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Background The JEV genome is a positive-sense RNA with a highly structured capped 5′UTR, 3′UTR and a large open reading frame. 3′UTR is the untranslated region of flavivirus and has various important functions during viral replication, such as translation, replication and encapsidation. During viral replication, the 3′UTR interacts with viral proteins and host proteins and is required for viral RNA replication and translocation. Methods The expression level of FUBP3 was knocked down by siRNA and Flag-tagged FUBP3 overexpression plasmid was constructed for overexpression. BHK-21 cells were cultured and infected with JEV to investigate the functional role of FUBP3 in the viral infection cycle. Subcellular localization of FUBP3 and viral replication complexes was observed by dual immunofluorescence staining. Results Four host proteins were specifically associated with the 3′UTR of JEV, and FUBP3 was selected to further investigate its potential functional role in the JEV infection cycle. Knockdown of FUBP3 protein resulted in a significant decrease in JEV viral titer, whereas ectopic overexpression of FUBP3 resulted in increased JE viral infectivity. In cells stably knocked down for FUBP3 and then infected with JEV, we found almost no detectable viral NS5 protein. In contrast, when cells stably knocking-down of FUBP3 overexpressed FUBP3, we found a significant increase in viral RNA production over time compared to controls. We also demonstrated that FUBP3 re-localized in the cytoplasm after infection with JEV and co-localized with viral proteins. Exogenous overexpression of FUBP3 was also shown to be located in the JE replication complex and to assist viral replication after JEV infection. Conclusions The overall results suggest that FUBP3 regulates RNA replication of JEV and promotes subsequent viral translation and viral particle production.

Traditional Chinese Medicine (TCM) has been extensively used to ameliorate diseases in Asia for over thousands of years. However, owing to a lack of formal scientific validation, the absence of information regarding the mechanisms underlying TCMs restricts their application. After oral administration, TCM herbal ingredients frequently are not directly absorbed by the host, but rather enter the intestine to be transformed by gut microbiota. The gut microbiota is a microbial community living in animal intestines, and functions to maintain host homeostasis and health. Increasing evidences indicate that TCM herbs closely affect gut microbiota composition, which is associated with the conversion of herbal components into active metabolites. These may significantly affect the therapeutic activity of TCMs. Microbiota analyses, in conjunction with modern multiomics platforms, can together identify novel functional metabolites and form the basis of future TCM research.

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.

This study was conducted to investigate the energy mobilization and ionoregulation pattern of tilapia living recirculating aquaculture system (RAS) and cage culture environments. Three different groups of tilapia were compared as tilapia cultured in RAS (Group I - RAS), tilapia cultured in open water cage (Group II - Cage) and tilapia transferred from cage to RAS (Group III - Compensation) as physiology compromising model. Results revealed that Group II tilapia mobilized glycogen as primarily energy for daily exercise activity and promoted growth, whilst tilapia from Group I and III mobilised lipid to support gonadogenesis and protein reserved for somatogensis. The gills and kidney NKA activities remained relative stable to maintain balance homeostasis with a electrolytes level. As a remark, this study revealed that tilapia re-strategized their energy mobilization pattern in accessing glycogen as easy energy to support exercise metabolism and mobilized lipid and protein for growth and gonadal development.

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.

Steroid hormones have been proven as a key drive of sex change in sequentially hermaphroditic organisms. However, the upstream mechanism of sex steroid hormones regulation that affect sex change remain unknown. The main glucocorticoid in teleost fish is cortisol, which both regulates steroidogenesis and has antistress action. Thus, cortisol might be one of the prime factors in sex change. In this study, the glucocorticoid-induced leucine zipper (GILZ) gene, was proven to have a dramatic effect in orange-spotted groupers (Epinephelus coioides) during sex change at the early stage of gonadal transition. The specific action of the GILZ protein is at the pouch-shaped proliferative spermatogonia instead of the degenerative oocyte at the onset of sex change. Immunohistochemical (IHC) evidence revealed that GILZ performs intensively at undifferentiated spermatogonia in the early testis stage. These results imply that cortisol provokes a rise of GILZ through regulation caused by steroid hormones leading to sex change.

