Ferroptosis resists intracellular Vibrio splendidus AJ01 mediated by ferroportin in sea cucumber Apostichopus japonicus.

Ferroptosis, a kind of programmed cell death, is characterized with iron-dependent lipid ROS buildup, which is considered as an important cellular immunity in resisting intracellular bacterial infection in mammalian macrophages. In this process, lipid ROS oxidizes the bacterial biofilm to inhibit intracellular bacteria. However, the function of ferroptosis in invertebrate remains unknown. In this study, the existence of ferroptosis in Apostichopus japonicus coelomocytes was confirmed, and its antibacterial mechanism was investigated. First, our results indicated that the expression of glutathione peroxidase (AjGPX4) was significantly inhibited by 0.21-fold (p < 0.01) after injecting A. japonicus with the ferroptosis inducer RSL3, and the contents of MDA (3.93-fold, p < 0.01), ferrous iron (1.40-fold, p < 0.01), and lipid ROS (3.10-fold, p < 0.01) were all significantly increased under this condition and simultaneously accompanied with mitochondrial contraction and disappearance of cristae, indicating the existence of ferroptosis in the coelomocytes of A. japonicus. Subsequently, the contents of ferrous iron (1.40-fold, p < 0.05), MDA (2.10-fold, p < 0.01), ROS (1.70-fold, p < 0.01), and lipid ROS (2.50-fold, p < 0.01) were all significantly increased, whereas the mitochondrial membrane potential and GSH/GSSG were markedly decreased by 0.68-fold (p < 0.05) and 0.69-fold (p < 0.01) under Vibrio splendidus (AJ01) infection. This process could be reversed by the iron-chelating agent deferoxamine mesylate, which indicated that AJ01 could induce coelomocytic ferroptosis. Moreover, the results demonstrated that the intracellular AJ01 load was clearly decreased to 0.49-fold (p < 0.05) and 0.06-fold (p < 0.01) after treating coelomocytes with RSL3 and ferrous iron, which indicated that enhanced ferroptosis could inhibit bacterial growth. Finally, subcellular localization demonstrated that ferrous iron efflux protein ferroportin (AjFPN) and intracellular AJ01 were co-localized in coelomocytes. After AjFPN interference (0.58-fold, p < 0.01), the signals of ferrous iron and lipid ROS levels in intracellular AJ01 were significantly reduced by 0.38-fold (p < 0.01) and 0.48-fold (p < 0.01), indicating that AjFPN was an important factor in the introduction of ferroptosis into intracellular bacteria. Overall, our findings indicated that ferroptosis could resist intracellular AJ01 infection via AjFPN. These findings provide a novel defense mechanism for aquatic animals against intracellular bacterial infection.

Midgut microbiota affects the intestinal barrier by producing short-chain fatty acids in Apostichopus japonicus.

Introduction: The intestinal microbiota participates in host physiology and pathology through metabolites, in which short-chain fatty acids (SCFAs) are considered principal products and have extensive influence on intestine homeostasis. It has been reported that skin ulceration syndrome (SUS), the disease of Apostichopus japonicus caused by Vibrio splendidus, is associated with the alteration of the intestinal microbiota composition. Method: To investigate whether the intestinal microbiota affects A. japonicus health via SCFAs, in this study, we focus on the SCFA profiling and intestinal barrier function in A. japonicus treated with V. splendidus. Results and discussion: We found that V. splendidus could destroy the mid-intestine integrity and downregulate the expression of tight junction proteins ZO-1 and occludin in A. japonicus, which further dramatically decreased microorganism abundance and altered SCFAs contents. Specifically, acetic acid is associated with the largest number of microorganisms and has a significant correlation with occludin and ZO-1 among the seven SCFAs. Furthermore, our findings showed that acetic acid could maintain the intestinal barrier function by increasing the expression of tight junction proteins and rearranging the tight junction structure by regulating F-actin in mid-intestine epithelial cells. Thus, our results provide insights into the effects of the gut microbiome and SCFAs on intestine barrier homeostasis and provide essential knowledge for intervening in SUS by targeting metabolites or the gut microbiota.

NEDD4 activates mitophagy by interacting with LC3 to restrain reactive oxygen species and apoptosis in Apostichopus japonicus challenged with Vibrio splendidus.

