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.

A Novel and Effective Therapeutic Method for Treating Aeromonas schubertii Infection in Channa maculata.

Aeromonas schubertii is a pathogen that severely affects aquatic animals, including the snakehead, Channa maculata. Lytic bacteriophages have been recognized as effective alternatives to antibiotics for controlling bacterial infections. However, there have been no reports of A. schubertii phages as far as we know. In this study, a lytic bacteriophage SD04, which could effectively infect A. schubertii, was isolated from pond water cultured with diseased snakehead. The SD04 phage formed small, round plaques on Petri dishes. Electron microscopy revealed a hexagonal head and a contractile tail. Based on its morphology, it may belong to the Myoviridae family. Two major protein bands with molecular weights of 50 and 38 kilodaltons were observed after the phage was subjected to SDS-PAGE. The phage showed a large average burst size, high specificity, and a broad host range. When stored at 4 °C, phage SD04 had high stability over 12 months and showed almost no variation within the first six months. All fish were healthy after both intraperitoneal injection and immersion administration of SD04, indicating the safety of the phage. After treatment with SD04, Channa maculata in both phage therapy groups and prevention groups showed high survival rates (i.e., 83.3 ± 3.3% and 100 ± 1.3%, respectively). Phage therapy inhibits bacterial growth in the liver, the target organ of the infected Channa maculat. The experimental results indicate the potential use of phage SD04 for preventing A. schubertii infection in Channa maculata.

The Stability and Efficency of CPB Cells Were Acclimated for Virus Proliferation.

BACKGROUND: Vaccinations are still the most effective means of preventing and controlling fish viral diseases, and cells are an important substrate for the production of a viral vaccine. Therefore, the rapid-stable growth and virus sensitivity of cells are urgently needed. METHODS: Chinese perch brain 100th passage (CPB p100) were acclimated in a low serum with 5% FBS L-15 for 50 passages, then transferred to 8% FBS L-15 for 150 passages. Additionally, the morphology and cell type of CPB 300th passage (CPB p300) cells were identified. We analyzed the transfection efficiency and virus sensitivity of CPB p300 cells, and then optimized the conditions of ISKNV, SCRV, and LMBV multiplication in CPB cells. RESULTS: CPB p300 cells were more homogeneous, and the spread diameter (20-30) µm in CPB p300 cells became the dominant population. The doubling time of CPB p300 was 1.5 times shorter than that of CPB p100.However, multiplication rate of CPB p300 was 1.37 times higher than CPB p100. CPB p300 cells were susceptible to ISKNV, SCRV, and LMBV, and the optimal conditions of ISKNV, SCRV, and LMBV multiplication were simultaneous incubation, 0.6 × 105 cells/cm2 and MOI = 0.1; infection at 48 h, 0.8 × 105 cells/cm2 and MOI = 0.01; simultaneous incubation, 0.7 × 105 cells/cm2 and MOI = 0.05, respectively. The time and economic costs of ISKNV, SCRV, and LMBV multiplication in CPB p300 cells were significantly reduced. CONCLUSIONS: The acquisition of CPB p300 cells laid a good material foundation for the production of ISKNV, SCRV, and LMBV vaccines.

An Avirulent Largemouth Bass Birnavirus Vaccine Candidate Protects Largemouth Bass against Birnavirus Infection.

BACKGROUND: Largemouth bass birnavirus (LBBV) disease outbreaks in largemouth bass fingerlings lead to high mortality in China. Therefore, the development of immersion immunization strategies is paramount. METHODS: An avirulent LBBV strain was screened using a fish challenge assay. The proliferation dynamics of the avirulent strain were determined in vitro and in vivo. The efficacy of the avirulent vaccine was evaluated using immune gene expression, viral load, and a virus challenge, and the safety was also assessed using a reversion to virulence test. RESULTS: An avirulent virus strain, designated as largemouth bass birnavirus Guangdong Sanshui (LBBV-GDSS-20180701), was selected from five fish birnavirus isolates. The proliferation peak titer was 109.01 TCID50/mL at 24 hpi in CPB cells and the peak viral load was 2.5 × 104 copies/mg at 4 dpi in the head kidneys and spleens of largemouth bass. The largemouth bass that were immersed within an avirulent vaccine or injected with an inactivated vaccine were protected from the virulent LBBV challenge with a relative percent survival (RPS) of 75% or 42.9%, respectively. The expression levels of IL-12, MHCI, MHCII, CD8, CD4, and IgM in the avirulent group were significantly upregulated at a partial time point compared to the inactivated vaccine group. Moreover, the viral load in the avirulent vaccine group was significantly lower than those in the inactivated vaccine group and control group using real-time PCR. CONCLUSIONS: LBBV-GDSS-20180701 is a potential live vaccine candidate against LBBV disease.

