Abstract Rock bream iridovirus (RBIV) causes severe mass mortality in Korean rock bream (Oplegnathus fasciatus) populations. To date, immune defense mechanisms of rock bream against RBIV are unclear. While red blood cells (RBCs) are known to be involved in the immune response against viral infections, the participation of rock bream RBCs in the immune response against RBIV has not been studied yet. In this study, we examined induction of the immune response in rock bream RBCs after RBIV infection. Each fish was injected with RBIV, and virus copy number in RBCs gradually increased from 4 days post-infection (dpi), peaking at 10 dpi. A total of 318 proteins were significantly regulated in RBCs from RBIV-infected individuals, 183 proteins were upregulated and 135 proteins were downregulated. Differentially upregulated proteins included those involved in cellular amino acid metabolic processes, cellular detoxification, snRNP assembly, and the spliceosome. Remarkably, the MHC class I-related protein pathway was upregulated during RBIV infection. Simultaneously, the regulation of apoptosis-related proteins, including caspase-6 (CASP6), caspase-9 (CASP9), Fas cell surface death receptor (FAS), desmoplakin (DSP), and p21 (RAC1)-activated kinase 2 (PAK2) changed with RBIV infection. Interestingly, the expression of genes within the ISG15 antiviral mechanism-related pathway, including filamin B (FLNB), interferon regulatory factor 3 (IRF3), nucleoporin 35 (NUP35), tripartite motif-containing 25 (TRIM25), and karyopherin subunit alpha 3 (KPNA3) were downregulated in RBCs from RBIV-infected individuals. Overall, these findings contribute to the understanding of RBIV pathogenesis and host interaction. Keywords: rock bream, RBIV, red blood cells, erythrocyte, proteome, MHC class I, apoptosis, ISG15 Introduction Rock bream iridovirus (RBIV) is a dsDNA virus that belongs to family Iridoviridae, genus Megalocytivirus ([33]1). This virus causes severe mass mortality in Korean rock bream (Oplegnathus fasciatus) populations. RBIV was first reported in the summer of 1998 in southern coastal areas of Korea ([34]2). Since then, high mortality resulting from RBIV occurs every year, causing important economic losses in rock bream aquaculture. RBIV is known to cause strong pathogenicity in rock bream individuals ([35]3–[36]7). To date, the immune response of rock bream with RBIV infection remains unclear, although it represents an important aquaculture health concern. Therefore, it is necessary to further detail the immune response mechanisms underlying the RBIV infection process in rock bream. Over the years, a considerable number of studies have investigated the immune response of rock bream at both physiological and molecular levels by transcriptomic and microarray analyses ([37]8, [38]9). Recently, an increasing number of studies have been focused on the transcriptional immune responses of rock bream against RBIV ([39]10–[40]15). However, most have focused on kidney-mediated immune responses to determine the pathways responsible for fish mortality or survivability. Therefore, evaluation of the immune response or immune defense mechanisms in different organs is useful for