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.

Epidermal growth factor receptor promotes infectious spleen and kidney necrosis virus invasion via PI3K-Akt signaling pathway.

Infectious spleen and kidney necrosis virus disease (ISKNVD) caused significant economic losses to the fishery industry. Epidermal growth factor receptor (EGFR), phosphatidylinositide 3-kinase (PI3K) played an important role in ISKNV invasion. However, the molecular regulatory mechanisms among EGFR, PI3K-Akt, and ISKNV invasion are not clear. In this study, ISKNV infection rapidly induced EGFR activation. While, EGFR activation promoted virus entry, but EGFR inhibitors and specific RNA (siRNA) decreased virus invasion. The PI3K-Akt as downstream signalling of EGFR was activated upon ISKNV infection. Consistent with the trends of EGFR, Akt activation increased ISKNV entry into cells, Akt inhibition by specific inhibitor or siRNA decreased ISKNV invasion. Akt silencing combination with EGFR activation showed that EGFR activation regulation ISKNV invasion is required for activation of the Akt signalling pathway. Those data demonstrated that ISKNV-induced EGFR activation positively regulated virus invasion by PI3K-Akt pathway and provided a better understanding of the mechanism of EGFR-PI3K-Akt involved in ISKNV invasion.

Mandarin Fish (Siniperca chuatsi) p53 Regulates Glutaminolysis Induced by Virus via the p53/miR145-5p/c-Myc Pathway in Chinese Perch Brain Cells.

p53, as an important tumor suppressor protein, has recently been implicated in host antiviral defense. The present study found that the expression of mandarin fish (Siniperca chuatsi) p53 (Sc-p53) was negatively associated with infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV) proliferation as well as the expression of glutaminase 1 (GLS1) and glutaminolysis pathway-related enzymes glutamate dehydrogenase (GDH) and isocitrate dehydrogenase 2 (IDH2). This indicated that Sc-p53 inhibited the replication and proliferation of ISKNV and SCRV by negatively regulating the glutaminolysis pathway. Moreover, it was confirmed that miR145-5p could inhibit c-Myc expression by targeting the 3' untranslated region (UTR). Sc-p53 could bind to the miR145-5p promoter region to promote its expression and to further inhibit the expression of c-Myc. The expression of c-Myc was proved to be positively correlated with the expression of GLS1 as well. All these suggested a negative relationship between the Sc-p53/miR145-5p/c-Myc pathway and GLS1 expression and glutaminolysis. However, it was found that after ISKNV and SCRV infection, the expressions of Sc-p53, miR145-5p, c-Myc, and GLS1 were all significantly upregulated, which did not match the pattern in normal cells. Based on the results, it was suggested that ISKNV and SCRV infection altered the Sc-p53/miR145-5p/c-Myc pathway. All of above results will provide potential targets for the development of new therapeutic strategies against ISKNV and SCRV. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV) as major causative agents have caused a serious threat to the mandarin fish farming industry (J.-J. Tao, J.-F. Gui, and Q.-Y. Zhang, Aquaculture 262:1-9, 2007, https://doi.org/10.1016/j.aquaculture.2006.09.030). Viruses have evolved the strategy to shape host-cell metabolism for their replication (S. K. Thaker, J. Ch'ng, and H. R. Christofk, BMC Biol 17:59, 2019, https://doi.org/10.1186/s12915-019-0678-9). Our previous studies showed that ISKNV replication induced glutamine metabolism reprogramming and that glutaminolysis was required for efficient replication of ISKNV and SCRV. In the present study, the mechanistic link between the p53/miR145-5p/c-Myc pathway and glutaminolysis in the Chinese perch brain (CPB) cells was provided, which will provide novel insights into ISKNV and SCRV pathogenesis and antiviral treatment strategies.

PI3K/AKT/p53 pathway inhibits infectious spleen and kidney necrosis virus infection by regulating autophagy and immune responses.

The PI3K/AKT/p53 signaling pathway is activated by various types of cellular stimuli or pathogenic infection, and then regulates fundamental cellular functions to combat these stimulations. Here, we studied the meaningful roles of PI3K/AKT/p53 in regulating cellular machine such as autophagy, immune responses, as well as antiviral activity in Chinese perch brain (CPB) cells infected by infectious spleen and kidney necrosis virus (ISKNV), which is an agent caused devastating losses in mandarin fish (Siniperca chuatsi) industry. We found that ISKNV infection induced up-regulation of host PI3K/AKT/p53 axis, but inhibited autophagy in CPB cells. Interestingly, activation of PI3K/AKT/p53 axis factors trough agonists or overexpression dramatically decreased host autophagy level, inhibited ISKNV replication, and elevated the expression of immune-related genes in CPB cells. In contrast, suppression of PI3K/AKT/p53 pathway by inhibitors or small interfering RNA (siRNA)-mediated gene silence increased the autophagy and ISKNV replication, but down-regulated immune responses in CPB cells. All these results indicate that PI3K/AKT/p53 pathway plays an important role in anti-ISKNV infection and can be used as a new target for controlling ISKNV disease.