Mitophagy, the selective degradation of damaged mitochondria by autophagy, plays a crucial role in the survival of coelomocytes in Apostichopus japonicus following Vibrio splendidus infection by suppressing the generation of reactive oxygen species (ROS) and attenuating cell apoptosis. A recent study revealed that reducing the expression of the neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4), an enzyme 3 (E3) ubiquitin ligase, significantly affects mitochondrial degradation. Prior to the present study, the functional role of NEDD4 in marine invertebrates was largely unexplored. Therefore, we investigated the role of NEDD4 in the activation of mitophagy, modulation of ROS levels, and induction of apoptosis in A. japonicus infected with V. splendidus. The results demonstrated that V. splendidus infection and lipopolysaccharide (LPS) challenge significantly increased the mRNA levels of NEDD4 in A. japonicus coelomocytes, which was consistent with changes in mitophagy under the same conditions. Knockdown of AjNEDD4 using specific small interfering RNAs (siRNAs) impaired mitophagy and caused accumulation of damaged mitochondria, as observed using transmission electron microscopy (TEM) and confocal microscopy. Furthermore, AjNEDD4 was localized to the mitochondria in both coelomocytes and HEK293T cells. Simultaneously, coelomocytes were treated with the inhibitor indole-3-carbinol (I3C) to confirm the regulatory role of AjNEDD4 in mitophagy. The accumulation of AjNEDD4 in the mitochondria and the level of mitophagy decreased. Subsequent investigations demonstrated that AjNEDD4 interacts directly with the microtubule-associated protein light chain 3 (LC3), a key regulator of autophagy and mitophagy, indicating its involvement in the mitophagy pathway. Moreover, AjNEDD4 interference hindered the interaction between AjNEDD4 and LC3, thereby impairing the engulfment and subsequent clearance of damaged mitochondria. Finally, AjNEDD4 interference led to a significant increase in intracellular ROS levels, followed by increased apoptosis. Collectively, these findings suggest that NEDD4 acts as a crucial regulator of mitophagy in A. japonicus and plays a vital role in maintaining cellular homeostasis following V. splendidus infection. NEDD4 suppresses ROS production and subsequent apoptosis by promoting mitophagy, thereby safeguarding the survival of A. japonicus under pathogenic conditions. Further investigation of the mechanisms underlying NEDD4-mediated mitophagy may provide valuable insights into the development of novel strategies for disease control in aquaculture farms.

Ring Finger Protein 34 Facilitates Nervous Necrosis Virus Evasion of Antiviral Innate Immunity by Targeting TBK1 and IRF3 for Ubiquitination and Degradation in Teleost Fish.

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

Evolution of mitogen-activated protein kinase family and their immune function in Apostichopus japonicus.

The mitogen-activated protein kinase family plays an important role in cell differentiation, growth, proliferation, and survival. However, the current research on the mitogen-activated protein kinase (MAPK) family in invertebrates is limited to the individual gene, and the analysis has not been conducted at the family level. In the present study, echinoderm MAPK family was identified by genomic screening, and five members, including three ERK subfamily members, one c-Jun N-terminal kinase (JNK) subfamily, and one p38-MAPK member were detected. Phylogenetic analysis showed that three MAPK subfamilies were separated into three separated clusters, and ERK subfamily appeared earlier than the other two subfamilies. Synteny analysis revealed that the p38 subfamily might be derived from the continuous gene duplication events of MAPK14 subfamily in invertebrates, which displayed genome expansion via gene duplication in vertebrates. The role of MAPK family in echinoderm immune defense was determined by investigating the expression profiles of MAPKs in Vibrio splendidus-challenged Apostichopus japonicus and LPS-exposed coelomocytes. The result showed that five MAPK members displayed induced expression profiles both in vitro and in vivo, and the peak expression was detected at different time points. Our study provides new insights into the evolutionary history of the MAPK family and show the similar immune function among MAPK members.

Mesentery AjFGF4-AjFGFR2-ERK pathway modulates intestinal regeneration via targeting cell cycle in echinoderms.