the understanding host-RBIV interactions. In contrast to mammalian red blood cells (RBCs) or erythrocytes, which lack a cell nucleus and organelles ([41]16), nonmammalian RBCs are nucleated and contain organelles in their cytoplasm ([42]17). Although the main physiological role for RBCs is the transportation of respiratory gases, their role in the antiviral response has recently been uncovered ([43]18). Importantly, teleost RBCs can induce toll-like receptor (TLR) and peptidoglycan recognition protein (PGRP) receptor families ([44]19), pathogen presentation to macrophages ([45]20), and cytokine or interferon production ([46]21–[47]25). In addition, transcriptomic and proteomic studies of rainbow trout (Oncorhynchus mykiss) showed that nucleated RBCs contribute to several immune functions such as antigen presentation, leukocyte activation or immune cytokine production ([48]26, [49]27). To date, the impact of RBIV on rock bream RBCs in the global fish immune response has not been studied yet. In the present study, we aimed to investigate the differentially expressed proteins (DEPs) in rock bream RBCs upon RBIV in vivo infection in order to understand the molecular contribution of this cell type in the fish immune response against RBIV infection. Proteomic profiling of RBCs from RBIV-infected fish revealed upregulation of apoptosis, antigen processing, and presentation of peptide antigen via MHC class I (MHC-I) pathways. However, the ISG15 antiviral mechanism pathway appeared to be downregulated. Materials and Methods Isolation of RBIV RBIV was obtained from naturally infected rock bream individuals as previously described ([50]11). RBIV major capsid protein (MCP) gene copy number was quantified from supernatant preparations by quantitative real-time polymerase chain reaction (RT-qPCR). Virus titer was calculated as 1.1 × 10^7/100 μL MCP gene copies. Although some studies have demonstrated the use of cell lines to culture Megalocytivirus ([51]28, [52]29), RBIV does not replicate well in in vitro cell culture conditions, so the TCID[50] method was not used in this study. Quantification of RBIV Viral Copy Number RBIV-free rock bream individuals were obtained from a local farm. Thirty fish (11.2 ± 1.2 cm, 28.1 ± 3.2 g) were maintained at 23°C in an aquarium containing 250 L of UV-treated seawater. Fish were injected intraperitoneally (i.p.) with RBIV (100 μL/fish, 1.1 × 10^7 MCP gene copies) or phosphate-buffered saline (PBS) (100 μL/fish) as a control. Blood (200 μL/fish) and organs (spleen, kidney, and liver) were collected from RBIV-infected rock bream individuals at 1, 2, 4, 7, and 10 days post infection (dpi) (4 fish per time point). RBCs were isolated from blood (100 μL/fish) and purified by 2 consecutive density gradient centrifugations (7,206 g, Ficoll 1.007, Sigma-Aldrich). For RBIV copy number analysis, genomic DNA was isolated from the RBCs, blood, spleen, kidney, and liver of each fish using High Pure PCR Template Preparation Kit (Roche) following standard protocol. A standard curve was generated to determine RBIV MCP gene copy number by RT-qPCR as described previously ([53]11). Virus copy number was determined from 100 μL of total genomic