Molecular cloning,characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides): Serum amyloid A

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.     

miR-homoHSV of Singapore Grouper Iridovirus (SGIV) Inhibits Expression of the SGIV Pro-apoptotic Factor LITAF and Attenuates Cell Death

Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-α (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.     

Singapore grouper iridovirus,a large DNA virus,induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling

Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (ΔΨm) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.     

Characterization of an envelope gene VP19 from Singapore grouper iridovirus

Background

Viral envelope proteins are always proposed to exert important function during virus infection and replication. Vertebrate iridoviruses are enveloped large DNA virus, which can cause great economic losses in aquaculture and ecological destruction. Although numerous iridovirus envelope proteins have been identified using bioinformatics and proteomic methods, their roles in virus infection remained largely unknown.

Methods

Using SMART and TMHMM programs, we investigated the structural characteristics of Singapore grouper iridovirus (SGIV) VP19. A specific antibody against VP19 was generated and the expression profile of VP19 was clarified. The subcellular localization of VP19 in the absence or presence of other viral products was determined via transfection and immune fluorescence assay. In addition, Western blot assay and electron microscopy examination were performed to demonstrate whether SGIV VP19 was an envelope protein or a capsid protein.

Results

Here, SGIV VP19 was cloned and characterized. Among all sequenced iridoviruses, VP19 and its orthologues shared common features, including 19 invariant cysteines, a proline-rich motif and a predicted transmembrane domain. Subsequently, the protein synthesis of VP19 was only detected at the late stage of SGIV infection and inhibited obviously by treating with AraC, confirming that VP19 was a late expressed protein. Ectopic expression of EGFP-VP19 in vitro displayed a punctate pattern in the cytoplasm. In SGIV infected cells, the newly synthesized VP19 protein was initially localized in the cytoplasm in a punctate pattern, and then aggregated into the virus assembly site at the late stage of SGIV infection, suggesting that other viral protein products were essential for VP19’s function during SGIV infection. In addition, Western blot assay and electron microscopy observation revealed that SGIV VP19 was associated with viral envelope, which was different from major capsid protein (MCP).

Conclusion

Taken together, the current data suggested that VP19 represented a conserved envelope protein in iridovirus, and might contribute greatly to virus assembly during virus infection.

     

Establishment and characterization of a brain‐cell line from kelp grouper Epinephelus moara

A new brain‐cell line, EMB, was developed from kelp grouper Epinephelus moara, a cultured marine fish. The EMB cells were subcultured for more than 60 passages. The cells were cultured in Leibovitz's L‐15 medium (L15) supplemented with antibiotics, foetal bovine serum (FBS), 2‐mercaptoethanol (2‐ME) and basic fibroblast growth factor (bFGF). The cells could grow at 18–30° C, with the maximum growth between 24 and 30° C. The optimum FBS concentration for the cells growth ranged between 15 and 20%. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 45. After being transfected with pEGFP‐N3 plasmid, the cells could successfully express green fluorescence protein (GFP), implying that this cell line can be used for transgenic studies. A significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV) and the viral replication was confirmed by quantitative real‐time PCR (qrt‐PCR) assay, which suggested EMB's application potential for studies of SGIV and RGNNV.     

Antiviral effects of β-defensin derived from orange-spotted grouper (Epinephelus coioides)

Defensins are a group of small antimicrobial peptides playing an important role in innate host defense. In this study, a β-defensin cloned from liver of orange-spotted grouper, Epinephelus coioides, EcDefensin, showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (VNNV). The expression profiles of EcDefensin were significantly (P < 0.001) up-regulated after challenging with Lipopolysaccharide (LPS), SGIV and Polyriboinosinic Polyribocytidylic Acid (polyI:C) in vivo. Immunofluorescence staining observed its intracellular innate immune response to viral infection of SGIV and VNNV. EcDefensin was found to possess dual antiviral activity, inhibiting the infection and replication of SGIV and VNNV and inducting a type I interferon-related response in vitro. Synthetic peptide of EcDefensin (Ec-defensin) incubated with virus or cells before infection reduced the viral infectivity. Ec-defensin drastically decreased SGIV and VNNV titers, viral gene expression and structural protein accumulation. Grouper spleen cells over-expressing EcDefensin (GS/pcDNA-EcDefensin) support the inhibition of viral infection and the upregulation of the expression of host immune-related genes, such as antiviral protein Mx and pro-inflammatory cytokine IL-1β. EcDefensin activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. Reporter genes of IFN-Luc and ISRE-Luc were significantly up-regulated in cells transfected with pcDNA-EcDefenisn after infection with SGIV and VNNV. These results suggest that EcDefensin is importantly involved in host immune responses to invasion of viral pathogens, and open the new avenues for design of antiviral agents in fisheries industry.     

