA5, LGP2, Fas, PKR and PKZ. Previous research

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showed that RIG-1, MDA5, and LGP2 act as homologs of the RLRs to activate the host IFN

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system (similar to mammals) upon recognition of the viral PAMPs in the cytoplasm [48, 49].

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RLRs have been found to be essential viral sensors in the cytoplasm and mediate type I IFN

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induction in response to viral infection. It has been reported that the RLRs including RIG-I

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and MDA5 was up-regulated post NNV infection in zebrafish and activation of RLRs

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pathway inhibited the replication of Hepatitis B Virus [49, 50]. However, LGP2 in fish has

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different regulation effects on RIG-I and MDA5 in different species [51]. Our data showed

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that the expression of MDA5 and RIG-I have been shown to increase following infection by

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CyHV-2 in crucian carp indicating that the host's herpes simplex infection pathway and RIG-I

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like receptor signaling pathway was activated. Activated RIG-I and MDA5 act on

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ACCEPTED MANUSCRIPT mitochondria through the interaction between RIG-I/MDA5 and mitochondrial antiviral

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signal protein (MAVS/IPS-1), a receptor of mitochondria connecting RIG-I and MDA5,

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facilitating phosphorylation of interferon regulatory factors (IRF) 3 and 7 which triggering the

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production of IFN [48, 52]. The results seem to indicate that the host induces up-regulation of

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RIG-I and MDA5 after receiving CyHV-2 injection to induce IFN production to fight the

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virus. Our results showed that significant up-regulation of multiple interferon-induced

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proteins after CyHV-2 infection appears to warrant interferon production. IFN plays a key

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role in vertebrate innate immunity because it can induce vertebrate cells to enter antiviral state

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and cause necroptosis to prevent virus replication [15]. Studies have shown that p53 and Fas

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are involved in promoting apoptosis and that Fas is involved in the apoptosis of the

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exogenous pathway caused by p53. Fas is a prototypical death receptor that induces apoptosis

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of the extrinsic pathway in the tumor necrosis factor receptor (TNFR) superfamily and

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apoptosis is vital for antiviral defense of infected cells [53]. Fas in brain cells of Siniperca

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chuatsi is significantly up-regulated after infectious spleen and kidney necrosis virus (ISKNV)

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infection [50], which is similar to our results. It is well known that apoptosis is an active

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suicide and energy-consuming process that requires ATP to provide energy. As our results

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shown that Fas in the head kidney was significantly up-regulated after 3 d of CyHV-2

459

infection, accompanied by up-regulation of multiple energy metabolism related proteins, such

460

as ATP synthase subunit beta of mitochondria in table 2, indicating that the extrinsic pathway

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was probably activated by CyHV-2 infection in head kidneys. Fas ligand-producing T

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lymphocytes can induce apoptosis in target cells infected by pathogens and damaged cells can

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cause their own apoptosis by producing Fas ligand and related proteins by themselves. The

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ACCEPTED MANUSCRIPT results may indicate that the host activated apoptosis through Fas activation of Necroptosis

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pathway after infection with CyHV-2. At the same time, IFNs can also cause necroptosis,

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further illustrating the activation of RLRs-related pathways after CyHV-2 infection. The latest

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research results indicate the synergy between IFN and TNF signaling can induce the

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necroptosis downstream of multiple innate immune sensing pathways [54]. Therefore, we

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hypothesized that after CyHV-2 infection, the necroptosis was induced by the synergy

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between IFN and TNF signaling (the downstream motif of the above pathways) to resist

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CyHV-2 invasion.

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PKR plays a significant role in IFN-induced antiviral responses, and is also involved in

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intracellular signaling pathways, including the apoptosis, transcription and proliferation

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pathways. Fish have a PKR-like protein that named PKZ because of its Z-DNA binding

475

domain. And PKZ in zebrafish is able to phosphorylate alpha subunit of eukaryotic initiation

476

factor 2 (eIF2a) and may play a role, like PKR, in host defense against virus infection [55].

