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The efficient use of livestock and poultry manure waste has become a global challenge, with microorganisms playing an important role. To investigate the impact of novel ammonifying microorganism cultures (NAMC) on microbial community dynamics and carbon and nitrogen metabolism, five treatments [5% (v/w) sterilized distilled water, Amm-1, Amm-2, Amm-3, and Amm-4] were applied to cow manure compost. Inoculation with NAMC improved the structure of bacterial and fungal communities, enriched the populations of the functional microorganisms, enhanced the role of specific microorganisms, and promoted the formation of tight modularity within the microbial network. Further functional predictions indicated a significant increase in both carbon metabolism (CMB) and nitrogen metabolism (NMB). During the thermophilic phase, inoculated NAMC treatments boosted carbon metabolism annotation by 10.55%–33.87% and nitrogen metabolism annotation by 26.69%–63.11. Structural equation modeling supported the NAMC-mediated enhancement of NMB and CMB. In conclusion, NAMC inoculation, particularly with Amm-4, enhanced the synergistic interaction between bacteria and fungi. This collaboration promoted enzymatic catabolic and synthetic processes, resultng in positive feedback loops with the endogenous microbial community. Understanding these mechanisms not only unravels how ammonifying microorganisms influence microbial communities but also paves the way for the development of the composting industry and global waste management practices. [Display omitted] • Novel ammonifying microorganism cultures (NAMC) increased the stability of the composting • Inoculation with NAMC improved the structure of bacterial and fungal communities. • Inoculation with NAMC enhanced the complementary effects of bacteria and fungi in compost • Inoculated NAMC promoted carbon metabolism and nitrogen metabolism in composting. [ABSTRACT FROM AUTHOR]

Abstract The lysosomal cysteine protease cathepsin C plays a pivotal role in regulation of inflammatory and immune responses. However, the function of fish cathepsin C in virus replication remains largely unknown. In this study, cathepsin C gene (Ec-CC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CC cDNA was composed of 2077 bp. It contained an open reading frame (ORF) of 1374 bp and encoded a 458-amino acid protein which shared 89% identity to cathepsin C from bicolor damselfish (Stegastes partitus). Amino acid alignment analysis showed that Ec-CC contained an N-terminal signal peptide, the propeptide region and the mature peptide. RT-PCR analysis showed that Ec-CC transcript was expressed in all the examined tissues which abundant in spleen and head kidney. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the relative expression of EC-CC was significantly increased at 24 h post-infection. Subcellular localization analysis revealed that Ec-CC was distributed mainly in the cytoplasm. Further studies showed that overexpression of Ec-CC in vitro significantly delayed the cytopathic effect (CPE) progression evoked by SGIV and inhibited the viral genes transcription. Moreover, overexpression of Ec-CC significantly increased the expression of proinflammatory cytokines during SGIV infection. Taken together, our results demonstrated that Ec-CC might play a functional role in SGIV infection by regulating the inflammation response. Highlights • Cathepsin C gene from Epinephelus coioides (Ec-CC) was identified and characterized. • The expression of Ec-CC was up-regulated in the spleen of grouper challenged with SGIV. • Overexpression of Ec-CC significantly inhibited SGIV replication. • Overexpression of Ec-CC regulated inflammation response during SGIV infection. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly( l -lysine)-b-poly( l -leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis. [ABSTRACT FROM AUTHOR]

