Function analysis of fish PACT gene in response to virus infection.

PACT (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a cellular protein which can activate PKR in dsRNA-independent manner. However, the role of PACT in fish virus infection remains largely unknown. In this study, a PACT homologue from grouper (Epinephelus coioides)(EcPACT) was cloned and characterized. The open reading frame of EcPACT has a full length of 924 bp and encodes a protein of 307 amino acids with a predicted molecular weight of 33.29 kDa. Similar to mammals, EcPACT contains three dsRBD domains. EcPACT shares 99.67 % homology with E. lanceolatus. Real-time fluorescence quantitative PCR results showed that EcPACT mRNA was widely expressed in all tissues and abundantly expressed in brain, blood, head kidney and kidney. In addition, SGIV and RGNNV infection significantly upregulated the transcript levels of EcPACT. Subcellular localization analysis showed that EcPACT was mainly distributed in the nucleus. Overexpression of EcPACT inhibited the replication of SGIV and RGNNV in vitro and positively regulated the expression of interferon (IFN) and pro-inflammatory factors. The results provide a better understanding of the relationship between PACT and viral infection in fish.

Isolation and identification of a megalocytivirus strain (SKIV-TJ) from cultured spotted knifejaw (Oplegnathus punctatus) in China and its pathogenicity analysis.

The spotted knifejaw (Oplegnathus punctatus) has recently emerged as a highly economically significant farmed fish in China. However, due to increasing environmental pollution and breeding density, a range of infectious diseases, including the iridovirus pathogen, have begun to spread widely. In this study, we isolated and identified a strain of Megalocytivirus, SKIV-TJ, from cultured spotted knifejaw in Tianjin, China. We observed significant cytopathic effects (CPE) in SKIV-TJ-infected spotted knifejaw brain (SKB) cells, and electron microscopy showed numerous virus particles in the cytoplasm of SKB cells 6 days post-infection. The annotated complete genome of SKIV-TJ (GenBank accession number ON075463) contained 112,489 bp and 132 open reading frames. Based on the multigene association evolutionary tree using 26 iridovirus core genes, SKIV-TJ was found to be most closely related to Rock bream iridovirus (RBIV). Cumulative mortality of spotted knifejaw infected with SKIV-TJ reached 100% by day 9. A transcriptomic analysis were conducted and a total of 5517 differentially expressed genes were identified, including 2757 upregulated genes and 2760 downregulated genes. The upregulated genes were associated with viral infection and immune signaling pathways. Our findings provide a valuable genetic resource and a deeper understanding of the immune response to SKIV infection in spotted knifejaw.

Establishment and characterization of a brain tissue cell line from spotted knifejaw (Oplegnathus punctatus) and its susceptibility to several fish viruses.

Cells are important in the study of virus isolation and identification, viral pathogenic mechanisms and antiviral immunity. The spotted knifejaw (Oplegnathus punctatus) is a significant farmed fish in China that has been greatly affected by diseases in recent years. In this study, a new cell line derived from the spotted knifejaw brain (SKB) was established and characterized. SKB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28°C. Chromosome analysis revealed that modal chromosome number was 48 for SKB. SKB cells exhibit susceptibility to several fish viruses, such as a largemouth bass virus, red grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV), Singapore grouper iridovirus (SGIV) and spotted knifejaw iridovirus isolate (SKIV-TJ), as shown by cytopathic effect and increased viral titers. Electron microscopy results showed that the cytoplasm contained a large number of vacuoles, and many virus particles existed at the edge of the vacuoles in RGNNV-infected cells and numerous viral particles were scattered throughout the cytoplasm in both ISKNV- and SKIV-TJ-infected cells. These results suggest that SKB is an ideal tool for studying host-virus interactions and potential vaccine development.

Grouper cGAS is a negative regulator of STING-mediated interferon response.