477

PKR also affects various transcriptional factors such as p53, IRF1, STATs. In other fish like

478

Paralichthys olivaceus and Gobiocypris rarus, either PKZ or PKR was reported [56]. Our

479

data showed that PKR and PKZ were up-regulated after CyHV-2 infection. This seems to

480

indicate that eIF2a is phosphorylated and results in a blockade on translation initiation to

481

prevent viral replication and inhibits normal cell ribosome function. When the cellular DNA is

482

damaged, the p53 signaling pathway of the host will be activated to induce the cell cycle

483

arrest and when viral infection causes DNA damage to be difficult to repair, cells will initiate

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apoptosis procedures [57]. In conclusion, when CyHV-2 infects the head kidney, the host

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activates the p53 signaling pathway through the up-regulation of PKR/PKZ to promote

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apoptosis and thereby achieve the purpose of resisting the virus. The data provide significant

487

evidence for the PKR-mediated antiviral pathway in fish. Among 42 down-regulated cDGEs, multiple genes involved in the phagosome pathway.

489

Endocytosis is a fundamental cellular process that is used by the mostly of animal virus

490

families to introduce their genetic material to the cell interior, such as human herpes

491

virus-(HHV-)6A and HHV-6B [58], adenovirus [59], vesicular stomatitis virus (VSV) [60]

492

and white spot syndrome virus (WSSV) [61]. Studies have shown that the first step in viral

493

infection is the binding of the virus to cell surface receptors and a common family of viral

494

receptors in the integrin family. It has been demonstrated that integrin αvβ3 can promote

495

herpes simplex virus (HSV) type 2 entry into human genital tract epithelial cells [62]. Our

496

data showed that Integrin α and Integrin β2 are significantly down-regulated after CyHV-2

497

infection. We hypothesized that Integrin α and Integrin β2 may mediate the entry of CyHV-2

498

into cells as the cell surface receptors. Therefore, the down regulation of Integrin α and

499

Integrin β2 may prevent the invasion of the CyHV-2. NCF4 is the component of the

500

nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase) and NCF2 regulates

501

superoxide via regulating electron flow from NADPH to oxygen. Digestion of

502

microorganisms (including bacteria and viruses) within phagosome is a complicated process,

503

in which NADPH-oxidase plays an important role. Our results showed that NCF2 and NCF4

504

was significantly down-regulated after CyHV-2 infection, indicating that the phagosome

505

pathway

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columnare-infected Culter alburnus [63]. This is contrary to our results, which may be due to

507

different pathogens causing different reactions after pathogenic infection of the host.

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was

inhibited.

However,

NCF2

was

up-regulated

in

Flavobacterium

ACCEPTED MANUSCRIPT In conclusion, there are 128 cDGEs in the crucian carp head kidney at 3 d post CyHV-2

509

infection, when compared with the control group. These data are helpful in promoting the

510

immunology research with crucian carp. We have found that integrins may mediate the

511

invasion of the virus as a membrane surface receptor when infected with CyHV-2. And after

512

CyHV-2 infection, herpes simplex infection pathway, RIG-I like receptor signaling pathway,

513

p53 signaling pathway, necroptosis pathway and phagosome pathway play a major defensive

514

role. With these findings, tools for more detailed investigations about the diseases caused by

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CyHV-2, including elucidation of pathogenesis, interaction of hosts and pathogens, and

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defense mechanisms, can also be obtained. This study also provided important information for

517

the prevention and treatment of fish diseases.

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Acknowledgments

The current study was supported by grants from the National Natural Sciences

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Foundation of China (NSFC Nos. 31570176; 31602198), the Natural Science Foundation of

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Jiangsu Higher Education institutions of China (Grant No. 16KJD240002), the Natural

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Science Foundation of Jiangsu Province (Grant No. BK20151545), Project for Aquaculture in

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Jiangsu Province (Grant Nos. Y2016-28; Y2017-34) and the project funded by the Priority

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Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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[48] M. Chang, B. Collet, P. Nie, K. Lester, S. Campbell, C.J. Secombes, et al., Expression and functional characterization of the RIG-I-like receptors MDA5 and LGP2 in Rainbow trout (Oncorhynchus mykiss), J Virol. 85 (2011) 8403-8412.

[49] H.Y. Chen, W. Liu, S.Y. Wu, P.P. Chiou, Y.H. Li, Y.C. Chen, et al., RIG-I specifically mediates group II type I IFN activation in nervous necrosis virus infected zebrafish cells, Fish Shellfish Immunol. 43 (2015)

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427-435.