Development of a New Marine Fish Continuous Cell Line Derived from Brain of Red Sea Bream (Pagrosomus major) and Its Application to Fish Virology and Heavy Metal Toxicology.

Red sea bream (Pagrosomus major) is one of the most popular farmed marine teleost fish species. Fish cell lines are becoming important research tool in the aquaculture field, and they are suitable models to study fish virology, immunology and toxicology. To obtain a Pagrosomus major cell line for biological studies, a continuous cell line from brain of red sea bream (designated as RSBB cell line) was established and has been successfully subcultured over 100 passages. The RSBB cell line predominantly consisted of fibroblast-like cells and multiplied well in M199 medium supplemented with 10% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome numbers of RSBB cells was 48. After transfection with pEGFP-N1, RSBB cells showed bright green fluorescence with a transfection efficiency approaching 8%. For toxicology study, it was demonstrated that metal Cd could induce cytotoxic effects of RSBB cells, accompanied with a dose-dependent MTT conversion capacity. Morphologically, cells treated with metal Cd produced rounding, shrinking and detaching and induced both cell apoptosis and necrosis. For virology study, the RSBB cells were highly susceptible to Nervous necrosis virus (NNV) and Singapore grouper iridovirus (SGIV) with steady titers (i.e., 108.0~8.3 TCID50 mL-1 and 107.0~7.2 TCID50 mL-1 respectively). Furthermore, an obvious cytopathic effect (CPE) could be observed in RSBB cells infected with Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdoviruses (SCRV). Meanwhile, all the infections were confirmed by polymerase chain reaction. The new brain cell line developed and characterized from red sea bream in this study could be used as an in vitro model for fish studies in the fields of toxicology and virology.

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.

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.

The composition and antiviral activity of scTRIM59 in Mandarin fish.

The tripartite motif (TRIM) proteins play critical roles in viral infection by modulating innate immunity. However, the molecular and antiviral activity of TRIM59 in mandrain fish is not fully understood. In present study, we cloned and sequenced the TRIM59 core sequence and explored its characteristics in Mandarin fish. The Siniperca chuatsi TRIM59 (scTRIM59) showed relatively high expression in immune-related organs. scTRIM59 expression was significantly down-regulated post ISKNV infection in vivo and vitro, but up-regulated at the early stages of SCRV infection in CPB cells. The overexpression of scTRIM59 inhibited ISKNV and SCRV infection, but decreased the expression of IRF3/IRF7-mediated signal genes. However, knockdown of scTRIM59 promoted the ISKNV and SCRV infection, but increased the expression of IRF3/IRF7-mediated signal genes. Those results indicated that scTRIM59 negatively regulated ISKNV, SCRV infection and IRF3/IRF7-mediated signal genes. This study provided new ideas about the function of scTRIM59.

Antimicrobial and immunoregulatory activities of the derived peptide of a natural killer lysin from black rockfish (Sebastes schlegelii).

Natural killer lysin (NK-lysin) is a small molecule antimicrobial peptide secreted by natural killer cells and T lymphocytes. In this study, we characterized a cDNA sequence encoding an NK-lysin homologue (SsNKL1) from black rockfish, Sebastes schlegelii. The open reading frame (ORF) of SsNKL1 encodes a putative protein of 149 amino acids and shares 44%-87% overall sequence identities with other teleost NK-lysins. SsNKL1 possesses conserved NK-lysin family features, including a signal sequence and a surfactant-associated protein B (SapB) domain, sequence analysis revealed that SsNKL1 is most closely related to false kelpfish (Sebastiscus marmoratus) NK-lysin (with 87% sequence identity). SsNKL1 transcripts were detected in all the tested tissues, with the highest level in the kidney, followed by the spleen and gills. Upon Listonella anguillarum infection, the mRNA expression of SsNKL1 in the black rockfish was significantly up-regulated in the liver and kidney. The derived peptide SsNKLP27 from SsNKL1 was synthesized, and its biological function was studied. SsNKLP27 showed direct antibacterial activity against Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, L. anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus. SsNKLP27 treatment facilitated the bactericidal process of erythromycin by enhancing the permeability of the outer membrane. In the process of interaction with the target bacterial cells, SsNKLP27 changed the permeability and retained the morphological integrity of the cell membrane, then penetrated into the cytoplasm, and induced the degradation of genomic DNA and total RNA. In vivo studies showed that administration of SsNKLP27 before bacterial and viral infection significantly reduced the transmission and replication of pathogens in tissues. In vitro analysis showed that SsNKLP27 could enhance the respiratory burst ability and regulate the expression of some immune-related genes of macrophages. In summary, these results provided new insights into the function of NK-lysins in teleost fish and support that SsNKLP27 is a new broad-spectrum antimicrobial peptide that has a potential application prospect in aquaculture against pathogenic infection.

