Red-spotted Grouper Nervous Necrosis Virus Infection Research Articles

Interferon regulatory factor 3 from sea perch (Lateolabrax japonicus) exerts antiviral function against nervous necrosis virus infection

Interferon (IFN) regulatory factor 3 (IRF3) is a major regulator contributing to the host away from viral infection. Here, an IRF3 gene from sea perch (LjIRF3) was identified and its role in regulating early apoptosis signaling and IFN response was investigated during red spotted grouper nervous necrosis virus (RGNNV) infection. The cDNA of LjIRF3 encoded a putative 465 amino acids protein, containing a DNA binding domain, an IRF association domain and a serine-rich domain. Phylogenetic analysis suggested that LjIRF3 shared the closest genetic relationship with Epinephelus coioides IRF3. LjIRF3 was constitutively expressed in all examined tissues with the highest expression level in the liver. Upon RGNNV infection, mRNA transcript level of LjIRF3 was significantly up-regulated in vivo and in vitro, indicating the involvement of LjIRF3 in immune response to RGNNV infection. Furthermore, overexpression of LjIRF3 significantly suppressed RGNNV replication in vitro, meanwhile significantly up-regulating the expression of IFNI and IFN stimulated genes and resulting in the activation of caspase 3 and 9 proteases in the early stage of RGNNV infection. In short, these results demonstrated that LjIRF3 exerted antiviral function against RGNNV infection via triggering early apoptotic cell death and inducing IRF3-dependent IFN immune response.

Features of chicken egg yolk immunoglobulin (IgY) against the infection of red-spotted grouper nervous necrosis virus

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.

Functional characterization of tumor necrosis factor receptor-associated factor 3 of sea perch (Lateolabrax japonicas) in innate immune

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifunctional regulator implicated in both bacterial defense and antiviral immunity. Here, a TRAF3 gene from the seawater fish sea perch, designated as LjTRAF3, was characterized. The full-length cDNA of LjTRAF3 was 2972 bp including a 5′ untranslated region (UTR) of 243 bp, a 3′UTR of 941 bp and a putative open reading frame of 1608 bp encoding a putative protein of 536 amino acid. The deduced LjTRAF3 protein contained a RING finger, two zinc fingers, a coiled-coil, and a meprin and TRAF-C homology domain. Phylogenetic analysis showed that LjTRAF3 shared the closest genetic relationship with Larimichthys crocea TRAF3. Gene expression analyses suggested that LjTRAF3 mRNA was ubiquitously expressed in all the tissues tested, and was up-regulated post red spotted grouper nervous necrosis virus (RGNNV) infection in vivo and in vitro. Reporter gene assay showed that LjTRAF3 significantly activated zebrafish type I interferon (IFN) promoter in vitro. During RGNNV infection, ectopic expression of LjTRAF3 significantly reduced the RNA dependent RNA polymerase transcription of RGNNV, and enhanced the expression of RIG-I-like receptors (RLR), janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway related genes and IFN stimulated genes (ISGs), including ISG15, PKR, VIG and TRIM39. Taken together, our results suggested that LjTRAF3 might trigger the expression of various ISGs to counter RGNNV infection by regulating the RLR-induced IFN and JAK-STAT signaling pathways.

MicroRNA-146a promotes red spotted grouper nervous necrosis virus (RGNNV) replication by targeting TRAF6 in orange spotted grouper, Epinephelus coioides

MicroRNA-146a (miR-146a) has been demonstrated to function as a negative regulator of cellular immune responses against pathogens in mammals, however, little information focused on its functions in lower vertebrates. In this study, we investigated the regulatory roles of orange spotted grouper, Epinephelus coioides miR-146a during red spotted grouper nervous necrosis virus (RGNNV) infection. During RGNNV infection in grouper spleen (GS) cells, the endogenous expression level of miR-146a and tumor necrosis factor receptor-associated factor 6 (TRAF6) significantly increased along with the infection time. Overexpression of miR-146a significantly facilitated viral infection, evidenced by the increased transcription of viral CP and RdRp genes, while miR-146a knockdown by specific inhibitors decreased RGNNV replication. Using pMIR-REPORT Luciferase system, we found that the 3′ untranslated region (UTR) of grouper TRAF6 could be specifically targeted by miR-146a. Further studies showed that its downstream target gene pro-inflammatory cytokines, including TNF-α, IL-8 and IL-1β, were all significantly decreased in miR-146a mimic transfected cells, but increased in miR-146a inhibitors transfected cells during RGNNV infection. Thus, our results suggested and verified that holding the level of miR-146a exerted crucial roles in RGNNV infection through TRAF6-mediated inflammatory response.

