Identification and characterization of lncRNAs and the interaction of lncRNA-mRNA in Epinephelus coioides induced with Singapore grouper iridovirus infection.

Singapore grouper iridovirus (SGIV) is a highly pathogenic double-stranded DNA virus, and the fatality rate of SGIV-infected grouper is more than 90%. Up to now, there is no effective methods to control the disease. Long non-coding RNAs (lncRNAs) might play an important role in individual growth and development, immune regulation and other life processes. In this study, lncRNAs were identified in Epinephelus coioides, an important economic aquaculture marine fish in China and Southeast Asia, and the regulatory relationships of lncRNAs and mRNA response to SGIV infection were analyzed. A total of 11,678 lncRNAs were identified and classified from the spleen and GS (grouper spleen) cells. 105 differentially expressed lncRNAs (DElncRNAs) were detected during SGIV infection. The lncRNAs and the regulated mRNAs were analyzed using co-expression network, lncRNA target gene annotation and GO enrichment. At 24 and 48 h after SGIV infection, 118 and 339 lncRNA-mRNA pairs in GS cells were detected, and 728 and 688 differentially expressed lncRNA-mRNA pairs in spleen were obtained, respectively. GO and KEGG were used to predict the DE lncRNAs' target genes, and deduce the DE lncRNAs-affected signaling pathways. In GS cells, lncRNAs might participate in cell part, binding and catalytic activity; and lncRNAs might be involved in immune system process and transcription factor activity in spleen. These data demonstrated that lncRNAs could regulate the expression of immune-related genes response to viral infection, and providing a new insight into understanding the complexity of immune regulatory networks mediated by lncRNAs during viral infection in teleost fish.

Characterization and function analysis of Epinephelus coioides Hsp40 response to Vibrio alginolyticus and SGIV infection.

Heat shock protein 40 (Hsp40), a member of Heat shock proteins (Hsps) family, plays a crucial role in regulation of cell proliferation, survival and apoptosis in mammals. In this study, Hsp40, EcHsp40, was identified from Epinephelus coioides, an economically important marine-cultured fish in China and Southeast Asian counties. The full length of EcHsp40 was 2236 bp in length containing a 1026 bp open reading frame (ORF) encoding 341 amino acids, with a molecular mass of 37.88 kDa and a theoretical pI of 9.09. EcHsp40 has two conserved domains DnaJ and DnaJ_C. EcHsp40 mRNA was detected in all tissues examined, and the expression was significantly up-regulated response to challenged with Vibrio alginolyticus or Singapore grouper iridovirus (SGIV), one of the important pathogens of marine fish. EcHsp40 was distributed in both the cytoplasm and nucleus, over-expression of EcHsp40 can inhibit the activity of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), significantly promote SGIV-induced apoptosis, intracellular caspase-3 activity and viral replication, suggesting that the EcHsp40 may play an important role in pathogenic stimulation.

Molecular characterization and function analysis of Epinephelus coioides Hsp22 response to SGIV and Vribro alginolyticus infection.

Heat shock protein 22 (Hsp22) is an important regulatory factor response to various stresses in mammals. In this study, the full length cDNA of Epinephelus coioides Hsp22, which was 1680bp in length, with a 289 bp 5' UTR, a 725 bp 3'UTR, and a 666 bp open reading frame encoding 221 amino acids, was obtained. E. coioides Hsp22 contains a highly conserved α-crystallin domain. E. coioides Hsp22 mRNA was detected in all tissues examined by quantitative real-time PCR, with the highest expression in blood, followed by the spleen, skin, gill, head kidney, muscle, heart, liver, trunk kidney, stomach, pyloric caeca, intestine, brain and thymus. The expression patterns of E. coioides Hsp22 response to infection with Singapore grouper iridovirus (SGIV) and Vribro alginolyticus, the important pathogens of E. coioides, were studied. The expression levels of the gene were up-regulated in the tissues examined. Subcellular localization analysis demonstrated that E. coioides Hsp22 was distributed in both the cytoplasm and nucleus. In addition, E. coioides Hsp22 significantly inhibited the SGIV-induced cell apoptosis. In summary, the E. coioides Hsp22 might play a critical role in pathogenic stimulation.

