Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major).

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

Identification of a novel RIG-I isoform and its truncating variant in Japanese eel, Anguilla japonica.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic viral RNA sensor that triggers the production of type I interferons (IFNs) and proinflammatory cytokines during viral infection. RIG-I gene has been identified previously in Japanese eel, Anguilla japonica. In the present study, we have characterized a novel isoform of RIG-I (designated as AjRIG-Ib) and its truncated variant (AjRIG-Ibv). The AjRIG-Ib encodes 940 amino acids (aa) consisting of two N-terminal caspase activation and recruitment domains (CARDs), a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory domain (CTD). The AjRIG-Ibv encodes a protein of 843 aa, that shares similar structural organization with AjRIG-Ib, but lacking CTD. The gene expression analyses showed that AjRIG-Ib and AjRIG-Ibv were detectable in all tissues/organs examined, and AjRIG-Ib was the predominant form. The mRNA level of AjRIG-Ibv was upregulated rapidly at 8 h after the Poly I:C injection, and the significant increase of AjRIG-Ib was observed at 16 and 24 h post-injection (hpi). Laser confocal microscopy showed that AjRIG-Ib and AjRIG-Ibv were both located in cytoplasm. In addition, the overexpression of AjRIG-Ib or AjRIG-Ibv led to the increased activity of IFN promoter in transient transfection assay. Taken together, our results indicated that AjRIG-Ib and AjRIG-Ibv may play cooperative or somewhat complementary roles in coordinating the antiviral response in fish.

Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90ß) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C.

Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70, HSP90α and HSP90ß from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90ß were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90α and HSP90ß in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90α and HSP90ß were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70, HSP90α and HSP90ß in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach.

Comparative analysis of two ferritin subunits from blunt snout bream (Megalobrama amblycephala): Characterization, expression, iron depriving and bacteriostatic activity.

Iron is an essential microelement for almost all living organisms, while an excess of iron is toxic, thus maintenance of iron homeostasis is vital. As iron storage protein, ferritin plays an important role in iron metabolism. In the present study, we cloned and characterized the ferritin H subunit from Megalobrama amblycephala, termed as MamFerH. An iron-responsive element (IRE) was predicted in the 5' untranslated region (UTR) of MamFerH, while its bulge structural was different from that of the reported ferritin M subunit (MamFerM). The MamFerH and MamFerM genes exhibited similar expression patterns during early development with specifically high expression post hatching, whereas their tissue expression patterns were different. Specifically, MamFerM was highly expressed in the spleen, liver and kidney, while MamFerH was predominantly expressed in the blood and brain, indicating their different functions. In addition, the expression of the two genes was induced upon Aeromonas hydrophila infection at both transcriptional and translational levels, and MamFerH was more efficient. Immunohistochemistry and immunofluorescence analysis confirmed their significant changes at protein level and distribution in the liver post infection, indicating their participation in host immune response. Furthermore, bacteriostatic experiment revealed that recombinant MamFerH displayed more significant inhibitory effect on the growth of A. hydrophila.

Identification, ontogeny and expression analysis of a novel laboratory of genetics and physiology 2 (LGP2) transcript in Asian seabass, Lates calcarifer.

LGP2 (laboratory of genetics and physiology 2) is an important member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which plays a significant role in antiviral innate immunity. In this study, we have cloned the full-length cDNA sequence of LGP2 from Asian seabass, Lates calcarifer (AsLGP2). The complete AsLGP2 cDNA sequence consisted of 2586 nucleotides encoding a putative protein of 681 amino acids with a molecular mass of 77.6 kDa. From the AsLGP2 protein, four different conserved domains were predicted: a DExDc (DEAD/DEAH box helicase domain), a bacterial type III restriction enzyme domain (RES III), a HELICc (Helicase superfamily c-terminal domain and a RIG-I_C-RD (RIG-I C-terminal regulatory domain). The transcript of AsLGP2 could be detected in all the 11 tissues tested in healthy animals with high expression noticed in tissues facing external environment such as gill, hindgut and skin. The ontogenic expression profile of AsLGP2 implies a possible maternal transfer of this gene as it has been detected in all early embryonic developmental stages along with unfertilized eggs. Viral analogue, poly I:C, injection resulted in rapid up-regulated expression in different tissues with the highest modulation of expression observed in kidney followed by liver and gill. A rapid response of AsLGP2 expression was also observed in the different tissues of Vibrio alginolyticus-injected L. calcarifer, while significant change in expression was noticed following Staphylococcus aureus infection. Similarly, exposure to different pathogen-mimicking microbial analogues such as poly I:C, LPS and PGN resulted in enhanced expression of AsLGP2 in SISK cell-line. Taking together, these observations suggest that AsLGP2 can act as both antiviral and antibacterial cytosolic receptor and may play a significant role in embryonic and larval development in marine euryhaline teleosts like Asian seabass.

