Siniperca Chuatsi Rhabdovirus Research Articles

Transcriptomic profiling of the immune response to Siniperca chuatsi rhabdovirus (SCRV) in E11 cells

Transcriptomic profiling of the immune response to Siniperca chuatsi rhabdovirus (SCRV) in E11 cells

Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

CircMORC3-encoded novel protein negatively regulates antiviral immunity through synergizing with host gene MORC3.

The protein-coding ability of circRNAs has recently been a hot topic, but the role of protein-coding circRNAs in antiviral innate immunity of teleost fish has rarely been reported. Here, we identified a novel circRNA, termed circMORC3, derived from Microrchidia 3 (MORC3) gene in Miichthys miiuy. circMORC3 can inhibit the expression of antiviral cytokines. In addition, circMORC3 encodes a novel peptide with a length of 84 amino acids termed MORC3-84aa. MORC3-84aa not only significantly inhibited TRIF-mediated activation of IRF3 and NF-κB signaling pathways, but also effectively suppressed the expression of antiviral cytokines triggered by RNA virus Siniperca chuatsi rhabdovirus (SCRV). We found that MORC3-84aa directly interacted with TRIF and negatively regulated TRIF protein expression. In addition, host gene MORC3 attenuates SCRV-induced IFN and ISG expression. Mechanistically, MORC3-84aa promotes autophagic degradation of TRIF by enhancing K6-linked ubiquitination and inhibits TRIF-mediated activation of the type I interferon signaling pathway. And the host gene MORC3 not only repressed IRF3 protein expression but also inhibited IRF3 phosphorylation levels. Our study shows that circMORC3 and host gene MORC3 played a synergistic role in viral immune escape.

A novel protein encoded by circVPS13D attenuates antiviral innate immunity by targeting MAVS in teleost fish.

The expression of circVPS13D was upregulated with SCRV invasion, which proved that circVPS13D was involved in the regulation of the antiviral immune response. Our study revealed that the existence of circVPS13D promoted the replication of SCRV. Functionally, circVPS13D negatively regulates the antiviral responses of fish. Mechanistically, we confirmed that circVPS13D inhibited RLRs antiviral signaling pathway via the encoded protein VPS13D-170aa by targeting MAVS. Our study provided novel insights into the roles of protein-coding circRNAs and supported VPS13D-170aa as a negative regulator in the antiviral immune responses of teleost fish.

Smyd3 negatively regulates the anti-viral pathway by promoting TAK1 degradation in teleost fish.

In this study, we have found that the existence of Smyd3 promoted the replication of SCRV. Additionally, we report that Smyd3 negatively regulates the NF-κB and IRF3 signaling pathway by facilitating the degradation of TAK1 in fish. Our findings suggest that Smyd3 interacts with TAK1. Further investigations have revealed that Smyd3 specifically mediates K48-linked ubiquitination of TAK1 and enhances TAK1 degradation, resulting in a significant inhibition of the NF-κB and IRF3 signaling pathway. These results not only contribute to the advancement of fish anti-viral immunity but also provide new evidence for understanding the mechanism of TAK1 in mammals.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

Oxygen-Sensing Protein Cysteamine Dioxygenase from Mandarin Fish Involved in the Arg/N-Degron Pathway and Siniperca chuatsi Rhabdovirus Infection.

Mammalia cysteamine (2-aminoethanethiol) dioxygenase (ADO) controls the stability of the regulator of G protein signaling 4 (RGS4) through the Cys branch of the Arg/N-degron pathway, thereby affecting the response of the body to hypoxia. However, the oxygen-sensing function of ADO remains unknown in teleost fish. Mandarin fish (Siniperca chuatsi) is one of the most important freshwater economic fishes in China. As the scale of the rearing density continues to increase, hypoxia has become an important factor threatening the growth of mandarin fish. Herein, the molecular characterization, the oxygen-sensing enzyme function, and the role in virus infection of ADO from mandarin fish (scADO) were explored. Bioinformation analysis results showed that scADO had all the molecular foundations for achieving thiol dioxygenase function: three histidine residues coordinated with Fe(II), PCO/ADO domain, and a "jelly roll" β-barrel structure. The expression pattern analysis showed that scAdo was highly expressed in the immune-related tissues, liver, and kidneys and responded to hypoxia on the expression level. Protein degradation experiment results revealed that scADO could lead to the degradation of RGS4 protein through the Cys branch of the Arg/N-degron pathway. Furthermore, the expression levels of scADO responded to fish virus infection. scADO could significantly promote the replication of Siniperca chuatsi rhabdovirus, and this was associated with its thiol dioxygenase activity. These findings not only demonstrate scADO as an oxygen-sensing protein in teleost fish, but are also of considerable importance for clarifying the contribution of the mechanism of hypoxia to the outbreaks of fish viruses.