The double-stranded RNA-binding protein Staufen1 (Stau1) has multiple functions during RNA virus infection. In this study, we investigated the role of Stau1 in viral translation by using a combination of enterovirus 71 (EV-A71) infection, RNA reporter transfection, and in vitro functional and biochemical assays. We demonstrated that Stau1 specifically binds to the 5&prime, untranslated region of EV-A71 viral RNA. The RNA-binding domain 2-3 of Stau1 is responsible for this binding ability. Subsequently, we created a Stau1 knockout cell line using the CRISPR/Cas9 approach to further characterize the functional role of Stau1&rsquo, s interaction with viral RNA in the EV-A71-infected cells. Both the viral RNA accumulation and viral protein expression were downregulated in the Stau1 knockout cells compared with the wild-type na&iuml, ve cells. Moreover, dysregulation of viral RNA translation was observed in the Stau1 knockout cells using ribosome fractionation assay, and a reduced RNA stability of 5&prime, UTR of the EV-A71 was also identified using an RNA stability assay, which indicated that Stau1 has a role in facilitating viral translation during EV-A71 infection. In conclusion, we determined the functional relevance of Stau1 in the EV-A71 infection cycle and herein describe the mechanism of Stau1 participation in viral RNA translation through its interaction with viral RNA. Our results suggest that Stau1 is an important host factor involved in viral translation and influential early in the EV-A71 replication cycle.

Myostatin is a negative regulator of myogenesis and has been suggested to be an important factor in the development of muscle wasting during viral infection. The objective of this study was to characterize the main regulatory element of the grouper myostatin promoter and to study changes in promoter activity due to viral stimulation. In vitro and in vivo experiments indicated that the E-box E6 is a positive cis-and trans-regulation motif, and an essential binding site for MyoD. In contrast, the E-box E5 is a dominant negative cis-regulatory. The characteristics of grouper myostatin promoter are similar in regulation of muscle growth to that of other species, but mainly through specific regulatory elements. According to these results, we conducted a study to investigate the effect of viral infection on myostatin promoter activity and its regulation. The nervous necrosis virus (NNV) treatment significantly induced myostatin promoter activity. The present study is the first report describing that specific myostatin motifs regulate promoter activity and response to viral infection.

Betanodaviruses cause viral nervous necrosis in numerous fish species, but some species are resistant to infection by these viruses. It is essential to fully characterize the immune responses that underlie this protective response. Complete characterization of the immune responses against nodaviruses may allow the development of methods that stimulate fish immunity and of an effective betanodavirus vaccine. Such strategies could include stimulation of specific immune system responses or blockage of factors that decrease the immune response. The innate immune system clearly provides a front-line defense, and this includes the production of interferons and other cytokines. Interferons that are released inside infected cells and that suppress viral replication may be the most ancient form of innate immunity. This review focuses on the immune responses of fish to betanodavirus infection.

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.

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.

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.

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.

Marine heterotrophic microalgal species which are potentially rich in docosahexaenoic acid (DHA, C22:6n-3) have been found in Taiwan; however, there was a lack of detailed analysis and characterization of these indigenous algae which is needed for the development of commercial applications. Hence, the objective of this study was to screen DHA-rich heterotrophic microalgae species indigenous to Taiwan for commercial purposes. Heterotrophic microalgae from a variety of marine habitats were isolated, cultivated, and then identified according to their 18S rRNA gene sequences and morphological characteristics. A comparison was made of their fatty acid profiles, fatty acid content, and amount of biomass. For the strain with highest DHA yield, the optimal growth conditions were determined in order to establish the best fermentation conditions for scale-up. In this study, 25 heterotrophic microalgal strains were successfully isolated from marine habitats around Taiwan. All of the isolated strains showed a close phylogenic relationship with the Thraustochytriaceae family according to their 18S rRNA gene sequences. GC/MS analysis discerned seven distinctive fatty acid profiles of these strains, with the production of eicosapentaenoic acid (C20:5n-3) ranging from 0.02 to 2.61 mg L(-1), and DHA ranging from 0.8 to 18.0 mg L(-1). An Aurantiochytrium strain BL10 with high DHA production was subsequently chosen for further manipulation. Under optimal growth conditions it could produce up to 59.0 g of dry biomass per liter of culture, with dry biomass containing 73% total fatty acid and 29% DHA, revealing BL10 as an excellent source of microbial DHA.