Transcription factor ATF1 is a member of the ATF/CREB family of the CREB subfamily and is involved in physiological processes such as tumorigenesis, organ development, reproduction, cell survival, and apoptosis in mammals. However, studies on ATF1 in fish have been relatively poorly reported, especially on its role in antiviral immunity in fish. In this study, ATF1 from orange-spotted grouper (named EcATF1) were cloned and characterized. Molecular characterization analysis showed that EcATF1 encodes a 307-amino-acid protein, containing PKID and bZIP_CREB1 domains. Homology analysis showed that had the highest homology with E. lanceolatus (88.93%). Tissue expression pattern showed that EcATF1 was extensively distributed in twelve selected tissues, with higher expression in the skin, gill, liver and spleen. Subcellular localization analysis showed that EcATF1 was distributed in the nucleus of GS cells. EcATF1 overexpression inhibits Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by a diminished degree of CPE induced by SGIV and RGNNV and a reduction in the level of viral gene transcription and viral capsid protein expression. Furthermore, EcATF1 overexpression upregulated interferon pathway-related genes and proinflammatory factors, and increased the promoter activities of IFN, IFN stimulated response element (ISRE), and nuclear factor κB(NFκB). Meanwhile, EcATF1 overexpression positive regulate the MHC-I signaling pathway, and upregulated the promoter activity of MHC-I. Collectively, these data demonstrate that EcATF1 plays an important role during the host antiviral immune response. This study provides insights into the function of ATF1 in the immune system of lower vertebrates. • We cloned and characterized ATF1 (EcATF1) from grouper. • EcATF1 inhibit SGIV and RGNNV replication in GS cells. • EcATF1 upregulated IFN pathway and proinflammatory factors. • EcATF1 positive regulate the MHCI pathway. [ABSTRACT FROM AUTHOR]

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. • MARCO gene from Epinephelus coioides (Ec-MARCO) was identified and characterized. • The transcription of Ec-MARCO was up-regulated in the GS cells stimulated with SGIV. • Ec-MARCO knockdown positively regulated inflammatory responses and interferon-stimulated genes. • Ec-MARCO knockdown promoted the activation of reporter genes. [ABSTRACT FROM AUTHOR]

Regulatory factor X 5 (RFX 5) is a member of the RFX family, and it forms the transcription factor complex RFX with RFXANK/B and RFXAP. The RFX complex can activate MHC expression by binding to the MHC promoter. However, the regulate mechanism of RFX in fish species is not been fully elucidated. In this study, we investigated the transcriptional regulation of Epinephelus akaara RFX5 (EaRFX5) on EaMHCI, and its effect on immune pathways. The genomic sequence of EaRFX5 was 35,774 bp and consisted of ten exons and nine introns. The length of EaRFX5 ORF sequence is 2,160 bp, encoding 719 amino acids. By qRT-PCR, EaRFX5 was detected constitutively expressed in twelve selected tissues, showing a wide range of expression. EaRFX5 expression parttern in response to poly (I:C), LPS, Zymosan A, SGIV, and NNV challenges showed that EaRFX5 plays a differentiated immunomodulatory role in response to various stimuli in different tissues, and EaRFX 5 was most significantly upregulated in the kidney after challenge with SGIV. Subcellular localization assays showed that EaRFX5 is a typical nuclear protein. Based on the in vitro overexpression experiments, EaRFX5 appeared to promote the expression of EaMHCIa gene, interferon signalling pathway and inflammatory cytokine. Luciferase reporter assay showed that the −267 bp to +82 bp region of EaMHCIa promoter was the core region where EaRFX5 modulated. Additionally, point mutations and electrophoretic mobility shift assays indicating M3 is the EaRFX5 binding sites in the EaMHCIa promoter. These results contribute to elucidating the function of EaRFX5 in fish immune response, and provide the first evidence of positive regulation of MHCIa expression by RFX5 in fish. • The molecular characteristics of EaRFX5 were analysed. • EaRFX5 positively promotes the transcription expression of many cytokines. • RFX5 activates MHC Iα expression by binding M3 site on its promoter. [ABSTRACT FROM AUTHOR]

Rab32 is a member of the Rab GTPase family that is involved in membrane trafficking and immune response, which are crucial for controlling pathogen infection. However, the role of Rab32 in virus infection is not well understood. In this study, we focused on the regulation of Rab32 on virus infection and the host immunity in orange-spotted grouper, Epinephelus coioides. EcRab32 encoded a 213-amino acid polypeptide, which shared a high sequence identity with other Rab32 proteins from fishes to mammals. In healthy orange-spotted grouper, the mRNA of EcRab32 was expressed in all the detected tissues, with the more expression levels in the head kidney, liver and gill. Upon SGIV infection, the expression of EcRab32 was significantly up-regulated in vitro , indicating its potential role in viral infection. EcRab32 was observed to be distributed in the cytoplasm as punctate and vesicle-like structures. EcRab32 overexpression was found to notably inhibit SGIV infection, while the interruption of EcRab32 significantly promoted SGIV infection. In addition, using single particle imaging analysis, we found that EcRab32 overexpression prominently reduced the attachment and internalization of SGIV particles. Furthermore, the results demonstrated that EcRab32 played a positive role in regulating the interferon immune and inflammatory responses. Taken together, these findings indicated that EcRab32 influenced SGIV infection by regulating the host immune response, providing an overall understanding of the interplay between the Rab32 and innate immunity. • Rab32 gene from Epinephelus coioides (EcRab32) was identified and characterized. • EcRab32 exerted an antiviral role during SGIV infection. • EcRab32 involved in the attachment and internalization of SGIV. • EcRab32 positively regulated the interferon immune and inflammatory responses. [ABSTRACT FROM AUTHOR]

