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Interferon-induced transmembrane proteins (IFITMs) are novel viral restriction factors which inhibit numerous virus infections by impeding viral entry into target cells. To investigate the roles of IFITMs during fish virus infection, we cloned and characterized an IFITM1 homolog from orange spotted grouper (Epinephelus coioides) (EcIFITM1) in this study. EcIFITM1 encodes a 131-amino-acid polypeptide, which shares 64 and 43% identity with Seriola dumerili and Homo sapiens , respectively. The multiple sequence alignment showed that EcIFITM1 contained five domains, including NTD (aa 1–45), IMD (aa 46–67), CIL (aa 68–93), TMD (aa 94–119), and CTD (aa 120–131). In vitro , the level of EcIFITM1 mRNA expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV), or red-spotted grouper nervous necrosis virus (RGNNV) infection. EcIFITM1 encoded a cytoplasmic protein, which was partly colocalized with early endosomes, late endosomes, and lysosomes. The ectopic expression of EcIFITM1 significantly inhibited the replication of SGIV or RGNNV, which was demonstrated by the reduced virus production, as well as the levels of viral gene transcription and protein expression. In contrast, knockdown of EcIFITM1 using small interfering RNAs (siRNAs) promoted the replication of both viruses. Notably, EcIFITM1 exerted its antiviral activity in the step of viral entry into the host cells. Furthermore, the results of non-targeted lipometabolomics showed that EcIFITM1 overexpression induced lipid metabolism remodeling in vitro. All of the detected ceramides were significantly increased following EcIFITM1 overexpression, suggesting that EcIFITM1 may suppress SGIV entry by regulating the level of ceramide in the lysosomal system. In addition, EcIFITM1 overexpression positively regulated both interferon-related molecules and ceramide synthesis-related genes. Taken together, our results demonstrated that EcIFITM1 exerted a bi-functional role, including immune regulation and lipid metabolism in response to fish virus infections. [ABSTRACT FROM AUTHOR]

Biomarkers have important roles in various physiological functions and disease pathogenesis. As a nucleocytoplasmic DNA virus, Singapore grouper iridovirus (SGIV) causes high economic losses in the mariculture industry. Aptamer-Q5-complexed major capsid protein (MCP) in the membrane of SGIV-infected cells can be used as a specific molecular probe to investigate the crucial events of MCP endocytosis into SGIV-infected host cells during viral infection. Chlorpromazine blocks clathrin-mediated endocytosis, and MCP endocytosis into SGIV-infected cells decreased significantly when the cells were pretreated with chlorpromazine. The disruption of cellular cholesterol by methyl-β-cyclodextrin also significantly reduced MCP endocytosis. In contrast, inhibitors of key regulators of caveolae/raft-dependent endocytosis and macropinocytosis, including genistein, Na+/H+ exchanger, p21-activated kinase 1 (PAK1), myosin II, Rac1 GTPase, and protein kinase C (PKC), had no effect on MCP endocytosis. The endocytosis of the biomarker MCP is dependent on low pH and cytoskeletal actin filaments, as shown with various inhibitors (chloroquine, ammonia chloride, cytochalasin D). Therefore, MCP enters SGIV-infected host cells via clathrin-mediated endocytosis, which is dependent on dynamin, cholesterol, low pH, and cytoskeletal actin filaments. This is the first report of a specific aptamer-based probe used to analyze MCP endocytosis into SGIV-infected host cells during viral infection. This method provides a convenient strategy for exploring viral pathogenesis and facilitates the development of diagnostic tools for and therapeutic approaches to viral infection. [ABSTRACT FROM AUTHOR]

Abstract Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro , Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1β and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV. Highlights • Cystatin C gene from Epinephelus coioides (Ec-CysC) was identified and characterized. • The transcription of Ec-CysC was up-regulated in the spleen of grouper stimulated with SGIV. • Overexpression of Ec-CysC significantly inhibited SGIV infection in GS cells. • Overexpression of Ec-CysC suppressed SGIV induced apoptosis in FHM cells. [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]