Cyclic GMP-AMP synthase (cGAS) is one of the classical pattern recognition receptors that recognizes mainly intracytoplasmic DNA. cGAS induces type I IFN responses to the cGAS-STING signaling pathway. To investigate the roles of cGAS-STING signaling pathway in grouper, a cGAS homolog (named EccGAS) was cloned and identified from orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EccGAS is 1695 bp, encodes 575 amino acids, and contains a Mab-21 typical structural domain. EccGAS is homologous to Sebastes umbrosus and humans at 71.8% and 41.49%, respectively. EccGAS mRNA is abundant in the blood, skin, and gills. It is uniformly distributed in the cytoplasm and colocalized in the endoplasmic reticulum and mitochondria. Silencing of EccGAS inhibited the replication of Singapore grouper iridovirus (SGIV) in grouper spleen (GS) cells and enhanced the expression of interferon-related factors. Furthermore, EccGAS inhibited EcSTING-mediated interferon response and interacted with EcSTING, EcTAK1, EcTBK1, and EcIRF3. These results suggest that EccGAS may be a negative regulator of the cGAS-STING signaling pathway of fish.

Isolation, Characterization, and Transcriptome Analysis of an ISKNV-Like Virus from Largemouth Bass.

Largemouth bass (Micropterus salmoides) is an important commercial fish farmed in China. Challenges related to diseases caused by pathogens, such as iridovirus, have become increasingly serious. In 2017, we detected iridovirus-infected diseased largemouth bass in Zunyi, Guizhou Province. The isolated virus was identified as an infectious spleen and kidney necrosis virus (ISKNV)-like virus (ISKNV-ZY). ISKNV-ZY induces a cytopathic effect after infecting mandarin fish brain (MFB) cells. Abundant hexagonal virus particles were observed in the cytoplasm of ISKNV-ZY-infected MFB cells, using electron microscopy. The whole genome of ISKNV-ZY contained 112,248 bp and 122 open reading frames. Phylogenetic tree analysis showed that ISKNV-ZY was most closely related to BCIV, indicating that it is an ISKNV-like megalocytivirus. ISKNV-ZY-infected largemouth bass started to die on day six and reached a death peak on days 7-8. Cumulative mortality reached 100% on day 10. Using RNA sequencing-based transcriptome analysis after ISKNV-ZY infection, 6254 differentially expressed unigenes (DEGs) were identified, of which 3518 were upregulated and 2673 downregulated. The DEGs were associated with endocytosis, thermogenesis, oxidative phosphorylation, the JAK-STAT signaling pathway, the MAPK signaling pathway, etc. These results contribute to understanding the molecular regulation mechanism of ISKNV infection and provide a basis for ISKNV prevention.

Characterization and functional analysis of GSK3ß from Epinephelus coioides in Singapore grouper iridovirus infection.

Glycogen synthase kinase 3ß (GSK3ß), a serine/threonine protein kinase, is a crucial regulator of several signaling pathways and plays a vital role in cell proliferation, growth, apoptosis, and immune responses. However, the role of GSK3ß during viral infection in teleosts remains largely unknown. In the present study, a GSK3ß homologue from Epinephelus coioides (EcGSK3ß) was cloned and characterized. The open reading frame of EcGSK3ß consists of 1323 bp, encoding a 440 amino acid protein, with a predicted molecular mass of 48.23 kDa. Similar to its mammalian counterpart, EcGSK3ß contains an S_TKc domain. EcGSK3ß shares 99.77% homology with the giant grouper (Epinephelus lanceolatus). Quantitative real-time PCR analysis indicated that EcGSK3ß mRNA was broadly expressed in all tested tissues, with abundant expression in the skin, blood, and intestines. Additionally, the expression of EcGSK3ß increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that EcGSK3ß is mainly distributed in the cytoplasm. EcGSK3ß overexpression promoted SGIV replication during viral infection in vitro. In contrast, silencing of EcGSK3ß inhibited SGIV replication. EcGSK3ß significantly downregulated the activities of interferon-ß, interferon-sensitive response element, and NF-κB. Taken together, these findings are important for a better understanding of the function of GSK3ß in fish and reveal its involvement in the host response to viral immune challenge.

Critical Roles of G3BP1 in Red-Spotted Grouper Nervous Necrosis Virus-Induced Stress Granule Formation and Viral Replication in Orange-Spotted Grouper (Epinephelus coioides).

