Development and immune evaluation of LAMP1 chimeric DNA vaccine against Singapore grouper iridovirus in orange-spotted grouper, Epinephelus coioides.

Grouper is one of the most important and valuable mariculture fish in China, with a high economic value. As the production of grouper has increased, massive outbreaks of epidemic diseases have limited the development of the industry. Singapore grouper iridovirus (SGIV) is one of the most serious infectious viral pathogens and has caused huge economic losses to grouper farming worldwide due to its rapid spread and high lethality. To find new strategies for the effective prevention and control of SGIV, we constructed two chimeric DNA vaccines using Lysosome-associated membrane protein 1 (LAMP1) fused with major capsid proteins (MCP) against SGIV. In addition, we evaluated the immune protective effects of vaccines including pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, chimeric DNA vaccine pcDNA3.1-MLAMP and pcDNA3.1-LAMCP by intramuscular injection. Our results showed that compared with groups injected with PBS, pcDNA3.1-3HA, pcDNA3.1-LAMP1 or pcDNA3.1-MCP, the antibody titer significantly increased in the chimeric vaccine groups. Moreover, the mRNA levels of immune-related factors in groupers, including IRF3, MHC-I, TNF-α, and CD8, showed the same trend. However, MHC-II and CD4 were significantly increased only in the chimeric vaccine groups. After 28 days of vaccination, groupers were challenged with SGIV, and mortality was documented for each group within 14 days. The data showed that two chimeric DNA vaccines provided 87 % and 91 % immune protection for groupers which were significantly higher than the 52 % protection rate of pcDNA3.1-MCP group, indicating that both forms of LAMP1 chimeric vaccines possessed higher immune protection against SGIV, providing the theoretical foundation for the creation of novel DNA vaccines for fish.

Curcumin Alleviates Singapore Grouper Iridovirus-Induced Intestine Injury in Orange-Spotted Grouper (Epinephelus coioides).

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.

Modulatory effects of curcumin on Singapore grouper iridovirus infection-associated apoptosis and autophagy in vitro.

Singapore grouper iridovirus (SGIV) with high pathogenicity can cause great economic losses to aquaculture industry. Thus, it is of urgency to find effective antiviral strategies to combat SGIV. Curcumin has been demonstrated effective antiviral activity on SGIV infection. However, the molecular mechanism behind this action needs to be further explanations. In view of the fact that apoptosis (type I programmed cell death) and autophagy (type II programmed cell death) were key regulators during SGIV infection, we aimed to investigate the relevance between antiviral activity of curcumin and SGIV-associated programmed and clarify the role of potential signaling pathways. Our results showed that curcumin suppressed SGIV-induced apoptosis. At the same time, the activities of caspase-3/8/9 and activating protein-1 (AP-1), P53, nuclear factor-κB (NF-ΚB) promoters were inhibited. Besides, the activation of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen activate protein kinase (p38 MAPK) signal pathways were suppressed in curcumin-treated cells. On the other hand, curcumin down-regulated protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway to promote autophagy representing by increased LC3 II and Beclin1 expression. Curcumin also hindered the transition of cells from G1 to S phase, as well as down-regulating the expression of CyclinD1. Our findings revealed the resistance curcumin induced to the effects of DNA virus on cell apoptosis and autophagy and the insights gained from this study may be of assistance to understand the molecular mechanism of curcumin against DNA virus infection.

Functional analysis of the Cystatin F gene response to SGIV infection in orange-spotted grouper, Epinephelus coioides.

Cystatin F (CyF), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyF and its latent molecular mechanism during virus infection in fish remain vacant. In our research, we cloned the open reading frame (ORF) of CyF homology from orange-spotted grouper (Ec-CyF) consisting of 342 nucleotides and encoding a 114-amino acid protein. Ec-CyF included two cystatins family sequences containing one KXVXG sequence without the signal peptide, and a hairpin ring containing proline and tryptophan (PW). Tissue distribution analysis indicated that Ec-CyF was highly expressed in spleen and head kidney. Besides, further analysis showed that the expression of Ec-CyF increased during SGIV infection in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyF was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyF demoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was impeded, as well as the restraint of caspase 3/7 and caspase 8. In addition, Ec-CyF overexpression up-regulated the expression of IFN related molecules including ISG15, IFN, IFP35, IRF3, IRF7, MYD88 and down-regulated proinflammatory factors such as IL-1ß, IL-8 and TNF-α. At the same time, Ec-CyF-overexpressing increased the activity of IFN3 and ISRE promoter, but impeded NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyF was involved in innate immunity response and played a key role in DNA virus infection.

Functional analysis of the cystatin A gene response to SGIV infection in orange-spotted grouper, Epinephelus coioides.

Cystatin A (CyA), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyA and its potential molecular mechanism during virus infection in fish remain unknown. In our study, we cloned the open reading frame (ORF) of CyA homology from orange-spotted grouper (Ec-CyA) consisting of 303 nucleotides and encoding a 101-amino acid protein. Ec-CyA included two conserved sequences containing one N-terminal glycine fragment and one QXVXG sequence (48aa-52aa) without the signal peptide. Tissue distribution analysis showed that Ec-CyA was highly expressed in spleen and head kidney. Moreover, further analysis indicated that the expression of Ec-CyA increased during SGIV simulation in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyA was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyA promoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was facilitated, as well as the activation of caspase-3/7, caspase-9. In addition, Ec-CyA overexpression down-regulated the expression of interferon (IFN) related molecules including ISG15, IFN, IRF3, MAVS, MyD88, TRAF6 and up-regulated proinflammatory factors such as IL-1ß, IL-8 and TNF-α. At the same time, Ec-CyA-overexpressing inhibited the activity of IFN and ISRE promoter, but induced NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyA was involved in innate immune response and played a key role in DNA virus infection.