The use of rockfish Sebastiscus marmoratus as a sentinel species to assess petroleum hydrocarbons pollution: A case study in Quanzhou Bay, China.

To monitor the biological effects of marine pollution, choosing a native fish species and establishing suitable biomarkers are required. In this study, the full-length cDNA of cyp1a1 was cloned from Sebastiscus marmoratus (SM-CYP1A1). Then the dose-response and time-course induction of hepatic CYP1A1 mRNA by the crude oil water-soluble fraction (WSF) were determined. Subsequently, SM-CYP1A1 mRNA was applied to investigate the biological effect of petroleum hydrocarbon pollution in Quanzhou Bay, China. The transcription levels of hepatic CYP1A1 were significantly elevated in fish caged in the polluted sites for 2weeks compared with those of the reference site, which were correlated with the concentrations of petroleum hydrocarbon and polycyclic aromatic hydrocarbon (PAH) in the surface seawaters. The results suggest that S. marmoratus is a potential sentinel organism to monitor marine pollutants and the hepatic CYP1A1 mRNA can serve as a sensitive biomarker to organic xenobiotics in aquatic environments.

Molecular characterization and functional analysis of four teleostean K+ channels in macrophages of sea perch (Lateolabrax japonicas).

Potassium ion channels are one of the most diversely and widely distributed channels, which are involved in all kinds of physiological functions in both excitable and non-excitable cells. The expression of voltage-gated potassium ion (Kv) channels is highly variable according to the state of macrophages activation. Macrophages have an important function in innate immunity against intruding pathogens. They produce a variety of inflammatory and immunoactive molecules that modulate imflammatory responses. Here we show that blockade of K+ channels by non-selective Kv channel inhibitor tetraethylammonium chloride (TEA), and 4-aminopyridine (4-AP) inhibited proinflammatory cytokines expression, cell proliferation, and reactive oxygen species (ROS) production in LPS-stimulated macrophages of Sea perch (Lateolabrax japonicas). Then we isolated four Kv channels genes (spKv1.1, spKv1.2, spKv1.5 and spKv3.1) in LPS-activated fish macrophages. These channels genes were up-regulated after LPS stimulation except spKv3.1, which remained unchanged during the test. The results of this study indicate that Kv channels could be required for modulating the immune function of fish macrophages.

Copper-zinc-superoxide dismutase (CuZnSOD), an antioxidant gene from seahorse (Hippocampus abdominalis); molecular cloning, sequence characterization, antioxidant activity and potential peroxidation function of its recombinant protein.

Copper-zinc-superoxide dismutase (CuZnSOD) from Hippocampus abdominalis (HaCuZnSOD) is a metalloenzyme which belongs to the ubiquitous family of SODs. Here, we determined the characteristic structural features of HaCuZnSOD, analyzed its evolutionary relationships, and identified its potential immune responses and biological functions in relation to antioxidant defense mechanisms in the seahorse. The gene had a 5' untranslated region (UTR) of 67 bp, a coding sequence of 465 bp and a 3' UTR of 313 bp. The putative peptide consists of 154 amino acids. HaCuZnSOD had a predicted molecular mass of 15.94 kDa and a theoretical pI value of 5.73, which is favorable for copper binding activity. In silico analysis revealed that HaCuZnSOD had a prominent Cu-Zn_superoxide_dismutase domain, two Cu/Zn signature sequences, a putative N-glycosylation site, and several active sites including Cu(2+) and Zn(2+) binding sites. The three dimensional structure indicated a ß-sheet barrel with 8 ß-sheets and two short α-helical regions. Multiple alignment analyses revealed many conserved regions and active sites among its orthologs. The highest amino acid identity to HaCuZnSOD was found in Siniperca chuatsi (87.4%), while Maylandia zebra shared a close relationship in the phylogenetic analysis. Functional assays were performed to assess the antioxidant, biophysical and biochemical properties of overexpressed recombinant (r) HaCuZnSOD. A xanthine/XOD assay gave optimum results at pH 9 and 25 °C indicating these may be the best conditions for its antioxidant action in the seahorse. An MTT assay and flow cytometry confirmed that rHaCuZnSOD showed peroxidase activity in the presence of HCO3(-). In all the functional assays, the level of antioxidant activity of rHaCuZnSOD was concentration dependent; metal ion supplementation also increased its activity. The highest mRNA expressional level of HaCuZnSOD was found in blood. Temporal assessment under pathological stress showed a delay response by HaCuZnSOD. Our findings demonstrated that HaCuZnSOD is an important antioxidant, which might be involved in the host antioxidant defense mechanism against oxidative stress.