DNA. Statistical analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software, USA). One-way analysis of variance (ANOVA) was performed between conditions, with Tukey's multiple comparison test. P < 0.05 were considered to indicate statistical significance. Experimental Infection for RBC Proteomic Analysis Fish (11.0 ± 0.8 cm, 29.3 ± 4.7 g) were randomly divided into two groups (20 fish per group): a virus-injected group and a PBS-injected group. The experimental group was injected i.p. with RBIV (100 μL/fish) containing 1.1 × 10^7 MCP gene copies, and the control group was injected i.p. with PBS (100 μL/fish). Each group of fish were maintained at 23°C in the aquarium containing 250 L of UV-treated seawater. Blood (100 μL/fish) was collected from 8 fish at 7 dpi. Then, RBCs were purified by 2 consecutive density gradient centrifugations (7,206 g, Ficoll 1.007, Sigma-Aldrich). All rock bream experiments were carried out in strict accordance with the recommendations of the Institutional Animal Care and Use Committee of Chonnam National University (permit number: CNU IACUC-YS-2015-4). Proteomic Analysis Ficoll-purified RBCs from 5 fish in each group were pelletized by centrifugation (1,600 rpm). The cell pellet was washed with PBS, digested, cleaned-up/desalted, and pooled for each group (2 control groups and 2 RBIV-infected fish groups). Then, samples were subjected to liquid chromatography and mass spectrometry analysis (LC-MS) as previously described ([54]26), except that the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Inc.) was used and 3 peptide fractions were collected. Progenesis QI v4.0 (Nonlinear Dynamics, Newcastle, UK) was used for protein differential expression analysis according to “between-subject design.” Log[2] peptide ratios followed a normal distribution that was fitted using least squares regression. Mean and standard deviation values were derived from Gaussian fit and were used to estimate P-values and false discovery rates (FDRs). The confidence interval for protein identification was set to ≥95% (P ≤ 0.05). Only proteins having ≥2 quantitated peptides were considered. Peptides with an individual ion score above the 1% FDR threshold were considered correctly identified. Pathway Enrichment Analysis DEP pathway enrichment analysis was performed using ClueGO ([55]30), CluePedia ([56]31), and Cytoscape ([57]32). The GO Biological Process, GO Immune Process, Kegg, Reactome, and Wikipathways databases were used. A P ≤ 0.05 and Kappa score of 0.4 were used as threshold values. Proteins were identified by sequence homology with Homo sapiens using Blast2GO version 4.1.9 ([58]33). Quantitative Real-Time PCR Analysis of Gene Expression For immune gene expression analysis, total RNA was extracted from RBCs using RNAiso Plus reagent (TaKaRa) following standard protocol. Total RNA was treated with DNase I (TaKaRa) and reverse transcribed using a ReverTra Ace qPCR RT Kit (Toyobo) according to manufacturer's protocol. Real-time PCR was carried out in an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer) using an AccuPre® 2x Greenstar qPCR Master Mix (Bioneer) as described previously ([59]11). Each assay was performed in duplicate