Search Results (12)

Haploid embryonic stem cells (ESCs), which combine the properties of haploidy and pluripotency, hold significant potential for advancing developmental biology and reproductive technology. However, while previous research has largely focused on haploid ESCs in freshwater species like Japanese medaka (Oryzias latipes), little is known about their counterparts in marine species. This study hypothesizes that haploid ESCs from marine fish could offer unique insights and tools for genetic and virological research. To address this, we successfully established and characterized a novel haploid ESC line, hMMES1, derived from marine medaka (Oryzias melastigma). The hMMES1 cells contain 24 chromosomes, exhibit core stem cell characteristics, and express key pluripotency markers. In vitro, hMMES1 cells form embryonic bodies (EBs) capable of differentiating into the three germ layers. In vivo, hMMES1 cells were successfully transplanted into marine medaka and zebrafish, resulting in the generation of interspecies and interordinal chimeras. Additionally, hMMES1 cells demonstrate high efficiency in transfection and transduction, and show susceptibility to major aquaculture viruses, nodavirus (NNV) and iridovirus (SGIV). These findings suggest that hMMES1 cells represent a valuable model for genetic manipulation and virological studies in marine fish species. Full article

Groupers are valuable economic fish in the southern sea area of China, but the threat of disease is becoming more and more serious. Vibrio harveyi, V. parahaemolyticus, and Singapore grouper iridovirus (SGIV) are three important pathogens that cause disease in groupers, and infection with either a single one or a mix of these pathogens poses a serious threat to the healthy development of grouper culture. To enhance the rapid diagnosis and screening in the early stages, it is necessary to develop rapid detection methods of these pathogens. To simultaneously and rapidly detect the three pathogens, in this study, we utilized the TolC of V. harveyi, DNAJ of V. parahaemolyticus, and RAD2 of SGIV as the target genes and established a triple visual loop-mediated isothermal amplification (LAMP) method. This LAMP method showed a detection time as fast as 30 min and a high sensitivity of 100 fg/μL. Moreover, this method exhibited strong specificity and no cross-reaction with seven types of Vibrio and Staphylococcus aureus, as well as five common viruses in aquatic animals. Then, the LAMP products were enzymically cut, and three characteristic strips were used to identify the pathogen species. The results of the clinical trials demonstrated that the method could accurately and specifically detect V. harveyi, V. parahaemolyticus, and SGIV in grouper tissues. In summary, this study successfully established a triple visual LAMP rapid detection method for V. harveyi, V. parahaemolyticus, and SGIV. The method offers several advantages including simple equipment, easy operation, rapid reaction, high specificity, high sensitivity, and visual results. It is suitable for the early and rapid diagnosis of groupers infected with V. harveyi, V. parahaemolyticus, and SGIV, thereby providing useful technical support for further application in the large-scale disease surveillance of aquaculture animals. Full article

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., 10^8.0~8.3 TCID₅₀ mL⁻¹ and 10^7.0~7.2 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. Full article

Singapore grouper iridovirus (SGIV) is a virus with high fatality rate in the grouper culture industry. The outbreak of SGIV is often accompanied by a large number of grouper deaths, which has a great impact on the economy. Therefore, it is of great significance to find effective drugs against SGIV. It has been reported that edaravone is a broad-spectrum antiviral drug, most widely used clinically in recent years, but no report has been found exploring the effect of edaravone on SGIV infections. In this study, we evaluated the antiviral effect of edaravone against SGIV, and the anti-SGIV mechanism of edaravone was also explored. It was found that the safe concentration of edaravone on grouper spleen (GS) cells was 50 µg/mL, and it possessed antiviral activity against SGIV infection in a dose-dependent manner. Furthermore, edaravone could significantly disrupt SGIV particles and interference with SGIV binding to host cells, as well as SGIV replication in host cells. However, edaravone was not effective during the SGIV invasion into host cells. This study was the first time that it was determined that edaravone could exert antiviral effects in response to SGIV infection by directly interfering with the processes of SGIV infecting cells, aiming to provide a theoretical basis for the control of grouper virus disease. Full article

