Citation: Wanmeng Liu, Ming Kuang, Ze Zhang, Yuanan Lu, Xueqin Liu. Molecular Characterization and Expression Analysis of ftr01, ftr42, and ftr58 in Zebrafish (Danio rerio) .VIROLOGICA SINICA, 2019, 34(4) : 434-443.  http://dx.doi.org/10.1007/s12250-019-00112-5

Molecular Characterization and Expression Analysis of ftr01, ftr42, and ftr58 in Zebrafish (Danio rerio)

  • Corresponding author: Xueqin Liu, xueqinliu@mail.hzau.edu.cn, ORCID: 0000-0002-0012-4084
  • Received Date: 25 December 2018
    Accepted Date: 21 February 2019
    Published Date: 15 April 2019
    Available online: 01 August 2019
  • Tripartite motif (TRIM) proteins were shown to play an important role in innate antiviral immunity. FinTRIM (ftr) is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates. However, little is known about its function. In this study, we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish (Danio rerio) ftr01, ftr42, and ftr58, as well as qualitative real-time PCR to examine their expression patterns in zebrafish embryonic fibroblast (ZF4) cells and zebrafish tissues. Sequence analysis showed that the three finTRIMs are highly conserved, and all contain a RING domain, B-box domain, and SPRY-PRY domain. In addition, ftr42 and ftr58 had one coiled-coil domain (CCD), whereas ftr01 had two CCDs. Tissue expression analysis revealed that the mRNA level of ftr01 was the highest in the liver, whereas those of ftr42 and ftr58 were the highest in the gill; the expression of these finTRIMs was clearly upregulated not in the eyes, but in the liver, spleen, kidney, gill, and brain of zebrafish following spring viremia of carp virus (SVCV) infection. Similarly, the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection. Our study revealed that ftr01, ftr42, and ftr58 may play an important role in antiviral immune responses, and these findings validate the need for more in-depth research on the finTRIM family in the future.

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    Molecular Characterization and Expression Analysis of ftr01, ftr42, and ftr58 in Zebrafish (Danio rerio)

      Corresponding author: Xueqin Liu, xueqinliu@mail.hzau.edu.cn
    • 1. College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
    • 2. Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, China
    • 3. Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Wuhan 430070, China
    • 4. School of Life Sciences, Beijing Normal University, Beijing 100875, China
    • 5. National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing 102206, China
    • 6. Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, HI, USA

    Abstract: Tripartite motif (TRIM) proteins were shown to play an important role in innate antiviral immunity. FinTRIM (ftr) is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates. However, little is known about its function. In this study, we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish (Danio rerio) ftr01, ftr42, and ftr58, as well as qualitative real-time PCR to examine their expression patterns in zebrafish embryonic fibroblast (ZF4) cells and zebrafish tissues. Sequence analysis showed that the three finTRIMs are highly conserved, and all contain a RING domain, B-box domain, and SPRY-PRY domain. In addition, ftr42 and ftr58 had one coiled-coil domain (CCD), whereas ftr01 had two CCDs. Tissue expression analysis revealed that the mRNA level of ftr01 was the highest in the liver, whereas those of ftr42 and ftr58 were the highest in the gill; the expression of these finTRIMs was clearly upregulated not in the eyes, but in the liver, spleen, kidney, gill, and brain of zebrafish following spring viremia of carp virus (SVCV) infection. Similarly, the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection. Our study revealed that ftr01, ftr42, and ftr58 may play an important role in antiviral immune responses, and these findings validate the need for more in-depth research on the finTRIM family in the future.

    • Innate immunity is the body's immune defense function in the process of development and evolution (Fearon and Locksley 1996; Beutler 2004). Previous studies showed that innate immunity plays an important role in resistance against infection of pathogenic microorganisms, antitumor mechanism, self-tolerance maintenance, and specific immunity (Smyth et al. 2001; Germain 2004).