OBJECTIVES: The purpose of the study aims to understand the regeneration process and its cytology mechanism in economic echinoderms. MATERIALS AND METHODS: The intestine regeneration process of Apostichopus japonicus was investigated by immunohistochemistry and the cell proliferation was detected by immunofluorescence and flow cytometry. Fibroblast growth factor 4 of A. japonicus (AjFGF4) was screened by RNA-seq analysis and validated to regulate cell proliferation by siAjFGF4 and recombinant-AjFGF4 treatment. The binding and co-localization of AjFGF4 and AjFGFR2 were verified by Co-IP, GST-pull down, and immunofluorescence. Then, the AjFGF4-AjFGFR2-ERK-cell cycle axis was examined by western blot, immunofluorescence, and flow cytometry techniques. RESULTS: The mesentery was served as the epicenter of intestinal regeneration via activating cell proliferation and other cellular events. Mechanically, AjFGF4-mediated cell proliferation was dependent on the binding to its receptor AjFGFR2, and then triggered the conserved ERK-MAPK pathway but not JNK and p38 pathway. The activated ERK-MAPK subsequently mediated the expression of cell cycle regulatory proteins of CDK2, Cyclin A, and Cyclin B to promote cell proliferation. CONCLUSIONS: We provide the first functional evidence that AjFGF4-AjFGFR2-ERK-cell cycle axis mediated cell proliferation was the engine for mesentery-derived intestine regeneration in echinoderms.

Phenotypic and functional characterization of two coelomocyte subsets in Apostichopus japonicus.

The hemocytes of invertebrates are composed of different cell subsets with different morphologies and structures. Different cell subsets have different immune functions, which play an important role in innate immune response against pathogens. However, the understanding of the classification of Apostichopus japonicus coelomocytes and the molecular basis of immune function of different cell subsets is very limited. In this study, two coelomocyte subpopulations of A. japonicus were isolated by Percoll density gradient centrifugation. They were identified from their morphological and structural characteristics, namely, spherical cells with a size of 10-12 µm spherical in shape and a large number of small granules inside; lymphocyte-like cells with a size of 4-5 µm spherical or oval in shape, and 1-3 filopodia. Functionally, the phagocytic capacity and lysosomal activity in spherical cells were significantly greater than those in lymphocyte-like cells. The results suggest that spherical cells may play a more critical role in the immune responses. Meanwhile, transcriptome sequencing analysis was performed to further clarify the functional differences between the two cell subsets. The data indicated significantly different gene expression patterns in them. Spherical cells tend to participate in immune defense, whereas lymphocyte-like cells tend to participate in energy metabolism. In addition, lymphocyte-like cells may convert oxidative phosphorylation to glycolysis by changing the manner of energy metabolism to quickly adapt to the energy demand of external stimuli. Spherical cells may respond to LPS stimulation through phagocytosis, and their response time is slower than that of lymphocyte-like cells. The expression of genes involved in endocytosis, phagocytosis, and lysosomal and humoral immunity in spherical cells was significantly higher than that in lymphocyte-like cells. These data provide valuable information for understanding the molecular basis of cellular and humoral immunity in A. japonicus.

The Capsid Protein of Nervous Necrosis Virus Antagonizes Host Type I IFN Production by a Dual Strategy to Negatively Regulate Retinoic Acid-Inducible Gene-I-like Receptor Pathways.

Nervous necrosis virus (NNV), a highly pathogenic RNA virus, is a major pathogen in the global aquaculture industry. To efficiently infect fish, NNV must evade or subvert the host IFN for their replication; however, the precise mechanisms remain to be elucidated. In this study, we reported that capsid protein (CP) of red-spotted grouper NNV (RGNNV) suppressed the IFN antiviral response to promote RGNNV replication in Lateolabrax japonicus brain cells, which depended on the ARM, S, and P domains of CP. CP showed an indirect or direct association with the key components of retinoic acid-inducible gene-I-like receptors signaling, L. japonicus TNFR-associated factor 3 (LjTRAF3) and IFN regulatory factor (LjIRF3), respectively, and degraded LjTRAF3 and LjIRF3 through the ubiquitin-proteasome pathway in HEK293T cells. Furthermore, we found that CP potentiated LjTRAF3 K48 ubiquitination degradation in a L. japonicus ring finger protein 114-dependent manner. LjIRF3 interacted with CP through the S domain of CP and the transcriptional activation domain or regulatory domain of LjIRF3. CP promoted LjIRF3 K48 ubiquitination degradation, leading to the reduced phosphorylation level and nuclear translocation of LjIRF3. Taken together, we demonstrated that CP inhibited type I IFN response by a dual strategy to potentiate the ubiquitination degradation of LjTRAF3 and LjIRF3. This study reveals a novel mechanism of RGNNV evading host immune response via its CP protein that will provide insights into the complex pathogenesis of NNV.