Viral infection causes changes in the internal environment of host cells, and a series of stress responses are generated to respond to these changes and help the cell survive. Stress granule (SG) formation is a type of cellular stress response that inhibits viral replication. However, the relationship between red-spotted grouper nervous necrosis virus (RGNNV) infection and SGs, and the roles of the SG marker protein RAS GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) in viral infection remain unclear. In this study, RGNNV infection induced grouper spleen (GS) cells to produce SGs. The SGs particles co-located with the classic SG marker protein eIF3η, and some SGs depolymerized under treatment with the translation inhibitor, cycloheximide (CHX). In addition, when the four kinases of the eukaryotic translation initiation factor 2α (eIF2α)-dependent pathway were inhibited, knockdown of HRI and GCN2 with small interfering RNAs and inhibition of PKR with 2-aminopurine had little effect on the formation of SGs, but the PERK inhibitor significantly inhibited the formation of SGs and decreased the phosphorylation of eIF2α. G3BP1 of Epinephelus coioides (named as EcG3BP1) encodes 495 amino acids with a predicted molecular weight of 54.12 kDa and 65.9% homology with humans. Overexpression of EcG3BP1 inhibited the replication of RGNNV in vitro by up-regulating the interferon and inflammatory response, whereas knockdown of EcG3BP1 promoted the replication of RGNNV. These results provide a better understanding of the relationship between SGs and viral infection in fish.

Isolation and Characterization of a Newly Discovered Phage, V-YDF132, for Lysing Vibrio harveyi

A newly discovered lytic bacteriophage, V-YDF132, which efficiently infects the pathogenic strain of Vibrio harveyi, was isolated from aquaculture water collected in Yangjiang, China. Electron microscopy studies revealed that V-YDF132 belonged to the Siphoviridae family, with an icosahedral head and a long noncontractile tail. The phage has a latent period of 25 min and a burst size of 298 pfu/infected bacterium. V-YDF132 was stable from 37 to 50 °C. It has a wide range of stability (pH 5-11) and can resist adverse external environments. In addition, in vitro the phage V-YDF132 has a strong lytic effect on the host. Genome sequencing results revealed that V-YDF132 has a DNA genome of 84,375 bp with a GC content of 46.97%. In total, 115 putative open reading frames (ORFs) were predicted in the phage V-YDF132 genome. Meanwhile, the phage genome does not contain any known bacterial virulence genes or antimicrobial resistance genes. Comparison of the genomic features of the phage V-YDF132 and phylogenetic analysis revealed that V-YDF132 is a newly discovered Vibrio phage. Multiple genome comparisons and comparative genomics showed that V-YDF132 is in the same genus as Vibrio phages vB_VpS_PG28 (MT735630.2) and VH2_2019 (MN794238.1). Overall, the results indicate that V-YDF132 is potentially applicable for biological control of vibriosis.

The roles of grouper TAK1 in regulating the infection of Singapore grouper iridovirus.

Transforming growth factor-ß activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase family. It is an upstream factor of the IκB kinase, which activates IKKα and IKKß. TAK1 is a key factor in the induction of nuclear factor κB (NF-κB) and plays a crucial role in the activation of inflammatory responses. However, the roles of TAK1 during viral infection in teleost fish are largely unknown. In this study, we cloned a TAK1 homolog (HgTAK1) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀). The open reading frame of HgTAK1 consists of 1728 nucleotides encoding 575 amino acids, and the predicted molecular weight is 64.32 kDa HgTAK1 has an S_TKc domain, which consists of a serine/threonine protein kinase and a catalytic domain. Expression pattern analysis showed that HgTAK1 was distributed in all tested tissues, with abundant contents in the heart, head kidney, and blood. Additionally, HgTAK1 was distributed in the cytoplasm of grouper spleen (GS) cells. After Singapore grouper iridovirus (SGIV) infection, the expression of HgTAK1 increased in GS cells. Overexpression of HgTAK1 could promote the replication of SGIV in GS cells and inhibit the activation of NF-κB and IFN stimulated response elements (ISRE) in reporter assay. When co-expressed with IRF3 or HgIRF7 in GS cells, HgTAK1 obviously down-regulated IRF3- or IRF7-mediated the NF-κB and ISRE promoter induction. The interaction between HgTAK1 and IRF3 or IRF7 has been identified by co-immunoprecipitation assay. These findings provide a basis for understanding the innate immune mechanism of the grouper response to viral infection.