using β-actin genes as the endogenous control. The primers used are listed in [60]Table 1. Relative gene expression was determined by the 2^−ΔΔCt method ([61]34). Statistical analyses were performed using GraphPad Prism software. Unpaired T-tests were performed between conditions. P < 0.05 were considered to indicate statistical significance. Data are represented as mean ± standard deviation. Table 1. List of primers used. Name Sequence Accession number β-actin F CAGGGAGAAGATGACCCAGA R CATAGATGGGCACTGTGTGG [62]FJ975145 MCP F GTGTCTAAAGGGACTGAACATCG R CCCTCAAACGTTACTGGATACTG [63]AY849394 IRF3 F TGGGAGTAACCCTTATGTCCTG R CTTCCTCGTCTGTTCCTTCTTG [64]KF267453.1 MHC class I F AGATTACTGGGAAAAAGGCACA R TCATTCGTTTCATCAGGATGTC [65]KC193602 Fas F GTTTCGTGCGTCGTTTATCA R CAAACCTGCAGCACACAGACA [66]AB619804 Caspase 9 F TCTTGGAGAGACACCCAGTCG R GCCCTTTTGCAGAGTTTTGG [67]KF501038 [68]Open in a new tab Results RBIV Levels in Rock Bream RBCs RBIV copy number was quantified in RBC, blood, spleen, kidney, and liver samples. At 2, 4, 7, and 10 dpi, increased viral copy numbers were observed in the spleen, kidney, and liver. The maximum copy number for all samples was reached at 10 dpi (average value of 4.99 × 10^7 in the spleen, 2.56 × 10^7 in the kidney, and 2.44 × 10^7/100 μL in the liver) ([69]Figures 1A–C). Figure 1. [70]Figure 1 [71]Open in a new tab RBIV MCP gene copy number in different rock bream organs. Fish i.p. injected with RBIV (1.1 × 10^7) were maintained at 23°C. Virus copy number in spleen (A), kidney (B), liver (C), blood (D), and RBCs (E) were analyzed at 1, 2, 4, 7, and 10 days post infection (dpi). One-way analysis of variance (ANOVA) was performed between conditions, with Tukey's multiple comparison test. Different superscript letters denote significant differences (P < 0.05). a≠ b. Data are represented as individual values. Line represents mean value. In blood samples, the viral transcription level was 7.16 × 10^1/100 μL at 1 dpi, gradually increased to 3.81 × 10^2/100 μL at 2 dpi, and reached maximum values of 9.36 × 10^3/100 μL at 7 dpi and 2.04 × 10^4/100 μL at 10 dpi ([72]Figure 1D). In Ficoll-purified RBCs from fish at 1, 2, 4, 7, and 10 dpi, virus copy numbers gradually increased with time; the average number of virus copies was 1.25 × 10^2, 2.31 × 10^2, 8.42 × 10^2, 9.22 × 10^3, and 3.54 × 10^4/100 μL, respectively ([73]Figure 1E). Protein Profiling of RBCs From RBIV-Infected Rock Bream Cytoscape pathway enrichment analysis was performed in order to evaluate the functional pathways involved in the response of rock bream RBCs to RBIV ([74]Figure 2). Proteins with a FDR < 0.001 and−1.5>log[2] Fold Change (FC)>1.5 were selected for functional network analysis. A total of 318 proteins were differentially regulated at a significant level in RBCs from RBIV-infected individuals: 183 proteins were upregulated and 135 were downregulated. Upregulated pathways were categorized into 13 main categories, while downregulated pathways were categorized into 2 ([75]Figures 2–[76]6 and [77]Tables 2–[78]4). Within upregulated pathways, proteins were involved in synthesis of active ubiquitin, E1 and E2 enzymatic roles, pyridine-containing compound metabolic processes, RNA