Effects of antimicrobial peptides on serum biochemical parameters, antioxidant activity and non-specific immune responses in Epinephelus coioides.

Antimicrobial peptides (AMPs) are small proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens (bacteria, fungi and viruses). In this study, the effects of AMPs from Bacillus subtilis on Epinephelus coioides were examined. E. coioides were fed with diets containing AMPs (0, 100, 200, 400 or 800 mg/kg) for four weeks. Results showed that the levels of total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose (GLU) and lipopolysaccharide (LPS) in the serum of E. coioides changed than those of the control group; compared to the control group, the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and lysozyme (LZM) levels in E. coioides fed with different dosages AMP diets were also different; in addition, the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin-1-beta (IL-1ß), and heat shock protein 90 (Hsp90) in the tissues of E. coioides were measured, the three genes in the tissues examined were significantly upregulated. The results demonstrated that diets containing AMPs can enhance the antioxidant capacity and innate immune ability of E. coioides, indicating that AMPs might be a potential alternative to antibiotics in E. coioides.

A novel MKK gene (EcMKK6) in Epinephelus coioides: Identification, characterization and its response to Vibrio alginolyticus and SGIV infection.

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.

Integrated multi-omics analysis reveals liver metabolic reprogramming by fish iridovirus and antiviral function of alpha-linolenic acid.

Iridovirus poses a substantial threat to global aquaculture due to its high mortality rate; however, the molecular mechanisms underpinning its pathogenesis are not well elucidated. Here, a multi-omics approach was applied to groupers infected with Singapore grouper iridovirus (SGIV), focusing on the roles of key metabolites. Results showed that SGIV induced obvious histopathological damage and changes in metabolic enzymes within the liver. Furthermore, SGIV significantly reduced the contents of lipid droplets, triglycerides, cholesterol, and lipoproteins. Metabolomic analysis indicated that the altered metabolites were enriched in 19 pathways, with a notable down-regulation of lipid metabolites such as glycerophosphates and alpha-linolenic acid (ALA), consistent with disturbed lipid homeostasis in the liver. Integration of transcriptomic and metabolomic data revealed that the top enriched pathways were related to cell growth and death and nucleotide, carbohydrate, amino acid, and lipid metabolism, supporting the conclusion that SGIV infection induced liver metabolic reprogramming. Further integrative transcriptomic and proteomic analysis indicated that SGIV infection activated crucial molecular events in a phagosome-immune depression-metabolism dysregulation-necrosis signaling cascade. Of note, integrative multi-omics analysis demonstrated the consumption of ALA and linoleic acid (LA) metabolites, and the accumulation of L-glutamic acid (GA), accompanied by alterations in immune, inflammation, and cell death-related genes. Further experimental data showed that ALA, but not GA, suppressed SGIV replication by activating antioxidant and anti-inflammatory responses in the host. Collectively, these findings provide a comprehensive resource for understanding host response dynamics during fish iridovirus infection and highlight the antiviral potential of ALA in the prevention and treatment of iridoviral diseases.

Relationship of environmental factors in pond water and dynamic changes of gut microbes of sea bass Lateolabrax japonicus.

The effect of structure of gut microbes on the health of host has attracted increasing attention. Sea bass Lateolabrax japonicus is an important farmed fish in China. The relationship of the dynamic changes of intestinal bacterial communities in L. japonicus and the cultural water environment is very important for healthy culture. Here, the diversity and abundance of the gut microbial communities of L. japonicus were evaluated during the culture using 16S rRNA Illumina sequencing. Both the opportunistic pathogens Aeromonas (1.68%), Vibrio (1.59%), and Acinetobacter (1.22%); and the potential probiotics Lactobacillus (2.27%), Bacillus (1.16%), and Lactococcus (0.37%) were distributed in the gut of L. japonicus. The increasing concentration of nitrogen of water environments with the increase of culture time significantly correlated with shifts in the microbial community structure: 40.04% of gut microbial changes due to nitrogen concentration. Higher concentrations of nitrogen showed a significantly negative correlation with intestinal probiotics in L. japonicus. The results indicate that the abundance of intestinal bacteria of L. japonicus is mainly driven by the changes of environmental factors (e.g., nitrogen), and it's very important that the linking environmental parameters with bacterial data of guts could be used as an early warning indicator in L. japonicus heath culture.