Grouper TRAF3 inhibits nodavirus infection by regulating the STING-mediated antiviral signaling pathway.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are major signal transducers for the TNF and interleukin-1/Toll-like receptor superfamilies that transduce signals from various immune receptors. To investigate the interaction of TRAF3 and other proteins in signaling pathways and to identify its antiviral function in teleosts, we cloned and characterized a TRAF3 homolog from orange-spotted grouper (Epinephelus coioides) (EcTRAF3). The open reading frame of EcTRAF3 consists of 1767 base pairs encoding a 588 amino acid protein, and the predicted molecular mass is 66.71 kDa EcTRAF3 shares 99.83% identity with TRAF3 of Epinephelus lanceolatus. Expression analysis revealed that EcTRAF3 was broadly distributed in examined tissues and was up-regulated under polyinosinic-polycytidylic acid and red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vivo. EcTRAF3 was identified as a cytosolic protein based on fluorescence microscopy analysis. Overexpression of EcTRAF3 inhibited RGNNV replication in grouper spleen cells, and it interacted with the coat protein of RGNNV. Overexpression of EcTRAF3 also induced the activation of interferon ß (IFN-ß), IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). EcTRAF3 co-transfected with Stimulator of Interferon Genes (STING) of grouper (EcSTING) induced a significantly higher level of IFN-ß promoter activity. Moreover, EcTRAF3 interacted with EcSTING, implying that EcTRAF3 may function as an enhancer in EcSTING-mediated signaling. Taken together, our results suggest that EcTRAF3 negatively regulates the RGNNV-induced cellular antiviral response and plays an important role in the immune response system of fish.

Grouper TIA-1 functions as a crucial antiviral molecule against nervous necrosis virus infection.

T-cell intracellular antigen (TIA)-1 is a prion-related RNA-binding protein involved in splicing and translational repression, and regulates translation in response to stress conditions by isolating target mRNAs in stress granules (SGs). However, little is known about the potential roles of fish TIA-1 and how it works in viral infection. In this study, the TIA-1 (EcTIA-1) homolog from orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) sequence of EcTIA-1 encoded a 388 amino acid protein with predicted molecular mass of 42.73 kDa. EcTIA-1 contains three conserved domains of RNA recognition motif (RRM) that may interact with RNA via its second and third RRMs. Overexpression of EcTIA-1 inhibited red-spotted grouper nervous necrosis virus (RGNNV) replication and positively regulated interferon immune response, which was increased by knockdown of EcTIA-1. RGNNV induced formation of SGs in cells with EcTIA-1 overexpression. These results provide a novel insight into understanding the roles of fish TIA-1 in response to RNA viruses.

Grouper TRAF5 exerts negative regulation on antiviral immune response against iridovirus.

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an intracellular protein that binds to the cytoplasmic portion of tumor necrosis factor receptors and mediates the activation of downstream nuclear factor-kappa B (NF-κB), interferon regulatory factor 3, and mitogen activated protein kinase signaling pathways. Compared with other TRAF proteins, TRAF5 is largely unknown in teleosts. In the present study, a TRAF5 homologue (HgTRAF5) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) was cloned and characterized. The open reading frame of HgTRAF5 consists of 1743 nucleotides encoding a 581 amino acid protein with a predicted molecular mass of 64.90 kDa. Similar to its mammalian counterpart, HgTRAF5 contains an N-terminal RING finger domain, a zinc finger domain, and a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. HgTRAF5 shares 99.83% identity with giant grouper (Epinephelus lanceolatus) TRAF5. Quantitative real-time PCR analysis indicated that HgTRAF5 mRNA was broadly expressed in all examined tissues. The expression of HgTRAF5 increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that the full-length HgTRAF5 protein mainly distributed in the cytoplasm. HgTRAF5 overexpression also promoted SGIV replication during viral infection in vitro. HgTRAF5 significantly promoted the activities of interferon-ß, interferon-sensitive response element, and NF-κB. Taken together, these results are important for a better understanding of the function of TRAF5 in fish and reveal its involvement in the host response to immune challenge by SGIV.