Cyprinid herpesvirus type 3 (CyHV-3), also called Koi herpesvirus (KHV), which leads to mass cyprinid mortality and enormous economic losses. To establish an infection, CyHV-3 needs to counteract host antiviral responses. CD81 belongs to the evolutionary conserved tetraspanin family of proteins. Several studies have shown that different members of the tetraspanin superfamily modulates different virus infectious processes. Here we aimed at analysing the role of CD81 in CyHV-3 infection. In this study, we cloned and characterized the CD81 of Common Carp, the open reading frame of Cc CD81 gene was 702 bp, which encoded 234 amino acids with four transmembrane domains (TM1 to TM4), a small extracellular loop (SEL), and a large extracellular loop (LEL). Tissue distribution analysis showed that Cc CD81 was widely expressed in all the tested tissues with the highest expression in head kidney, followed by a high expression in brain. Subsequently, expression levels of Cc CD81 were significantly increased in CCB cells within the first 3h after infection, meanwhile, the expression of viral gene VP136 was reduced after Cc CD81 knockdown in CCB cells post CyHV-3 infection. Furthermore, Cc CD81 knockdown can significantly reduce the autophagy process and increase the promoter activity of ISRE and IFN-1 in the CCB cells after viral infection, as well as other genes involved in the IFN signaling pathway, including RIG-1、MDA5、MAVS、TBK1 and IRF3. Taking the data together, we revealed that Cc CD81 mediates autophagy and blocks RIG-1-mediated antiviral signaling and negatively regulates the promoter activity of type I interferon (IFN) promoting virus replication. These results reveal a new link between autophagy and four-transmembrane-domain protein superfamily and contribute to elucidate the mechanism of CyHV-3 infection. • The tetraspanin protein CD81 of Common Carp was cloned and characterized. • Cc CD81 promoting virus replication. • Cc CD81 could mediates autophagy process significantly. • Cc CD81 blocks RIG-1-mediated antiviral signaling and negatively regulates the promoter activity of type I interferon (IFN). [ABSTRACT FROM AUTHOR]