      The tripartite motif (TRIM) protein family has attracted the attention of many researchers in recent years for various reasons. This family is known to include conserved domain-containing proteins existing in the cytoplasm. Members of the TRIM family contained a RING finger domain linked with one or two B-box domain and a predicted coiled-coil (CC) region, as well as a highly variable C-terminus, such as the PRY/SPRY domain, named the B30.2 domain or the NHL domain. The TRIM family is also known as the Ring finger, B box, coiled-coil (RBCC) family (Reddy and Etkin 1991; Reddy et al. 1992; Borden 1998; Reymond et al. 2001; Uchil et al. 2008). Studies showed that many TRIM proteins play an essential role in antiviral immune signaling pathways in mammals. TRIM5α is the first TRIM protein found to play a role in viral infection. Particularly, the PRY-SPRY domain of TRIM5α can bind directly to the human immunodeficiency virus (HIV) capsid protein and act as a direct antiviral agent (Matthew et al. 2004). In addition, the PRY-SPRY domain of TRIM5α was shown to bind to encephalomyocarditis virus (EMCV) 3C protease and to the nucleoprotein of influenza virus, ultimately degrading viral proteins via the ubiquitin proteasome pathway (Barr et al. 2008; Eldin et al. 2009; Pietro et al. 2013). TRIM52 was reported to inhibit viral replication by degrading Japanese encephalitis virus (JEV)'s NS2A (Fan et al. 2016). In addition, except binding with viral proteins directly, the TRIM proteins TRIM11, TRIM22, TRIM28, and TRIM32 are also reported to inhibit virus replication by interfering viral transcription (Rajsbaum et al. 2014). In addition to participating in antiviral response directly, TRIM proteins have recently been shown to play an important role in regulating antiviral innate immune signaling pathway either positively, such as TRIM4, TRIM21, TRIM25, and TRIM56, or negatively, such as TRIM11, TRIM27, TRIM28, TRIM38, and TRIM59 (Rajsbaum et al. 2014).

      Many studies have shown that, in addition to that of mammals, TRIM proteins play an important role in the antiviral immune system of fish. Fish TRIM8 exerts antiviral roles by regulating proinflammatory factors and interferon signaling (Huang et al. 2016b). TRIM32 and TRIM39 can inhibit the replication of iridovirus and nodavirus (Wang et al. 2016a, b; Yu et al. 2017), and some fish TRIM proteins play a negative regulatory role in antiviral signaling pathways. For example, TRIM16 negatively regulates interferon immune response and promotes the replication of DNA virus (Yu et al. 2016). On the other hand, orange-spotted grouper (Epinephelus coioides) TRIM62 promotes the replication of Redspotted grouper nervous necrosis virus (RGNNV), a positive singlestranded RNA (ssRNA) virus (Yang et al. 2016b).

      A new fish TRIM family was identified in zebrafish (Danio rerio), and named finTRIM (ftr), which is a novel gene family unique to teleost fishes with no obvious orthologs in higher vertebrates. FinTRIMs are specifically induced by viruses and poly (I:C), and therefore, may be involved in the teleost antiviral natural immune processes (Van der Aa et al. 2009, 2012). Little is currently known about the role of finTRIM in the fish immune system. Recent studies have shown that FTR36 triggers the IFN pathway and mediates the inhibition of spring viremia of carp virus (SVCV) replication (Chen et al. 2019). Overexpression of ftr83 promotes the infection of some RNA viruses, including Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and SVCV. However, ftr82 did not play any role in viral infection, although engaged in zebrafish vascular patterning (Chang et al. 2017; Langevin et al. 2017).

      In our previous study, RNA sequencing (RNA-Seq) technology was employed to identify differentially expressed genes in zebrafish after SVCV infection (Wang et al. 2017). Three ftr genes, namely finTRIM family, member 1, 42, and 58 (ftr01, ftr42, and ftr58, respectively), were obviously upregulated, and thus selected based on transcriptome analysis results. This study aimed to: (1) characterize the molecular structure of the ftr01, ftr42, and ftr58 genes of zebrafish; (2) analyze the spatial expression patterns of the three genes using quantitative real-time PCR; (3) investigate the mRNA expression dynamics of the three genes in vivo and in vitro. The results of this study will enhance our understanding of the potential function of these putative finTRIM proteins in the antiviral system of zebrafish.

    • Zebrafish embryonic fibroblast (ZF4) cells (ATCC CRL-2050) derived from 1-day-old zebrafish embryos were routinely maintained in a humidified chamber (28 ℃, 5% CO2) with Dulbecco minimal Eagle's/F-12 medium (Hyclone, Logan, UT, USA) containing 10% (v/v) fetal bovine serum (FBS, Gibco, Melbourne, Australia), streptomycin (100 μg/mL), and penicillin (100 μg/mL) (Gibco). SVCV (ATCC: VR-1390) was used for infection; the virus titer was determined using a 50% tissue culture infectious dose (TCID50) assay and calculated basing on the Reed-Muench method (Pizzi 1950). Adult wild-type (WT) AB strain male zebrafish were purchased from the China Zebra-fish Resource Centre (CZRC) to ensure a clear genetic background. The care, breeding, and feeding of zebrafish followed established protocols (http://www.zfish.cn/inforscan/251.html). Next, zebrafish were allowed to acclimate to a lower temperature by reducing the water temperature by 1 ℃ every 24 h until the water temperature reached 17 ℃. Subsequently, they were maintained at this temperature for ≥ 7 days (d) before the virus infection assay (Ahne et al. 2002; Sanders et al. 2003; Novoa et al. 2006).

