Characterization of orange-spotted grouper (Epinephelus coioides) interferon regulatory factor 4 regulated by heat shock factor 1 during heat stress in response to antiviral immunity.

Interferon regulatory factor 4 (IRF4), in conjunction with thermogenic regulation, is a negative regulator of immune responses. Therefore, we examined whether temperature changes regulated the antiviral response of IRF4 in nervous necrosis virus (NNV)-infected orange-spotted groupers. We found that osgIRF4 mRNA expression was responsive to poly I:C stimulation and NNV infection. In vitro overexpression of osgIRF4 caused a marked decrease in the promoter activity of the antiviral protein Mx1, and magnified NNV replication. Notably, we showed that the IAD domain of osgIRF4 exerted a dominant inhibitory effect on the Mx1 promoter. Furthermore, on exposure to high temperatures, the action of osgIRF4 was dependent on heat shock factor 1 (HSF1) expression. Additionally, small interfering RNA knockdown of HSF1 abrogated high temperature-mediated osgIRF4 activity. These findings suggest that osgIRF4 is an essential negative regulator of innate antiviral immunity and enhances viral replication during heat stress in the orange-spotted grouper.

Cloning and characterisation of type I interferon receptor 1 in orange-spotted grouper (Epinephelus coioides) for response to nodavirus infection.

Grouper is known as a highly economical teleost species in the Asian aquaculture industry; however, intensive culture activities easily cause disease outbreak, especially viral disease. For the prevention of viral outbreaks, interferon (IFN) is among the major defence systems being studied in different species. Fish type I IFNs are known to possess antiviral properties similar to mammalian type I IFNs. In order to stimulate antiviral function, IFN will bind to its cognate receptor, the type I interferon receptor (IFNAR), composed of heterodimeric receptor subunits known as IFNAR1 and IFNΑR2. The binding of type I interferon to receptors assists in the transduction of signals from the external to internal environments of cells to activate biological responses. In order to study the function of IFN, we first need to understand IFN receptors. In this study, we cloned and identified IFNAR1 in orange-spotted grouper (osgIFNAR1) and noted the up-regulated mRNA expression of the receptor and downstream effectors in the head kidney cells with cytokine treatment. The transcriptional expression of osgIFNAR1, which is characterised using polyinosinic-polycytidylic acid (poly[I:C]) and lipopolysaccharide (LPS) treatments, indicated the involvement of osgIFNAR1 in the immune response of grouper. The subcellular localisation of osgIFNAR1 demonstrated scattering across the grouper cell. Viral infection showed the negative feedback regulation of osgIFNAR1 in grouper larvae. Further loss of function of IFNAR1 showed a decreased expression of the virus. This study reported the identification of osgIFNAR1 and characterisation of receptor sensitivity towards immunostimulants, cytokine response, and viral challenge in the interferon pathway of orange-spotted grouper and possible different role of the receptor in viral production. Together, these results provide a frontline report of the potential function of osgIFNAR1 in the innate immunity of teleost.

Characterization of betanodavirus quasispecies influences on the subcellular localization and expression of tumor necrosis factor (TNF).

The aim of this study was to investigate the influence of variant coat proteins (CPs) from different quasispecies of betanodavirus on diverse aspects of nodavirus-induced pathogenesis. It is known that variant CPs can acquire either nuclear or cytoplasmic localization, depending on the nodavirus CP genotype, and this variation may arise during viral replication and influence the regulation of host and viral gene transcription. To investigate the role of these variant CPs in pathogenesis, six variant CP expression plasmids were constructed, each containing different quasispecies CP variants from nodavirus genotype red spotted grouper nervous necrosis virus (RGNNV). The CP expression plasmids were transiently transfected into grouper GF-1 cells. At different times, the cell cycle and cell proliferation were assayed using flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. The proportion of G2/M-phase GF-1 cells transfected with CP expression plasmids was higher than that of cells transfected with the blank plasmid, especially in regards to quasispecies 2 (QS2). The proliferation ratio of cells transfected with the CP expression plasmids was significantly higher than that of cells transfected with the blank plasmid, with the exception of QS6. We also found that the different quasispecies CPs downregulated the promoter activity of the tumor necrosis factor (TNF) gene to different degrees. In addition, this is the first report showing the betanodavirus CP derived from different quasispecies of RGNNV provide evidence of a chronically nodavirus-infected grouper. Overall, this study represents the first comprehensive analysis of variant CPs from grouper with persistent nodavirus infections and their effects on different aspects of pathogenesis.