Rab1, a GTPase, is present in all eukaryotes, and is mainly involved in vesicle trafficking between the endoplasmic reticulum and Golgi, thereby regulating many cellular activities and pathogenic infections. However, little is known of how Rab1 functions in fish during virus infection. Groupers (Epinephelus spp.) are high in economic value and widely cultivated in China and Southeast Asia, although they often suffer from diseases. Red-spotted grouper nervous necrosis virus (RGNNV), a highly pathogenic RNA virus, is a major pathogen in cultured groupers, and causes huge economic losses. A series of host cellular proteins involved in RGNNV infection was identified. However, the impact of Rab1 on RGNNV infection has not yet been reported. In this study, a novel Rab1 homolog (EcRab1) from Epinephelus coioides was cloned, and its roles during virus infection and host immune responses were investigated. EcRab1 encoded a 202 amino acid polypeptide, showing 98% and 78% identity to Epinephelus lanceolatus and Homo sapiens , respectively. After challenge with RGNNV or poly(I:C), the transcription of EcRab1 was altered both in vitro and in vivo , implying that EcRab1 was involved in virus infection. Subcellular localization showed that EcRab1 was displayed as punctate structures in the cytoplasm, which was affected by EcRab1 mutants. The dominant negative (DN) EcRab1, enabling EcRab1 to remain in the GDP-binding state, caused EcRab1 to be diffusely distributed in the cytoplasm. Constitutively active (CA) EcRab1, enabling EcRab1 to remain in the GTP-binding state, induced larger cluster structures of EcRab1. During the late stage of RGNNV infection, some EcRab1 co-localized with RGNNV, and the size of EcRab1 clusters was enlarged. Importantly, overexpression of EcRab1 significantly inhibited RGNNV infection, and knockdown of EcRab1 promoted RGNNV infection. Furthermore, EcRab1 inhibited the entry of RGNNV to host cells. Compared with EcRab1, overexpression of DN EcRab1 or CA EcRab1 also promoted RGNNV infection, suggesting that EcRab1 regulated RGNNV infection, depending on the cycles of GTP- and GDP-binding states. In addition, EcRab1 positively regulated interferon (IFN) immune and inflammatory responses. Taken together, these results suggest that EcRab1 affects RGNNV infection, possibly by regulating host immunity. Our study furthers the understanding of Rab1 function during virus infection, thus helping to design new antiviral strategies. • EcRab1 was obviously downregulated after RGNNV infection. • EcRab1 significantly inhibited RGNNV infection. • EcRab1 inhibits RGNNV entry, which was the first report of the effect of Rab1 on virus entry. • EcRab1 has a positive regulatory effect on host immunity. [ABSTRACT FROM AUTHOR]

Peroxisome proliferative–activated receptor α (PPARα) belongs to the superfamily of nuclear receptors (NR). Studies have demonstrated that PPARα functions in energy metabolism, hepatic function, immune response, cell cycle, and apoptosis. In teleost fish, few studies have investigated the role of PPARα in the immune response. In this study, the grouper PPARα gene (EcPPARα) was investigated for its role in viral infection. The open reading frame of EcPPARα encoded a protein of 469 amino acids and contained an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Phylogenetic analysis revealed that EcPPARα was most closely related to homologous genes in Sander lucioperca and Perca flavescens. Upon challenge with SGIV (Singapore grouper iridovirus) and RGNNV (Red-spotted grouper nervous necrosis virus), EcPPARα expression levels were significantly upregulated in different tissues. Subcellular localization analysis showed that the EcPPARα protein localized throughout the cytoplasm and nucleus with diffuse intracellular expression patterns, which is consistent with the localization pattern of mammalian PPARs. Based on morphological observation of cytopathic effect (CPEs), viral gene expression mRNAs, and virus titer assays, the results presented here showed that an overexpression of EcPPARα promoted SGIV production in grouper spleen cells. Overexpression of EcPPARα significantly inhibited the expression of several cytokines, including interferon-related genes (IFN-γ , ISG15 , MXI , MXII , MAVS and MDA5), inflammatory cytokines (IL-1β , IL-6 , IL-8 , TNF-α) and Toll like receptor adaptors (TRAF6 and MyD88). Luciferase activity of IFN-α , IFN-γ , ISRE and NF-κB promoters was also significantly decreased in EcPPARα overexpression cells. Due to these detected interferon-related genes and inflammatory cytokines play important antiviral effect against SGIV in grouper, we speculated that the promotion effect of EcPPARα on SGIV replication may be caused by down-regulation of interferon and inflammatory response. In addition, through apoptotic body observation, capspase-3 activity detection, and flow cytometry analysis, it was found that overexpression of EcPPARα promoted SGIV-induced apoptosis in fathead minnow (FHM) cells. These data may increase an understanding of the role of PPARα in fish antiviral immune responses and apoptosis. • We characterized EcPPARα and illuminated its roles in fish against virus infection. • EcPPARα seems to promote SGIV infection in vitro. • Overexpression of EcPPARα promoted SGIV-induced apoptosis in FHM cells. [ABSTRACT FROM AUTHOR]