Fish DDX3X exerts antiviral function against grouper nervous necrosis virus infection

Human DEAD box ATP-dependent RNA helicase DDX3X has been demonstrated to exert crucial functions in carcinogenesis and antiviral immune response. However, to our knowledge, few information focused on the functions of fish DDX3X. In this study, we cloned and characterized a DDX3X homolog from orange spotted grouper (Epinephelus coioides) (EcDDX3X). EcDDX3X encoded a 733-amino acid protein which shared 97% and 76% identity to spiny damselfish (Acanthochromis polyacanthus) and human (Homo sapiens), respectively. Amino acid alignment analysis showed that EcDDX3X contained conserved DExDc and Helic C domains. The transcription levels of EcDDX3X were significantly increased in poly I:C transfected cells and red-spotted grouper nervous necrosis virus (RGNNV) infected cells. Under fluorescence microscopy, the green fluorescence was observed evenly in the cytoplasm in EcDDX3X transfected cells. The ectopic expression of EcDDX3X significantly inhibited the replication of RGNNV, evidenced by the decreased numbers of the vacuoles evoked by RGNNV infection, and the reduced transcription levels of RGNNV coat protein (CP) and RNA-dependent RNA polymerase (RdRp) genes. In contrast, the replication of Singapore grouper iridovirus (SGIV) in grouper spleen (GS) cells was not significantly affected by EcDDX3X overexpression. Further studies showed that overexpression of EcDDX3X in vitro significantly increased the expression levels of several interferon associated cytokines or effectors. Moreover, the regulatory effect of EcDDX3X on interferon immune response was dependent on its N terminal region, but not the DExDc and Helic C domain. In addition, we also found that overexpression of EcDDX3X significantly increased the interferon promoter activity, and the activation of interferon immune response was regulated by both IRF3 and IRF7. Together, our results firstly showed that fish DDX3X exerted crucial roles in antiviral immunity against RNA virus infection via upregulating interferon antiviral responses.

The antiviral role of heat shock protein 27 against red spotted grouper nervous necrosis virus infection in sea perch

Heat shock protein 27 (HSP27), functioning as a stress induced protective protein, has been reported to participate in various biological processes, including apoptosis, thermal protection, and virus infection. In this study, a HSP27-like gene from the seawater fish sea perch, designated as LjHSP27, was characterized. The 1361 bp full-length cDNA of LjHSP27 encoded a 221 amino acid protein containing a conserved α-crystallin domain, two variable amino- and carboxy-terminal extensions, a WD/EPF motif, two serine phosphorylation sites, and two putative actin binding regions. Phylogenetic analysis showed that LjHSP27 shared the closest genetic relationship with HSP27 of the Asian seabass Lates calcarifer. LjHSP27 mRNA was ubiquitously expressed in all tissues examined, but significantly up-regulated in spleen and kidney and down-regulated in brain post red spotted grouper nervous necrosis virus (RGNNV) infection. In vitro, LjHSP27 transcript was remarkably reduced post RGNNV infection, but rapidly increased after polyinosinic-polycytidylic acid treatment. Up-regulation and down-regulation of LjHSP27 inhibited and promoted RGNNV replication in cultured LJB cells, respectively. Luciferase assay indicated that LjHSP27 could enhance the promoter activities of zebrafish interferon (IFN)1 and IFN3, suggesting its potential role in innate immune responses. Moreover, overexpression of LjHSP27 inhibited RGNNV-induced apoptosis, as indicated by the up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes, while KNK437 caused down-regulation of LjHSP27 dramatically led to opposite results, suggesting that LjHSP27 might exert its anti-RGNNV activities by regulating the apoptosis signaling pathway. Our results would provide a new insight into the underlying molecular mechanism of HSP and RGNNV interaction.

Rapid and sensitive detection of redspotted grouper nervous necrosis virus (RGNNV) infection by aptamer-coat protein-aptamer sandwich enzyme-linked apta-sorbent assay (ELASA).