Novel subunit vaccine with linear array epitope protect giant grouper against nervous necrosis virus infection.

Viral nervous necrosis caused by nervous necrosis virus (NNV) is one of the most severe diseases resulting in high fish mortality rates and high economic losses in the giant grouper industry. Various NNV vaccines have been evaluated, such as inactivated viruses, virus-like particles (VLPs), recombinant coat proteins, synthetic peptides of coat proteins, and DNA vaccines. However, a cheaper manufacturing process and effective protection of NNV vaccines for commercial application are yet to be established. Hence, the present study developed a novel subunit vaccine composed of a carrier protein, receptor-binding domain of Pseudomonas exotoxin A, and tandem-repeated NNV coat protein epitopes by using the structural basis of epitope prediction and the linear array epitope (LAE) technique. On the basis of the crystal structure of the NNV coat protein, the epitope was predicted from the putative target cell receptor-binding region to elicit neutralizing immune responses. The safety of the LAE vaccine was evaluated, and all vaccinated fish survived without any physiological changes. The coat protein-specific antibody titers in the vaccinated fish increased after vaccine administration and exerted NNV-neutralizing effects. The efficacy tests revealed that the relative percent survival (RPS) of LAE antigen formulated with adjuvant was above 72% and LAE vaccine was effective for preventing NNV infection in giant grouper. This study is the first to develop an NNV vaccine by using epitope repeats, which provided effective protection to giant grouper against virus infection. The LAE construct can be used as a vaccine design platform against various pathogenic diseases.

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection.

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35-217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35-338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214-338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.

Nutrient pulses driven by internal solitary waves enhance heterotrophic bacterial growth in the South China Sea.

This study demonstrated the potential effects of internal waves (IWs) on heterotrophic bacterial activities for the first time. Nine anchored studies were conducted from 2009-2012 in the South China Sea areas with different physical conditions, i.e. areas subjected to elevation IWs, to depression IWs, and to weak/no IWs. The latter two areas were treated as the Control sites. Field survey results indicated that within the euphotic zone, the minima of the depth-averaged bacterial production (IBP; ∼1.0 mgC m-3 d-1 ) and growth rate (IBµ; ∼0.1 d-1 ) at all sites were similar. Except for one case, the maxima of IBP (6-12 mgC m-3 d-1 ) and IBµ (0.55-1.13 d-1 ) of the elevation IWs areas were ∼fivefolds higher than those of the Control sites (IBP 1.7-2.1 mgC m-3 d-1 ; IBµ 0.13-0.24 d-1 ). Replicate surveys conducted at the north-western area of the Dongsha atoll during spring-to-neap (NW1 survey) and neap-to-spring (NW2 survey) tide periods showed a great contrast to each other. Low variation and averages of IBµ in NW1 survey were similar to those of the Control sites, while those in NW2 were similar to the other elevation IWs sites with larger variation and higher averages of IBµ. This finding suggests that bacterial activities may be a function of the lunar fortnightly (14-day) cycle. Enrichment experiments suggested more directly that the limiting inorganic nutrients introduced by the elevation waves (EIWs) may contribute a higher IBµ within the euphotic zone.

Molecular cloning of orange-spotted grouper (Epinephelus coioides) heat shock transcription factor 1 isoforms and characterization of their expressions in response to nodavirus.

Heat shock transcription factor 1 (HSF1) regulates heat shock proteins (HSPs), which assist in protein folding and inhibit protein denaturation following stress. HSF1 was firstly cloned from orange-spotted grouper and exists as two isoforms, one with (osgHSF1a) and one without (osgHSF1b) exon 11. Heat exposure increased the expression of osgHSF1b while cold exposure increased that of osgHSF1a. Both isoforms were mainly expressed in the brains, eyes, and fins. Expression of osgHSF1b was higher than osgHSF1a during development. Poly I:C and LPS could also induce osgHSF1 isoforms expression differentially. Exposure to nervous necrosis virus (NNV) increased the level of both osgHSF1 isoforms at 12 h. GF-1 cells with overexpression of osgHSF1 isoforms enhanced viral loads within 24 h, whereas both pharmacological inhibition and RNA interference of HSF1 reduced virus infection. This study shows that osgHSF1 can support the early stage of virus infection and provides a new insight into the molecular regulation of osgHSF1 between the influence of temperatures and immunity.