    • To examine the tissue distribution of ftr01, ftr42, and ftr58, 8 healthy zebrafish (4 males and 4 females) were sampled. The fishes were anesthetized with MS-222 (300 mg/L) before dissection. Brain, eyes, gill, heart, liver, spleen, kidney, swim bladder, skin, and muscle tissues were collected for RNA isolation.

      To examine the immune responses of those genes in different immune-related tissues after SVCV infection, 120 juvenile zebrafish were collected at the laboratory of Huazhong Agricultural University. In total, 20 zebrafish was randomly sampled, injected with MEM medium (Hyclone, Logan, UT, USA), and used as a control group. The other group was injected intraperitoneally with 10 μL of SVCV at a dose of 105 TCID50, which was determined by our preliminary experiment in this study. In total, 10 fishes was randomly sampled from the experimental group at 0, 1, 2, 3, 4, and 5 days post-infection (dpi). The fishes were anesthetized with MS-222, and then the spleen, head kidney, spleen, liver, gill, brain, and eye were collected for RNA extraction.

    • To examine the immune responses of the ftr01, ftr42, and ftr58 genes in zebrafish ZF4 cells, the cells were infected with SVCV at 5 MOI and then collected by centrifugation at 6, 12, 24, and 36 h after infection.

    • Total RNA was extracted from various tissues and cells using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer's protocol. One microgram of total RNA was used for reverse transcription reaction using PrimeScriptTM RT reagent Kit with a gDNA Eraser (TaKaRa). cDNA products were stored at - 20 ℃.

    • Phylogenetic trees were constructed by the neighboringjoining method by resampling (1000 replicates) using the MEGA7 software (Kumar et al. 2016). The number at each branch of the phylogenetic tree represents the bootstrap value (1000 replicates). The protein structures of the target genes were predicted using the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) (Letunic and Bork 2017); the multiple sequence alignment was performed with the CLUSTALW program (Thompson et al. 1994), and the 3D structural models of the ftr01, ftr42, and ftr58 B30.2 domains, including PRY and SPRY, were built with the Swiss-model program using the high-resolution structure of mammalian TRIM25 as a template (PDB 4B8E) (Waterhouse et al. 2018). The Chimera program was used for structural analysis and B30.2 superposition (Pettersen et al. 2004).

    • Quantitative real time PCR (qRT-PCR) was used to detect the mRNA expression levels of the ftr01, ftr42, and ftr58 genes in various tissues of the affected fish and ZF4 cells at different times post-infection using a 7300 RT-PCR system (Applied Biosystems, USA). The gene-specific primer pairs for qPCR were designed based on the coding DNA sequence of the ftr01, ftr42, and ftr58 genes. In addition, the Primer-BLAST (Ye et al. 2012) was used to assess nontarget amplification using the forward and reverse primer sequences and the entire NCBI nucleotide collection. The TATA-Box Binding Protein (TBP) gene was also used as an internal control gene (Liu et al. 2015). The PCR reaction mixture, at a total volume of 20 μL, consisted of 10 μL LightCycler®480 SYBR Green Ⅰ Master (Roche, Germany), 6.4 μL ddH2O, 2 μL cDNA (5 times dilution of the template), and 0.8 μL of either gene-specific primer (10 μmol/L). The primers used for qRT-PCR amplification of finTRIMs are listed in Supplementary Table S1. The qRT-PCR of each sample was performed in triplicate according to the following conditions: 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s, 58 ℃ for 20 s, and 72 ℃ for 20 s. At the end of each PCR reaction, amplification curve and melting curve analyses were performed to check the integrity of the reaction and the quality of the product, respectively. To compare the gene expressions of ftr01, ftr42, and ftr58, the 2-ΔΔCT method was adopted to calculate the relative expression levels of the target genes (Livak and Schmittgen 2001). All qPCR data were expressed as mean ± standard error (SE). One-way ANOVA and Duncan's post hoc test were conducted using the SPSS 17.0 software to examine the differences in relative gene expression among different samples. The t test was performed to examine the differences in gene expression level between the control and experimental groups after stimulation. A difference was considered statistically significant if P < 0.05 (*).