Redspotted grouper nervous necrosis virus (RGNNV) is one of the most devastating pathogens in the aquaculture of the grouper, Epinephlus sp., worldwide. The early and rapid diagnosis of RGNNV is important for the prevention of RGNNV infection. In this study, an aptamer (A10)-based sandwich enzyme-linked apta-sorbent assay (ELASA) was developed for RGNNV diagnosis. This sandwich ELASA showed high specificity for the RGNNV coat protein (CP) and virions in virus-infected cells and tissues. At the optimized working concentration of 200 nM of aptamer, the ELASA could detect RGNNV in the lysates of as few as 4×103 RGNNV-infected GB cells. Incubation for 10 min was sufficient to produce accurate results. The sandwich ELASA was most stable at incubation temperatures of 4-25°C, but could still distinguish RGNNV-infected samples from the controls at 37°C. It could detect RGNNV infection in brain lysates diluted 1/10, with results consistent with those of reverse transcription PCR, although with 10% less sensitivity. The main equipment required includes dissection tools, a water bath, Pierce™ Streptavidin Coated Plates and a microplate reader. The sandwich ELASA has great potential utility for the rapid and sensitive diagnosis of RGNNV in its early stages by fish farmers.

Characterization of microRNAs in orange-spotted grouper (Epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF-1) cells infected with red-spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non-infected groups, 51 and 16 differentially expressed miRNAs (DE-miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE-miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE-miRNAs covered a wide range of functions, such as regulation of transcription, oxidation-reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE-miRNAs including miR-1, miR-30b, miR-150, and miR-184 on RGNNV replication were evaluated, and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection.

Transcriptomic profiles of striped snakehead fish cells (SSN-1) infected with red-spotted grouper nervous necrosis virus (RGNNV) with an emphasis on apoptosis pathway

Nervous necrosis virus (NNV), the causative agent of viral nervous necrosis (VNN) disease, has caused mass mortality of cultured marine and freshwater fish worldwide, resulting in enormous economic losses in the aquaculture industry. However, the molecular mechanisms underlying the pathogenicity of NNV are still poorly understood. In this study, the transcriptomic profiles of striped snakehead fish (Channa striatus) cells (SSN-1) infected with red-spotted grouper NNV (RGNNV) were investigated using deep RNA sequencing technique. From 254,955,234 raw reads, a total of 253,338,544 clean reads were obtained and they were assembled into 93,372 unigenes. Differentially expressed genes (DEGs) were identified from RGNNV-infected or mock-infected SSN-1 cells, including 1184 up-regulated and 1456 down-regulated genes at 3 h (h) post of infection (poi), and 1138 up-regulated and 2073 down-regulated genes at 24 h poi, respectively. These DEGs were involved in many pathways related to viral pathogenesis, including retinoic acid-inducible gene I (RIG-I) like receptors pathway, apoptosis pathway, oxidative phosphorylation, PI3K-Akt signaling pathway, and MAPK signaling pathway. Subsequent analysis focusing on the apoptosis pathway showed that the expression of Endonuclease G (EndoG) was up-regulated upon RGNNV infection at both 3 and 24 h poi. Therefore, EndoG gene was cloned and its function was further characterized. The results showed that over-expression of EndoG could also induce cellular apoptosis in SSN-1 cells, indicating that RGNNV infection might induce apoptosis of SSN-1 cells via EndoG-associated mitochondrial pathway. These results will shed a new light on the pathogenesis of NNV.

Glutamine is required for red-spotted grouper nervous necrosis virus replication via replenishing the tricarboxylic acid cycle