Analysis of interferon gamma protein expression in zebrafish (Danio rerio).

IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.

A member of the immunoglobulin superfamily, orange-spotted grouper novel immune gene EcVig, is induced by immune stimulants and type I interferon.

A novel grouper immune gene, EcVig was identified in orange-spotted grouper (Epinephelus coioides). We recently determined that EcVig expression can be induced by infection with nervous necrosis virus (NNV, an RNA virus), whereas NNV replication may be suppressed when EcVig was overexpressed. Although EcVig appeared to be involved in grouper antiviral activity, its immune effects have not been well characterized. In the present study, two PAMPs (pathogen-associated molecular patterns; lipopolysaccharides [LPS] and synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid [poly(I:C)]), as well as fish DNA virus (red sea bream iridovirus, RSIV; grouper iridovirus, GIV), were used to study EcVig responses in orange-spotted grouper. In addition, groupers were given recombinant type I interferon to determine whether EcVig expression was induced. Poly(I:C) rapidly induced substantial expression of EcVig, whereas LPS stimulation did not appear to have any effect in grouper intestine. Expression levels of total EcVig and other IFN-stimulated genes (ISGs) were all significantly increased after RSIV and GIV infection. Furthermore, stimulation of recombinant type I IFN also increased EcVig expression. We conclude that EcVig may be a novel IFN-stimulated gene that demonstrates an antiviral immune response.

Salmonids have an extraordinary complex type I IFN system: characterization of the IFN locus in rainbow trout oncorhynchus mykiss reveals two novel IFN subgroups.

Fish type I IFNs are classified into two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into four subgroups, termed IFN-a, -b, -c, and -d. Salmonids possess all four subgroups, whereas other teleost species have one or more but not all groups. In this study, we have discovered two further subgroups (IFN-e and -f) in rainbow trout Oncorhynchus mykiss and analyzed the expression of all six subgroups in rainbow trout and brown trout Salmo trutta. In rainbow trout RTG-2 and RTS-11 cells, polyinosinic-polycytidylic acid stimulation resulted in early activation of IFN-d, whereas the IFN-e subgroup containing the highest number of members showed weak induction. In contrast with the cell lines, remarkable induction of IFN-a, -b, and -c was detected in primary head kidney leukocytes after polyinosinic-polycytidylic acid treatment, whereas a moderate increase of IFNs was observed after stimulation with resiquimod. Infection of brown trout with hemorrhagic septicemia virus resulted in early induction of IFN-d, -e, and -f and a marked increase of IFN-b and IFN-c expression in kidney and spleen. IFN transcripts were found to be strongly correlated with the viral burden and with marker genes of the IFN antiviral cascade. The results demonstrate that the IFN system of salmonids is far more complex than previously realized, and in-depth research is required to fully understand its regulation and function.

Molecular cloning and characterization of orange-spotted grouper (Epinephelus coioides) CXC chemokine ligand 12.

Chemokines are a family of soluble peptides that can recruit a wide range of immune cells to sites of infection and disease. The CXCL12 is a chemokine that binds to its cognate receptor CXCR4 and thus involved in multiple physiological and pathophysiological processes. In this study, we cloned and characterized CXCL12 from Epinephelus coioides (osgCXCL12). We found that the open reading frame of osgCXCL12 consists of 98 amino acid residues with the small cytokine C-X-C domain located between residues 29 and 87. Higher expression levels for osgCXCL12 were detected at the kitting stage, compared with the prolarva and larva shape stages. The expression patterns revealed that osgCXCL12 may play a key role in early grouper development. We detected mRNA transcripts for osgCXCL12 in healthy tissues and found the highest osgCXCL12 expression in the head kidney. Furthermore, a time-course analysis revealed significantly increased osgCXCL12 and osgCXCR4 expression levels after the nervous necrosis virus (NNV) challenge. In addition, expression of osgCXCL12 was affected by injection with microbial mimics [LPS and poly(I:C)]. These results suggest that osgCXCL12 is associated with inflammatory and developmental processes in the grouper.

Development of pedigree classification using microsatellite and mitochondrial markers for Giant grouper broodstock (Epinephelus lanceolatus) management in Taiwan.