Determination and Characterization of a Novel Birnavirus Associated with Massive Mortality in Largemouth Bass.

Largemouth bass (Micropterus salmoides) is an important and fast-growing aquaculture species in China. In 2017, an epidemic associated with severe mortality occurred in fingerlings of largemouth bass in Guangdong, China. The causative pathogen was identified and named as largemouth bass Birnavirus (LBBV) by virome analysis, viral isolation, electron microscopy, genome sequencing, Western blot, indirect immunofluorescence, experimental challenge, and so on. Virome sequencing results showed that the relative abundance reads related to the family Birnaviridae were the highest, occupied ∼25% of the total viral reads. Electron microscopy revealed large numbers of nonenveloped virus particles in the spleen of diseased fish with a diameter of about 53 nm. LBBV was isolated and propagated in Chinese perch brain cells and induced a typical cytopathic effect. LBBV was stable to chloroform, heat, and 5-bromo-2'-deoxyuridine, but sensitive to acid (pH 3.0). The complete genome of LBBV was comprised of segment A with a size 3525 bp and segment B with a size 2737 bp. Phylogenetic analysis basing on RdRp and VP2 protein sequences revealed that LBBV were clustered into one clade with Lates calcarifer Birnavirus (LCBV), sharing 98.7% or 91.9% sequence identity with LCBV, respectively, but only sharing 59.7% and 52.7% sequence identity with Blosnavirus, suggesting that LBBV and LCBV probably belonged to a new genus. Challenge experiments results indicated that clinical disease symptoms similar to those observed naturally were replicated and the cumulative mortality reached 100% at 3 dpi by i.p. injection. The investigation of prevalence of LBBV infection showed that 41.5% (17/41) sample pools collected from diseased ponds was positive during 2017-2020, indicating that an emerging outbreak of this disease may be spreading within the largemouth bass in China. Above results confirmed that LBBV is a novel Birnavirus associated with massive mortality for fingerlings of largemouth bass. This provides a basis for prevention and control of this emerging viral disease. IMPORTANCE Pathogen isolation and identification are vital for emerging infectious outbreaks. Here we report the isolation, determination and characterization of a novel largemouth bass Birnavirus (LBBV) associated with massive mortality in largemouth bass. And genome of LBBV is determined and analyzed. Based on phylogenetic and alignment analysis of genome, we suggest LBBV belongs to a new genus (designated as Perbirnavirus genus) in Birnaviridae family. Our findings will provide a basis for the further study on prevention and control of this emerging viral disease.

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.

The dynamic immune responses of Mandarin fish (Siniperca chuatsi) to ISKNV in early infection based on full-length transcriptome analysis and weighted gene co-expression network analysis.