Glutamine, one of the most important nutrients, plays a vital role in carbon metabolic pathway and has been reported to be required for the replication of several human DNA viruses. However, whether glutamine is required for RNA virus replication and the related mechanism remains elusive. Nervous necrosis virus (NNV), a positive-stranded RNA virus, can infect a number of important aquatic species and has caused great economic losses in aquaculture industry worldwide. In this study, the effects of glutamine on red-spotted grouper nervous necrosis virus (RGNNV) replication were investigated. The results showed that lack of glutamine did not affect the cell viability, but dramatically inhibited RGNNV replication, indicating that glutamine was required for RGNNV replication. Glutamine can be converted to α-ketoglutarate (α-KG) by glutaminase (GLS) to join in the tricarboxylic acid (TCA) cycle. Inhibiting the activity of GLS by a GLS inhibitor: bis-2-5-phenylacetamido-1,3,4-thiadiazol-2-ethyl sulfide (BPTES) significantly inhibited RGNNV replication, while adding the TCA cycle intermediates: α-KG, oxaloacetic acid (OAA), or pyruvate significantly restored RGNNV replication in glutamine-free medium, indicating that the requirement of glutamine for RGNNV replication was due to replenishing the TCA cycle. Taken together, these data revealed that glutamine could regulate RGNNV replication via TCA cycle, which will pave a new way for the prevention of the RGNNV infection in the future.

Negative regulation of the antiviral response by grouper LGP2 against fish viruses.

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 invitro 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.

Fish TRIM8 exerts antiviral roles through regulation of the proinflammatory factors and interferon signaling.

The tripartite motif (TRIM)-containing proteins usually exert important regulatory roles during multiple biological processes. TRIM8 has been demonstrated to be a RING domain-containing E3 ubiquitin ligase which plays critical roles in inflammation and cancer. In this study, a TRIM8 homolog from grouper, Epinephelus coioides (EcTRIM8) was cloned, and its effects on fish virus replication were investigated. The full-length EcTRIM8 cDNA encoded a polypeptide of 568 amino acids with 92% identity to TRIM8 homolog from large yellow croaker (Larimichthys crocea). Sequence alignment analysis indicated that EcTRIM8 contained conserved RING finger, B-box and coiled-coil domain. Expression patterns analysis showed that EcTRIM8 was predominant in kidney, gill, fin, liver, spleen and brain. After challenging with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the EcTRIM8 transcript was significantly increased at the early stage of injection. Under fluorescence microscopy, we observed different distribution patterns of EcTRIM8 in grouper spleen (GS) cells, including punctate fluorescence evenly situated throughout the cytoplasm and bright aggregates. The ectopic expression of EcTRIM8 in vitro significantly inhibited the replication of SGIV and red spotted grouper nervous necrosis virus (RGNNV), evidenced by the obvious reduction in the severity of cytopathic effect (CPE) and the significant decrease in viral gene transcription and protein synthesis. Moreover, the transcription of the proinflammatory factors and interferon related immune factors were differently regulated by EcTRIM8 during SGIV or RGNNV infection. In addition, overexpression of EcTRIM8 significantly increased the transcription of interferon regulator factor 3 (IRF3) and IRF7, and enhanced IRF3 or IRF7 induced interferon-stimulated response element (ISRE) promoter activity. Together, our results firstly demonstrated that fish TRIM8 could exert antiviral function through the regulation of the expression of proinflammatory cytokines and interferon related transcription factors in response to fish viruses.

Antiviral function of grouper MDA5 against iridovirus and nodavirus.

Melanoma differentiation-associated gene 5 (MDA5) is a critical member of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family which can recognize viral RNA and enhances antiviral response in host cells. In this study, a MDA5 homolog from orange spotted grouper (Epinephelus coioides) (EcMDA5) was cloned, and its roles on grouper virus infection were characterized. The full-length EcMDA5 cDNA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homolog from rock bream (Oplegnathus fasciatus). Amino acid alignment analysis indicated that EcMDA5 contained three functional domains: two caspase activation and recruitment domain (CARDs), a DEAD box helicase-like (DExDc) domain, a helicase superfamily C-terminal domain (HELICc), and a C-terminal regulatory domain (RD). Upon challenge with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the transcript of EcMDA5 was significantly up-regulated especially at the early stage post-injection. Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. Interestingly, during virus infection, the distribution pattern of EcMDA5 was significantly altered in SGIV infected cells, but not in red spotted grouper nervous necrosis virus (RGNNV) infected cells, suggested that EcMDA5 might interact with viral proteins during SGIV infection. The ectopic expression of EcMDA5 invitro obviously delayed virus infection induced cytopathic effect (CPE) progression and significantly inhibited viral gene transcription of RGNNV and SGIV. Moreover, overexpression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (ISRE) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. In addition, the transcription level of the proinflammatory factors, including TNF-α, IL-6 and IL-8 were differently altered by EcMDA5 overexpression during SGIV or RGNNV infection, suggesting that the regulation on proinflammatory cytokines by EcMDA5 were also important for RGNNV infection. Together, our results demonstrated for the first time that the inhibitory effect of fish MDA5 on iridovirus replication might be mainly through the regulation of proinflammatory cytokines.