    • Amino acid alignment analysis showed that ftr01, ftr42, and ftr58 shared 51.16%–71.79% identity; the full-length cDNA of ftr01 encoded a polypeptide of 556 amino acids that shared 56.01%–54.03% identity with ftr42 and ftr58. Homo sapiens TRIM25 shared 23%–25% amino acid identity to zebrafish ftr01, ftr42, and ftr58. Most of fin-TRIMs contain four conserved domains, including a RING/B-Box/coiled-coil domain and a typical B30.2 domain (Fig. 1A). To further confirm the identities of the three finTRIM genes, a phylogenetic tree was constructed (Fig. 1B). Homo sapiens TRIM25 was alone in the tree, which was separated from all zebrafish finTRIMs. The others were divided into two groups, with the first group containing ftr01 and its homologous gene, whereas the second group contained ftr42, ftr58, and other similar genes. As shown in Fig. 1B, ftr42 showed the closest genetic relationship with ftr58, instead of ftr01.

      Figure 1.  A Multiple sequence alignment of zebrafish ftr01, ftr42, and ftr58, and Homo sapiens TRIM25 amino acid sequences, as analyzed by ClustalW using default parameters. The conserved domains, including a RING-finger domain, B box/coiled-coil domain, and B30.2 domain (PRY and SPRY), were indicated with different colors. B Phylogenetic analysis. The Neighbor-Joining tree was constructed using MEGA7. A tree including zebrafish finTRIM genes and Homo sapiens TRIM25. The bootstrap values from 10, 000 replicates were indicated at the branches.

    • To reveal the conserved and diverse domains in these genes, the structural drawings of ftr01, ftr42, and ftr58, and mammalian TRIM25 were shown with boxes representing the distinct domains. The domain structures of finTRIMs were shown schematically with RING, B-box, coiled-coil, and B30.2 domain (PRY and SPRY). The results indicated that ftr42, ftr58, and TRIM25 held a typical TRIM motif distribution, whereas ftr01 had two coiled-coil motifs (Fig. 2A). The 3D structure of the B30.2 domains of ftr01, ftr42, and ftr58 were predicted by the Swiss-model (Fig. 2B). For better presentation, different colors were used in the figure. Golden color represents the structure of the template, the B30.2 domain of mammalian TRIM25, and blue color represents the predicted structure of finTRIM proteins. The results showed that the three finTRIMs shared high sequence similarity with mammalian TRIM25.

      Figure 2.  A Typical domain analysis of zebrafish ftr01, ftr42, and ftr58, and Homo sapiens TRIM25 predicted by the SMART online server. The different domains are labeled and presented with different colors and shapes. B 3D structure of the B30.2 domains of ftr01, ftr42, and ftr58 were built with the Swiss-Model program using the high-resolution structure of mammalian TRIM25 as a template (PDB 4B8E). Model superposition was performed with the Chimera program. Golden color represents the structure of the template, the B30.2 domain of mammalian TRIM25, and blue color represents the predicted structure of finTRIM proteins.

    • qRT-PCR was used to quantify the three finTRIM genes in the brain, eyes, gill, heart, liver, spleen, kidney, swim bladder, skin, and muscle. As shown in Fig. 3, ftr01, ftr42, and ftr58 were detected in all test tissues. ftr01 was expressed at the highest level in the liver. ftr42 and ftr58 were expressed at the highest level in the gill, and highly expressed in several immune-related organs, such as the liver.

      Figure 3.  Basal expression of ftr01, ftr42, and ftr58 mRNA in various tissues of zebrafish. The mRNA levels of the ftr01, ftr42, and ftr58 genes were normalized to those of TBP (internal control) in the same samples. The lowest expression was detected for ftr01 in the eye (A), for ftr42 in the swim bladder (B), and for ftr58 (C) the in skin, and these were selected as calibration values. The relative expression of the ftr01, ftr42, and ftr58 genes in other tissues are represented as fold-changes compared to the calibration values. The results are expressed as mean ± SE (bars in the graph) from 6 fish.

