Iridovirus has become a great threat to global aquaculture due to its high mortality, while the molecular events in virus pathogenesis are not well elucidated. Here, the multi-omics approach was performed with Singapore grouper iridovirus (SGIV)-infected groupers, and the roles of key metabolites were investigated. SGIV induced obvious histopathological damage and changes of metabolic enzymes in liver. Besides, SGIV significantly decreased the contents of lipid droplets, triglycerides, cholesterol, and lipoprotein. Metabolome analysis showed that that the differentially metabolites were enriched in 19 pathways, and lipid metabolites, including glycerophosphates and alpha-linolenic acid (ALA) were down-regulated, which is consistent with the disturbed lipid homeostasis in liver. Combining with transcriptomic and metabolomic data, the top enriched pathways were related to cell growth and death, nucleotide, carbohydrate, amino acid, and lipid metabolisms, supporting the conclusion that SGIV infection induced liver metabolic reprogramming. Integrative transcriptomic and proteomic analysis indicated that SGIV infection activated the crucial molecular events in phagosome-immune depression-metabolism dysregulation-necrosis signaling cascade. Of note, integrative multi-omics analysis demonstrated that metabolites ALA and linoleic acid (LA) were consumed, while L-glutamic acid (GA) was accumulated, accompanied by the alteration of immune, inflammation, and cell death related genes. Further experimental data showed that ALA, but not GA suppressed SGIV replication by activating host antioxidant and anti-inflammatory effects. Together, our findings for the first time provide a comprehensive resource to understand the landscape of host response dynamics during fish iridovirus infection, and highlight the antiviral roles of ALA in the prevention and treatment for iridovirus diseases.

Largemouth bass virus (LMBV), belonging to the genus Ranavirus, causes high mortality and heavy economic losses in largemouth bass aquaculture. In the present study, a novel cell line, designated as MsF, was established from the fin of largemouth bass (Micropterus salmoides), and applied to investigate the characteristics of cell death induced by LMBV. MsF cells showed susceptibility to LMBV, evidenced by the occurrence of a cytopathic effect (CPE), increased viral gene transcription, protein synthesis, and viral titers. In LMBV-infected MsF cells, two or more virus assembly sites were observed around the nucleus. Notably, no apoptotic bodies occurred in LMBV-infected MsF cells after nucleus staining, suggesting that cell death induced by LMBV in host cells was distinct from apoptosis. Consistently, DNA fragmentation was not detected in LMBV-infected MsF cells. Furthermore, only caspase-8 and caspase-3 were significantly activated in LMBV-infected MsF cells, suggesting that caspases were involved in non-apoptotic cell death induced by LMBV in host cells. In addition, the disruption of the mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) generation were detected in both LMBV-infected MsF cells and fathead minnow (FHM) cells. Combined with our previous study, we propose that cell death induced by LMBV infection was cell type dependent. Although LMBV-infected MsF cells showed the characteristics of non-apoptotic cell death, the signal pathways might crosstalk and interconnect between apoptosis and other PCD during LMBV infection. Together, our results not only established the in vitro LMBV infection model for the study of the interaction between LMBV and host cells but also shed new insights into the mechanisms of ranavirus pathogenesis.

Nervous necrosis virus (NNV) is the etiological agent of viral nervous necrosis (VNN), also known as viral encephalopathy and retinopathy (VER), which results in heavy economic losses to the aquaculture industry worldwide. Dramatic cytoplasmic vacuoles were observed during NNV infection both in vitro and in vivo; however, the origin and mechanism of cytoplasmic vacuolization remains unknown. In this report, we found that the cytoplasmic vacuole morphology became fused and enlarged during infection with red spotted grouper nervous necrosis virus (RGNNV), which was accompanied by increased cell death. Notably, Lyso-Tracker, but not Mito-Tracker or ER-Tracker, was accumulated in the vacuoles, and abnormal lysosome swelling was observed in RGNNV-infected cells, suggesting that the cytoplasmic vacuoles originated from lysosomal organelles. Cytoplasmic vacuolization and cell death in RGNNV-infected cells was completely blocked by the vacuolar H⁺-ATPase inhibitor (bafilomycin A1), and was significantly weakened by chloroquine (CQ), a lysosomotropic agent that induces the acidification of the lysosomes. This suggests that lysosome acidification was essential for vacuole formation. Significant inhibitory effects on vacuolization and cell death were also observed in the RGNNV-infected cells following treatment with nigericin and monensin (ionophores that uncouple the proton gradient present in lysosomes). This indicated that lysosome function was tightly associated with RGNNV infection-induced cell death. In addition, vacuoles were found to be partially co-localized with GFP-LC3II punctate dots during RGNNV infection. Moreover, the severity of vacuolization and cell death were both significantly decreased after treatment with the autophagy inhibitor, 3-MA, suggesting that autophagy was involved in lysosomal vacuolization and cell death evoked by RGNNV infection. Thus, our results demonstrate that autophagy participates in lysosomal vacuolation-mediated cell death during RGNNV infection, and provides new insight into our understanding of the potential mechanisms underlying nodavirus pathogenesis in vitro.

TRIM (tripartite motif) proteins have been demonstrated to exert critical roles in host defense against different microbial pathogens. Among them, TRIM23 acts as an important regulatory factor in antiviral immune and inflammatory responses, but the roles of fish TRIM23 against virus infection still remain largely unknown. Here, we investigated the characteristics of TRIM23 homolog from orange spotted grouper (Epinephelus coioides) (EcTRIM23). EcTRIM23 encoded a 580 amino acid peptide, which shared 93.1%, 89.73% and 86.36% identity with golden perch (Perca flavescens), zebrafish (Danio rerio) and human (Homo sapiens), respectively. The transcription levels of EcTRIM23 were significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTRIM23 overexpression in vitro significantly inhibited RGNNV and SGIV replication, evidenced by the delayed cytopathic effect (CPE) progression and the decreased expression of viral core genes. EcTRIM23 significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines, as well as the promoter activities of IFN and NF-κB, suggesting that EcTRIM23 exerted antiviral function by positively regulating host IFN response. Exogenous EcTRIM23 exhibited either diffuse or aggregated localization in grouper cells. After co-transfection, TANK binding kinase 1 (TBK1), TNF receptor associated factor (TRAF) 3 and TRAF4, TRAF5 and TRAF6 were found to interact with EcTRIM23 in grouper cells. Moreover, these proteins could be recruited and co-localized with EcTRIM23 in vitro. Together, our results demonstrated that fish TRIM23 exerted antiviral activity against fish viruses by interacting with multiple host proteins to regulate immune responses.