[50] X. Hu, X. Fu, N. Li, X. Dong, L. Zhao, J. Lan, et al., Transcriptomic analysis of Mandarin fish brain cells infected with infectious spleen and kidney necrosis virus with an emphasis on retinoic acid-inducible gene 1-like receptors and apoptosis pathways, Fish Shellfish Immunol. 45 (2015)

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619-629.

[51] X. Chen, C. Yang, J. Su, Y. Rao, T. Gu, LGP2 plays extensive roles in modulating innate immune responses in Ctenopharyngodon idella kidney (CIK) cells, Dev Comp Immunol. 49 (2015) 138-148. [52] P.F. Zou, M.X. Chang, Y. Li, S. Huan Zhang, J.P. Fu, S.N. Chen, et al., Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish, Fish Shellfish Immunol. 43 (2015) 13-24. [53] Q. Fu, T.M. Fu, A.C. Cruz, P. Sengupta, S.K. Thomas, S. Wang, et al., Structural Basis and Functional Role of Intramembrane Trimerization of the Fas/CD95 Death Receptor, Mol Cell. 61 (2016) 602-613. [54] M. Brault, T.M. Olsen, J. Martinez, D.B. Stetson, A. Oberst, Intracellular Nucleic Acid Sensing Triggers Necroptosis through Synergistic Type I IFN and TNF Signaling, J Immunol. 200 (2018) 2748-2756. [55] J. Su, Z. Zhu, Y. Wang, Molecular cloning, characterization and expression analysis of the PKZ gene

ACCEPTED MANUSCRIPT

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in rare minnow Gobiocypris rarus, Fish Shellfish Immunol. 25 (2008) 106-113. [56] R. Zhu, Y.B. Zhang, Q.Y. Zhang, J.F. Gui, Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus, J Virol. 82 (2008) 6889-901. [57] H. Wang, Y. Ye, JH. Chui, ZL.Yu, Oridonin induces G2/M cell cycle arrest and apoptosis through MAPK and p53 signaling pathways in HepG2 cells, Oncol Rep. 24 (2010) 647-651. Virol. 2013 (2013) 469538.

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[58] A.V. Nicola, H.C. Aguilar, J. Mercer, B. Ryckman, C.M. Wiethoff, Virus entry by endocytosis, Adv [59] C.J. Burckhardt, M. Suomalainen, P. Schoenenberger, K. Boucke, S. Hemmi, U.F. Greber, Drifting motions of the adenovirus receptor CAR and immobile integrins initiate virus uncoating and membrane lytic protein exposure, Cell Host Microbe. 10 (2011) 105-117.

[60] D.K. Cureton, R.H. Massol, S. Saffarian, T.L. Kirchhausen, S.P. Whelan, Vesicular stomatitis virus internalization, PLoS Pathog. 5 (2009) e1000394.

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enters cells through vesicles incompletely coated with clathrin that depend upon actin for [61] H. Duan, S. Jin, Y. Zhang, F. Li, J. Xiang, Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways, Dev Comp Immunol. 46

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(2014) 186-193.

[62] N. Cheshenko, J.B. Trepanier, P.A. Gonzalez, E.A. Eugenin, W.R. Jacobs, B.C. Herold, Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin v 3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells, J Virol. 88 (2014) 10026-10038. [63] L. Zhao, J. Tu, Y. Zhang, J. Wang, L. Yang, W. Wang, et al., Transcriptomic analysis of the head kidney of Topmouth culter (Culter alburnus) infected with Flavobacterium columnare with an emphasis on phagosome pathway, Fish Shellfish Immunol. 57 (2016) 413-418.

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666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688

Fig. 1. GO distribution of differentially expressed genes and proteins in head kidney after

692

CyHV-2 infection, (A) up-regulated genes of biological process, (B) up-regulated genes of

693

cellular component, (C) up-regulated genes of molecular function, (D) down-regulated genes

694

of biological process, (E) down-regulated genes of cellular component, (F) down-regulated

695

genes of molecular function, (G) up-regulated proteins of biological process, (H) up-regulated

696

proteins of cellular component, (I) up-regulated proteins of molecular function, (J)

697

down-regulated proteins of biological process, (K) down-regulated proteins of cellular

698

component, (L) down-regulated proteins of molecular function.

699

Fig. 2. Quantitative analysis of differentially expressed genes and proteins between samples.

700

Enrichment based on GO categorization in each of the high- and low-abundance gene and

701

protein groups. The GO categories are represented as (A) up-regulated genes, (B)

702

down-regulated genes, (C) up-regulated proteins and (D) down-regulated proteins.