Most giant groupers in the market are derived from inbred stock. Inbreeding can cause trait depression, compromising the animals' fitness and disease resistance, obligating farmers to apply increased amounts of drugs. In order to solve this problem, a pedigree classification method is needed. Here, microsatellite and mitochondrial DNA were used as genetic markers to analyze the genetic relationships among giant grouper broodstocks. The 776-bp fragment of high polymorphic mitochondrial D-loop sequence was selected for measuring sibling relatedness. In a sample of 118 giant groupers, 42 haplotypes were categorized, with nucleotide diversity (π) of 0.00773 and haplotype diversity (HD) of 0.983. Furthermore, microsatellites were used for investigation of parentage. Six out of 33 microsatellite loci were selected as markers based on having a high number of alleles and compliance with Hardy-Weinberg equilibrium. Microsatellite profiles based on these loci provide high variability with low combined non-exclusion probability, permitting practical use in aquaculture. The method described here could be used to improve grouper broodstock management and lower the chances of inbreeding. This approach is expected to lead to production of higher quality groupers with higher disease resistance, thereby reducing the need for drug application.

Detection of viruses directly from the fresh leaves of a Phalaenopsis orchid using a microfluidic system.

Early detection of pathogens is crucial for the effective surveillance of diseases. Many efforts have been made to explore methods which can detect these pathogens within a short period of time without requiring a tedious protocol. However, these developed methods have disadvantages such as they are relatively time-consuming or require specialized laboratory facilities. In this work, we have developed an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis from fresh Phalaenopsis orchid leaves. The entire protocol, including ribonucleic acid (RNA) purification, reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection by measuring changes in turbidity was performed on a single chip. This is the first time that an integrated microfluidic system for the detection of viruses infecting the Phalaenopsis orchid has been demonstrated. The sensitivity of the developed system was also explored in this study to validate its performance. FROM THE CLINICAL EDITOR: In this study, the authors report the development of an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis of fresh Phalaenopsis orchid leaves, performing the 3-step protocol using a single chip. Similar methods may find clinical application for fast and accurate detection of viral infections.

Effects of Salinity on Abiotic Aggregation of Organic Matter and Subsequent Microbial Responses.

Studies of marine aggregation have focused on determining formation rates of larger particles from small particles. However, it has been shown that particles can form from the dissolved phase, which includes colloidal material. The purpose of this study was to investigate the effect of aggregation on the chemical composition of both the dissolved and particulate phases in two salinity regimes: (1) the coast of Avery Point, Connecticut, USA (AP; salinity of 30.1 psu); (2) the estuary of Thames River, Connecticut, USA (TR; salinity of 5.0 psu). The samples were incubated on a roller table for two days in the dark at a speed of 8 rpm. The mixed collision mechanism of shear and differential sedimentation provided by the roller table enhanced the gross aggregation of particulate organic carbon (POC; 0.75 µM d-1 and 1.04 µM d-1 in AP and TR, respectively). Subsequent microbial degradation led to a negative net aggregation of POC (-5.20 µM d-1 and -1.19 µM d-1 in AP and TR, respectively). Although bacterial abundance remained in a narrow range in this study, the aggregation of organic matter (OM) enhanced planktonic community respiration (CR; CR increased 5.1 mg-C m-3 d-1 and 205.4 mg-C m-3 d-1 in AP and TR, respectively). The collision also led to a gross aggregation of uncharacterized particulate organic matter (POM) transferred from uncharacterized dissolved organic matter (DOM; 0.62 µM-C d-1 and 0.56 µM-C d-1 in AP and TR, respectively). The aggregated, uncharacterized POM could be biologically refractory. The C- and N-yields and enrichment factor (EF) analysis indicated that the organic substrate dynamics in this study were complicated.

Viral shunt in tropical oligotrophic ocean.

Viruses cause massive bacterial mortality and thus modulate bacteria-governed carbon transfer and nutrient recycling at global scale. The viral shunt hypothesis states the crucial role of viral lysis in retaining microbial carbon into food web processes, while its applicability to nature has not been well identified for over two decades. Here, we conducted nine diel surveys in the tropical South China Sea and suggested that the time scale adopted in sampling and system trophic status determine the "visibility" of the viral shunt in the field. Specifically, viral abundance (VA), bacterial biomass (BB), and bacterial specific growth rate (SGR) varied synchronously and presented the significant VA-BB and VA-SGR linkages at an hourly scale, which reveals direct interactions between viruses and their hosts. The differential responses of the viral shunt to temperature, i.e., looser VA-SGR coupling in warm and tighter VA-SGR coupling in cold environments, imply an altered carbon cycling in tropical oceans under climatic warming.