E3 Ubiquitin Ligase RNF114 Inhibits Innate Immune Response to Red-Spotted Grouper Nervous Necrosis Virus Infection in Sea Perch by Targeting MAVS and TRAF3 to Mediate Their Degradation.

RIG-I-like receptor (RLR)-mediated antiviral signaling is critical to trigger the immune response to virus infection; however, the antiviral responses are also tightly regulated to avoid uncontrolled production of type I IFN by various mechanisms, including ubiquitination. In this study, an E3 ubiquitin ligase ring finger protein 114 (RNF114) from sea perch (Lateolabrax japonicus) (LjRNF114) was identified as a suppressor of RLR signaling pathways during red-spotted grouper nervous necrosis virus (RGNNV) infection. RGNNV infection promoted the expression of LjRNF114. Overexpression of LjRNF114 enhanced RGNNV replication, whereas knockdown of LjRNF114 led to opposite effects. Type I IFN production induced by RGNNV was suppressed by LjRNF114, which is dependent on its ubiquitin ligase activity. Moreover, LjRNF114 inhibited IFN promoter activation induced by key signaling molecules in RLR signaling pathways. We observed the interactions between LjRNF114 and both sea perch mitochondrial antiviral signaling protein (MAVS) and TNFR-associated factor 3 (TRAF3). Domain mapping experiments indicated that the RING and ubiquitin interacting motif domains of LjRNF114 were required for its interaction with TRAF3 and MAVS. We found that LjRNF114 targeted MAVS and TRAF3 for K27- and K48-linked ubiquitination and degradation, resulting in the inhibition of IFN production. Taken together, our study reveals, to our knowledge, a novel mechanism that LjRNF114 targets and promotes K27- and K48-linked ubiquitination of MAVS and TRAF3 to negatively regulate the RLR signaling pathways, promoting viral infection.

Marine medaka heat shock protein 90ab1 is a receptor for red-spotted grouper nervous necrosis virus and promotes virus internalization through clathrin-mediated endocytosis.

Nervous necrosis virus (NNV) can infect many species of fish and causes serious acute or persistent infection. However, its pathogenic mechanism is still far from clear. Specific cellular surface receptors are crucial determinants of the species tropism of a virus and its pathogenesis. Here, the heat shock protein 90ab1 of marine model fish species marine medaka (MmHSP90ab1) was identified as a novel receptor of red-spotted grouper NNV (RGNNV). MmHSP90ab1 interacted directly with RGNNV capsid protein (CP). Specifically, MmHSP90ab1 bound to the linker region (LR) of CP through its NM domain. Inhibition of MmHSP90ab1 by HSP90-specific inhibitors or MmHSP90ab1 siRNA caused significant inhibition of viral binding and entry, whereas its overexpression led to the opposite effect. The binding of RGNNV to cultured marine medaka hMMES1 cells was inhibited by blocking cell surface-localized MmHSP90ab1 with anti-HSP90ß antibodies or pretreating virus with recombinant MmHSP90ab1 or MmHSP90ab1-NM protein, indicating MmHSP90ab1 was an attachment receptor for RGNNV. Furthermore, we found that MmHSP90ab1 formed a complex with CP and marine medaka heat shock cognate 70, a known NNV receptor. Exogenous expression of MmHSP90ab1 independently facilitated the internalization of RGNNV into RGNNV impenetrable cells (HEK293T), which was blocked by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. Further study revealed that MmHSP90ab1 interacted with the marine medaka clathrin heavy chain. Collectively, these data suggest that MmHSP90ab1 is a functional part of the RGNNV receptor complex and involved in the internalization of RGNNV via the clathrin endocytosis pathway.

Ubiquitin-specific protease 5 was involved in the interferon response to RGNNV in sea perch (Lateolabrax japonicus).