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ACCEPTED MANUSCRIPT Fig. 3. KEGG pathway enrichment analysis of differentially expressed proteins. The pathway

704

enrichment statistics were performed by Fisher's exact test, and those with a corrected p< 0.05

705

were considered the most significant pathways. The KEGG enrichments are represented as (A)

706

up-regulated protein, (B) down-regulated protein.

707

Fig. 4. The correlation between the protein level and mRNA level of genes. (A) Scatter

708

diagram of the transcriptome and proteome quantification relationship. (B) Bar diagram of

709

transcriptome and proteome differentially expressed proteins or mRNA. “-” indicates

710

unchanged protein and mRNA; “Down-” indicates down-regulated protein and unchanged

711

mRNA; “-Down” indicates unchanged protein and down-regulated mRNA; “Down-Down”

712

indicates co-down-regulated protein and mRNA; “Up-” indicates up-regulated protein and

713

unchanged mRNA; “-Up” indicates unchanged protein and up-regulated mRNA; “Up-Down”

714

indicates up-regulated protein and down-regulated mRNA and “Up-Up” indicates

715

co-up-regulated protein and mRNA.

716

Fig. 5. GO assignment for cDEGs after CyHV-2 infection. (A)The GO analysis for the

717

co-up-regulated genes and proteins upon CyHV-2. (B) The GO analysis for the

718

co-down-regulated genes and proteins upon CyHV-2 infection.

719

Fig. 6. KEGG pathway enrichment analysis of cDEGs. The KEGG enrichments are

720

represented as (A) co-up-regulated genes and proteins，(B) co-down-regulated genes and

721

proteins.

722

Fig. 7. GO and KEGG functional enriched clustering of genes and proteins in the head kidney

723

after CyHV-2 infection. GO enrichment of genes and proteins based on biological process (A),

724

cellular component (B) and molecular function (C). (D) KEGG enrichment of genes and

725

proteins. The clustered GO and KEGG biological process terms enriched in 8 groups are

726

depicted on the heat map. The red arrow corresponds to up-regulated genes and proteins, the

727

green arrow, down-regulated, and the yellow symbol, unchanged. High (red) and low (blue) in

728

the heat map represent statistical over- or under-representation, respectively.

729

Fig. 8. Verification of transcriptome and proteome genes by qRT-PCR. The Figure shows

730

comparative expressions analysis of 10 genes determined by qRT-PCR (white bars),

731

transcriptome (gray bars) and proteome (black bars) in the head kidneys infected by CyHV-2

732

at 3 d. Relative transcriptional and translational levels of genes are given in fold changes and

AC C

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703

ACCEPTED MANUSCRIPT 733

were determined by qRT-PCR, using β-actin as the internal control. Data shown are the mean

734

± SD (n=3).

735

Table 1 Co-up-regulated genes in crucian carp head kidneys with a 2-fold change

737

post-injection with CyHV-2.

738

Table 2 Co-down-regulated genes in crucian carp head kidneys with a 2-fold change

739

post-injection with CyHV-2.

740

RI PT

736

Table S1 The primers used for Real-time PCR in the experiment.

742

Table S2 Non-co-expression genes in crucian carp head kidneys post-injection with CyHV-2.

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ACCEPTED MANUSCRIPT

Coverage

Peptide

Expression fold change

Transcription fold change

XP_018958339 XP_018973417 XP_006797808 XP_018978897

4.009 22.437 6.5912 5.8085

7.4 9 18.5 16.4

SC

2 6 4 2

2.24 2.25 2.62 5.69

3.36 3.97 6.23 12.21

64.023

10.2

5

2.71

3.51

84.198 44.41

31.1 28.5

3 4

2.76 2.99

79.34 48.84

XP_016428868 KTF99322 XP_018946159

40.868 5.5003 11.228

20.3 9.1 21

5 3 11

2.07 2.15 2.19

4.35 14.52 4.38

XP_018976752

3.1766

3.3

1

2.25

3.51

70.41 71.306 63.489 36.195 123.25

21.3 21.7 21.7 25.8 27.4

4 7 12 3 16

2.37 2.46 2.96 2.99 3.44

3.48 4.00 5.54 2.99 20.11

NP_957055

TE D

XP_016329955 XP_016352137

M AN U

Score

AC C

Cytoskeleton/extracellular proteins src substrate cortactin-like influenza virus NS1A-binding protein natterin-3-like tropomodulin-2-like Transport proteins mitochondrial import inner membrane translocase subunit TIM14 receptor-transporting protein 3-like cytoglobin-1-like Immunologic proteins galectin-9-like dsRNA-dependent protein kinase (PKR) optineurin tumor necrosis factor receptor superfamily member 6-like (FAS) C-type lectin cell surface glycoprotein 1-like MDA5 beta-2-microglobulin LGP2