Molecular characterization and gene expression of ferritin in blunt snout bream (Megalobrama amblycephala).

Ferritins are conserved iron storage proteins that exist in most living organisms and play an essential role in iron homeostasis. In this study, we reported the identification and analysis of a ferritin middle-chain (M) subunit, MaFerM, from blunt snout bream, Megalobrama amblycephala. The full length cDNA of MaFerM contains a 5'-untranslated region (UTR) of 152 bp, an open reading frame (ORF) of 522 bp and a 3'-UTR of 270 bp. The ORF encodes a putative protein of 174 amino acids, which shares extensive sequence identities with the M ferritins of several fish species. In silico analysis identified both the ferroxidase center of mammalian heavy-chain (H) ferritins and the iron nucleation site of mammalian light-chain (L) ferritins in MaFerM. Quantitative real-time reverse transcription polymerase chain reaction analysis indicated that MaFerM expression was highest in the liver and lowest in the heart and responded positively to experimental challenges with Aeromonas hydrophila. The exposure of cultured M. amblycephala to treatment with stress inducers (iron and H2O2) significantly up-regulated the expression of MaFerM in a dose-dependent manner. Iron chelation analysis showed that recombinant MaFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that MaFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and immune stimulus.

A C-type lectin that inhibits bacterial infection and facilitates viral invasion in black rockfish, Sebastes schlegelii.

C-type lectins (CTLs) are important pattern recognition receptors (PRRs) that play vital roles in innate immunity. In teleosts, a number of CTLs have been reported, but their in vivo effects on host defense are still limited. In this study, a CTL homolog (SsLec1) was identified from black rockfish, Sebastes schlegelii, and its structure, expression and biological function was analyzed. The open reading frame of SsLec1 is 633 bp, with a 5'- untranslated region (UTR) of 36 bp and a 3'- UTR of 117 bp. The deduced amino acid sequence of SsLec1 shares the highest overall identity (73.20%) with the CTL of Oplegnathus fasciatus. SsLec1 possesses conserved CTL features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, the mannose-type carbohydrate-binding motif, the conserved calcium binding sites and a putative signal peptide. The expression of SsLec1 was highest in liver and could be induced by experimental infection with Listonella anguillarum. Recombinant SsLec1 (rSsLec1) purified from E. coli was able to bind and agglutinate the Gram-negative fish pathogens Vibrio ichthyoenteri and Vibrio vulnificus. The agglutinating ability of rSsLec1 was abolished in the presence of mannose or ethylenediaminetetraacetic acid. Further analysis showed that rSsLec1 could enhance phagocytosis by macrophages. In vivo experiments indicated that rSsLec1 could inhibit bacterial infection and promote viral invasion. Taken together, these results suggest that SsLec1 is a novel CTL that possesses apparent immunoregulation property and plays a critical role in host defense against pathogens invasion.

Genomic organization, expression and antimicrobial activity of a hepcidin from taimen (Hucho taimen, Pallas).

Hepcidin, an antimicrobial peptide, plays a crucial role in innate immune system of teleost fish. As a cysteine-rich peptide, hepcidin possesses a dual function including iron regulation and innate immunity. In the present study, a full-length hepcidin cDNA (HtHep) was cloned and characterized by RT-PCR and RACE techniques from taimen (Hucho taimen, Pallas), which is a type of rare, precious and cold-water fish species in China. The cDNA contains an open reading frame (ORF) of 267 bp encoding 88 amino acid (aa), with 170 bp located in the 5(') untranslated region (UTR) and 151 bp in the 3' UTR. The genomic sequences analysis showed that the HtHep gene consisted of three exons and two introns (with the length 94 and 251 bp, respectively). With a predicted molecular mass of 2881.4 Da and a theoretical pI of 8.53, the deduced amino acid encodes a signal peptide of 24 aa, prodomain of 39 aa and mature peptide of 25 aa. The signal peptidase (SA-VP) and the motif RX (K/R)R of propeptide convertase suggested the cleavage site of signal and mature peptide. Eight conserved cysteine residues were also identified and formed four disulfide bonds. Pair-wise alignments showed that HtHep clustered together with two fish species of Salmonidae family (Salmo salar and Oncorhynchus mykiss) in HAMP1 branch. Quantitative RT-PCR analysis indicated that the mRNA levels of HtHep were detected in a wide range of tissues and the highest level was detected in the liver. Its expression was also detected early during embryonic stage and could be up-regulated in the liver when challenged with pathogenic bacteria (Yersinia ruckeri). The recombinant HtHep (rHtHep) had antimicrobial activity against both gram-positive (Micrococcus lysodeikticus and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). Our results suggested that HtHep might be involved in the innate immune defense against bacterial pathogens in taimen.