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.

We cloned and sequenced 2C I-IFN, a two-cysteine containing type I interferon (I-IFN) gene, in orange-spotted grouper (Epinephelus coioides). The cDNA has 769 base pairs, the protein has 172 amino acids, and the predicted signal peptide has 18 amino acids with two cysteines. This gene is similar to I-FNs from sea bass and other teleosts. 2C I-IFN has 5 exons and 4 introns, also similar to other teleost I-IFNs. Immunohistochemical (IHC) analysis indicated that expression is predominantly membrane-localized in healthy grouper, but has a zonal distribution in nodavirus-infected grouper. Grouper infected with nodavirus had elevated levels of 2C I-IFN at 72 h and Mx at days 6-7. Recombinant 2C I-IFN activated grouper Mx, leading to upregulated antiviral activity. The grouper Mx promoter was highly induced after treatment with recombinant 2C I-IFN. The present results suggest that expression of grouper 2C I-IFN may participate in the immunologic barrier function against nodavirus.

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.

Heat shock transcription factor 1 (HSF1) mainly coordinates stress-induced transcription and is the key transcriptional activator of heat shock proteins (HSPs). In the study, two alternatively spliced isoforms of orange-spotted grouper HSF1 were identified as osgHSF1a and osgHSF1b, which differ in the inclusion (osgHSF1a) or exclusion (osgHSF1b) of exon 11. The osgHSF1 isoforms showed the highly conserved DNA binding domain, whereas the oligomerization domain exhibited more variations compared with other species. The expression level of osgHSF1a decreased dramatically upon heat exposure while that of osgHSF1b increases upon heat and cold exposure. From tissue distribution of grouper juvenile examined, the expression level of osgHSF1 isoforms was higher in brain, eye and fin than that in other tissues. Furthermore, high expression levels of osgHSF1b were observed during the early developmental stages of grouper larva. Upon microbial mimics challenge, expression level of both isoforms increased after poly(I:C) treatment. However, after grouper larva were challenged with betanodavirus, osgHSF1 isoforms were downregulated at 6 hours significantly. This results may suggest that virus infection inhibited osgHSF1 expression to enhance virus replication at the early infection stage. To further investigate the role of osgHSF1 during betanodavirus infection, a specific inhibitor of HSF1, KRIBB11 will be used. Taken together, the present results provided an insight that osgHSF1 isoform may play important roles at the early stage of virus infection.

Autophagy is a conserved cellular process in response to stress, sustain normal cell growth by degrade and recycle unnecessary cytosolic components in autophagosome and autolysosome. Microtubuleassociated protein light chain 3 known as LC3 is a major constituent of the autophagosome and used as a reliable marker for autophagy. In this study, a full length of orange-spotted grouper LC3 (osgLC3) mRNA had been cloned with a size of 1,440 bp including the sequence encoded for a 126 a.a. protein. The phylogenetic relationship and relative tissue mRNA expression levels for osgLC3 has been evaluated. The punctate structures increased in the GF-1 cells with over-expressed osgLC3 under nutrient deprivation revealed that starvation induced autophagy formation and the colocalization of punctate structures and lysosome indicated autolysosome formation during autophagy induction. Additionally, it has found that autophagy formation in GF-1 cells induced by nervous necrosis virus (NNV) infection through quantitation of endogenous osgLC3-positive punctate structures. In grouper larvae, the mRNA expression of osgLC3 were increased under NNV infection and the osgLC3 was able to convert to lipidated form of LC3 (osgLC3-II) for synthesis of autophagosome. Moreover, it has been verified that Hsp90 plays a role in autophagy and here the osgLC3 and Hsp90AB had higher FRET efficiency that implied the interaction between osgLC3 and Hsp90AB. Based on these results, osgLC3 contributed to the autophagy formation in response to NNV infection in grouper and the working mechanisms of osgLC3 and Hsp90AB during virus infection need further studies.