Accession

EP

Protein name

RI PT

Table 1 Co-up-regulated genes in crucian carp head kidneys with a 2-fold change post-injection with CyHV-2.

JX477183 XP_016389658 AIX47136 CAD44966 KM374816

68.277 52.895 10.341 9.0579

12.2 21.2 17 24.9

5 3 2 25

3.57 3.79 5.29 5.78

21.56 9.85 23.75 19.03

XP_016139654

254.95

26.1

11

5.92

12.38

XP_016123938

50.29

25.1

10

6.66

29.45

XP_016428232

1.5246

6.6

1

10.06

86.82

20.336 45.772 51.824 8.302 54.055 37.877 8.3489 9.0429 30.01 166.39 24.987 8.7544 130.5

19 4.6 8.8 18.7 32.1 45.5 22.4 7.3 4.5 36.4 17.2 10.5 23.1

5 3 6 3 9 20 13 13 11 10 10 3 8

2.02 2.06 2.09 2.14 2.18 2.34 2.36 2.37 2.45 2.60 2.63 2.66 2.66

3.10 19.70 10.41 9.00 4.86 6.73 8.40 41.93 10.41 3.18 191.34 49.52 17.27

28.55

5.5

1

2.71

14.42

EP

M AN U

TE D

BAA19849 XP_016113729 XM_016260546 XP_016361257 XP_016383738 XP_016364083 XP_018972844 XP_016425750 XP_016097277 XP_016301772 XP_016129071 XP_016303085 XM_016464208 XP_018968452

RI PT

XP_016420903 XP_016354723 XM_016510701 ADZ55452

AC C

Z-DNA binding protein kinase PKZ GTPase IMAP family member 8-like C-X-C motif chemokine 11-like retinoic acid-inducible protein I interferon-induced protein with tetratricopeptide repeats 5-like interferon-induced protein with tetratricopeptide repeats 1-like C-C motif chemokine 3-like Physiologic proteins Y box protein 1 calpain-2 catalytic subunit-like patched domain-containing protein 3-like phospholipid scramblase 1-like aminopeptidase N-like nicotinamide phosphoribosyl transferase E3 ubiquitin/ISG15 ligase TRIM25-like interferon-induced very large GTPase 1-like helicase with zinc finger domain 2-like perilipin-2-like L-amino-acid oxidase-like ubl carboxyl-terminal hydrolase 18-like secernin-3 cat eye syndrome critical region protein 5 homolog