The transcription factor nuclear factor kappa B (NF-κB) is a critical regulator of immune and inflammatory responses with crucial roles in various pathophysiologic conditions involving cell survival and death. Recent studies in mammals showed that NF-κB was also involved in peroxisome proliferator-activated receptors (PPARs)-mediated immune responses However, the mechanism by which NF-κB regulates PPARδ in teleosts remains unclear. In the present study, we analyzed the potential role of NF-κB/p65 (Ecp65) in the immune response stimulated by various pathogens in the grouper Epinephelus coioides. Ecp65 expression was significantly induced soon after infection with lipopolysaccharide, nervous necrosis virus, poly(I:C), and zymosan A. We also analyzed the promoter to determine the regulatory effect of Ecp65 on PPARδ expression, using progressive EcPPARδ promoter deletion mutations. Among the five truncated mutants, the luciferase reporter activity of the PPARδ-5 promoter region was highest in response to Ecp65, indicating that the core p65-binding region was located in the PPARδ-5 promoter region (+122 bp to +383 bp). Mutation analyses indicated that the luciferase reporter activity of the EcPPARδ promoter was dramatically decreased by mutation of the M3 (+305 bp to +324 bp) and M4 (+346 bp to +365 bp) binding sites, respectively. We further confirmed that Ecp65 bound to the M3 and M4 binding sites in the 5′-untranslated region of EcPPARδ by electrophoretic mobility shift assay. Finally, overexpression of Ecp65 in vitro notably promoted the transcription of EcPPARδ , interferon-related genes, and several inflammatory cytokines. This study demonstrated that Ecp65 plays an important role in modulating the innate immune responses in groupers. These results also further our understanding of the mechanisms involved in the transcriptional regulation of PPARs by p65 in bony fish. • We analyzed role of Ecp65 in the immune response against various pathogens. • Ecp65 could bind to the promoter of EcPPAR-δ and regulate its transcription. • Ecp65 could promote the transcription level of many cytokines in grouper cells. [ABSTRACT FROM AUTHOR]

The Kruppel-like factor 6 (KLF6) is a member of Kruppel-like factor family, which belong to the Zinc finger family of transcription factors that mediates various cellular processes, such as proliferation, differentiation, development, and programmed cell death. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily and they regulate numerous genes through ligand-dependent transcriptional activation and repression. In this study, we focus on the role of KLF6 gene in virus infection and the regulation of KLF6 on PPAR-δ in orange-spotted grouper (Epinephelus coioides). The ORF sequence of Ec KLF6 was 846 bp, encoding a polypeptide of 282 amino acids with three conserved Zinc finger (type Cys 2 -His 2) domain in the C-terminal region. Basing on the detection of the mRNA levels of viral genes, western blotting of MCP protein, and morphological CPEs, we found that the overexpression of Ec KLF6 suppressed the replication of Singapore grouper iridovirus (SGIV), exerting its antiviral activity against fish virus. Moreover, promoter analysis was performed to investigate whether Ec KLF6 was a regulator of Ec PPAR-δ. The luciferase reporter assay and real time PCR results indicated a negative regulatory role of Ec KLF6 on Ec PPAR-δ transcription in grouper. Further experimental analysis shows that the potential Ec KLF6 binding sites may locate in the Ec PPAR-δ-4-M3 (+133 to +154) and Ec PPAR-δ-4-M4 (+354 to +368) region of the Ec PPAR-δ promoter. Electrophoretic mobile shift assays (EMSAs) verified that Ec KLF6 interacted with the binding site of the Ec PPAR-δ-4-M4 promoter region. In addition, we also found that KLF6 promotes inflammatory responses in GS cells. Considering that KLF6 and PPAR-δ play opposite roles in regulating inflammatory responses, we speculated the promoting effect of KLF6 on inflammatory response may be related to its negative regulation on Ec PPAR-δ. In conclusion, the present study provides the first evidence of the negative regulation of Ec PPAR-δ transcription by Ec KLF6 and contributes to a better understanding of the transcriptional mechanisms of Ec KLF6 in fish. • We characterized EcKLF6 and illuminated its roles in fish against virus infection. • EcKLF6 seems to inhibit SGIV infection in vitro. • EcKLF6 could bind to the promoter of EcPPAR-δ and regulate its transcription. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor δ (PPAR-δ), also called PPAR-β or PPAR-β/δ, is a member of the peroxisome proliferator-activated receptor (PPAR) family, which belongs to the nuclear steroid receptor superfamily. Activated PPARs participate in the regulation of lipid and glucose metabolism and also affect cellular proliferation, differentiation, and apoptosis, and the immune responses. To investigate the roles of PPAR-δ in Singapore grouper iridovirus (SGIV) infection, we cloned and characterized the gene encoding a PPAR-δ homologue from the orange-spotted grouper, Epinephelus coioides (EcPPAR-δ). EcPPAR-δ encodes a 514-amino-acid polypeptide, with 95.29% and 74.76% homologue to the Seriola dumerili and human proteins, respectively. EcPPAR-δ contains a typical DNA-binding domain and a ligand-binding domain. Its expression was induced by SGIV infection in vitro. A subcellular localization analysis showed that EcPPAR-δ localizes throughout the cytoplasm and nucleus, with a diffuse intracellular expression pattern. SGIV replication was reduced by EcPPAR-δ overexpression, which was evident in the reduced severity of the cytopathic effect, reduced viral gene transcription, and the reduced expression of the viral capsid protein. The replication of SGIV increased with the knockdown of EcPPAR-δ. The overexpression and silencing of EcPPAR-δ in grouper spleen cells showed that EcPPAR-δ plays a positive role in the regulation of the interferon signaling pathway, but has an anti-inflammatory effect on the inflammatory response. The anti-inflammatory effect of EcPPAR-δ may be related to its function in maintaining cell homeostasis. Because the interferon signaling pathway plays an important role in antiviral immune responses, we speculate that the activation of the interferon signaling pathway by EcPPAR-δ overexpression underlies its inhibitory effect on SGIV replication. Together, our data greatly extend our understanding of the roles of the EcPPAR-δ family members in the pathogenesis of fish viruses. • We characterized EcPPAR-δ and illuminated its roles in fish against virus infection. • EcPPAR-δ seems to inhibit SGIV infection in vitro. • EcPPAR-δ plays different role in grouper interferon related genes and inflammatory factors. [ABSTRACT FROM AUTHOR]