Real-time quantitative PCR assay for monitoring of nervous necrosis virus infection in grouper aquaculture.

Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.

Molecular cloning and functional analysis of an orange-spotted grouper (Epinephelus coioides) secreted protein acidic and rich in cysteine (SPARC) and characterization of its expression response to nodavirus.

Mammalian secreted protein acidic and rich in cysteine (SPARC) is the primary regulator of cell shape and cell adhesion to fibronectin. We, for the first time, report the complete sequencing of SPARC cDNA from orange-spotted grouper. Despite the difference in the lengths of the SPARC transcripts, all of the SPARC molecules encoded a signal peptide, follistain-like copper binding sequence (KGHK) domain, and extracellular domain. The grouper SPARC gene was differentially expressed in vivo and contributed differently to high-level expression of SPARC in muscle. Immunohistochemical staining demonstrated a decreased level of SPARC in nodavirus-infected grouper compared with healthy grouper. Comparative real-time polymerase chain reaction analyses of eye tissues of viral nervous necrosis grouper and healthy grouper were performed. Recombinant SPARC produced changes in grouper cell shape 24 h after treatment. The results provide new insight into the pathogenesis of nodavirus, and demonstrate an experimental rationale for SPARC characterization in nodavirus-infected grouper.

Identification of antigens for the development of a subunit vaccine against Photobacterium damselae ssp. piscicida.

Photobacterium damselae ssp. piscicida (Ph.d.p.), the causative agent of photobacteriosis, is among the most important pathogens affecting finfish aquaculture globally. With the emergence of recombinant technology, subunit vaccines have been actively pursued, but mostly for viral diseases. Bacterial subunit vaccines are more difficult to develop since the bacterial genome is more complex, with numerous candidate antigens, leading to a lengthy and laborious screening process. Immunoproteomics, using western blotting on protein analyzed with 2DE and LC-MS/MS to isolate immune-reactive proteins and acquire amino acid sequences, followed by recombinant technology to clone the candidate gene, identified eight candidate antigens from Ph.d.p., which have been cloned and expressed in Escherichia coli BL21(DE3). These proteins were purified and used as antigens in an efficacy trial. Three, rHSP60, rENOLASE, and rGAPDH proteins, elicited higher specific antibody titers and stronger protective immunity than the other five and an inactivated Ph.d.p. whole bacterial vaccine. These three antigens may be candidates for the development of a subunit vaccine against Ph.d.p.

The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae.

The tumour necrosis factor (TNF) super-family is a group of important cytokines involved in inflammation, apoptosis, cell proliferation, and the general stimulation of the immune system. The TNF gene has been cloned in some bony fish; however, its counterparts are still unidentified in the majority of fish species. In this study, we cloned gTNF-1 and gTNF-2 from the orange-spotted grouper (Epinephelus coioides), an economically important farmed fish. Both genes include 4 exons and 3 introns and encoded 253 and 241 amino acid proteins with a molecular weight of approximately 27 and 26 kDa, respectively. The identity of the putative amino acid sequences between gTNF-1 and gTNF-2 was only 38%. The positions of cysteine residues, a protease cleavage site, and a transmembrane domain sequence derived from gTNF-1 and gTNF-2 were similar to those in other fish and mammalian TNF-α. The mRNA expression levels of the 2 gTNF molecules were evaluated in unstimulated/stimulated peripheral blood leukocytes, various organs, and fish larvae. Following lipopolysaccharide (LPS) treatment, gTNF-2 was expressed at higher levels, was up-regulated more quickly, and was more sensitive to the immune response than gTNF-1. gTNF-1 was constitutively expressed in the thymus, brain, and spleen, but it was also expressed in the heart, head kidney, and trunk kidney after LPS stimulation. gTNF-2 was constitutively expressed in the thymus, head kidney, trunk kidney, spleen, and intestine; further, gTNF-2 was highly expressed in all organs post-LPS stimulation. Finally, the gTNF expression levels were evaluated at various developmental stages in grouper larvae. A higher variation of gTNF expression levels was observed in fish larvae from a contaminated hatchery. This study revealed the different expression patterns of gTNF-1 and gTNF-2. In addition, gTNF-2 was more sensitive to pathogens than gTNF-1; therefore, it may be an appropriate marker for pathogen invasion and the evaluation of the larval rearing environment.