In previous studies, natural killer lysin (NK-lysin) emerged as a crucial antimicrobial peptide (AMP) discharged by NK cells and CTLs. The sequence of NK-lysin was cloned and discovered in some fishes, but its function remains unclear. In our study, we obtained a copy of NK-lysin from the spleen of the healthy clownfish (Amphiprion ocellaris; AoNK-lysin) through cloning and proceeded to investigate its potential functions and activities. The findings showed that the AoNK-lysin gene’s open reading frame (ORF) had a length of 465 base pairs (bp) and encoded 154 amino acids (aa), which included a saposin B domain and six cysteine residues that are highly conserved, forming three intrachain disulfide bonds to carry out antimicrobial activity. The AoNK-lysin gene was widely present in different tissues, with the skin showing the highest expression, followed by the eye, intestine, and muscle. Additionally, the expression of AoNK-lysin was significantly upregulated in the immune organs (spleen, gill, intestine, and head kidney) of A. ocellaris after being challenged by Singapore group iridovirus (SGIV). Furthermore, a 399 base pair cDNA sequence that encodes the fully developed peptide of AoNK-lysin was successfully inserted into a secretion plasmid called pPIC9K. Subsequently, a significant amount of the recombinant AoNK-lysin protein was efficiently manufactured using the Pichia pastoris expression system. The antibacterial test demonstrated that the AoNK-lysin protein significantly suppressed the growth of various pathogens, particularly Streptococcus agalactiae, Streptococcus iniae, Salmonella typhi, Shigella sonnei, Pseudomonas aeruginosa, and Aeromonas caviae. The minimal inhibitory concentration (MIC) was found to be 7.81 μg/mL. Further analysis of antiviral assays showed all the viral mRNA of SGIV to be significantly reduced after AoNK-lysin protein stimuli in FHM cells. Collectively, these discoveries indicate that AoNK-lysin exhibits features of both direct pathogen-killing abilities and inhibited virus replication. Full article

The dual-specificity phosphatase (DUSP) family plays an important role in response to adverse external factors. In this study, the DUSP5 from Epinephelus coioides, an important marine fish in Southeast Asia and China, was isolated and characterized. As expected, E. coioides DUSP5 contained four conserved domains: a rhodanese homology domain (RHOD); a dual-specificity phosphatase catalytic domain (DSPc); and two regions of low compositional complexity, indicating that E. coioides DUSP5 belongs to the DUSP family. E. coioides DUSP5 mRNA could be detected in all of the examined tissues, and was mainly distributed in the nucleus. Infection with Singapore grouper iridovirus (SGIV), one of the most important pathogens of marine fish, could inhibit the expression of E. coioides DUSP5. The overexpression of DUSP5 could significantly downregulate the expression of the key SGIV genes (MCP, ICP18, VP19, and LITAF), viral titers, the activity of NF-κB and AP-I, and the expression of pro-inflammatory factors (IL-6, IL-8, and TNF-α) of E. coioides, but could upregulate the expressions of caspase3 and p53, as well as SGIV-induced apoptosis. The results demonstrate that E. coioides DUSP5 could inhibit SGIV infection by regulating E. coioides immune-related factors, indicating that DUSP5 might be involved in viral infection. Full article

Singapore grouper iridovirus (SGIV) is a new ranavirus species in the Iridoviridae family, whose high lethality and rapid spread have resulted in enormous economic losses for the aquaculture industry. Curcumin, a polyphenolic compound, has been proven to possess multiple biological activities, including antibacterial, antioxidant, and antiviral properties. This study was conducted to determine whether curcumin protected orange-spotted grouper (Epinephelus coioides) from SGIV-induced intestinal damage by affecting the inflammatory response, cell apoptosis, oxidative stress, and intestinal microbiota. Random distribution of healthy orange-spotted groupers (8.0 ± 1.0 cm and 9.0 ± 1.0 g) into six experimental groups (each group with 90 groupers): Control, DMSO, curcumin, SGIV, DMSO + SGIV, and curcumin + SGIV. The fish administered gavage received DMSO dilution solution or 640 mg/L curcumin every day for 15 days and then were injected intraperitoneally with SGIV 24 h after the last gavage. When more than half of the groupers in the SGIV group perished, samples from each group were collected for intestinal health evaluation. Our results showed that curcumin significantly alleviated intestine damage and repaired intestinal barrier dysfunction, which was identified by decreased intestine permeability and serum diamine oxidase (DAO) activity and increased expressions of tight junction proteins during SGIV infection. Moreover, curcumin treatment suppressed intestinal cells apoptosis and inflammatory response caused by SGIV and protected intestinal cells from oxidative injury by enhancing the activity of antioxidant enzymes, which was related to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Moreover, we found that curcumin treatment restored the disruption of the intestinal microbiota caused by SGIV infection. Our study provided a theoretical basis for the functional development of curcumin in aquaculture by highlighting the protective effect of curcumin against SGIV-induced intestinal injury. Full article

