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.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper (Epinephelus coioides) (EcCH25H). EcCH25H encoded a 271-amino-acid polypeptide, with 86 and 59% homology with yellow croaker (Larimichthys crocea) and humans, respectively. EcCH25H contained a conserved fatty acid (FA) hydroxylase domain and an ERG3 domain. EcCH25H expression was induced by RGNNV or SGIV infection, lipopolysaccharide (LPS) or poly (I:C) treatment in vitro. Subcellular localization showed that EcCH25H and mutant EcCH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum. SGIV and RGNNV replication was decreased by EcCH25H overexpression, which was reflected in the reduced severity of the cytopathic effect and a decrease in viral gene transcription, but replication of both viruses was increased by knockdown of EcCH25H. Besides, the antiviral activity was dependent on its enzymatic activity. Treatment with 25HC significantly inhibited replication of SGIV and RGNNV. EcCH25H overexpression positively regulated the IFN-related molecules and proinflammatory cytokines, and increased both IFN and ISRE promoter activities. Moreover, 25HC treatment significantly suppressed SGIV and RGNNV entry into host cells. The similar inhibitory effect on SGIV entry was observed in EcCH25H overexpression cells. Taken together, our findings demonstrated that EcCH25H inhibited SGIV and RGNNV infection by regulating IFN signaling molecules, and might also influence viral entry via an effect on cholesterol.

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.