    • In our study, tissue-specific differences in the levels of the three finTRIMs were observed in the examined tissues following SVCV infection. RT-PCR was used to confirm whether zebrafish was infected with SVCV, and the results showed that virus was detected in all tissues (Fig. 4A), and in accordance with the result of our previous experiment, the amount of SVCV increased with time (Chen et al. 2019). As shown in Fig. 4B, the expression of ftr01 mRNA in the kidney, spleen, and eye was upregulated from 1 to 5 dpi, and it reached the highest value at 5 dpi. In the liver and brain, the expression significantly increased from 0 to 4 dpi, and reached a peak value at 4 dpi before decreasing significantly at 5 dpi.

      Figure 4.  Expression pattern of ftr01, ftr42, and ftr58 in the zebrafish tissues infected with SVCV. A Detection of the SVCV-G gene using RT-PCR in the eye, gill, spleen, kidney, brain, and liver samples from SVCV-infected zebrafish and the control group. At 1, 2, 3, 4, and 5 dpi, different tissues were collected and subjected to total RNA extraction and real-time PCR analysis. The mRNA levels of ftr01 (B), ftr42 (C), and ftr58 (D) were normalized to those of TBP, and the relative expression was represented as fold-changes compared to the mock group (M). The data are shown as mean ± SD, and statistical significances were determined by the Student's t-test. *P < 0.05.

      The expression of ftr42 mRNA in the kidney, spleen, and eye was upregulated from 1 to 5 dpi, and it reached a peak value at 5 dpi. In the liver and brain, the expression was significantly upregulated from 1 to 4 dpi, with a peak value at 4 dpi. The expression was significantly downregulated afterwards at 5 dpi (Fig. 4C). The expression level of ftr58 mRNA in the spleen and eye increased from 0 to 5 dpi, and reached a peak value at 5 dpi. In the kidney and brain, the expression significantly increased from 0 to 4 dpi, and reached a peak value at 4 dpi before significantly decreasing at 5 dpi. In the gill, the expression was significantly upregulated from 1 to 3 dpi, and it reached the highest value at 3 dpi; moreover, it slightly decreased at 4 dpi and increased again from 4 to 5 dpi. In the liver, the expression significantly increased from 1 dpi, and increased again from 3 to 5 dpi, with a slight decrease at 4 dpi (Fig. 4D).

    • The relative expression of ftr01, ftr42, and ftr58 mRNAs in ZF4 cells was detected after infection with SVCV at 5 MOI. As shown in Fig. 5A, the mRNA expression of ftr01 was significantly upregulated between 6 h and 24 h postinjection time. The expression level of ftr42 and ftr58 mRNA was upregulated during the entire post-infection time (36 h), and it reached a peak value at 36 h (Figs. 4C and 5B).

      Figure 5.  Expression pattern of ftr01, ftr42, and ftr58 in ZF4 cells infected with SVCV at 5 MOI. Gene expression was normalized to that of TBP, and is presented as fold-change compared to the respective controls. The data are shown as mean ± SD, and statistical significances were determined by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

    • The TRIM family is an E3 ubiquitin ligase group that has attracted more and more attention in recent years owing to its effects on many important cellular processes, such as apoptosis, cell cycle regulation, differentiation, metabolic pathway, and cell response to viral infection (Meroni and Diez-Roux 2005). Increasing number of studies have shown that members of the TRIM family can regulate and coordinate innate immunity and antiviral responses (Chang 2008; Uchil et al. 2008, 2013; Versteeg et al. 2014). In addition, a few recent studies have indicated that the TRIM family play an important role in fish innate immunity; for example, orange-spotted grouper TRIM25 can inhibit the replication of Singapore grouper iridovirus (SGIV) and RGNNV through interferon immune response (Yang et al. 2016a), and common carp TRIM32 can inhibit the replication of SVCV (Wang et al. 2016a, b). However, little is currently known about the finTRIM family in terms of its biological function.