SC

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

70.876 225.84

13.5 38.8

XP_016316343

40.729

9.3

XP_016347281 XP_020317282 XP_016344419 XP_016117153

323.31 83.738 10.659 57.17

70.9 9.9 12.5 22.1

SC

M AN U

XP_016097162

12 8

2.80 2.83

4.47 41.07

10

2.95

5.94

35 10 3 6

3.24 3.31 3.36 3.48

28.25 4.72 43.41 11.88

RI PT

XP_016102986 XP_018971289

6.05

3.6

3

4.19

8.57

136.39 21.957 323.31 323.31 29.218 323.31 75.24 69.123

21.9 13 11.8 67.7 15 23.6 32.8 17.6

11 7 79 34 2 89 19 10

4.24 4.25 4.45 3.48 4.45 4.51 4.84 5.01

41.93 34.54 8.57 6.87 18.38 9.51 137.19 36.76

121.14

34.7

6

5.64

328.56

XP_016329651 AAF17609

41.813 90.277

7 40.4

3 6

5.65 7.64

29.04 60.13

XP_018920582

9.9445

14

4

2.05

12.21

EP

TE D

XP_018920423 XP_016395339 XP_016388594 XP_016145021 XP_016310090 XP_016424105 XP_016110885 XP_018972420 XP_018958746

AC C

putative helicase mov-10-B.1 saxitoxin and tetrodotoxin-binding protein MORC family CW-type zinc finger protein 3-like fibrinogen beta chain-like coagulation factor V-like probable E3 ubiquitin-protein ligase ARI7 interferon-induced protein 44-like probable ATP-dependent RNA helicase YTHDC2 galectin-3-binding protein A-like probable E3 ubiquitin-protein ligase HERC4 microtubule-actin cross-linking factor 1-like fibrinogen gamma chain magnesium-dependent phosphatase 1 sacsin-like E3 ubiquitin-protein ligase TRIM21-like GTPase IMAP family member 4-like urokinase plasminogen activator surface receptor-like phospholipase D4-like ubiquitin-like protein Intracellular proteins lysosome-associated membrane glycoprotein 3-like

XP_016308764 NP_001107919

42.4 67.218

55.4 13.3

XP_018919004

33.963

XP_016408145

XP_016420043 XP_020772775 XP_699055

2.15

3.58

11 10

2.16 2.38

50.56 4.29

8.5

2

2.89

13.18

35.452

6.8

14

3.00

24.93

35.389

22.4

6

3.55

3.84

29.307

11.9

5

3.72

17.63

12.319 116.16

63.5 27

14 8

4.07 4.58

3.48 26.54

27.3 25 22.2 17.6 16.9 13.6 21.6 32.7 8.1 10.4

4 4 7 3 4 3 32 10 3 8

2.19 2.36 2.48 2.59 2.66 2.97 2.99 3.33 3.51 3.51

28.05 13.55 64.00 29.45 13.83 8.94 16.68 22.78 3.14 39.12

M AN U

XP_016150245

KTF81920 XM_016557259 XP_018973791 XM_016229368 XP_016388068 XP_016374881 XM_016527853 XP_016416989 XP_018934868 XP_018928494

23.945 19.799 23.101 32.609 9.6159 5.0247 253.89 43.652 21.132 20.442

2

RI PT

5.6

AC C

uncharacterized protein LOC109072823 uncharacterized LOC107743829 uncharacterized protein LOC109104919 uncharacterized LOC107548613 uncharacterized protein LOC107723855 uncharacterized protein LOC107713783 uncharacterized LOC107720215 uncharacterized protein LOC107747078 uncharacterized protein LOC109062232 uncharacterized protein LOC109055762

10.098

TE D

Unkown/hypothetical proteins

XP_018940062

EP

ribosome biogenesis regulatory protein homolog ES1 protein homolog, mitochondrial-like poly [ADP-ribose] polymerase 14 structural maintenance of chromosomes flexible hinge domain-containing protein 1-like NFX1-type zinc finger-containing protein 1-like nuclear autoantigen Sp-100-like spermatogenesis-associated serine-rich protein 2-like ATP synthase subunit beta, mitochondrial UMP-CMP kinase 2, mitochondrial

SC

ACCEPTED MANUSCRIPT

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XP_018958371 XM_016268492 XM_016471498

EP AC C

24 22 2 6 5 5 12 9 6 4

RI PT

20.5 35.9 2.7 42.9 20.1 38.8 33.9 52.9 28.8 15.8

SC

XP_016111839 KTF81917 XP_016409302 XP_018928792

154.38 295.74 18.176 25.281 18.139 90.613 125.98 113.82 55.99 149.87

M AN U

XP_016296337

TE D

uncharacterized protein LOC107653992 hypothetical protein uncharacterized protein LOC107570153 hypothetical protein uncharacterized protein LOC107741190 uncharacterized protein LOC109056037 hypothetical protein uncharacterized protein LOC109088671 uncharacterized LOC107582590 uncharacterized LOC107676816

3.67 4.12 4.38 5.03 5.04 5.31 6.01 6.35 6.86 7.64

13.64 116.16 63.56 72.50 5.17 49.18 5792.62 18.38 64.00 27.67

ACCEPTED MANUSCRIPT

Table 2 Co-down-regulated genes in crucian carp head kidneys with a 2-fold change post-injection with CyHV-2.