Deubiquitinases are widely involved in the regulation of the virus-triggered type I interferon (IFN) signaling. Here, we found sea perch (Lateolabrax japonicus) ubiquitin-specific protease 5 (LjUSP5) was a negative regulatory factor of the red-spotted grouper nervous necrosis virus (RGNNV)-triggered IFN response. LjUSP5 encoded a polypeptide of 830 amino acids, containing a zinc finger UBP domain (residues 197-270 aa), two ubiquitin-associated domains (residues 593-607 aa; 628-665 aa), and one UBP domain (residues 782-807 aa), and shared the closest genetic relationship with the USP5 of Larimichthys crocea. Quantitative RT-PCR analysis showed that LjUSP5 was ubiquitously expressed and up-regulated significantly in all inspected tissues post RGNNV infection, and its transcripts significantly increased in brain, liver and kidney tissues post RGNNV infection. LjUSP5 was up-regulated in cultured LJB cells after poly I:C and RGNNV treatments. In addition, overexpression of LjUSP5 significantly inhibited the activation of zebrafish IFN 1 promoter and promoted RGNNV replication in vitro. Furthermore, LjUSP5 inhibited the activation of zebrafish IFN 1 promoter induced by key genes of retinoic acid-inducible gene I-like receptors signaling pathway. Our findings provides useful information for further elucidating the mechanism underlying NNV infection.

MiR-202-5p Inhibits RIG-I-Dependent Innate Immune Responses to RGNNV Infection by Targeting TRIM25 to Mediate RIG-I Ubiquitination.

The RIG-I-like receptors (RLRs) signaling pathway is essential for inducing type I interferon (IFN) responses to viral infections. Meanwhile, it is also tightly regulated to prevent uncontrolled immune responses. Numerous studies have shown that microRNAs (miRNAs) are essential for the regulation of immune processes, however, the detailed molecular mechanism of miRNA regulating the RLRs signaling pathway remains to be elucidated. Here, our results showed that miR-202-5p was induced by red spotted grouper nervous necrosis virus (RGNNV) infection in zebrafish. Overexpression of miR-202-5p led to reduced expression of IFN 1 and its downstream antiviral genes, thus facilitating viral replication in vitro. In comparison, significantly enhanced levels of IFN 1 and antiviral genes and significantly low viral burden were observed in the miR-202-5p-/- zebrafish compared to wild type zebrafish. Subsequently, zebrafish tripartite motif-containing protein 25 (zbTRIM25) was identified as a target of miR-202-5p in both zebrafish and humans. Ectopic expression of miR-202-5p suppressed zbTRIM25-mediated RLRs signaling pathway. Furthermore, we showed that miR-202-5p inhibited zbTRIM25-mediated zbRIG-I ubiquitination and activation of IFN production. In conclusion, we demonstrate that RGNNV-inducible miR-202-5p acts as a negative regulator of zbRIG-I-triggered antiviral innate response by targeting zbTRIM25. Our study reveals a novel mechanism for the evasion of the innate immune response controlled by RGNNV.

Maternal miR-202-5p is required for zebrafish primordial germ cell migration by protecting small GTPase Cdc42.

In many lower animals, germ cell formation, migration, and maintenance depend on maternally provided determinants in germ plasm. In zebrafish, these processes have been extensively studied in terms of RNA-binding proteins and other coding genes. The role of small non-coding RNAs in the regulation of primordial germ cell (PGC) development remains largely unknown and poorly investigated, even though growing interests for the importance of miRNAs involved in a wide variety of biological processes. Here, we reported the role and mechanism of the germ plasm-specific miRNA miR-202-5p in PGC migration: (i) both maternal loss and knockdown of miR-202-5p impaired PGC migration indicated by the mislocalization and reduced number of PGCs; (ii) cdc42se1 was a direct target gene of miR-202-5p, and overexpression of Cdc42se1 in PGCs caused PGC migration defects similar to those observed in loss of miR-202-5p mutants; (iii) Cdc42se1 not only interacted with Cdc42 but also inhibited cdc42 transcription, and overexpression of Cdc42 could rescue PGC migration defects in Cdc42se1 overexpressed embryos. Thus, miR-202-5p regulates PGC migration by directly targeting and repressing Cdc42se1 to protect the expression of Cdc42, which interacts with actin to direct PGC migration.

Zebrafish TRIM25 Promotes Innate Immune Response to RGNNV Infection by Targeting 2CARD and RD Regions of RIG-I for K63-Linked Ubiquitination.