Molecular cloning and characterization of two novel hepcidins from orange-spotted grouper, Epinephelus coioides.

Orange-spotted grouper, Epinephelus coioides is one of the most important economic species of marine-cultured fish in China and Southeast Asia countries. However, very little information of the innate immune mechanisms against microbial pathogens is available in grouper, Epinephelus sp. Hepcidin, as an antimicrobial peptide (AMP), is a very important component in the innate immune system and widespread in fish. In this study, two novel types of hepcidin gene (designated EC-hepcidin1 and EC-hepcidin2) were cloned from E. coioides. They consist of open reading frames (ORFs) of 267 bp and 263 bp encoding the putative peptides of 88 and 87 amino acids, respectively. The homologous identity of deduced amino acid sequences between EC-hepcidin1 and EC-hepcidin2 is up to 79%, and predicted mature regions of both them have four cysteines residues. Genomic DNAs of both EC-hepcidin1 and EC-hepcidin2 consist of three exons and two introns. RT-PCR results showed that EC-hepcidin1 transcript was most abundant in liver and less in stomach. However, the transcript of EC-hepcidin2 was only detected in liver. The expressions of both EC-hepcidins were up-regulated by microbial and viral challenges, and iron overload, respectively, and EC-hepcidin1 was more responsive. The growth of Gram-negative bacterium of Vibrio vulnificus and Gram-positive bacterium of Staphylococcus aureus was inhibited by synthetic EC-hepcidins, and EC-hepcidin1 displayed stronger antimicrobial activity. The replication of Singapore grouper iridovirus (SGIV) was inhibited in the EC-hepcidin1 and EC-hepcidin2 over-expressed stable transfected fish cell lines (GS/pcDNA-Hep1, GS/pcDNA-Hep2) indicative of the antiviral activity of EC-hepcidins. These data should offer important information on the antimicrobial and antiviral roles of EC-hepcidins, and will be help to the better understanding of molecular mechanisms of grouper innate immunity.

Molecular cloning, inducible expression with SGIV and Vibrio alginolyticus challenge, and function analysis of Epinephelus coioides PDCD4.

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

The Roles of Epinephelus coioides miR-122 in SGIV Infection and Replication.

In mammals, mature miR-122 is 22 nucleotides long and can be involved in regulating a variety of physiological and biological pathways. In this study, the expression profile and effects of grouper Epinephelus coioides miR-122 response to Singapore grouper iridovirus (SGIV) infection were investigated. The sequences of mature microRNAs (miRNAs) from different organisms are highly conserved, and miR-122 from E. coioides exhibits high similarity to that from mammals and other fish. The expression of miR-122 was up-regulated during SGIV infection. Up-regulation of miR-122 could significantly enhance the cytopathic effects (CPE) induced by SGIV, the transcription levels of viral genes (MCP, VP19, LITAF and ICP18), and viral replication; reduce the expression of inflammatory factors (TNF-a, IL-6, and IL-8), and the activity of AP-1 and NF-κB, and miR-122 can bind the target gene p38α MAPK to regulate the SGIV-induced cell apoptosis and the protease activity of caspase-3. The results indicated that SGIV infection can up-regulate the expression of E. coioides miR-122, and up-regulation of miR-122 can affect the activation of inflammatory factors, the activity of AP-1 and NF-κB, and cell apoptosis to regulate viral replication and proliferation.