Rapid and sensitive detection of Singapore grouper iridovirus by loop-mediated isothermal amplification

Aims:  The aim of this paper was to develop a loop-mediated isothermal amplification (LAMP) method for rapid, sensitive and inexpensive detection of Singapore grouper iridovirus (SGIV) in grouper (GP), Epinephelus sp.
Methods and Results:  A set of six specific primers was designed by targeting the SGIV ORF-014L. With Bst DNA polymerase large fragment, the target DNA can be amplified as early as 20 min at 65°C in a simple water bath. The detection limit is about 0·02 fg (equivalent to 6·3 copies) of plasmid ORF-014L. LAMP products could be judged with three different methods. There were no cross-reactions with seven other aquatic animal viruses indicating high specificity of the LAMP. The LAMP method was applied to detect SGIV in virus-infected GP cells and GP tissues effectively.
Conclusions:  The LAMP described in this study is a cheap, sensitive, specific and rapid protocol for the detection of SGIV in cells and in GP tissues.
Significance and Impact of the Study:  The developed LAMP method can be simply applied both in field condition and in laboratory operation for specific detection of SGIV infection.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

Subcellular redistribution and sequential recruitment of macromolecular components during SGIV assembly

Virus infection consists of entry, synthesis of macromolecular components, virus assembly and release. Understanding of the mechanisms underlying each event is necessary for the intervention of virus infection in human healthcare and agriculture. Here we report the visualization of Singapore grouper iridovirus (SGIV) assembly in the medaka haploid embryonic stem (ES) cell line HX1. SGIV is a highly infectious DNA virus that causes a massive loss in marine aquaculture. Ectopic expression of VP88GFP, a fusion between green fluorescent protein and the envelope protein VP088, did not compromise the ES cell properties and susceptibility to SGIV infection. Although VP88GFP disperses evenly in the cytoplasm of non-infected cells, it undergoes aggregation and redistribution in SGIV-infected cells. Real-time visualization revealed multiple key stages of VP88GFP redistribution and the dynamics of viral assembly site (VAS). Specifically, VP88GFP entry into and condensation in the VAS occurred within a 6-h duration, a similar duration was observed also for the release of VP88GFP-containing SGIV out of the cell. Taken together, VP088 is an excellent marker for visualizing the SGIV infection process. Our results provide new insight into macromolecular component recruitment and SGIV assembly.     

Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina

A large icosahedral virus was isolated from diseased grouper Epinephelus tauvina. The virus grew well in several cultured fish cell lines, with stable and high infectivity after serial passages in grouper cell line (GP). The virus was sensitive to both acid and heat treatments. Virus replication was inhibited by 5-iodo-2-deoxyuridine (IUDR), indicative of a DNA-containing genome. The virus infectivity was reduced with ether treatment, suggesting that the virus was lipid-enveloped. Electron micrographs showed abundant cytoplasmic icosahedral virons in the virus-infected GP cells. The size of the intracellular nucleocapsid was 154 nm between the opposite sides, or 176 nm between the opposite vertices with an inner electron-dense core of 93 nm. Virus particles were released through budding from plasma membranes with a size of 200 nm in diameter. SDS-PAGE of purified virus revealed 20 structural protein bands and a major capsid protein (MCP) of 49 kDa. A DNA fragment of approximately 500 nucleotides was successfully amplified by polymerase chain reaction (PCR) using the primers from conserved regions of the MCP gene of frog virus 3 (FV3), the type species of Ranavirus. Subsequent multiple alignment and phylogenetic analysis showed that the newly isolated grouper virus was closely related to largemouth bass virus (LMBV), FV3 and Regina ranavirus (RRV). Our data suggests that the virus isolate is a novel member of genus Ranavirus, family Iridoviridae. We tentatively name the virus as Singapore grouper iridovirus (SGIV). SGIV was able to cause serious systemic disease capable of killing 96% of grouper fry.