RIG-I-like receptors (RLRs) play important roles in response to virus infection by regulating host innate immune signaling pathways. Meanwhile, the RLR signaling pathway is also tightly regulated by host and virus to achieve the immune homeostasis between antiviral responses and virus survival. Here, we found that zebrafish TRIM25 (zbTRIM25) functioned as a positive regulator of RLR signaling pathway during red spotted grouper nervous necrosis virus (RGNNV) infection. Post-RGNNV infection, zbTRIM25 expression was obviously inhibited and ectopic expression of zbTRIM25 led to enhanced expression of RLR signaling pathway-related genes. Overexpression and knockdown analysis revealed that zbTRIM25 promoted zebrafish RIG-I (zbRIG-I)-mediated IFN signaling and inhibited RGNNV replication. Mechanistically, zbTRIM25 bound to zbRIG-I; in particular, the SPRY domain of zbTRIM25 interacted with the tandem caspase activation and recruitment domains (2CARD) and repressor domain (RD) regions of zbRIG-I. zbTRIM25 promoted the K63 polyubiquitination of 2CARD and RD regions of zbRIG-I. Furthermore, zbTRIM25-mediated zbRIG-I activation of IFN production was enhanced by K63-linked ubiquitin, indicating that zbTRIM25-mediated zbRIG-I polyubiquitination was essential for RIG-I-triggered IFN induction. In conclusion, these findings reveal a novel mechanism that zbTRIM25 positively regulates the innate immune response by targeting and promoting the K63-linked polyubiquitination of zbRIG-I.

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China.

Fish rhabdoviruses are a family of viruses responsible for large-scale fish die-offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT-PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT-PCR. Electron microscopy showed the presence of bullet-shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole-genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen-free largemouth bass juveniles. VHSVLB2018-infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%-93.3% by 7 days post-infection. VHSVLB2018 was re-isolated from dead fish and confirmed by RT-PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.

Comparative transcriptome analysis reveals the role of p53 signalling pathway during red-spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells.

Nervous necrosis virus (NNV) is one of the fish pathogens that have caused mass mortalities of many marine and freshwater fishes in the world. To better comprehend the molecular immune mechanism of sea perch (Lateolabrax japonicus) against NNV infection, the comparative transcriptome analysis of red-spotted grouper nervous necrosis virus (RGNNV)-infected or mock-infected L. japonicus brain (LJB) cells was performed via RNA sequencing technology. Here, 1,969 up-regulated genes and 9,858 down-regulated genes, which were widely implicated in immune response pathways, were identified. Furthermore, we confirmed that p53 signalling pathway was repressed at 48 hr post-RGNNV infection, as indicated by up-regulation of Mdm2 and down-regulation of p53 and its downstream target genes, including Bax, Casp8 and CytC. Overexpression of L. japonicus p53 (Ljp53) significantly inhibited RGNNV replication and up-regulated the expression of apoptosis-related genes, whereas the down-regulation caused by pifithrin-α led to the opposite effect, suggesting Ljp53 might promote cell apoptosis to repress virus replication. Luciferase assay indicated that Ljp53 could enhance the promoter activities of zebrafish interferon (IFN)1, indicating that Ljp53 could exert its anti-RGNNV activities by enforcing the type I IFN response. This study revealed the potential antiviral role of p53 during NNV infection.

Molecular characterization of vasa homologue in marbled goby, Oxyeleotris marmorata: Transcription and localization analysis during gametogenesis and embryogenesis.

Identification of germ cell markers is important for investigating reproduction biology in fish. Vasa is one of the most studied germ cell markers in mammals and lower vertebrates including fish. Here, we characterized a vasa homologue from the fish marbled goby (Oxyeleotris marmorata), termed omvasa. The full length of omvasa cDNA is 2344 bp and encodes 658 amino acids, sharing high identities with Vasa homologues of other vertebrates. OmVasa protein contains 15 RG/RGG repeats at N-terminus, 2 ATPase motifs, as well as RNA unwinding and RNA binding domains at C-terminus. Phylogenetic tree showed that omVasa had the closest relationship with the Vasa homologue from the fish Boleophthalmus pectinirostris, the great blue-spotted mudskipper. qRT-PCR analysis indicated that omvasa was specifically transcribed in gonads, and the transcription level was gradually increased during oocyte development. The germ cell-specific distribution of omvasa mRNA was revealed by fluorescent in situ hybridization. In ovary, the signal of omvasa RNA displayed strong-weak-strong dynamics from oogonia over pre-vitellogenic oocytes to vitellogenic oocytes. In testis, omvasa signal was strong in spermatogonia, modest in spermatocytes but undetectable in spermatids and somatic cells. During embryogenesis, the transcription of omvasa mRNA was high at blastula stage, gradually decreased from gastrula stage and maintained at a low level in later developmental stages. Whole mount in situ hybridization indicated that omvasa mRNA was specific to primordial germ cells (PGCs). In summary, marbled goby vasa is a germ cell-specific transcript during gametogenesis, and can be used as an ideal marker for tracing PGC formation and migration, which is pivotal to germ cell manipulation in this species.