Dynamic distribution and tissue tropism of nervous necrosis virus in juvenile pompano (Trachinotus ovatus) during early stages of infection

Abstract The distribution of red-spotted grouper nervous necrosis virus (RGNNV) in marine fish during the earlier stages of infection has not yet been described. In this study, we investigated the dynamic distribution and tissue tropism of RGNNV in experimentally infected juvenile pompano ( Trachinotus ovatus ) using quantitative real-time polymerase chain reaction (qPCR). Our findings showed that the brain, kidney, spleen, gill, heart, and muscle tissues were the first sites in which RGNNV was detected as early as 4 h post-injection (hpi), whereas the virus was difficult to detect in liver and intestinal tissue until 96 hpi. The pattern of host-virus dynamics in the tissues was U-shaped from 4 to 96 hpi. Infection of juvenile pompano with RGNNV led to a higher viral load in the brain compared to other tissues, which corresponds to the results of the histopathological and immunohistochemical tests. The results provide a foundation for early monitoring of RGNNV infection and indicate that there is a distinct dynamic distribution of the viral genome in juvenile pompanos during the early infection, suggesting that viral replication is affected by the host innate immune response.

Involvement of fish signal transducer and activator of transcription 3 (STAT3) in nodavirus infection induced cell death

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is an important signaling pathway activated by interferons in response to virus infection. Fish STAT3 has been demonstrated to be involved in Singapore grouper iridovirus (SGIV) infection and virus induced paraptosis, but its effects on the replication of other fish viruses still remained uncertain. Here, the roles of grouper STAT3 (Ec-STAT3) in red spotted grouper nervous necrosis virus (RGNNV) infection were investigated. The present data showed that the distribution of phosphorylated Ec-STAT3 was altered in RGNNV infected fish cells, and the promoter activity of STAT3 was significantly increased during virus infection, suggesting that STAT3 activation was involved in RGNNV infection. Using STAT3 specific inhibitor, we found that inhibition of Ec-STAT3 in vitro did not affect the transcription and protein synthesis of RGNNV coat protein (CP), however, the severity of RGNNV induced vacuolation and autophagy was significantly increased. Meanwhile, at the late stage of virus infection, RGNNV induced necrotic cell death was significantly decreased after inhibition of Ec-STAT3. Further studies indicated that Ec-STAT3 inhibition significantly increased the transcript level of autophagy related genes, including UNC-51–like kinase 2 (ULK2) and microtubule-associated protein 1 light chain 3-II (LC3-II) induced by RGNNV infection. Moreover, the expression of several pro-inflammatory factors, including TNFα, IL-1β and IL-8 were mediated by Ec-STAT3 during RGNNV infection. Together, our results not only firstly revealed that STAT3 exerted novel roles in response to fish virus infection, but also provided new insights into understanding the roles of STAT3 in different forms of programmed cell death.

Identification of orange-spotted grouper (Epinephelus coioides) interferon regulatory factor 3 involved in antiviral immune response against fish RNA virus