Iridoviruses are large DNA viruses that infect a wide range of invertebrates and lower vertebrates, causing serious threats to ecological security and aquaculture industry worldwide. However, the mechanisms underlying intracellular transport of iridovirus remain unknown. In this study, the transport of Singapore grouper iridovirus (SGIV) in early endosomes (EEs) and late endosomes (LEs) was explored by single-particle tracking technology. SGIV employs EEs to move rapidly from the cell membrane to the nucleus, and this long-range transport is divided into "slow-fast-slow" stages. SGIV within LEs mainly underwent oscillatory movements near the nucleus. Furthermore, SGIV entered newly formed EEs and LEs, respectively, possibly based on the interaction between the viral major capsid protein and Rab5/Rab7. Importantly, interruption of EEs and LEs by the dominant negative mutants of Rab5 and Rab7 significantly inhibited the movement of SGIV, suggesting the important roles of Rab5 and Rab7 in virus transport. In addition, it seems that SGIV needs to enter clathrin-coated vesicles to move from actin to microtubules before EEs carry the virus moving along microtubules. Together, our results for the first time provide a model whereby iridovirus transport depending on EEs and LEs, helping to clarify the mechanism underlying iridovirus infection, and provide a convenient tactic to investigate the dynamic infection of large DNA virus. [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 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]

Mammalian studies have shown that the nuclear transcription factor Y (NFYC) regulates the expression of major histocompatibility complex (MHC) by binding to CCAAT-box on promoters. However, few studies have focused on the regulatory mechanisms of NFYC in MHC pathway in fish. To explore the transcriptional regulatory mechanism of MHCIa in fish, we characterized NFYC and MHCIa of red-spotted grouper (Epinephelus akaara) (named EaNFYC and EaMHCIa, respectively). The EaNFYC genome sequence is 13,796 bp and contains 1,065 bp open reading frame. It is composed of ten exons and nine introns and encode a 354 amino acid sequence. The putative EaNFYC protein sequence shared 67.2–99.4% identity to vertebrate NFYC and possesses a typically conserved domain (histone- or haem-associated protein 5 domain (HAP5)) at the N-terminus. Transcripts of both EaNFYC and EaMHCIa were ubiquitously expressed in all detect tissues, and higher mRNA levels were detected in immune-relevant tissues (middle-kidney). EaNFYC expression increased after treatment with polyinosinic: polycytidylic acid, lipopolysaccharide, nervous necrosis virus, zymosan A, and Singapore grouper iridovirus. Analysis of subcellular localization indicated that EaNFYC was localized at the cell nucleus only. Furthermore, overexpression of EaNFYC significantly stimulated the expression of EaMHCIa , interferon signalling molecules and inflammatory cytokine. The region −878 bp to +82 bp of EaMHCIa promoter was identified to be the core promoter which EaNFYC take effect on. Additionally, point mutations and electrophoretic mobility shift assays verified that NFYC activate MHCIa expression by binding at the M1 and M2 binding sites that do not contain CCAAT-box. These results contribute to elucidating the function of fish NFYC on MHC transcriptional mechanisms, and provide the first evidence of positive regulation of MHCIa expression by NFYC in fish. • NFYC and MHCIa of E. akaara are functionally characterized. • Expression of these two genes is the highest in kidney. • NFYC activate MHCIa expression by binding with no CCAAT-box on its promoter. [ABSTRACT FROM AUTHOR]

Rab20, a member of the Rab GTPase family, associates with membrane trafficking and immune response, which play important roles in bacteria control. However, little is known about the role of Rab20 during virus infection. Groupers (Epinephelus spp.) with high economic value are widely cultured in China and Southeast Asian countries. However, the cultured groupers have suffered from diseases caused by viruses such as Singapore grouper iridovirus (SGIV) resulting in huge economic losses. In this study, the roles of a novel Rab20 homolog (EcRab20), from orange-spotted grouper (Epinephelus coioides), on the infection of SGIV and host immune response were investigated. EcRab20 showed high sequence identity with other Rab20 from mammals to fishes. In healthy groupers, EcRab20 was predominantly expressed in immune organs, such as kidney, liver and spleen. After SGIV infection, the expression of EcRab20 in spleen was significantly up-regulated. Confocal imaging showed that EcRab20 distributed in the cytoplasm as punctate and vesicle-like structures, which was destructed by overexpression of the dominant negative EcRab20 (DN EcRab20). Moreover, EcRab20 notably colocalized with Golgi and early endosomes, partly colocalized with endoplasmic reticulum and late endosomes, but did not colocalize with mitochondria or lysosome. Ultimately, overexpression of EcRab20 significantly inhibited SGIV infection, whereas overexpression of DN EcRab20 promoted SGIV infection. Furthermore, using single particle imaging analysis, we found that EcRab20 or DN EcRab20 had no effect on the entry of SGIV. In addition, EcRab20 positively regulated the IFN immune and inflammatory responses. Taken together, these results demonstrated that EcRab20 affect SGIV infection by regulating the host immunity, providing a new idea of the anti-virus mechanisms of Rab20 against SGIV infection and contributing to design new antiviral strategies. • It is the first time to explore the interaction between Rab20 and virus infection. • After SGIV infection in spleen, EcRab20 is significantly up-regulated. • EcRab20 significantly inhibits SGIV infection, without affecting SGIV entry. • The host immunity is positively regulated by EcRab20. [ABSTRACT FROM AUTHOR]