Cloning and expression of two hepcidin genes in the mudskipper (Boleophthalmus pectinirostris) provides insights into their roles in male reproductive immunity.

The mudskipper Boleophthalmus pectinirostris is a burrow-dwelling fish inhabiting intertidal mudflats. During the spawning season, in a spawning chamber located at the center of their burrow, a pair of male and female fish mate and fertilized eggs adheres onto the inner walls and ceiling with filamentous attachments. During 5 days of incubation, the fertilized eggs are kept clean and hatch with a very high hatching rate under the natural conditions filled with microorganisms. This suggests that the male and/or female reproductive tract may synthesize antimicrobial substances to offer protection against microorganisms that may be deleterious to fertility. To study the antimicrobial strategy of this fish in the spawning season, we first cloned the two hepcidin isoforms from B. pectinirostris, and designated them as Hepcidin-1 and Hepcidin-2 based on phylogenetic analyses. Both of these hepcidin isoforms were highly expressed in the liver, but only Hepcidin-1 showed significant change in response to iron overload. Interestingly, these two hepcidin isoforms were expressed in male reproductive tracts, i.e. the testes and seminal vesicles. The monthly expression pattern indicated that Hepcidin-1 transcript levels showed a peak point only in March (before spawning) in the seminal vesicle, while Hepcidin-2 transcript levels were correlated with male reproductive status and reached their highest level in May (the peak spawning period). Under experimental conditions, the expression of these two hepcidin isoforms showed no response to iron overload in the male gonad. However, after lipopolysaccharide injection, the Hepcidin-1 transcript level was significantly up-regulated in the testes and seminal vesicle 6 h post injection, while Hepcidin-2 transcript levels exhibited a clear time-course dependent upregulation pattern and reached the highest levels 24 h post injection. More interestingly, after injection with LHRH-A3, the expression of Hepcidin-2 was significantly up-regulated in both testes and seminal vesicle. Results from in situ hybridization showed that Hepcidin-2 was expressed in the Leydig cells of the testes and in the epithelium of the seminal vesicle. Taken together, the results from our study indicated that these two hepcidin isoforms in the mudskipper may have different functions: Hepcidin-1 may play a dual role in both iron metabolism regulation in the liver and a short antimicrobial response in male reproductive tracts, while Hepcidin-2 is more specialized in reproductive immunity in male reproductive tracts.

Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP release in fish.

Identification of a retinoic acid-inducible gene I from Japanese eel (Anguilla japonica) and expression analysis in vivo and in vitro.

RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognition receptors (PRRs) for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). The full-length cDNA sequence of RIG-I (AjRIG-I) in Japanese eel (Anguilla japonica) was identified and characterized in this article. The full-length cDNA of AjRIG-I was 3468 bp, including a 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 617 bp and an open reading frame (ORF) of 2799 bp encoding a polypeptide of 933 amino acid residues with a calculated molecular mass of 106.2 kDa. NCBI CDD analysis showed that the AjRIG-I protein had the typical conserved domains, including two adjacent caspase activation and recruitment domains (CARDs), a DEXDc domain, a HELICc domain and a C-terminal regulatory domain (RD). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjRIG-I in a wide range of tissues, with the predominant expression in liver, followed by the gills, spleen, kidney, intestine, skin, and the very low expression in muscle and heart. The AjRIG-I expressions in liver, spleen and kidney were significantly induced following injection with LPS, the viral mimic poly I:C, and Aeromonas hydrophila infection. In vitro, the AjRIG-I transcripts of Japanese eel liver cells were significantly enhanced by poly I:C and PGN stimulation, down-regulated with CpG-DNA treatment whereas no change of the expression level was found post LPS challenge. These results collectively suggested AjRIG-I transcripts expression possibly play an important role in fish defense against viral and bacterial infection.

Cloning, characterization, and expression analysis of LGP2 cDNA from goose, Anser cygnoides.