Krϋppel-like factor 9 (KLF9) is a member of the SP/KL family, which are transcription factors implicated in several biological processes, including cell proliferation, differentiation, development and apoptosis. Studies have focused on the function of KLF9 in mammalian disease and the immune system, such as its regulatory role in the growth of tumors and its impact on interferon-related genes and inflammatory cytokines. In fish, little is known about the role of KLF9, especially its regulatory function in the innate antiviral immune response. In this study, we characterized the grouper KLF9 gene (EcKLF9) and investigated its role in viral infection. Amino acid alignment analysis showed that EcKLF9 was approximately 228 amino acids long and contained a typical three-tandem Krϋppel-like zinc fingers. Phylogenetic tree analysis revealed that EcKLF9 clustered with three fish species: Amphiprion ocellaris, Acanthochromis pollyacanthus and Stegastes partitus. Comparison analyses showed that the three Kruppel-like zinc finger domains of KLF9 were highly conserved in different fish species. Tissue expression analysis showed that EcKLF9 was constitutively expressed in all 12 tissues tested, in the healthy grouper, the highest expression being detected in the gonads. The relative expression levels of EcKLF9 in the head kidney, spleen and brain was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infections. Using fluorescence microscopy, EcKLF9 was primarily localized to the nucleus and cytoplasm. The in vitro ectopic expression of EcKLF9 significantly increased the severity of vacuoles induced by RGNNV and the cytopathic effect progression evoked by SGIV infection. Real-time PCR results showed that the transcription levels of viral genes, such as the Singapore grouper iridovirus infection genes, MCP (major capsid protein), LITAF (lipopolysaccharide induced TNF-α factor), VP19 (envelop protein) ICP-18 (infected cell protein-18) and the red-spotted grouper nervous necrosis virus genes, CP (coat protein), RdRp (RNA-dependent RNA polymerase), were all significantly increased in EcKLF9 overexpressing cells, when compared to control cells. Furthermore, western blotting analyses showed that protein levels of the RGNNV gene, CP and the SGIV gene, MCP were also increased in EcKLF9 overexpressing cells, suggesting EcKLF9 may promote viral activity against iridovirus and nodavirus, in vitro. Moreover, the overexpression of EcKLF9 significantly inhibited the expression of several interferon related cytokines and several inflammatory cytokines. Accordingly, we speculate that EcKLF9 may exert stimulatory effects on RGNNV and SGIV replication, through the negative regulation of host immune and inflammation responses. • This study characterized the grouper KLF9 gene (EcKLF9) and illuminated its roles in fish against virus infection. • EcKLF9 promotes iridovirus and nodavirus infection in vitro. • EcKLF9 significantly inhibited the expression of several interferon related cytokine and inflammatory cytokine factors. [ABSTRACT FROM AUTHOR]