Mandarin fish (Siniperca chuatsi) been seriously harmed by infectious spleen and kidney necrosis virus (ISKNV) in recent years, but the early immune response mechanism of infection is still unknown. Here, we performed RNA sequencing on the spleens of mandarin fish infected with ISKNV at 0, 12, 24, 48, and 72 h post-infection (hpi) using short-read Illumina RNA sequencing and long-read Pacific Biosciences isoform sequencing to generate a full-length transcriptome. The immune responses of mandarin fish infected with ISKNV at the molecular level were characterized by RNA-seq analysis and weighted gene co-expression network analysis (WGCNA). A total of 26,528 full-length transcript sequences were obtained. There were 2,729 (1,680 up-regulated and 1,112 down-regulated), 1,874 (1,136 up-regulated and 738 down-regulated), 2,032 (1,158 up-regulated and 847 down-regulated), and 4,176 (2,233 up-regulated and 1,943 down-regulated) differentially expressed genes (DEGs) in mandarin fish at 12, 24, 48, and 72 hpi, compared with uninfected fish, respectively. A total of four modules of co-expressed DEGs identified by WGCNA were significantly positively correlated to the four time points after infection, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the immune-related DEGs in all these modules were mainly enriched in Phagosome, Endocytosis, Herpes simplex infection, and Cytokine-cytokine receptor interaction pathways. Further analysis showed that oher signaling pathways, including CAMs, NOD-like receptor and ER protein processing, Intestinal immune network for IgA production, TLR pathway, and Apoptosis significantly enriched in four modules corresponding to 12, 24, 48, and 72 hpi respectively, had specifically participated in the immune response. Hub genes identified based on the high-degree nodes in the WGCN, including CAM3, IL-8, CCL21, STING, SNX1, PFR and TBK1, and some DEGs such as MHCI, MHCII, TfR, STING, TNF α, TBK1, IRF1, and NF-kB, BCR, IgA and Bcl-XL had involved in dynamic molecular response of mandarin fish to ISKNV infection. In sum, this study provides a set of full-length transcriptome of the spleen tissue of mandarin fish for the first time and revealed a group of immune genes and pathways involved in different temporal responses to ISKNV infection, which has implications for resource conservation and aiding the development of strategies to prevent virus early infection for mandarin fish.

The mTOR/PGC-1α/SIRT3 Pathway Drives Reductive Glutamine Metabolism to Reduce Oxidative Stress Caused by ISKNV in CPB Cells.

Under oxidative stress, viruses prefer glycolysis as an ATP source, and glutamine is required as an anaplerotic substrate to replenish the TCA cycle. Infectious spleen and kidney necrosis virus (ISKNV) induces reductive glutamine metabolism in the host cells. Here we report that ISKNV infection the increased NAD+/NADH ratio and the gene expression of glutaminase 1 (GLS1), glutamate dehydrogenase (GDH), and isocitrate dehydrogenase (IDH2) resulted in the phosphorylation and activation of mammalian target of rapamycin (mTOR) in CPB cells. Inhibition of mTOR signaling attenuates ISKNV-induced the upregulation of GLS1, GDH, and IDH2 genes expression, and exhibits significant antiviral activity. Moreover, the expression of silent information regulation 2 homolog 3 (SIRT3) in mRNA level is increased to enhance the reductive glutamine metabolism in ISKNV-infected cells. And those were verified by the expression levels of metabolic genes and the activities of metabolic enzymes in SIRT3-overexpressed or SIRT3-knocked down cells. Remarkably, activation of mTOR signaling upregulates the expression of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene, leading to increased expression of SIRT3 and metabolic genes. These results indicate that mTOR signaling manipulates reductive glutamine metabolism in ISKNV-infected cells through PGC-1α-dependent regulation of SIRT3. Our findings reveal new insights on ISKNV-host interactions and will contribute new cellular targets to antiviral therapy. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of farmed fish disease that has caused huge economic losses in fresh and marine fish aquaculture. The redox state of cells is shaped by virus into a favorable microenvironment for virus replication and proliferation. Our previous study demonstrated that ISKNV replication induced glutamine metabolism reprogramming, and it is necessary for the ISKNV multiplication. In this study, the mechanistic link between the mTOR/PGC-1α/SIRT3 pathway and reductive glutamine metabolism in the ISKNV-infected cells was provided, which will contribute new insights into the pathogenesis of ISKNV 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.

Development of a Double-Antibody Sandwich ELISA for Rapid Detection of the MCP Antigen Concentration in Inactivated ISKNV Vaccines.

Infectious spleen and kidney necrosis virus (ISKNV) resulted in severe systemic diseases with high morbidity and mortality in Siniperca chuatsi. Vaccination is the primary method for effective prevention and control of these diseases. The development of inactivated ISKNV vaccines made some progress, but the technique of quality evaluation is scarce. Herein, a measurement of the MCP (major capsid protein) antigen concentration for the inactivated ISKNV vaccine was developed by double-antibody sandwich ELISA. Firstly, mouse monoclonal antibodies against ISKNV particles and MCP were generated. Then, a double-antibody sandwich ELISA was developed using the monoclonal antibody 1C8 1B9 as the capture antibody and Biotin-3B12 6B3 as the detection antibody. A standard curve was generated using the MCP concentration versus OD value with the linear range of concentration of 4.69~300 ng/mL. The assay sensitivity was 0.9 ng/mL. The antigen content of three batches of inactivated ISKNV vaccines was quantitatively detected using the double-antibody sandwich ELISA. The results showed that MCP antigen contents of inactivated ISKNV vaccines were positively correlated with the viral titers. The newly established double-antibody sandwich ELISA provided a useful tool for the detection of antigen quality for ISKNV inactivated vaccines.