Infection tracing and organ tropism of Siniperca chuatsi rhabdovirus expressing enhanced green fluorescent protein

Siniperca chuatsi Rhabdovirus (SCRV) is one of the important pathogens that infect mandarin fish. Recovery of recombinant viruses expressing fluorescent protein is an important method for virology research. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted between the leader and the N gene of the SCRV genome. The recombinant SCRV of rS3-LeNEGFP expressing the EGFP was rescued, and the virus titer was as high as 2 × 106 PFU/mL in EPC cells. The insertion of the EGFP gene in the genome of SCRV was stable. EGFP was expressed efficiently after 10 passages in EPC cells. In vitro growth characterization, pathogenicity and organ tropism in vivo were studied. Compared to the parental rS3, rS3-LeNEGFP was defective in vitro, but not attenuated in vivo. The mortality of red zebrafish caused by rS3-LeNEGFP was the same as the parental SCRV rS3. The earliest expression of EGFP was detectable at 2 h, and clear fluorescence spots appeared at 8 h post-infection in EPC cells. The liver, heart, and brain were the major organ targets of rS3-LeNEGFP in vivo, and the kidney, gill, and spermatocyte were also invaded. The organ infection in vitro was different from that in vivo. This study is of great significance for revealing the pathogenesis of SCRV and developing vaccines and antiviral drugs for SCRV in the future.

METTL3-Mediated m6A Modification of TRIF and MyD88 mRNAs Suppresses Innate Immunity in Teleost Fish, Miichthys miiuy.

Methyltransferase (METTL3), the most important N6-methyladenosine (m6A) writer, plays a vital role in regulating immune-related signaling pathways. However, the underlying mechanism of METTL3 action remains largely unknown, especially in lower vertebrates. The results of this study show that METTL3 inhibits innate immune response and promotes the infection of miiuy croaker, Miichthys miiuy, by Siniperca chuatsi rhabdovirus and Vibrio anguillarum. Significantly, the function of METTL3 in inhibiting immunity depends on its methylase activity. Mechanistically, METTL3 increases the methylation level of trif and myd88 mRNA, rendering them sensitive to degradation by the YTHDF2/3 reader proteins. By contrast, we found that the YTHDF1 reader protein promotes the translation of myd88 mRNA. In summary, these results indicate that METTL3-mediated m6A modification of trif and myd88 mRNAs suppresses innate immunity by inhibiting the TLR pathway, unveiling a molecular mechanism by which RNA methylation controls innate immunity to pathogens in the teleost fish.

Interaction of Nmi and IFP35 Promotes Mutual Protein Stabilization and IRF3 and IRF7 Degradation to Suppress Type I IFN Production in Teleost Fish.

IFN-stimulated genes (ISGs) can act as effector molecules against viral infection and can also regulate pathogenic infection and host immune response. N-Myc and STAT interactor (Nmi) is reported as an ISG in mammals and in fish. In this study, the expression of Nmi was found to be induced significantly by the infection of Siniperca chuatsi rhabdovirus (SCRV), and the induced expression of type I IFNs after SCRV infection was reduced following Nmi overexpression. It is observed that Nmi can interact with IRF3 and IRF7 and promote the autophagy-mediated degradation of these two transcription factors. Furthermore, Nmi was found to be interactive with IFP35 through the CC region to inhibit IFP35 protein degradation, thereby enhancing the negative role in type I IFN expression after viral infection. In turn, IFP35 is also capable of protecting Nmi protein from degradation through its N-terminal domain. It is considered that Nmi and IFP35 in fish can also interact with each other in regulating negatively the expression of type I IFNs, but thus in enhancing the replication of SCRV.