Characterization and function of mandarin fish c-Myc during viral infection process.

c-Myc is a transcription factor and master regulator of cellular metabolism, and plays a critical role in virus replication by regulating glutamine metabolism. In this study, the open-reading frame (ORF) of c-Myc, designated as Sc-c-Myc, was cloned and sequenced. Multiple alignment of the amino acid sequence showed that the conserved domain of Sc-c-Myc, including the helix-loop-helix-zipper (bHLHzip) domain and Myc N-terminal region, shared high identities with other homologues from different species. Sc-c-Myc mRNA was widely expressed in the examined tissues of mandarin fish, and the higher mRNA levels was expressed in hind kidney. Moreover, mRNA and protein level of Sc-c-Myc was significantly increased in the Chinese perch brain (CPB) cells and spleen of mandarin fish post infection with infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Sc-c-Myc overexpression promoted ISKNV and SCRV replication, on the contrary, knocking down Sc-c-Myc restrained ISKNV and SCRV replication. These results indicated that Sc-c-Myc involved in ISKNV and SCRV replication and proliferation, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

Characterization of mandarin fish (Siniperca chuatsi) IL-6 and IL-6 signal transducer and the association between their SNPs and resistance to ISKNV disease.

In fish, interleukin-6 (IL-6) is a very important immune-regulatory cytokine that plays a polyfunctional role in inflammation, metabolism, regeneration, and neural processes. IL-6 signal transducer (IL-6ST) is a specific receptor for IL-6 and expressed mainly in immune cells and hepatocytes. In this study, the complete cDNA and genomic DNA sequences of mandarin fish (Siniperca chuatsi) IL-6 and IL-6ST genes were identified and analyzed. Quantitative real-time PCR showed that IL-6 and IL-6ST were chiefly expressed in the immune organs. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the expression levels of IL-6 were significantly up-regulated after 6 h and 24 h in the head kidney and spleen, respectively (p < 0.01), the peak value for both reached at 72 h, IL-6ST increased significantly after 120 h with a peak at 168 h in the head kidney (p < 0.01) and improved markedly at 168 h in the spleen (p < 0.01). Besides, IL-6 and IL-6ST have been identified 3 and 8 single nucleotide polymorphisms (SNPs), respectively. Statistical analysis showed that one SNP locus (1625C/T) in the coding region of IL-6 was significantly related to the resistance of mandarin fish against ISKNV. The 1625C→T locus in the coding region of IL-6 is a synonymous mutation; compared with the susceptible group, the frequency of allele T in the disease resistance group was significantly higher, which may be due to the rare codon produced by the mutation affecting translation. The involvement of IL-6 and IL-6ST in response to ISKNV infection in mandarin fish clearly indicate that the role of SNP markers in IL-6 was associated with the ISKNV resistance, which was demonstrated for the first time in our results. Thus, the current study may provide fundamental information for further breeding of mandarin fish with resistance to ISKNV infection.

Molecular characterization and function of EGFR during viral infectionprocess in Mandarin fishSiniperca chuatsi.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase protein and plays a critical role in virus infection by modulating innate immunity. In this study, we cloned and sequenced the EGFR coding sequence of mandarin fish, designed as scEGFR, and explored its characteristics. scEGFR mRNA was widely expressed in the tested tissues of mandarin fish, and the higher mRNA levels were expressed in kidney and spleen. scEGFR expression was up-regulated in spleen and CPB cells at early stage of ISKNV and SCRV infection. Gefitinib (EGFR inhibitor) inhibited ISKNV and SCRV replication, and increased the expression of the interferon-stimulated genes (ISG). However the EGF (EGFR activator) promoted ISKNV and SCRV replication, and decreased the interferon-stimulated genes. Those results indicated that scEGFR and its signaling involved in ISKNV and SCRV infection, and EGFR activation negatively regulated the interferon response, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

Mandarin fish p53: Genomic structure, alternatively spliced variant and its mRNA expression after virus challenge.

A number of size variants of the p53 protein have been described in mammal, but little is known about alternative splicing of p53 expression and function in the fish. In our previous study, the immune defense and antiviral responses of p53 had been determined in mandarin fish (Siniperca chuatsi). However, the role of its splicing variants remains unknown. In the present study, the organization of mandarin fish p53 (Sc-p53) genome sequence was determined and a novel splice variant was characterized. The Sc-p53 genomic sequence was composed of 5543 bp, containing 11 exons and 10 introns, which was similar to other species. Then, a 1106 bp full-length cDNA of a novel splice variant p53 from mandarin fish (designed as Sc-p53I6) was cloned and characterized. Quantitative real-time PCR assays revealed that Sc-p53I6 was expressed in all tissues examined, and it was most abundant in the gill, hemocyte and hind kidney. Western blotting analysis revealed that Sc-p53I6 protein was abundant in liver, trunk kidney, hind kidney, stomach and heart. In addition, the regulation of Sc-p53I6 gene expression after virus infection was determined and characterized. The results showed twice rise expression pattern of Sc-p53I6 in CPB cells and spleen of mandarin fish in response to infectious kidney and spleen necrosis virus (ISKNV). However, a different expression pattern, once rise, of Sc-p53I6 in response to Siniperca chuatsi rhabdovirus (SCRV) infection was found. The mRNA expression of Sc-p53I6 was significantly up-regulated in CPB at 4 h and spleen of mandarin fish at 12 h post-infection. These results will shed a new light on antiviral response mechanisms of p53 in mandarin fish.