The antibacterial activity of erythrocytes from Clarias fuscus associated with phagocytosis and respiratory burst generation.

It is widely known that red blood cells (RBCs) are responsible for respiration and the transport of gas. However, recent reports have also described the immune properties of RBCs, therefore creating new understanding for the functionality of RBCs. However, little is known about the immunological role of RBCs in bony fish. In this study, we used RBCs from Clarias fuscus as a model and demonstrate that these cells exhibited phagocytic ability with both latex beads and bacteria. Scanning electron microscopy and transmission electron microscopy provided visual confirmation of the phagocytotic process in RBCs. In addition, we used flow cytometry and fluorescence microscopy to analyse the rate of phagocytosis in RBCs. We found that RBCs exhibited stable phagocytotic ability with latex beads ranging from 0.5 to 1.0 µm in size. In response to bacterial stimulation, RBCs produced reactive oxygen species (ROS) and nitric oxide synthase (NOS), which are harmful to bacteria. RBCs also have an antioxidant system. Under conditions of oxidative stress, the expression levels of antioxidant enzymes, and particularly those of superoxide dismutase（SOD） increased significantly. Our results show that the erythrocytes of bony fish are phagocytic and also produce ROS which are toxic to bacteria. In addition, RBCs have an antioxidant system that removes excess ROS production to protect cells from oxidative damage.

Large-Scale Microcarrier Culture of Chinese Perch Brain Cell for Viral Vaccine Production in a Stirred Bioreactor.

Mandarin fish (Siniperca chuatsi) is one of the important cultured fish species in China. Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca Chuatsi rhabdovirus (SCRV) have hindered the development of mandarin fish farming industry. Vaccination is the most effective method for control of viral diseases, however viral vaccine production requires the large-scale culture of cells. Herein, a suspension culture system of Chinese perch brain cell (CPB) was developed on Cytodex 1 microcarrier in a stirred bioreactor. Firstly, CPB cells were cultured using Cytodex 1 microcarrier in 125 mL stirring flasks. With the optimum operational parameters, CPB cells grew well, distributed uniformly, and could fully cover the microcarriers. Then, CPB cells were digested with trypsin and expanded step-by-step with different expansion ratios from the 125 mL stirring bottle to a 500 mL stirring bottle, and finally to a 3-L bioreactor. Results showed that with an expansion ratio of 1:3, we achieved a high cell density level (2.25 × 106 cells/mL) with an efficient use of the microcarriers, which also confirmed the data obtained from the 125 mL stirring flask. Moreover, obvious cytopathic effects (CPE) were observed in the suspended CPB cells post-infection with ISKNV and SCRV. This study provided a large-scale culture system of CPB cells for virus vaccine production.

Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus.

Tongue sole tissue factor pathway inhibitor 2 (TFPI-2) C-terminus derived peptide, TC38, has previously been shown to kill Vibrio vulnificus cells without lysing the cell membrane; thus, the remaining bacterial shell has potential application as an inactivated vaccine. Therefore, this study aimed to evaluate the immune response induced by the novel V. vulnificus vaccine. The protective potential of TC38-killed V. vulnificus cells (TKC) was examined in a turbot model. Fish were intramuscularly vaccinated with TKC or FKC (formalin-killed V. vulnificus cells) and challenged with a lethal-dose of V. vulnificus. The results showed that compared with FKC, TKC was effective in protecting fish against V. vulnificus infection, with relative percent of survival (RPS) rates of 53.29% and 63.64%, respectively. The immunological analysis revealed that compared with the FKC and control groups, the TKC group exhibited: 1) significantly higher respiratory burst ability and bactericidal activity of macrophages at 7 d post-vaccination; 2) increased alkaline phosphatase, acid phosphatase, lysozyme, and total superoxide dismutase levels post-vaccination; 3) higher serum agglutinating antibody titer with corresponding higher serum bactericidal ability, and a more potent serum agglutination effect, as well as an increased IgM expression level; 4) higher expression of immune relevant genes, which were involved in both innate and adaptive immunity. Taken together, this is the first study to develop a novel V. vulnificus inactivated vaccine based on AMP inactivation, and TKC is an effective vaccine against V. vulnificus infection for aquaculture.

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.