Role of asparagine biosynthesis pathway in Siniperca chuatsi rhabdovirus proliferation.

Viruses are non-living organisms that rely on host cellular metabolism to complete their life cycle. Siniperca chuatsi rhabdovirus (SCRV) has caused huge economic losses to the Chinese perch (Siniperca chuatsi) industry worldwide. SCRV replication is dependent on the cellular glutamine metabolism, while aspartate metabolism plays an important role in viral proliferation in glutamine deficiency. Herein, we investigated roles of asparagine metabolism in SCRV proliferation. Results showed that SCRV infection upregulated the expression of key enzymes in the aspartate metabolic pathway in CPB cells. And the key enzymes of malate-aspartic acid shuttle pathway upregulated during the virus invasion phase, and key enzymes of the asparagine biosynthesis pathway upregulated during the viral replication and release phase. When asparagine was added to the depleted medium, the SCRV copy number restored to 90% of those in replete medium, showing that asparagine and glutamine completely rescue the replication of SCRV. Moreover, inhibition of the aspartate- malate shuttle pathway and knockdown of the expression of key enzymes in the asparagine biosynthesis pathway significantly reduced SCRV production, indicating that the aspartic acid metabolic pathway was required to the replication and proliferation of SCRV. Above results provided references for elucidating pathogenic mechanism of SCRV by regulation of aspartate metabolism.

Thermal and environmental stability of Siniperca chuatsi Rhabdovirus

Siniperca chuatsi Rhabdovirus (SCRV) is an important pathogen that can cause huge economic losses and serious threats to aquaculture in mandarin fish and other economic fish. This study evaluated the stability of SCRV in liquids and on dry solid surfaces and explored the mechanism of SCRV inactivation by thermal treatment and UV irradiation. SCRV in the DMEM medium environment was very stable at 4 °C with a decrease of only 1 log10 for 6 months. The virus titer was still as high as 3 × 103 PFU/mL after 1 year. It survived for 14 days at 37 °C, and survived for 60 days and 150 days at 25 °C and 15 °C, respectively. The stability of SCRV in ddH2O was close to that in DMEM, and the stability in pond water was weaker than that in ddH2O and DMEM. On the surfaces of plastic (polystyrene), glass, and stainless steel, SCRV survived for 6 days at 25 °C and could survive for up to 1 month at 4 °C. The commonly used procedure of heat inactivation at 55 °C for 30 min is not suitable for SCRV. The complete thermal inactivation for SCRV was 60 °C for 30 min or 65 °C for 5 min. UV irradiation (wavelength 254 nm, intensity 400 mW/m2, irradiation distance 75 cm) for 120 s can effectively inactivate SCRV. RT-PCR analysis of the G gene indicated that the lost infectivity of SCRV was in part due to the disruption of genomic RNA. These findings are of great value for the prevention and control of SCRV outbreaks, and also have important reference value for disease control in other fisheries.

The m6A Reader YTHDF2 Modulates Antiviral and Antibacterial Activity by Suppressing METTL3 Methylation-Modified STING in Fish.

At present, N6-methyladenosine (m6A) modification has been proven to participate in a wide range of gene expression regulation, such as stability, translation, splicing, and output, among others, which has attracted much attention. Unlike mammals, however, the role of m6A in innate immunity of lower invertebrates has not yet been studied. In this study, we found that the total m6A level of Miichthys miiuy increased during Siniperca chuatsi rhabdovirus and Vibrio anguillarum infection, suggesting that m6A may play an important role in the immune process against pathogens in fish. In addition, our study shows that stimulator of IFN genes (STING) plays a dual immune function against viruses and bacteria in fish, and through degrading STING by identifying its m6A methylation site modified by methyltransferase-like 3 (METTL3), YTH domain family 2 (YTHDF2) can weaken the IRF3 and NF-κB-driven signaling pathway, thus weakening the innate immunity and promoting the infection of Siniperca chuatsi rhabdovirus and V. anguillarum to the M. miiuy. Although there have been reports on m6A modification of STING in mammals, it is still unclear whether there is also m6A modification in lower vertebrates, especially in fish. Therefore, our study provides a reference for filling the gap of m6A modification between fish and mammals.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

Siniperca chuatsi Rhabdovirus (SCRV)-Induced Key Pathways and Major Antiviral Genes in Fish Cells.