Nuclear-export-signal-dependent protein translocation of dUTPase encoded by Singapore grouper iridovirus.

The dUTPase is a ubiquitous and crucial enzyme responsible for regulating cellular levels of dUTP. In the present study, the expression pattern and translocation of a dUTPase homolog encoded by Singapore grouper iridovirus (SGIV) were elucidated. The SGIV ORF049R encodes a dUTPase homolog, which is a peptide of 155 amino acids that contains five conserved motifs. The temporal expression pattern during infection in vitro revealed that the SGIV dUTPase was an early transcript. A leucine-rich nuclear export signal (NES) at the C-terminus was predicted using CBS Online Servers. Subcellular location analysis showed that SGIV dUTPase is a cytoplasmic protein. Site-direct mutagenesis by overlap extension-PCR indicated that the NES is crucial for the translocation of SGIV dUTPase from the nucleus to the cytoplasm. We have discovered for the first time that the NES-dependent translocation of dUTPase is different for SGIV than for members of other species, which depend on a nuclear localization signal. These results provide new insights into the pathogenesis of fish iridoviruses.

Identification and characterization of a novel capsid protein encoded by Singapore grouper iridovirus ORF038L.

Singapore grouper iridovirus (SGIV) is an important pathogen isolated from grouper, Epinephelus tauvina, and characterized as a novel ranavirus. To better understand the function of viral structural genes involved in SGIV infection and virus-host interactions, a candidate gene, VP38 (ORF038L), was investigated in this study. SGIV VP38 was found to encode a 170-aa peptide containing an RGD motif, and it showed significant identity only to members of the genus Iridovirus, family Iridoviridae, except megalocytivirus. The VP38 gene was identified by temporal expression pattern analysis and drug inhibition assay as a late (L) gene. Immunofluorescence localization revealed that P38 was distributed predominately in the cytoplasm and that association of VP38 with viral factories increased at the late stage of SGIV infection. Consistent results from immunoelectron microscopy (IEM) and western blot analysis revealed that SGIV VP38 is a viral capsid protein. Furthermore, antibodies specific for SGIV VP38 exhibited substantial SGIV-neutralizing activity in vitro, suggesting that VP38 might play an important role in SGIV infectivity.

Prevention of in-stent restenosis with endothelial progenitor cell (EPC) capture stent placement combined with regional EPC transplantation: An atherosclerotic rabbit model.

BACKGROUND: Even with drug-eluting stents, the risk of in-stent restenosis (ISR) remains high. The goal of this study was to investigate the use of an endothelial progenitor cell (EPC) capture stent plus regional EPC transplantation to reduce the ISR rate. METHODS: Endothelial progenitor cell capture stents were fabricated using fibrin gel and anti-CD34 plus anti-VEGFR-2 dual antibodies. Twenty male New Zealand white rabbits established as an atherosclerotic model were randomly divided into two groups: group 1 (n = 10), in which EPC capture stents were deployed into the right iliac artery; and group 2 (n = 10), in which sirolimus-eluting stents were placed. In both groups, EPCs were transplanted into target vessels beyond the stents, with outflow blocked. Radiologic-pathologic correlation outcomes were reviewed after 2 months. RESULTS: The technical success rate of EPC capture stent placement plus EPC transplantation was 100%. The ISR rate in group 1 was lower than in group 2 (1/10 vs. 4/10; p > 0.05). Minimal luminal diameters were larger in group 1 than in group 2 (computed tomographic angiography, 1.85 ± 0.15 mm vs. 1.50 ± 0.20 mm; duplex ultrasound, 1.90 ± 0.10 mm vs. 1.70 ± 0.30 mm; p > 0.05). Transplanted EPCs were tracked positively only in group 1. Pathologic analysis demonstrated neointimal hyperplasia thickness of 0.21 ± 0.09 mm in group 1 vs. 0.11 ± 0.07 mm in group 2 (p < 0.05). CONCLUSION: Endothelial progenitor cell capture stent placement plus local EPC transplant decreases the ISR rate through thrombosis reduction rather than through neointimal hyperplasia inhibition.