transport, the spliceosome, cytosolic tRNA aminoacylation, the vitamin B6 biosynthetic process, snRNP assembly, cellular detoxification, the cholesterol biosynthetic process, the cellular amino acid metabolic process, the Parkin-Ubiquitin proteasomal system pathway, apoptosis, and antigen processing and presentation of peptide antigen via MHC class I ([79]Figures 2–[80]4 and [81]Tables 2, [82]3). Within downregulated pathways, proteins were mainly involved in the ISG15 antiviral mechanism and p130Cas linkage to MAPK signaling for integrins ([83]Figures 2, [84]5, [85]6 and [86]Table 4). Figure 2. [87]Figure 2 [88]Open in a new tab Cytoscape network analysis of differentially expressed protein (DEPs) in RBCs from RBIV-infected rock bream. DEPs in RBCs from RBIV-infected rock bream at 7 dpi, with −1.5 < log[2]FC < 1.5 and FDR P < 0.001. Overrepresented terms were identified by the Cytoscape ClueGo app, with GO Biological Process, Kegg, Reactome, and Wikipathways term databases. Red circles indicate upregulated/overrepresented terms, and green circles indicate downregulated/overrepresented terms. Gray circles indicate unspecific regulation. Color intensity represents the degree of overrepresentation. Figure 6. [89]Figure 6 [90]Open in a new tab Comparative protein levels in upregulated and downregulated overrepresented pathways in RBCs from RBIV-infected rock bream. Data represent the number of proteins represented in each pathway. Red bars indicate upregulated proteins and dashed bars indicate downregulated proteins. Table 2. List of upregulated pathways in RBCs from RBIV-infected rock bream. Category Accession Protein name Protein description Log[2]FC Synthesis of active ubiquitin: roles of E1 and E2 enzymes [91]A0A096M453 UCHL3 Ubiquitin C-terminal hydrolase L3 +4.54169 [92]A0A060YC09 UBE2L3 Ubiquitin conjugating enzyme E2 L3 +3.28977 [93]E7EXC7 USP9X Ubiquitin specific peptidase 9 X-linked +1.86819 [94]A0A1A8BMW9 USP5 Ubiquitin specific peptidase 5 +1.73518 [95]A0A1A7XFZ1 UBA6 Ubiquitin like modifier activating enzyme 6 −5.67014 Pyridine-containing compound metabolic process [96]A0A0P7UQB0 NUP98 Nucleoporin 98 +6.56510 [97]A0A060W490 PNPO Pyridoxamine 5′-phosphate oxidase +6.54472 [98]A0A1A8DQA8 PHGDH Phosphoglycerate dehydrogenase +5.96297 [99]A0A060X3S4 PDXK Pyridoxal kinase +3.52413 [100]A0A060X2R3 NUP93 Nucleoporin 93 +3.41500 [101]A0A023UJE3 ENO1 Enolase 1 +2.47019 [102]A0A060YZP7 MPC2 Mitochondrial pyruvate carrier 2 +2.35431 [103]A0A087XLW0 PGAM1 Phosphoglycerate mutase 1 +2.21249 [104]J3QRQ2 DCXR Dicarbonyl and L-xylulose reductase +1.89013 [105]A0A087Y0K3 PSAT1 Phosphoserine aminotransferase 1 +1.65664 [106]A0A087Y968 TPI1 Triosephosphate isomerase 1 −2.53038 [107]H3CAN5 GALK1 Galactokinase 1 −3.00322 [108]A0A146MRI7 NUP35 Nucleoporin 35 −6.06319 [109]A0A1A7XVE8 MDH1 Malate dehydrogenase 1 −7.96449 RNA transport [110]A0A0P7UQB0 NUP98 Nucleoporin 98 +6.56510 [111]A0A146RA28 EIF5B Eukaryotic translation initiation factor 5B +4.94553 [112]A0A087XQU0 PYM1 PYM homolog 1, exon junction complex associated factor +4.00632 [113]H2MNB4 EIF2B3 Eukaryotic translation initiation factor 2B subunit gamma +3.75534 [114]A0A060X2R3 NUP93 Nucleoporin 93 +3.41500 [115]C3KH96 RBM8 RNA binding motif protein 8A +3.16004 [116]A0A060WH91 PABPC1 Poly(A) binding protein cytoplasmic 1 +2.51108 [117]A0A1A7XKU0 RANGAP1 Ran GTPase activating protein 1 +2.40663 [118]A0A087XK21 TRNT1 tRNA nucleotidyl transferase 1 +1.52564 [119]A0A087XJ99 EIF3I Eukaryotic translation initiation factor 3 subunit I −2.81793 [120]A0A060XCL3 ALYREF Aly/REF export factor −3.15159 [121]H2LP66 EIF3J Eukaryotic translation initiation factor 3 subunit J −4.12793 [122]A0A146MRI7 NUP35 Nucleoporin 35 −6.06319 Spliceosome [123]A0A0P7XD74 SNRPF Small nuclear ribonucleoprotein polypeptide F +8.79320 [124]A0A087Y346 SNRPD1 Small nuclear ribonucleoprotein D1 polypeptide +4.98734 [125]I3KZX4 LSM3 LSM3 homolog, U6 small nuclear RNA and mRNA degradation associated +3.60900 [126]C3KH96 RBM8 RNA binding motif protein 8A +3.16004 [127]A0A060XGY3 SF3A3 Splicing factor 3a subunit 3 +2.23314 [128]A0A1L3A6A6 HSPA8 Heat shock protein family A (Hsp70) member 8 +1.81502 [129]A0A0P7UL65 SNRPG Small nuclear ribonucleoprotein polypeptide G +1.70328 [130]A0A087Y0E9 PPIH Peptidylprolyl isomerase H −3.11189 [131]A0A060XCL3 ALYREF Aly/REF export factor −3.15159 [132]H2RJ37 SNRPA1 Small nuclear ribonucleoprotein polypeptide A' −3.29532 Cytosolic tRNA aminoacylation [133]G3NSI9 FARSLA Phenylalanyl-tRNA synthetase subunit alpha +3.43435 [134]A0A1A7ZJC0 MARS Methionyl-tRNA synthetase +3.27543 [135]A0A087YJF0 EPRS Glutamyl-prolyl-tRNA synthetase +2.78295 [136]A0A060YC35 SARS Seryl-tRNA synthetase +2.61934 [137]A0A060WQF7 LARS Leucyl-tRNA synthetase −1.93372 [138]A0A060W490 PNPO Pyridoxamine 5′-phosphate oxidase +6.54472 [139]A0A060X3S4 PDXK Pyridoxal kinase +3.52413 [140]A0A087Y0K3 PSAT1 Phosphoserine aminotransferase 1 +1.65664 snRNP Assembly [141]A0A0P7XD74 SNRPF Small nuclear ribonucleoprotein polypeptide F +8.79320 [142]A0A0P7UQB0 NUP98 Nucleoporin 98 +6.56510 [143]A0A087Y346 SNRPD1 Small nuclear ribonucleoprotein D1 polypeptide +4.98734 [144]A0A060X2R3 NUP93 Nucleoporin 93 +3.41500 [145]A0A0P7UL65 SNRPG Small nuclear ribonucleoprotein polypeptide G +1.70328 [146]A0A146MRI7 NUP35 Nucleoporin 35 −6.06319 Cellular detoxification [147]A0A087YGW8 CLIC2 Chloride intracellular channel 2 +6.00740 [148]H2RV41 GSTM3 Glutathione S-transferase mu 3 +5.94070 [149]I3IV50 FAS Fas cell surface death receptor +5.88751 [150]W5KQL6 APOE Apolipoprotein E +4.62692 [151]A0A0S7HP87 FAM213B Family with sequence similarity 213 member B +4.13534 [152]B9MSR2 SOD1 Superoxide dismutase 1 +2.53220 [153]A0A087X9L9 TXNRD3 Thioredoxin reductase 3 +2.07657 [154]A0A060VRY4 XPA XPA, DNA damage recognition and repair factor +1.76996 [155]A0A087YMH6 ADH5 Alcohol dehydrogenase 5 (class III), chi polypeptide +1.57015 [156]W5NF82 NEFL Neurofilament light +1.50524 [157]A0A087YDB9 TRPM6 Transient receptor potential cation channel subfamily M member 6 −2.90258 [158]B3VTP4 APOA4 Apolipoprotein A4 −3.50052 [159]A0A087WSW9 TXNRD1 Thioredoxin reductase 1 −3.51362 [160]C9DTM6 EPX Eosinophil peroxidase −5.96073 [161]A0A0F8BVI8 MPO Myeloperoxidase −5.96073 Cholesterol biosynthetic process [162]W5KQL6 APOE Apolipoprotein E +4.62692 [163]W5NG17 GGPS1 Geranylgeranyl diphosphate synthase 1 +3.68607 [164]A0A0S7LJM9 CNBP