Glutamine and glutaminolysis are required for efficient replication of infectious spleen and kidney necrosis virus in Chinese perch brain cells.

Viruses rely on host cellular metabolism for energy and macromolecule synthesis during their replication. Infectious spleen and kidney necrosis virus (ISKNV) causes significant economic losses in the Chinese perch (Siniperca chuatsi) industry worldwide. However, little is known about the relationship between ISKNV replication and cellular metabolism. Using transcriptomic analysis, we observed that glutamine metabolism in Chinese perch brain (CPB) cells is altered during ISKNV infection. Moreover, ISKNV replication was decreased in CPB cells cultured in the glutamine-depleted medium. ISKNV replication was also inhibited in CPB cells cultured in the presence of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (an inhibitor of glutaminase), (-)-epigallocatechinmo nogallate (an inhibitor of glutamate dehydrogenase) or L-buthionine sulfoximine (an inhibitor of glutathione synthesis). However, virus replication was rescued by the addition of multiple tricarboxylic acid cycle intermediates, ATP, or glutathione reduced ethyl ester. ATP and reduced glutathione/oxidized glutathione levels were increased in CPB cells infected with ISKNV, but were decreased in CPB cells cultured in glutamine-depleted medium. These results indicate ISKNV infection induces glutaminolysis to accommodate the biosynthetic and energy needs for its efficient virus replication.

Molecular characterization and expression pattern of tumor suppressor protein p53 in mandarin fish, Siniperca chuatsi following virus challenge.

In recent years, the tumor suppressor protein p53, which is crucial for cellular defense against tumor development, has also been implicated in host antiviral defense. In the present study, a 1555 bp full-length cDNA of p53 from mandarin fish (Siniperca chuatsi) (Sc-p53) was cloned and characterized. Quantitative real-time PCR assays revealed that Sc-p53 was expressed in all tissues examined, and it was most abundant in the gill and kidney. Recombinant Sc-p53 fused with a His·Tag was expressed in Escherichia coli BL21 (DE3) cells and a rabbit polyclonal antibody was raised against recombinant Sc-p53. In addition, the regulation of Sc-p53 gene expression after experimental viral infection was determined and characterized. The mRNA and protein expression of Sc-p53 were significantly up-regulated in the Chinese perch brain (CPB) cell line and mandarin fish after infection with infectious kidney and spleen necrosis virus (ISKNV). The results showed a biphasic expression pattern of Sc-p53 protein in CPB. However, a different expression pattern of Sc-p53 in response to S. chuatsi rhabdovirus (SCRV) infection was found. The mRNA expression of Sc-p53 was significantly up-regulated in CPB at 6 h and spleen of mandarin fish at 24 h post-infection. The protein expression of Sc-p53 was significantly up-regulated in CPB at 1 h, remained elevated at 4 h, and then decreased to control level at 8 h post-infection by SCRV. All of these data suggested that Sc-p53 plays a critical role in immune defense and antiviral responses.

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.

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant economic losses in the cultured Mandarin fish (Siniperca chuatsi) industry. The molecular mechanisms that underlie the pathogenesis of the viral infection remain poorly understood. In this study, deep RNA sequencing technique was used to analyze the transcriptomic profiles of Mandarin fish brain cells (CPB) at progressive time points after ISKNV infection. A total of 96,206,040 clean data from 98,235,240 sequence reads were obtained. These raw data were assembled into 66,787 unigenes. Among these unigenes, 33,225 and 29,210 had significant hit the Nr and SwissProt databases where they matched 27,537and 19,638 unique protein accessions, respectively. In the samples harvested at 24 or 72 h post of the infection, a total of 10,834 or 7584 genes were differentially expressed in infected CPB cells compared to non-infected cells, including 5445 or 3766 up-regulated genes and 5389 or 3818 down-regulated genes, respectively. In addition, 12 differentially expressed genes (DEGs) were validated by quantitative PCR. These DEGs were involved in many pathways of viral pathogenesis. Further analysis of the major DEGs genes involved in the RLRs and apoptosis pathways revealed some interesting findings. In the RLRs pathway, ISKNV infection inhibited the activation of NF-κB via over expression of the IKKB-α and IKKB-ß and lessened expression of interleukin-1 receptor-associated kinase 4 (IRAK4). In the apoptosis pathway, ISKNV infection could induce apoptosis mainly via tumor necrosis factor (TNF) mediated extrinsic pathway. The cellular apoptosis induced by ISKNV infection was confirmed using annexinV-FITC/PI and DAPI staining methods.