Expression pattern, antiviral role and regulation analysis of interferon-stimulated gene 15 in black seabream, Acanthopagrus schlegelii.

Interferon stimulated gene 15 (ISG15) is an IFN inducible ubiquitin-like protein and plays a critical role in immune response against viral infection. In this study, an ISG15 gene (AsISG15) was cloned and characterized from the marine fish black seabream, Acanthopagrus schlegelii. The full-length cDNA of AsISG15 was 1302 bp and encoded 155 amino acids containing two ubiquitin-like motifs and a LRGG conjugation domain. Multiple alignment and phylogenetic tree showed that AsISG15 shared 31-70% amino acid identity with other known ISG15s and had a closer evolutionary relationship with teleost ISG15s. In vitro, AsISG15 expression was inducible by poly I:C, LPS and red spotted nervous necrosis virus (RGNNV) in cultured black seabream brain cells. In vivo, AsISG15 was ubiquitously expressed in all examined tissues with higher expression levels in eye and gill, and the expression was significantly up-regulated in most tissues post RGNNV infection, especially in liver, spleen and kidney. The testing of antiviral activity showed that silencing AsISG15 significantly increased RGNNV replication in RGNNV infected AsS cells, and the LRGG domain was crucial for the anti-RGNNV activity of AsISG15. By promoter-driven luciferase reporter assay, we demonstrated that two IFN-stimulated response elements within the promoter region of AsISG15 and the promoter-proximal intron were essential for AsISG15 expression. Furthermore, our results showed that the gamma-IFN activation sequence located in the intron was required for the intron mediated enhancement for AsISG15 expression. Our results would provide insights for understanding the underlying regulation mechanism of ISG15 in teleost.

Molecular characterization and expression analysis of interferon-gamma in black seabream Acanthopagrus schlegelii.

Interferon gamma (IFN-γ) is a major component in immunological signaling and plays a key role in resisting viral infection. In this study, we identified and characterized an IFN-γ gene (AsIFN-γ) in the marine fish black seabream (Acanthopagrus schlegelii). We cloned AsIFN-γ genomic sequence, which comprises four exons, three introns and an upstream promoter including several conserved regulatory elements. The complete cDNA of AsIFN-γ was 816 bp in length and encoded a putative 194 amino acids (aa) protein with a 22 aa signal peptide, six α-helices and one nuclear localization signal (NLS). Multiple alignment showed that AsIFN-γ protein shared 31-60% identity with IFN-γ of other fish but low identity with fish IFN-γrel and IFN-γ of other vertebrates. AsIFN-γ was constitutively expressed in all examined tissues with the highest expression level in immune organs, such as spleen, gill and kidney. In black seabream infected by red spotted nervous necrosis virus (RGNNV), the expression of AsIFN-γ was significantly up-regulated in most tissues, and RGNNV infection in vitro also induced significant up-regulation of AsIFN-γ, indicating that AsIFN-γ was involved in immune response to RGNNV infection. Overexpression of AsIFN-γ in cultured Acanthopagrus schlegelii brain (AsB) cells rapidly and transiently stimulated the expression of JAK-STAT signaling pathway related genes including STAT1, STAT2 and IRF9, as well as the downstream antiviral genes MX1 and ISG15. Furthermore, overexpression of AsIFN-γ was able to significantly inhibit RGNNV replication and virus production in AsB cells. In summary, we identified a conserved IFN-γ gene of black seabream, and demonstrated the rapid and strong antiviral activities of AsIFN-γ against RGNNV in black seabream.