Small ubiquitin-like modifiers (SUMOs), structurally similar to ubiquitin, ligated to lysine residues within sumoylation target proteins. Sumoylation and ubiquitination exhibit similar biological processes for post-translational modification regulation, apoptosis and protein stability which was participated in a number of cellular processes such as nuclear transport, transcriptional regulation, apoptosis and protein stability. In the present study, the cDNA of orange-spotted grouper SUMO, terms osgSUMO, was cloned by the combination of homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approaches. The SUMO1 and SUMO2-yellow fluorescent protein (YFP) were distributed in the cytoplasm. Our results show that SUMO1 was capable of decreasing coat protein (CP) of nucleus localization ratio, not SUMO2. Thus, we demonstrate here that SUMO1 was able to interact with CP by using analysis of fluorescence resonance energy transfer (FRET). Taken together, grouper SUMO can partially influence on localization of CP.

Groupers of the Epinephelus spp. are an important aquaculture species of high economic value in the Asia Pacific region. They are susceptible to piscine nodavirus infection, which results in viral nervous necrosis disease. In this study, a rapid and sensitive automated microfluidic chip system was implemented for the detection of piscine nodavirus; this technology has the advantage of requiring small amounts of sample and has been developed and applied for managing grouper fish farms. Epidemiological investigations revealed an extremely high detection rate of piscine nodavirus (89% of fish samples) from 5 different locations in southern Taiwan. In addition, positive samples from the feces of fish-feeding birds indicated that the birds could be carrying the virus between fish farms. In the present study, we successfully introduced this advanced technology that combines engineering and biological approaches to aquaculture. In the future, we believe that this approach will improve fish farm management and aid in reducing the economic loss experienced by fish farmers due to widespread disease outbreaks.

Chemokine (C-X-C motif) receptor 4 (CXCR4) from orange-spotted grouper (Epinephelus coioides) was identified and characterized in this study. gCXCR4 shared common features in protein sequence and predicted structure of CXCR4 family. This suggested that gCXCR4 is a member of G protein-coupled receptors with seven transmembrane domains. The expression patterns revealed that gCXCR4 may play a key role in early development of grouper. Furthermore, overexpression of gCXCR4-GFP for 48 h had significant effects on the GF-1 cell viability. gCXCR4 protein was mainly expressed in the marginal zone of head kidney and on the surface of intestinal villi. gCXCR4 expression can be detected in all the examined tissues and significantly up-regulated in eye and brain, which are the main targets for nervous necrosis virus (NNV) infection and replication. gCXCR4 gene expression can be induced in the spleen and eye by lipopolysaccharide and NNV, respectively. Our data suggested that gCXCR4 may not only play a role in the early immune response to microbial infection but also restrain to the immune system and central nervous system.

The N-terminal 200 amino acid residues of topoisomerase I (TopoN) is highly positive in charge and has DNA binding activity, without DNA sequence and topological specificity. Here, a fusion protein (6 × His-PTD-TopoN) containing a hexahistidine (6 × His) tag, a membrane penetration domain and TopoN (amino acid 3–200) was designed and developed. The protein can bind to different sizes (3.0–8.0 kb) and forms (circular and linear) of DNA and translocates the bound DNA to the nucleus. The protein also showed low cytotoxicity to GF-1 grouper fish fin cells that were previously very sensitive and difficult to transfect in vitro. Maintaining the hexahistidine tag increased the protein’s transfection efficiency in COS7 African green monkey kidney cells and simplified the purification process. The plasmid pEGFP-N1 was delivered into COS7 cells by the protein in ATP- and temperature-dependent manners. The results indicate that the binding ability of TopoN is very useful for DNA delivery and the carrier protein can be expressed in Escherichia coli without removal of the hexahistidine tag.

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.