      Bioinformatic analysis showed that ftr01, ftr42, and ftr58 all had the RING domain, as in other RING motifcontaining E3 ligases. The RING domain of TRIMs usually mediates interaction with ubiquitin-conjugating enzyme (E2) via zinc finger motifs (Ye and Rape 2009; Ebner et al. 2017; Esposito et al. 2017). Several studies have shown that the RING domain is essential for the antiviral activity of the TRIM members, including TRIM5α, TRIM56, TRIM41, TRIM25, and ftr83 (Zhang et al. 2013; Liu et al. 2014; Campbell et al. 2015; Yang et al. 2016a; Langevin et al. 2017). Several TRIM-like members, such as TRIM14 and TRIM16 lack a RING domain within the RBCC (Bell et al. 2012; Zhou et al. 2014), but fish TRIM16 has a RING domain while not possessing a B-Box domain (Yu et al. 2016). In this study, a B-Box domain existed in all three finTRIMs. The B-box domain is also crucial for TRIM proteins. A very recent study has indicated that the B-box domain is responsible for the self-association and protein– protein interactions TRIM, which can also promote the oligomerization of higher-order TRIM (Esposito et al. 2017). Analysis of the existing crystal structures of TRIM5α, TRIM20, TRIM25, and TRIM69 revealed that TRIM dimers are formed by coiled-coil antiparallel helical structures (Li et al. 2014; Sanchez et al. 2014; Weinert et al. 2015; Dawidziak et al. 2017; Esposito et al. 2017).

      Understanding the expression of genes in different tissues treated with different challenges is the basic method to examine the specific function of genes (Cui et al. 2010). In the present study, the expression of ftr01, ftr42, and ftr58 was detected in all tested tissues, and the overall expression patterns of the three finTRIMs were significantly higher in the liver and gill than in the other tissues tested. The head kidney, liver, gill, and hindgut of fish are generally considered as immune organs and central to the immune responses (Alvarez-Pellitero 2008). Thus, the expression levels of the three finTRIM genes are different in different tissues, indicating that they may exert their immune effect through tissue-specific mechanisms.

      To further understand the role of finTRIM genes in SVCV infection, we investigated the expression of ftr01, ftr42, and ftr58 at different post-infection times in SVCVinfected zebrafish. Similar to TRIM39 (Wang et al. 2016a, b), the three finTRIM genes were significantly upregulated in the late stage of infection, but to different degrees. However, there was no significant difference in the expression levels of these genes during the early stage of infection (B 24 h). We speculated that the virus titers in zebrafish were low during the early stages of SVCV infection, which had not caused large-scale immune response in vivo. This observation has also been reported recently for the TRIM16 gene (Yu et al. 2016). A host's immune response is activated to remove an invading pathogen, but excessive immune response is also unfavorable for the body, as shown by the results of this study; ftr01 and ftr42 expression in the liver of SVCV-infected fish reached a peak at 4 dpi and then dropped from day 4 to 5 post-infection. This decrease can effectively protect the body from excessive immune response. Such as the transcription of zebrafish ISGs, including IRF7, Mx were upregulated at the early stage of SVCV infection and downregulated at the later stage (Gong et al. 2018). Because many upregulated genes, such as TRIM8, TRIM13 and TRIM25, appeared to play a critical role in the fish immune system following a viral infection (Huang et al. 2016a, b; Yang et al. 2016a), it is reasonable to believe that these finTRIM genes in zebrafish may also play an important role in the immune system. In addition, we have also analyzed the expression of finTRIM genes in SVCV-infected ZF4 cells, and showed that all these three genes were upregulated during the 24 h of SVCV infection. In a previous study, ftr51 and ftr84 are also upregulated in SVCV-infected ZF4 cells (Luo et al. 2017), providing another supportive evidence that the finTRIM family may participate in antiviral innate immunity.

      In conclusion, we analyzed the sequence and characterized the structure of ftr01, ftr42, and ftr58 by bioinformatics, and showed that the expression of ftr01, ftr42, and ftr58 increased significantly after SVCV infection. The study results will serve as the baseline information and research ground for future studies to understand the essential role and mechanisms of these finTRIM proteins in fish innate immunity against viral infections.

    • This work was supported by the National Training Program of Innovation and Entrepreneurship for Undergraduates of Huazhong Agricultural University (2015308200403), the National Key Research and Development Program of China (2018YFD0900505), the Natural Science Foundation of China (31172433), and the Fundamental Research Funds for the Central Universities (2662018YJ022).

    • LX, LW and KM conceived the project, wrote and revised the manuscript. KM performed the experiments. LW, KM and ZZ did data analysis. LY, LX checked and finalized the manuscript. All authors read and approved the final manuscript.

    • The authors declare that they have no conflict of interest.

    • All animal procedures were conducted strictly in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All the animals used for viral infection were not endangered or protected species. Zebrafish were employed in in vivo experimental tests under the approval of the Animal Ethics Committee of Huazhong Agricultural University (HZAU). The infection and dissection experiments were performed under anesthesia with 3-aminobenzoic acid ethyl ester methane sulfonate (MS-222) (Sigma, USA) to minimize suffering in the fishes.

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