Fish rhabdoviruses, including Siniperca chuatsi rhabdovirus (SCRV), are epidemic pathogens that harm fish aquaculture. To clarify the interactions between SCRV and its host and explore antiviral targets, the present study performed transcriptome analysis in a cultured S. chuatsi skin cell line (SCSC) after SCRV infection at 3, 12, 24, and 36 h post-infection (hpi). Comparison with control obtained 38, 353, 896, and 1452 differentially expressed genes (DEGs) in the detected time points, respectively. Further analysis of the Go terms and KEGG pathways revealed the key pathways "Cytokine-cytokine receptor interaction" and "interferon related pathways" in SCSC cells responding to SCRV infection. The significantly up-regulated genes in the pathways were also verified by qPCR. Furthermore, gene cloning and overexpression revealed that five interferon-stimulated genes (ISGs) IFI4407, IFI35, Viperin, IFIT1, and IFIT5 had the ability to inhibit SCRV replication in FHM (Fathead minnow) cells, especially an inhibition efficiency more than 50% was observed in IFI35 overexpressed cells. In summary, current study revealed the main innate immune pathways in S. chuatsi cells induced by SCRV infection and the major ISGs of S. chuatsi in controlling SCRV replication.

FTO promotes innate immunity by controlling NOD1 expression via m6A-YTHDF2 manner in teleost.

N6-methyladenosine (m6A), the most abundant internal mRNA modification in eukaryotes, plays a vital role in regulating innate immunity. However, its underlying mechanism remains largely unknown, especially in lower vertebrates. The results of the present study show that fat-mass- and obesity-associated protein (FTO), also known as a m6A demethylase, improved the innate immunity and prevented Siniperca chuatsi rhabdo virus and Vibrio anguillarum infection in miiuy croaker. Significantly, FTO-promoted immunity was dependent on its m6A demethylase activity. In terms of mechanism, NOD1 has abundant methylation modification in its CDS and 3'UTR regions, and FTO can reduce its methylation level, thus avoiding its degradation by YTHDF2. In summary, our results indicate that the FTO-mediated m6A modification in NOD1 mRNA promotes innate immunity by activating the NOD-like receptor pathway, which provides a molecular mechanism for the regulation of immune response via RNA methylation in teleost.

Recovery of Siniperca chuatsi rhabdovirus from cloned cDNA.

Siniperca chuatsi rhabdovirus (SCRV) is an important pathogen that infects mandarin fish. A reverse genetics system is an important technical platform for virus research. In this study, the minigenome in which the enhanced green fluorescent protein gene is flanked by the viral genomic ends of SCRV and transcribed using a T7 promoter-terminator cassette was constructed. Co-transfection of the minigenome construct with SCRV-supporting plasmids of N, P, and L in BSRT7 cells resulted in the expression of the reporter gene. Transcription of a positive-strand RNA copy from cDNA of the SCRV genome along with the viral N, P, and L proteins resulted in the recovery of infectious SCRV in cells. Viral titre up to 108 PFU/ml was achieved. Recombinant SCRV was verified by the detection of a unique restriction site engineered into the SCRV genome. The phenotypes of the recombinant SCRV and the parental virus were evaluated by plaque size, replication kinetics in vitro, and pathogenicity in vivo. The recovered SCRV from cDNA showed similar phenotypes compared to the parental virus. The established reverse genetics system is of great significance and value for the functional genome study of SCRV and for laying a foundation for the development of the viral vector and SCRV vaccine.