Laboratory of genetics and physiology 2 ( LGP2: ) is a homologue of the retinoic acid inducible gene-I and melanoma differentiation associated gene 5 that lacks the caspase activation and recruitment domain required for signaling. It plays a pivotal role in host immune response. In this study, we cloned and characterized the full-length open reading frame ( ORF: ) sequence of LGP2 in the Qingyuan goose (Anser cygnoides) and evaluated the mRNA expression of this gene post infection with an H5N1 highly pathogenic avian influenza virus ( HPAIV: ). The full-length goose LGP2 ORF (2,028 bp) encoded a polypeptide of 675 amino acids. The deduced amino acid sequence contained 5 main overlapping structural domains-2 DEAD/DEAH box helicase domains, one conserved restriction domain of bacterial type III restriction enzyme, one helicase superfamily C-terminal domain and one C-terminal regulatory domain. Quantitative real-time PCR analysis indicated that goose LGP2 was constitutively expressed in all 19 investigated tissues, but the expression level was different among them. It was high expressed in the trachea, jejunum, bursa, kidney and heart, but low in the glandular stomach, lung, liver, spleen, crop and muscular stomach. A significant increase in the transcription of LGP2 was detected in the brain, spleen and lungs of geese post infection with H5N1 HPAIV versus uninfected tissues. These findings indicated that goose LGP2 was an important receptor that is involved in the host antiviral innate immune defense to H5N1 HPAIV in geese.

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 in vitro 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.

Molecular cloning, characterization and expression analysis of PPAR gamma in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge.

PPAR gamma was a key nuclear receptor, playing an important role in the immune defense and the anti-inflammatory mechanism. In this study, the full-length PPAR gamma (EcPPAR gamma) was obtained, containing a 5'UTR of 133 bp, an ORF of 1602 bp and a 3'UTR of 26 bp besides the poly (A) tail. The EcPPAR gamma gene encoded a protein of 533 amino acids with an estimated molecular mass of 60.02 KDa and a predicted isoelectric point (pI) of 6.26. The deduced amino acid sequence showed that EcPPAR gamma consisted of the conserved residues and the domains known to be critical for the PPAR gamma function. The quantitative real-time PCR analysis revealed that EcPPAR gamma transcript was expressed in all the examined tissue, while the strong expression was observed in intestine, followed by the expression in liver, gill, spleen heart, kidney and muscle. Vibrio challenge could stimulate the inflammatory response in grouper and induce a sharp increase of pro-inflammatory cytokines expression, lipid peroxidation and DNA damage, while the up-regulation of vibrio-induced inflammation could also increase the non-specific immune defense. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPPAR gamma transcript in immune tissues. Subcellular localization analysis revealed that EcPPAR gamma was distributed in the nucleus. Furthermore, overexpression of EcPPAR gamma could down-regulated the expression of IL1b, IL6, TNF1 and TNF2. In addition, the administration of PPAR gamma antagonist, GW9662, could up-regulate the expression of pro-inflammatory genes, including IL1b, IL6, TNF1 and TNF2. Together, these results indicated that EcPPAR gamma serving as a negative regulator of pro-inflammatory cytokines may play an important role in the immune defense against vibrio-induced inflammation in grouper.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish.

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-ß promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1ß and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

Molecular cloning, tissue distribution and expression analysis of a manganese superoxide dismutase in blunt snout bream Megalobrama amblycephala.

The full-length mitochondrial manganese superoxide dismutase cDNA of blunt snout bream Megalobrama amblycephala (denoted as MamMnSOD) was identified in liver using homology cloning and rapid amplification of cDNA ends. The full-length cDNA of MamMnSOD consisted of 986 bp, with an open reading frame encoding 224 amino acids, a 58-bp 5' untranslated region and a 256-bp 3' untranslated region. The deduced amino acid sequences of MamMnSOD showed high sequence homology to mitochondrial MnSODs from crustaceans. Several motifs, including three mitochondrial MnSOD signatures, amino acid residues responsible for coordinating the manganese, and the putative active center, were almost completely conserved in the deduced amino acid sequences of MamMnSOD. The mRNA expression of MamMnSOD in the tissues of heart, liver, spleen, kidney, muscle, intestine, and gill was examined by quantitative real-time PCR; the highest expression was in the liver. Transcription of MamMnSOD was kinetically modulated in response to nitrite stress in liver and gill tissues. The purified recombinant MamMnSOD showed potent antioxidant activity. Polyclonal antibodies generated from the recombinant product of MamMnSOD were used to specifically identify the native protein in liver of M. amblycephala. Collectively, the findings of this study strongly suggested that MamMnSOD combats oxidative stress and cellular damage induced by nitrite, by detoxifying harmful reactive oxygen species in M. amblycephala.