ABC69306

XP_016101863 XP_018930127

Expression fold change

RI PT

Peptide

65.6 9 49.8 40.3

Transcription fold change

34 23 75 19

0.418 0.396 0.371 0.376

0.1908 0.0114 0.0013 0.0085

4.7957

17

3

0.261

0.0013

179.42 19.863 243.28 280.31 61.278 71.025 73.328 33.324 73.767 187.6 178.97

28.6 14 68.5 47.5 33.8 19.1 14.1 36.2 21 19.9 26.2

12 3 9 7 5 9 4 6 6 20 19

0.475 0.474 0.48 0.21 0.332 0.486 0.43 0.321 0.474 0.477 0.41

0.0034 0.2500 0.0002 0.0007 0.0004 0.1571 0.1166 0.0004 0.0046 0.0292 0.0439

88.252 42.431

27 34.6

4 5

0.458 0.429

0.0306 0.0467

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XP_018924250 KTG39838 XP_018960242 XP_016316645 XP_018930635 XP_018948589 XP_016383511 XP_018974436 XP_016370541 XP_016368827 BAB39130

323.31 102.43 323.31 97.049

Coverage

SC

XP_016408711 XP_016414768 XP_020460185 XM_016443857

AC C

Cytoskeleton/extracellular proteins plastin-2 stabilin-1-like myosin-11-like vimentin A2 Transport proteins myoglobin Immunologic proteins complement C3-like immunoglobulin Z heavy chain ladderlectin-like leukocyte cell-derived chemotaxin-2-like lysozyme C-like neutrophil cytosol factor 2 neutrophil cytosol factor 4 ribonuclease-like 3 fucolectin-6-like integrin alpha-X-like integrin beta2-chain Physiologic proteins carboxypeptidase A1-like complement factor H-like

Score

M AN U

Accession

EP

Protein name

ACCEPTED MANUSCRIPT

EP

3 13 3 9 5 3 4 12 22 10 12 9 7 5 10 4 8 3 7

0.421 0.447 0.234 0.489 0.445 0.444 0.472 0.483 0.462 0.346 0.394 0.489 0.382 0.495 0.314 0.348 0.352 0.35 0.463

0.0040 0.0259 0.0007 0.0274 0.1550 0.2146 0.0284 0.0315 0.0012 0.0187 0.0012 0.0036 0.0157 0.1895 0.0111 0.0254 0.0769 0.0769 0.0129

SC

RI PT

14 41.3 17.7 25.5 20.9 24.6 26.5 25.7 37.9 44.6 58.9 15.6 27.8 9.1 66.8 25.5 38 10.1 41.6

M AN U

20.295 34.547 113.16 120.43 25.101 14.296 42.634 136.86 323.31 130.31 258.36 79.928 33.098 11.991 31.262 26.656 109.3 4.5891 103.39

TE D

BAO66551 AAC96093 XP_018932969 AAA21578 AAI54160 XP_018941534 XM_016256558 XP_018956544 XP_018925021 ABI31732 AAB62737 XP_016375259 XP_016410733 XP_016322103 XP_016133891 AAI52207 XP_018975835 AAS10175 XP_016328799

AC C

complement properdin creatine kinase M-type granulins-like kainate receptor alpha subunit potassium channel tetramerisation LIM domain-containing protein 2-like metalloproteinase inhibitor 3-like mucin-2-like myeloperoxidase-like myofibril-bound serine proteinase nephrosin precursor olfactomedin-4-like prostaglandin reductase 1-like rho GTPase-activating protein 15-like serine--pyruvate aminotransferase-like Thy1 protein tumor protein D52-like uncoupling protein 1 vitelline membrane outer layer protein 1-like Intracellular proteins chloride intracellular channel protein 1-like microfibril-associated glycoprotein 4-like Unkown/hypothetical proteins hypothetical protein hypothetical protein

XP_016305915 XM_019085863

199.51 39.305

54.4 24.5

10 5

0.495 0.439

0.2117 0.0836

KTF82305

137.74 77.705

44.7 20.4

7 6

0.352 0.476

0.0100 0.0728

ACCEPTED MANUSCRIPT

2.4712

2

2

EP

TE D

M AN U

SC

RI PT

XM_016556455

AC C

uncharacterized LOC107743289

0.428

0.1158

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

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EP

TE D

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights 7,085 differentially expressed transcriptions and 338 differentially expressed proteins were identified by RNA-seq and LC-MS/MS respectively.

RI PT

128 differentially co-expressed genes were identified after CyHV-2 infection. 10 immune proteins are verified for their immune roles in the CyHV-2 infection by Real-time PCR.

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Herpes simplex infection pathway and phagosome pathway play an important

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role in CyHV-2 infection.