Abstract Chemokine receptors are a superfamily of seven-transmembrane domain G-coupled receptors and have important roles in immune surveillance, inflammation, and development. In previous studies, a series of CXCRs in grouper (Epinephelus coioides) was identified; however, the function of CXCR in viral infection has not been studied. To better understand the effect of the CXCR family on the fish immune response, full-length CXCR1a was cloned, and its immune response to Singapore grouper iridovirus (SGIV) was investigated. Grouper CXCR1a shared a seven-transmembrane (7-TM) region and a G protein-coupled receptor (GPCR) family 1 that contained a triaa stretch (DRY motif). Phylogenetic analysis indicated that CXCR1a showed the nearest relationship to Takifugu rubripes , followed by other fish, bird and mammal species. Fluorescence microscopy revealed that CXCR1a was expressed predominantly in the cytoplasm. Overexpression of CXCR1a in grouper cells significantly inhibited the replication of SGIV, demonstrating that CXCR1a delayed the occurrence of cytopathic effects (CPE) induced by SGIV infection and inhibited viral gene transcription. Furthermore, our results also showed that CXCR1a overexpression significantly increased the expression of interferon-related cytokines and activated ISRE and IFN promoter activities. Taken together, the results demonstrated that CXCR1a might have an antiviral function against SGIV infection. Highlights • CXCR1a was significantly increase induced by SGIV infected fish in head kidney, kidney, skin and spleen tissues. • CXCR1a localized in cytoplasm. • CXCR1a overexpression significantly inhibited the replication of SGIV. • CXCR1a overexpression enhanced the expression of interferon-related cytokines and activated ISRE and IFN promoter. [ABSTRACT FROM AUTHOR]

Annexin A2 (AnxA2) is ubiquitous in vertebrates and has been identified as a multifunctional protein participating in a series of biological processes, such as endocytosis, exocytosis, signal transduction, transcription regulation, and immune responses. However, the function of AnxA2 in fish during virus infection still remains unknown. In this study, we identified and characterized AnxA2 (EcAnxA2) in Epinephelus coioides. EcAnxA2 encoded a 338 amino acids protein with four identical annexin superfamily conserved domains, which shared high identity with other AnxA2 of different species. EcAnxA2 was widely expressed in different tissues of healthy groupers, and its expression was significantly increased in grouper spleen cells infected with red-spotted grouper nervous necrosis virus (RGNNV). Subcellular locatio n analyses showed that EcAnxA2 diffusely distributed in the cytoplasm. After RGNNV infection, the spatial distribution of EcAnxA2 was unaltered, and a few EcAnxA2 co-localized with RGNNV during the late stage of infection. Furthermore, overexpression of EcAnxA2 significantly increased RGNNV infection, and knockdown of EcAnxA2 reduced RGNNV infection. In addition, overexpressed EcAnxA2 reduced the transcription of interferon (IFN)-related and inflammatory factors, including IFN regulatory factor 7 (IRF7), IFN stimulating gene 15 (ISG15), melanoma differentiation related gene 5 (MDA5), MAX interactor 1 (Mxi1) laboratory of genetics and physiology 2 (LGP2), IFN induced 35 kDa protein (IFP35), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 6 (IL-6). The transcription of these genes was up-regulated when EcAnxA2 was inhibited by siRNA. Taken together, our results showed that EcAnxA2 affected RGNNV infection by down-regulating the host immune response in groupers, which provided new insights into the roles of AnxA2 in fish during virus infection. • EcAnxA2 was remarkably up-regulated after RGNNV infection. • EcAnxA2 mainly distributed in the cytoplasm. • EcAnxA2 significantly affected RGNNV infection. • EcAnxA2 negatively regulated interferon-related and inflammatory factors. [ABSTRACT FROM AUTHOR]