CCHC-type zinc finger nucleic acid binding protein +3.65477 [165]A0A060X0E0 ERLIN2 ER lipid raft associated 2 +3.09663 [166]A0A060WK05 PMVK Phosphomevalonate kinase +3.00278 [167]C1BJ00 VDAC2 Voltage dependent anion channel 2 +2.784311 [168]B9MSR2 SOD1 Superoxide dismutase 1 +2.53220 [169]B3VTP4 APOA4 Apolipoprotein A4 −3.50052 [170]C1BKM7 APOA1 Apolipoprotein A1 −3.58118 [171]I6QFY3 CFTR Cystic fibrosis transmembrane conductance regulator −3.85295 Cellular amino acid metabolic process [172]A0A146NIL6 HNMT Histamine N-methyltransferase +7.33475 [173]Q19A30 ALDH9A1 Aldehyde dehydrogenase 9 family member A1 +7.08477 [174]H2M1L3 GCLC Glutamate-cysteine ligase catalytic subunit +7.05565 [175]A0A1A8DQA8 PHGDH Phosphoglycerate dehydrogenase +5.96297 [176]A0A087YCZ2 SBDS SBDS, ribosome maturation factor +5.38388 [177]H2SS02 PYCR3 Pyrroline-5-carboxylate reductase 3 +4.13757 [178]A0A0P7USQ3 PSMD11 Proteasome 26S subunit, non-ATPase 11 +3.83617 [179]W5UAL8 GSS Glutathione synthetase +3.49635 [180]A0A087X9P9 RPS28 Ribosomal protein S28 +3.46599 [181]G3NSI9 FARSLA Phenylalanyl-tRNA synthetase subunit alpha +3.43435 [182]A0A1A7ZJC0 MARS Methionyl-tRNA synthetase +3.27543 [183]A0A147AHI6 PSMB6 Proteasome subunit beta 6 +3.23477 [184]Q66HW0 COASY Coenzyme A synthase +2.88808 [185]A0A087YJF0 EPRS Glutamyl-prolyl-tRNA synthetase +2.78295 [186]A0A087WUL2 PSMB3 Proteasome subunit beta 3 +2.74293 [187]A0A060YC35 SARS Seryl-tRNA synthetase +2.61934 [188]H2VBD9 PSMD5 Proteasome 26S subunit, non-ATPase 5 +2.46929 [189]A0A060YZH5 RPS21 Ribosomal protein S21 +2.03250 [190]A0A0N8K350 ARG2 Arginase 2 +1.90666 [191]H2MN42 NIT2 Nitrilase family member 2 +1.87753 [192]Q45VN8 PSMB4 Proteasome subunit beta 4 +1.84703 [193]A0A087Y0K3 PSAT1 Phosphoserine aminotransferase 1 +1.65664 [194]A0A087XKC8 ALDH4A1 Aldehyde dehydrogenase 4 family member A1 −1.60365 [195]A0A0F8C9G0 AASDHPPT Aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase −1.8438 [196]W5M476 SARDH Sarcosine dehydrogenase −1.86083 [197]A0A060WQF7 LARS Leucyl-tRNA synthetase −1.93372 [198]A0A060Z3T7 MRI1 Methylthioribose-1-phosphate isomerase 1 −2.64492 [199]A0A087WSW9 TXNRD1 Thioredoxin reductase 1 −3.51362 Parkin-ubiquitin proteasomal system pathway [200]A0A146UQZ0 CCT3 Chaperonin containing TCP1 subunit 3 +4.20768 [201]A0A0P7USQ3 PSMD11 Proteasome 26S subunit, non-ATPase 11 +3.83617 [202]A0A060YC09 UBE2L3 Ubiquitin conjugating enzyme E2 L3 +3.28977 [203]A0A147AHI6 PSMB6 Proteasome subunit beta 6 +3.23477 [204]A0A146VFH4 TUBA4A Tubulin alpha-4A chain +2.86588 [205]A0A060WLR9 TUBA3C Tubulin alpha 3c +2.86588 [206]A0A087WUL2 PSMB3 Proteasome subunit beta 3 +2.74293 [207]H2VBD9 PSMD5 Proteasome 26S subunit, non-ATPase 5 +2.46929 [208]Q45VN8 PSMB4 Proteasome subunit beta 4 +1.84703 [209]A0A146PU69 ACTB Actin beta +1.83645 [210]A0A1L3A6A6 HSPA8 Heat shock protein family A (Hsp70) member 8 +1.81502 [211]A0A189JAM4 TUBA1C Tubulin alpha 1c −2.55283 [212]H6QXT0 CASP1 Caspase 1 −2.90548 [213]F2Z2E2 IQGAP3 IQ motif containing GTPase activating protein 3 −4.82941 Apoptosis [214]A0A0P7UQB0 NUP98 Nucleoporin 98 +6.56510 [215]I3IV50 FAS Fas cell surface death receptor +5.88751 [216]A0A060WPW9 RUVBL1 RuvB like AAA ATPase 1 +5.68115 [217]A0A060X986 CASP9 Caspase 9 +5.34643 [218]A0A096M453 UCHL3 Ubiquitin C-terminal hydrolase L3 +4.54169 [219]W5LA34 ABCB1 ATP binding cassette subfamily B member 1 +4.23220 [220]A0A146UQZ0 CCT3 Chaperonin containing TCP1 subunit 3 +4.20768 [221]A0A0P7USQ3 PSMD11 Proteasome 26S subunit, non-ATPase 11 +3.83617 [222]A0A060X2R3 NUP93 Nucleoporin 93 +3.41500 [223]A0A060YC09 UBE2L3 Ubiquitin conjugating enzyme E2 L3 +3.28977 [224]A0A147AHI6 PSMB6 Proteasome subunit beta 6 +3.23477 [225]A0A060X0E0 ERLIN2 ER lipid raft associated 2 +3.09663 [226]C1BJ00 VDAC2 Voltage dependent anion channel 2 +2.78431 [227]A0A087WUL2 PSMB3 Proteasome subunit beta 3 +2.74293 [228]A0A060XWP8 RPN2 Ribophorin II +2.51942 [229]A0A060WH91 PABPC1 Poly(A) binding protein cytoplasmic 1 +2.51108 [230]H2VBD9 PSMD5 Proteasome 26S subunit, non-ATPase 5 +2.46929 [231]A0A087XG68 HMGB2 High mobility group box 2 +2.41814 [232]A0A1A7XKU0 RANGAP1 Ran GTPase activating protein 1 +2.40663 [233]A0A146RM67 DSP Desmoplakin +2.25958 [234]A0A060VUK9 ACTL6A Actin like 6A +1.92039 [235]E7EXC7 USP9X Ubiquitin specific peptidase 9 X-linked +1.86819 [236]Q45VN8 PSMB4 Proteasome subunit beta 4 +1.84703 [237]A0A1L3A6A6 HSPA8 Heat shock protein family A (Hsp70) member 8 +1.81502 [238]A0A1A8BMW9 USP5 Ubiquitin specific peptidase 5 +1.73518 [239]H2MXM9 CASP6 Caspase 6 +1.65460 [240]A0A060W5L7 USP47 Ubiquitin specific peptidase 47 −1.85717 [241]A0A1A8GUB0 YWHAB Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta −2.17782 [242]A0A060WMK5 PAK2 p21 (RAC1) activated kinase 2 −2.39132 [243]G3NDG3 PLEC Plectin −3.20510 [244]G3NRU2 RNF146 Ring finger protein 146 −3.25047 [245]C1BKM7 APOA1 Apolipoprotein A1 −3.58118 [246]I6QFY3 CFTR Cystic fibrosis transmembrane conductance regulator −3.85294 [247]F2Z2E2 IQGAP3 IQ motif containing GTPase activating protein 3 −4.82941 [248]X1WEE8 TRIM25 Tripartite motif containing 25 −5.61605 [249]A0A146MRI7 NUP35 Nucleoporin 35 −6.06319 [250]Open in a new tab Table 4. List of downregulated pathways in RBCs from RBIV-infected rock bream. Category Accession Protein name Protein description Log[2]FC ISG15 antiviral mechanism [251]A0A0P7UQB0 NUP98 Nucleoporin 98 +6.56510 [252]A0A060X2R3 NUP93 Nucleoporin 93 +3.41500 [253]C7ATZ0 STAT1 Signal transducer and activator of transcription 1 +2.72893 [254]A0A060W790 KPNA3 Karyopherin subunit alpha 3 −1.55875 [255]A0A067ZTD7 IRF3 Interferon regulatory factor 3 −2.77578 [256]X1WEE8 TRIM25 Tripartite motif containing 25 −5.61605 [257]A0A087X811 FLNB Filamin B −5.77028 [258]A0A146MRI7 NUP35 Nucleoporin 35 −6.06319 p130Cas linkage to MAPK signaling for integrins [259]Q6PH06 CRK CRK proto-oncogene, adaptor protein +3.83669 [260]A0A146RM67 DSP Desmoplakin +2.25958 [261]A0A0F8ALN2 FGA Fibrinogen alpha chain −1.84245 [262]H2LW76 FGG Fibrinogen gamma chain −3.25828 [263]C1BKM7 APOA1 Apolipoprotein A1 −3.58117 [264]A0A087X4W0 FGB Fibrinogen beta chain −4.94492 [265]A0A0R4ICS1 ITGA4 Integrin subunit alpha 4 −5.35249 [266]Open in a new tab Figure 4. [267]Figure 4 [268]Open in a new tab GO Immune System Process terms in the proteome profile of RBIV-infected RBCs. Upregulated/overrepresented terms in DEPs of RBCs from RBIV-infected rock bream at 7 dpi, with −1.5