Isolation, genomic and biological characterizations of a rhabdovirus from mandarin fish (Siniperca chuatsi)

In this study, a mandarin fish (Siniperca chuatsi) rhabdovirus (SCRV) S3 was isolated, purified, and identified from live mandarin fish collected from a commercial market. The full-length genome of S3 is 11,582 bp and has a 36 bp insertion in the 5’-UTR of the genome. The genome homology between S3 and the representative species in the genera of Perhabdovirus, Spriviviruses, and Novirhabdoviruses is 29.6% ∼ 31.0%, 25.7% ∼ 25.8, and 0.3% respectively. The cell tropism study showed that S3 could not only infect fish-derived cells but also mammalian and human-derived cells at 28 °C. However, S3 had no infectivity to all the tested cells at 37 °C. The temperature was an important barrier to the infection of SCRV S3. The upper limit of temperature for S3 growth in cells is 32 °C and the lowest measurable virus replication temperature is 10 °C. The optimum growth temperature is 28 °C. The animal study showed that SCRV S3 exhibited strong pathogenicity to zebrafish. Mortality in blue zebrafish (Danio rerio) and red zebrafish (Danio rerio) at 28 °C was 53% and 73%, respectively. Zebrafish can be used as an infection model for SCRV. This study will aid in understanding the biological characterizations of SCRV and has important guiding significance for the effective prevention and control strategies of SCRV.

Establishment and characterization of a spinal cord tissue cell line from mandarin fish, Siniperca chuatsi and its susceptibility to several viruses.

In this study, we established and characterized a continuous cell line from the spinal cord tissue of mandarin fish, Siniperca chuatsi and assessed its susceptibility to infectious spleen and kidney necrosis virus (ISKNV), Siniperca chuatsi ranavirus (SCRaV) and Siniperca chuatsi rhabdovirus (SCRV). The cell line, named SCC, has been successively cultured up to 40 passages. The optimal growing conditions of SCC cells were in Leibovitz's L-15 medium supplemented with 20% foetal bovine serum (FBS) at 28°C. Karyotype analysis demonstrated 48 normal diploid chromosomes in the cells. The identity of S. chuatsi origin of SCC cells was confirmed by partial sequencing of the 16S rRNA and cytochrome oxidase I (COI) genes. Infection susceptibility assessment showed that ISKNV, SCRIV and SCRV and can be stably produced and transmitted in SCC cells, and the replication efficiency of ISKNV, SCRaV and SCRV ranged from 107.4 to 109.6 TCID50 /ml. In addition, transmission electron microscopy analysis of ISKNV, SCRAV and SCRV infected SCC cells showed numerous viral particles. In conclusion, the newly established SCC cells provide an important tool for isolation and production of viruses, as well as for molecular and cell biology studies.

Exon-Intron Circular RNA circRNF217 Promotes Innate Immunity and Antibacterial Activity in Teleost Fish by Reducing miR-130-3p Function.

Circular RNA (circRNA) is produced by splicing head to tail and is widely distributed in multicellular organisms, and circRNA reportedly can participate in various cell biological processes. In this study, we discovered a novel exon-intron circRNA derived from probable E3 ubiquitin-protein ligase RNF217 (RNF217) gene, namely, circRNF217, which was related to the antibacterial responses in teleost fish. Results indicated that circRNF217 played essential roles in host antibacterial immunity and inhibited the Vibrio anguillarum invasion into cells. Our study also found a microRNA miR-130-3p, which could inhibit antibacterial immune response and promote V. anguillarum invasion into cells by targeting NOD1. Moreover, we also found that the antibacterial effect inhibited by miR-130-3p could be reversed with circRNF217. In mechanism, our data revealed that circRNF217 was a competing endogenous RNA of NOD1 by sponging miR-130-3p, leading to activation of the NF-κB pathway and then enhancing the innate antibacterial responses. In addition, we also found that circRNF217 can promote the antiviral response caused by Siniperca chuatsi rhabdovirus through targeting NOD1. Our study provides new insights for understanding the impact of circRNA on host-pathogen interactions and formulating fish disease prevention to resist the severely harmful V. anguillarum infection.