Increased reports uncovered that mammalian tripartite motif-containing 62 (TRIM62) exerts crucial roles in cancer and innate immune response. However, the roles of fish TRIM62 in antiviral immune response remained uncertain. In this study, a TRIM62 gene was cloned from orange spotted grouper (EcTRIM62) and its roles in grouper RNA virus infection was elucidated in vitro. EcTRIM62 shared 99% and 83% identity to bicolor damselfish ( Stegastes partitus ) and human ( Homo sapiens ), respectively. Sequence alignment indicated that EcTRIM62 contained three domains, including a RING-finger domain, a B-box domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM62 was predominantly detected in brain and liver, followed by heart, skin, spleen, fin, gill, intestine, and stomach. Subcellular localization analysis indicated that bright fluorescence spots were observed in the cytoplasm of EcTRIM62-transfected grouper spleen (GS) cells. During red-spotted grouper nervous necrosis (RGNNV) infection, overexpression of EcTRIM62 significantly enhanced the severity of CPE and increased viral gene transcriptions. Furthermore, the ectopic expression of EcTRIM62 significantly decreased the transcription level of interferon signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), melanoma differentiation-associated protein 5 (MDA5), myxovirus resistance gene MXI, and MXII, suggesting that the negative regulation of interferon immune response by EcTRIM62 might directly contributed to its enhancing effect on RGNNV replication. Furthermore, our results also demonstrated that overexpression of EcTRIM62 was able to differently regulate the expression levels of pro-inflammation cytokines. In addition, we found the ectopic expression of EcTIRM62 negatively regulated MDA5-, but not mediator of IRF3 activation (MITA)-induced interferon immune response. Further studies showed that the deletion of RING domain and SPRY domain significantly affected the action of EcTRIM62, including the enhancing effect on virus replication and regulation of interferon immune response. Thus, our studies firstly demonstrated that EcTRIM62 negatively regulated the innate antiviral immune response against fish RNA viruses. [ABSTRACT FROM AUTHOR]

Laboratory of genetics and physiology 2 (LGP2), a member of RIG-I like receptor (RLR) family, plays crucial roles in modulating cellular antiviral response during viral infection. However, the detailed roles of LGP2 in different virus infection were controversial up to now. Here, we cloned a LGP2 gene from orange-spotted grouper (EcLGP2) and investigated its roles in response to grouper virus infection. EcLGP2 encoded a 678-aa protein which shared 83% identity to sea perch ( Lateolabrax japonicas ). Amino acid alignment showed that EcLGP2 contained three conserved domains, including a DEAD/DEAH box helicase domain, a helicase superfamily C-terminal domain and a C-terminal domain of RIG-I. In healthy grouper, the transcript of EcLGP2 could be predominantly detected in kidney, gill, fin, spleen and skin. Subcellular localization analysis showed that EcLGP2 distributed throughout the cytoplasm in grouper cells. Notably, the intracellular distribution of EcLGP2 was altered at the late stage of Singapore grouper iridovirus (SGIV) infection, but remained unchanged during red-spotted grouper nervous necrosis virus (RGNNV) infection. Moreover, overexpression of EcLGP2 in vitro significantly enhanced the viral replication of SGIV and RGNNV, evidenced by the acceleration of CPE occurrence and the up-regulation of the viral gene transcription or protein synthesis. Further studies indicated that overexpression of EcLGP2 decreased the expression level of interferon related molecules or effectors, including IRF3, IRF7, ISG15, IFP35, MXI, MXII, and MDA5, suggesting that the negative feedback of interferon immune response by EcLGP2 might contribute to the enhancement of RGNNV infection. Moreover, the expression levels of pro-inflammation cytokines, including IL-8 and TNFα were significantly decreased, but that of IL-6 was increased by the ectopic expression of EcLGP2. Thus, our results will contribute greatly to understanding the roles of fish LGP2 in innate immune response during iridovirus and nodavirus infection. [ABSTRACT FROM AUTHOR]