Scavenger receptor class B type 1 (SR-B1) is a cell surface transmembrane protein that plays a crucial role in the immune response against viral infections. To clarify the roles of fish SR-B1 in Singapore grouper iridovirus (SGIV) infection, we identified and characterized SR-B1 (Ec-SR-B1) in the orange-spotted grouper (Epinephelus coioides). The Ec-SR-B1 encoded a 487-amino acid protein with two transmembrane regions, CD36 domain and N -linked glycosylation sites, which shared high identities with reported SR-B1. The abundant transcriptional level of Ec-SR-B1 was found in intestine, liver, spleen and head kidney. And the Ec-SR-B1 expression was significantly up-regulated in grouper spleen (GS) cells after infection with SGIV in vitro. Subcellular localization analysis revealed that Ec-SR-B1 was distributed mainly in the cytoplasm and cell membrane. Overexpression of Ec-SR-B1 in vitro significantly inhibited SGIV infection in GS cells, as evidenced by reduced cytopathic effect (CPE), decreased SGIV major capsid protein (MCP) transcription and MCP protein expression. Further studies showed that overexpression of Ec-SR-B1 positively regulated proinflammatory cytokines, and enhanced IFN and NF-κB promoter activities. Moreover, overexpression of Ec-SR-B1 significantly inhibited SGIV entry into host cells. In summary, our results suggested that Ec-SR-B1 inhibited SGIV infection by regulating antiviral innate immune response and affecting viral entry. • SR-B1 gene from Epinephelus coioides (Ec-SR-B1) was identified and characterized. • The transcription of Ec-SR-B1 was up-regulated in the GS cells stimulated with SGIV. • Overexpression of Ec-SR-B1 significantly inhibited SGIV entry in GS cells. • Overexpression of Ec-SR-B1 significantly enhanced inflammatory response and IFN antiviral signaling. [ABSTRACT FROM AUTHOR]

Iridoviruses are well-known as causative agents of serious systemic diseases, and have been identified in more than 100 fish species worldwide in the last few decades. Singapore grouper iridovirus (SGIV) is a devastating viral pathogen of grouper aquaculture, and has caused substantial economic losses in China and Southeast Asia. However, there are currently no effective therapies against SGIV. Thus, rapid and sensitive diagnosis of SGIV infection is urgently needed for controlling virus infection. Lateral flow biosensors (LFBs) are widely designed for point-of-care use due to their properties of low cost, and simple and rapid operation. In the current study, we established and characterized a novel LFB combined with two previously screened DNA aptamers (Q2 and Q3) against SGIV-infected cells for SGIV detection. One aptamer was used for target isolation and the other was used for the strand displacement amplification reaction. The LFB rapidly detected SGIV infection in vitro and in vivo , with high specificity and sensitivity. It could specifically recognize SGIV-infected cells, other than other-virus-infected cells or uninfected cells. This biosensor detected SGIV infection in a number-dependent manner in SGIV-infected cells as low as 5 × 104 /mL. The LFB method did not need sophisticated manipulation and equipment, when compared to conventional PCR. It only needed shorter time for detection, with the complete process needing no more than 90 min. This is the first developed LFB combined with aptamers for detecting marine large DNA virus, and it could be a widely applicable tool in the grouper aquaculture industry. • It is the first time to detect iridoviruses by using LFB combined with aptamers instead of antibodies. • The limit of this LFB detection was as low as 5 × 104/mL SGIV-infected cells. • The detection process using LFB can be completed in less than 90 min. • This LFB works well and exhibits great potential application in the aquaculture industry. [ABSTRACT FROM AUTHOR]