Percutaneous mechanical thrombectomy for acute massive lower extremity deep venous thrombosis.

PURPOSE: To evaluate the clinical safety and effectiveness of percutaneous mechanical thrombectomy in patients with acute massive lower extremity deep venous thrombosis. MATERIALS AND METHODS: Twenty-five consecutive patients with acute massive lower extremity deep venous thrombosis were included in this retrospective study. An inferior vena cava filter was placed prophylactically to protect against pulmonary embolism in each patient. Percutaneous mechanical thrombectomy was performed using a 7F Amplatz thrombectomy device in an angiography suite through ipsilateral popliteal vein access. Anticoagulant therapy lasted for at least 6 months. Follow-up data included 1 year's color duplex sonography and clinical interviews. RESULTS: Successful placement of an inferior vena cava filter was achieved in all 25 (100%) patients. Twenty-two patients (88%) were clinically asymptomatic within 24 hours, whereas the remaining 3 patients (12%) showed moderate improvement within 48 hours. Venogram at discharge showed grade III lysis in 23 patients (92%) and grade II lysis in 2 patients (8%). No serious complications were reported during hospitalization in this study. At 1-year follow-up, no recurrent deep venous thrombosis was reported; 1 patient developed a mild postthrombotic syndrome. CONCLUSION: Percutaneous mechanical thrombectomy is a safe and effective treatment for acute massive lower extremity deep venous thrombosis and shows promising clinical mid-term results.

Percutaneous mechanical thrombectomy combined with catheter-directed thrombolysis in the treatment of symptomatic lower extremity deep venous thrombosis.

PURPOSE: To evaluate the efficacy of percutaneous mechanical thrombectomy (PMT) combined with catheter-directed thrombolysis (CDT) in the treatment of massive symptomatic lower limb deep venous thrombosis (DVT). MATERIALS AND METHODS: One hundred and three clinically confirmed DVT patients were discharged from our institution. Sixteen patients with massive lower limb DVT were included in this retrospective study. After prophylactic placement of inferior vena cava filters (IVCFs), percutaneous mechanical thrombectomy (ATD, n=10; Straub, n=6) and catheter-directed thrombolysis were performed in all patients. Complementary therapy included percutaneous transluminal venous angioplasty (PTA, n=3) and stent placement (n=1). The doses of thrombolytic agents, length of hospital stay, peri-procedure complications and discharge status were reviewed. Oral anticoagulation was continued for at least 6 months during follow-up. RESULTS: The average hospital stay was 7 days. The technical success rate (complete and partial lysis of clot) was 89%, the other 11% patients only achieved less than 50% clot lysis. The mean dose of urokinase was 3.3 million IU. There were no significant differences of clinical outcome between the ATD and Straub catheter group. The only major complication was an elderly male who experienced a fatal intracranial hemorrhage while still in the hospital (0.97%, 1/103). Minor complications consisted of three instances of subcutaneous bleeding. No transfusions were required. Vascular patency was achieved in 12 limbs during follow-up. No pulmonary emboli occurred. There is one recurrent DVT 4.5 months after the treatment. CONCLUSIONS: Percutaneous mechanical thrombectomy combined with catheter-directed thrombolysis is an effective and safe method for the treatment of symptomatic DVT. A randomized prospective study is warranted.

Identification and characterization of a novel envelope protein in Rana grylio virus.

Viral envelope proteins have been proposed to play significant roles in virus infection and assembly. In this study, an envelope protein gene, 53R, was cloned and characterized from Rana grylio virus (RGV), a member of the family Iridoviridae. Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blot detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found an association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R-GFP fusion protein in pEGFP-N3/53R-transfected cells. Furthermore, detergent extraction and Western blot detection confirmed that RGV 53R was associated with virion membrane. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that it may play an important role in virus assembly. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells.

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Deltapsim collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-kappaB activation and intracellular Ca(2+) increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.