(1) Background: Lysosomal aspartic protease Cathepsin D (CD) is a key regulator and signaling molecule in various biological processes including activation and degradation of intracellular proteins, the antigen process and programmed cell death. However, the function of fish CD in virus infection remains largely unknown. (2) Methods: The functions of the CD gene response to SGIV infection was determined with light microscopy, reverse transcription quantitative PCR, Western blot and flow cytometry. (3) Results: In this study, Ec-Cathepsin D (Ec-CD) was cloned and identified from the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of Ec-CD consisted of 1191 nucleotides encoding a 396 amino acid protein with a predicted molecular mass of 43.17 kDa. Ec-CD possessed typical CD structural features including an N-terminal signal peptide, a propeptide region and a mature domain including two glycosylation sites and two active sites, which were conserved in other CD sequences. Ec-CD was predominantly expressed in the spleen and kidneys of healthy groupers. A subcellular localization assay indicated that Ec-CD was mainly distributed in the cytoplasm. Ec-CD expression was suppressed by SGIV stimulation and Ec-CD-overexpressing inhibited SGIV replication, SGIV-induced apoptosis, caspase 3/8/9 activity and the activation of reporter gene p53 and activating protein-1 (AP-1) in vitro. Simultaneously, Ec-CD overexpression obviously restrained the activated mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In addition, Ec-CD overexpression negatively regulated the transcription level of pro-inflammatory cytokines and activation of the NF-κB promotor. (4) Conclusions: Our findings revealed that the Ec-CD possibly served a function during SGIV infection. Full article

Singapore grouper iridovirus (SGIV) causes high economic losses in mariculture. Effective drugs for managing SGIV infection are urgently required. Medicinal plant resources are rich in China. Medicinal plants have a long history and significant curative effects in the treatment of many diseases. Reverse-transcription quantitative real-time PCR is the most commonly used method for detecting virus infection and assessing antiviral efficacy with high accuracy. However, their applications are limited due to high reagent costs and complex time-consuming operations. Aptamers have been applied in some biosensors to achieve the accurate detection of pathogens or diseases through signal amplification. This study aimed to establish an aptamer-based high-throughput screening (AHTS) model for the efficient selection and evaluation of medicinal plants components against SGIV infection. Q2-AHTS is an expeditious, rapid method for selecting medicinal plant drugs against SGIV, which was characterized as being dram, high-speed, sensitive, and accurate. AHTS strategy reduced work intensity and experimental costs and shortened the whole screening cycle for effective ingredients. AHTS should be suitable for the rapid selection of effective components against other viruses, thus further promoting the development of high-throughput screening technology. Full article