It is known that the non-structural B2 protein of nervous necrosis virus (NNV) plays an important role in viral replication and can inhibit the RNA interference system of the host cell. Moreover, the mechanism of NNV B2 protein to inhibit RNAi is by sequestration and protection of double strand (ds) RNA. In the flock house virus (FHV), a model alphanodavirus, the structural and mutational analysis of B2 identified that the positively charged Arg54 of the alpha2 helix mediated the dsRNA-binding activity. According to the betanodavirus B2 protein alignment and modeling results, the amino acid sequences and the predicted structure of betanodavirus B2 are different from alphanodaviruses. It was suggested that the four Arg residues of alpha3 helix between amino residues 52-60 of B2 may be involved in dsRNA-binding activity. Thus, this study replaced these four Arg residues with Gln at position 52 (R52Q), 53 (R53Q), 59 (R59Q), and 60 (R60Q) by site-directed mutagenesis method. The dsRNA-binding assays of these B2 mutants demonstrated that mB2(R53Q) and mB2(R60Q) mutants are dsRNA-binding defective. Moreover, we have found mB2(R53Q) and mB2(R60Q) could not antagonize RNAi by using HeLa cell as an RNAi inhibition model. These results suggested that Arg53 and Arg60 of betanodavirus B2 protein may be similar to Arg54 of alphanodavirus FHV B2 protein and are critical for dsRNA binding and RNAi-inhibiting. This study may serve as an example where bioinformatic analysis of related viral genomes may lead to meaningful structural and functional clues for certain viral proteins.

Over-expression of grouper Mx negatively regulated nodavirus activity through direct interaction, likely via the binding and perturbation of the intracellular localization of nodavirus coat protein. Deletion analysis of grouper Mx indicated that the coat protein binds to the effector domain of Mx. The presence of grouper Mx in a poly [I:C] interferon system inhibited nodavirus infection, demonstrating that grouper Mx over-expression has an inhibitory effect on both coat protein and RNA-dependent RNA polymerase of nodavirus antigens, which results in reduced viral yields. We conclude that grouper Mx has a key role in cellular resistance to nodavirus infection.

Molecular cloning and nucleotide sequencing of cDNA encoding an orange-spotted grouper (Epinephelus coioides) homolog of Mx (‘‘OsgMx’’) was conducted and its possible role in fish immunity was analysed. Similar to mammalian Mx, the OsgMx are members of a family of interferon-inducible genes that are expressed by cells in response to nodavirus and iridovirus naturally-infected. Expression of OsgMx mRNA was noticeably upregulated in all tissues by nodavirus naturally-infected grouper. The transcription of OsgMx gene increased 6 h after intramuscular injection of nodavirus experimentally-infected fish and peaked at 72 h in their brains. Analysis of the 5#-flanking sequence of the gene shows that as in pufferfish and zebrafish, the OsgMx promoter contains two potential interferon-stimulated response element (ISRE) responsible for the induction of interferon-inducer polyinosinic-polycytidylic acid (Poly[I:C]). Transient transfection of grouper cells in gfp-reporter gene assays shows that the activation of the grouper Mx promoter fragment by Poly[I:C] is sufficient to allow the expression of green fluorescent protein (GFP). These results may provide a possible regulated pathway against nodavirus. 2005 Elsevier Ltd. All rights reserved.

Infection of virus (such as nodavirus and iridovirus) and bacteria (such as Vibrio anguillarum) in farmed grouper has been widely reported and caused large economic losses to Taiwanese fish aquaculture industry since 1979. The multiplex assay was used to detect dual viral infection and showed that only nervous necrosis virus (NNV) can be detected till the end of experiments (100% mortality) once it appeared. In addition, iridovirus can be detected in a certain period of rearing. The results of real-time PCR and in situ PCR indicated that NNV, in fact, was not on the surface of the eggs but present in the embryo, which can continue to replicate during the embryo development. The virus may be vertically transmitted by packing into eggs during egg development (formation) or delivering into eggs by sperm during fertilization. The ozone treatment of eggs may fail to remove the virus, so a new strategy to prevent NNV is needed.

Oxidative stress associated with nodavirus infection is poorly understood, especially pertaining to infection-mediated brain injury. Indirect evidence indicates that infection increases cellular abundance of reactive oxygen species (ROS) with consequent increase in cellular dityrosine production. The detection of dityrosine in nodavirus-infected grouper was demonstrated using immunohistochemical (IHC) staining. Proteomic analyses with eye tissues of healthy grouper revealed more abundant expression of crystallin protein in the eye than in various tissues, which was confirmed by real-time polymerase chain reaction and IHC analyses. Grouper crystallin belongs to a small heat shock protein family with chaperone-like function that prevents heat-induced and oxidative stress-induced protein aggregation. Recombinant crystallin induced nitric oxide (NO) production in RAW 264.7 cells after treatment. The results provide new insight into the pathogenesis of nodavirus and demonstrate an experimental rationale for antioxidant therapy research.