Functional Analysis of the Cathepsin D Gene Response to SGIV Infection in the Orange-Spotted Grouper, Epinephelus coioides.

(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.

High-density lipoproteins negatively regulate innate immunity and facilitate red-spotted grouper nervous necrosis virus entry via scavenger receptor B type 1.

Lipid metabolism plays an important role in viral infections, and it can directly or indirectly affect various stages of viral infection in cells. As an important component of lipid metabolism, high-density lipoprotein (HDL) plays crucial roles in inflammation, immunity, and viral infections. Scavenger receptor B type 1 (SR-B1), a receptor of HDL, cannot be ignored in the regulation of lipid metabolism. Here, we investigate, for the first time, the role of Epinephelus coioides SR-B1 (Ec-SR-B1) in red-spotted grouper nervous necrosis virus (RGNNV) infection. Our results indicate that Ec-SR-B1 could promote RGNNV infection. We also demonstrate that Ec-SR-B1 could facilitate viral entry and interact with capsid protein (CP) of RGNNV. As the natural ligand of SR-B1, HDL significantly increased RGNNV entry in a dose-dependent manner. However, we observed no effect of HDL on Ec-SR-B1 expression. The results of the micro-scale thermophoresis assay did not reveal an association between HDL and CP, suggesting that RGNNV does not enter target cells by using HDL as a ligand to bind to its receptor. In addition, block lipid transport-1, a compound that inhibits HDL-mediated cholesterol transfer, reduced the HDL-induced enhancement of RGNNV infection, indicating a role for lipid transfer in facilitating RGNNV entry. Furthermore, HDL inhibited the expression of pro-inflammatory factors and antiviral genes in a dose-dependent manner. These findings suggest that the HDL-induced enhancement of RGNNV entry involves the complex interplay between Ec-SR-B1, HDL, and RGNNV, as well as the regulation of innate antiviral responses by HDL. In summary, we highlight the crucial role of HDL in RGNNV entry, identify a possible molecular connection between RGNNV and lipoprotein metabolism, and indicate the role of Ec-SR-B1 in RGNNV infection.

Identification and functional characterization of Cystatin B in orange-spotted grouper, Epinephelus coioides.

Cystatin B is a cysteine protease inhibitor that plays a crucial role in immune response. Nevertheless, the molecular mechanism of fish Cystatin B in virus replication remains obscure. In this study, we identified and characterized Cystatin B (Ec-CysB) in the orange-spotted grouper (Epinephelus coioides). The Ec-CysB encoded a 100-amino acid protein with the conserved QXVXG motif, PC motif and cysteine protease inhibitory motif, which shared high identities with reported Cystatin B. The abundant transcriptional level of Ec-CysB was found in gill, intestine and head kidney. And the Ec-CysB expression was significantly up-regulated in spleen after infection with Singapore grouper iridovirus (SGIV) in vitro. Subcellular localization analysis revealed that Ec-CysB was distributed mainly in the cytoplasm and nucleus. Further studies showed that overexpression of Ec-CysB in vitro significantly increased SGIV replication and virus-induced cell apoptosis, but replication of SGIV was inhibited by knockdown or mutant of Ec-CysB. Moreover, overexpression of Ec-CysB significantly inhibited the interferon (IFN), interferon-stimulated response element (ISRE) promoter activities, and enhanced apoptosis-related transcription factors p53 promoter activities. Collectively, our results suggest that Ec-CysB affect viral replication and virus-induced cell apoptosis, which will help us to explore its potential functions during SGIV infection.

Characterization of scavenger receptor MARCO in orange-spotted grouper, Epinephelus coioides.

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor that plays a crucial role in the immune response against microbial infections. To clarify the roles of fish MARCO in Singapore grouper iridovirus (SGIV) infection, we identified and characterized Ec-MARCO in the orange-spotted grouper (Epinephelus coioides). The Ec-MARCO encoded a 370-amino acid protein with transmembrane region, coiled coil region and SR domain, which shared high identities with reported MARCO. The abundant transcriptional level of Ec-MARCO was found in spleen, head kidney and blood. And the Ec-MARCO expression was significantly up-regulated in grouper spleen (GS) cells after infection with SGIV in vitro. Subcellular localization analysis revealed that Ec-MARCO was mainly distributed in the cytoplasm and on the cell membrane. Ec-MARCO knockdown in vitro significantly inhibited SGIV infection in GS cells, as evidenced by reduced decreased SGIV major capsid protein (MCP) transcription and MCP protein expression. Further studies showed that Ec-MARCO knockdown positively regulated proinflammatory cytokines and interferon-stimulated genes, and enhanced IFN and ISRE promoter activities. However, overexpression of Ec-MARCO did not affect SGIV entry into host cells. In summary, our results suggested that Ec-MARCO affected SGIV infection by regulating antiviral innate immune response.