(1) Background: Singapore grouper iridovirus (SGIV) can cause extensive fish deaths. Therefore, developing treatments to combat virulent SGIV is of great economic importance to address this challenge to the grouper aquaculture industry. Green tea is an important medicinal and edible plant throughout the world. In this study, we evaluated the use of green tea components against SGIV infection. (2) Methods: The safe working concentrations of green tea components were identified by cell viability detection and light microscopy. Additionally, the antiviral activity of each green tea component against SGIV infection was determined with light microscopy, an aptamer (Q5c)-based fluorescent molecular probe, and reverse transcription quantitative PCR. (3) Results: The safe working concentrations of green tea components were green tea aqueous extract (GTAE) ≤ 100 μg/mL, green tea polyphenols (TP) ≤ 10 μg/mL, epigallocatechin-3-gallate (EGCG) ≤ 12 μg/mL, (-)-epigallocatechin (EGC) ≤ 10 μg/mL, (-)-epicatechin gallate (EGC) ≤ 5 μg/mL, and (-)-epicatechin (EC) ≤ 50 μg/mL. The relative antiviral activities of the green tea components determined in terms of MCP gene expression were TP > EGCG > GTAE > ECG > EGC > EC, with inhibition rates of 99.34%, 98.31%, 98.23%, 88.62%, 73.80%, and 44.31%, respectively. The antiviral effect of aptamer-Q5c was consistent with the results of qPCR. Also, TP had an excellent antiviral effect in vitro, wherein the mortality of fish in only the SGIV-injection group and TP + SGIV-injection group were 100% and 11.67%, respectively. (4) Conclusions: In conclusion, our results suggest that green tea components have effective antiviral properties against SGIV and may be candidate agents for the effective treatment and control of SGIV infections in grouper aquaculture. Full article

Considerable attention has been paid to the roles of lipid metabolism in virus infection due to its regulatory effects on virus replication and host antiviral immune response. However, few literature has focused on whether lipid metabolism is involved in the life cycle of lower vertebrate viruses. Singapore grouper iridovirus (SGIV) is the causative aquatic virus that extensively causes fry and adult groupers death. Here, the potential roles of cellular de novo fatty acid synthesis in SGIV infection was investigated. SGIV infection not only increased the expression levels of key enzymes in fatty acid synthesis in vivo/vitro, including acetyl-Coenzyme A carboxylase alpha (ACC1), fatty acid synthase (FASN), medium-chain acyl-CoA dehydrogenase (MCAD), adipose triglyceride lipase (ATGL), lipoprotein lipase (LPL) and sterol regulatory element-binding protein-1 (SREBP1), but it also induced the formation of lipid droplets (LDs), suggesting that SGIV altered de novo fatty acid synthesis in host cells. Using the inhibitor and specific siRNA of ACC1 and FASN, we found that fatty acid synthesis was essential for SGIV replication, evidenced by their inhibitory effects on CPE progression, viral gene transcription, protein expression and virus production. Moreover, the inhibitor of fatty acid β-oxidation could also reduce SGIV replication. Inhibition of fatty acid synthesis but not β-oxidation markedly blocked virus entry during the life cycle of SGIV infection. In addition, we also found that inhibition of ACC1 and FASN increased the IFN immune and inflammatory response during SGIV infection. Together, our data demonstrated that SGIV infection in vitro regulated host lipid metabolism and, in that process, cellular fatty acid synthesis might exert crucial roles during SGIV infection via regulating virus entry and host immune response. Full article

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, causes great economic losses in the aquaculture industry. Previous studies demonstrated the lipid composition of intracellular unenveloped viruses, but the changes in host-cell glyceophospholipids components and the roles of key enzymes during SGIV infection still remain largely unknown. Here, the whole cell lipidomic profiling during SGIV infection was analyzed using UPLC-Q-TOF-MS/MS. The lipidomic data showed that glycerophospholipids (GPs), including phosphatidylcholine (PC), phosphatidylserine (PS), glycerophosphoinositols (PI) and fatty acids (FAs) were significantly elevated in SGIV-infected cells, indicating that SGIV infection disturbed GPs homeostasis, and then affected the metabolism of FAs, especially arachidonic acid (AA). The roles of key enzymes, such as cytosolic phospholipase A2 (cPLA2), 5-Lipoxygenase (5-LOX), and cyclooxygenase (COX) in SGIV infection were further investigated using the corresponding specific inhibitors. The inhibition of cPLA2 by AACOCF3 decreased SGIV replication, suggesting that cPLA2 might play important roles in the process of SGIV infection. Consistent with this result, the ectopic expression of EccPLA2α or knockdown significantly enhanced or suppressed viral replication in vitro, respectively. In addition, the inhibition of both 5-LOX and COX significantly suppressed SGIV replication, indicating that AA metabolism was essential for SGIV infection. Taken together, our results demonstrated for the first time that SGIV infection in vitro disturbed GPs homeostasis and cPLA2 exerted crucial roles in SGIV replication. Full article