Interferon regulatory factor 3 (IRF3) is an important transcription factor which regulates the expression of interferon (IFN) and IFN-stimulated genes (ISGs) following virus recognition. In this study, a novel IRF3 gene was cloned from grouper Epinephelus coioides (EcIRF3) and its effects against Singapore grouper iridovirus (SGIV) and red spotted grouper nervous necrosis virus (RGNNV) was investigated. The full-length of EcIRF3 cDNA was composed of 2513 bp and encoded a polypeptide of 458 amino acids which shared 82% identity with European seabass (Dicentrarchus labrax). EcIRF3 contained three conserved domains including a DNA-binding domain (DBD), an IRF associated domain (IAD) and a serine-rich domain. Expression profile analysis revealed that EcIRF3 was abundant in head kidney, kidney, spleen and gill. Upon different stimuli in vitro, the transcript of EcIRF3 was significantly up-regulated after RGNNV infection or treatment with polyinosin-polycytidylic acid (poly I:C). During SGIV infection, the increase of the EcIRF3 transcription was only detected at the late stage, suggesting that EcIRF3 was differently regulated by different stimuli. Immune fluorescence assay indicated that the fluorescence signal of EcIRF3 was increased significantly after infection with RGNNV or treatment with poly I:C, but moderately at the late stage of SGIV infection. Reporter gene assay showed that EcIRF3 activated zebrafish type I IFN and type III IFN promoter in vitro. The viral gene transcription and virus production of RGNNV were significantly decreased in EcIRF3 overexpressing cells. However, the ectopic expression of EcIRF3 did not affect the gene transcription and virus production of SGIV. Moreover, the mRNA expression levels of type I IFN and IFN-inducible genes (MxI, ISG15 and ISG56) were increased in RGNNV infected EcIRF3 overexpressing cells compared to empty vector transfected cells. Together, our results demonstrated that IFN immune response mediated by grouper IRF3 was exerted crucial roles for fish RNA virus, but not for DNA virus replication.

Development of a new cell line from the snout of giant grouper, Epinephelus lanceolatus (Bloch), and its application in iridovirus and nodavirus pathogenesis

We developed and characterized a new marine fish cell line (ELGSN) from the snout of giant grouper, Epinephelus lanceolatus (Bloch). Two different phenotypes of cells, ELGSN(e), which consisted predominantly of epithelial-like cells, and ELGSN(f), which consisted predominantly of fibroblast-like cells, were isolated and cultured. Both ELGSN, and ELGSN(f) cells were subcultured for >80 passages and multiplied well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28 degrees C. Karyotyping analysis indicated that the modal chromosome numbers of ELGSN(e) and ELGSN(f) cells were 48 and 62, respectively. After transfection with EGFP-C1, bright green fluorescence was observed in these two cells and the transfection efficiency was 35% and 28%, respectively. Moreover, the two types of ELGSN cells both showed susceptibility to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), but not to soft-shelled turtle iridovirus (STIV) and spring viremia of carp virus (SVCV), indicated by the occurrence of severe cytopathic effect and increased viral titers. In SGIV-infected cells, paracrystalline arrays, different stages of virus particles and amorphous structures were observed under electron microscopy. In RGNNV-infected cells, numerous virus particles and circular virus-packed macropinosome-like structures were observed. All these data suggested that ELGSN cells could be used for effective virus propagation in vitro. In addition, we provided biochemical evidences that macropinocytosis was involved in RGNNV infection in ELGSN cells. Taken together, the new established ELGSN cell lines provided a useful tool for transgenic and genetic manipulation, as well as virus propagation and pathogenesis. (C) 2014 Elsevier B.V. All rights reserved.

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass (Dicentrarchus labrax) during the course of an experimental challenge

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain (cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.

Tissue distribution of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) genome in experimentally infected juvenile European seabass ( Dicentrarchus labrax)

The distribution of viral genome in the tissues of juvenile European seabass ( Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.

Required dose of fish nervous necrosis virus (NNV) for Poly(I:C) immunization of sevenband grouper Epinephelus septemfasciatus

It was recently reported that Poly(I:C) immunization of fish confers protection against viral infection. In this process, fish are immunized with a live pathogenic virus followed by administration of Poly(I:C), which induces a transient, non-specific antiviral state. As a result, fish survive the initial immunization with live virus, which would otherwise be lethal. In the present study, we tried to determine the exact dosage of red-spotted grouper nervous necrosis virus (RGNNV), the causative agent of viral nervous necrosis, required for Poly(I:C) immunization of sevenband grouper Epinephelus septemfasciatus. In Poly(I:C) immunization with more than 90% relative percentage survival (RPS) against RGNNV challenge with 104.3 TCID50/fish, approximately 104.0 TCID50/fish or greater of RGNNV was administered by intramuscular (IM) injection, whereas 105.3 TCID50/fish or greater of RGNNV was required for immersion methods. It was concluded that the degree of RGNNV infection must be similar to a fatal dose in order for the fish to become immune to RGNNV. Interestingly, antibody against RGNNV was not detected in the sera of fish immunized by immersion. Thus, Poly(I:C) immunization by immersion may have some interesting effects on the epidermal immune response in fish.