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.

Selection of reference genes for quantitative real-time RT-PCR on gene expression in Golden Pompano (Trachinotus ovatus).

Golden pompano (Trachinotus ovatus) is an important economically fish species. In this study, with an aim to identify reliable reference genes for quantitative real-time PCR (qRT-PCR) in golden pompano, we evaluated the expression stability of eight housekeeping genes in the presence and absence of poly I:C stimulation in eight tissues. The PCR data was analyzed by geNorm and NormFinder algorithms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations. When under normal physiological condition, geNorm and NormFinder identified B2M and 18S as suitable genes. When studying gene expression under conditions of poly I:C stimulation, the selection of the internal controls should be selected on a tissue basis. At 12 h stimulation, geNorm ranked Actin/UBCE, Actin/B2M, UBCE/B2M, Actin/UBCE, RPL13/B2M, UBCE/GAPDH, B2M/RPL13, and UBCE/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, intestine, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 48 h stimulation. Taken together, these results indicate that B2M and 18S are the most stable gene across tissue types under normal physiological conditions. However, during poly I:C stimulation, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types. If one gene is preferred, B2M, B2M, UBCE, Actin, B2M/RPL13, B2M, B2M, and RPL13 may be used in spleen, kidney, liver, gill, intestine, brain, muscle, and heart of golden pompano, respectively.

Establishment of a new cell line from the heart of giant grouper, Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility.

A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC50 ) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 µg mL(-1), respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.

[Risk factors for syncope in children with severe idiopathic pulmonary arterial hypertension].

Objective: To explore the risk factors for syncope in children with severe idiopathic pulmonary arterial hypertension (IPAH). Methods: Forty-four patients (age<18 years) with IPAH admitted to the Department of Pediatric Cardiology, Beijing Anzhen Hospital between May 2011 and October 2021 were retrospectively included. Patients were devided into syncope group and non-syncope group. Clinical manifestation and hemodynamic parameters including echocardiography, blood tests, right heart catheterization and acute pulmonary vascular expansion test were compared between two groups. Comparisons between groups were performed with unpaired Student t test, or Mann-Whitney U test or chi-square test. Logistic regression was used to calculate the odds ratio (OR) for parameters with statistically significant differences between groups and analyze the statistical correlation. Results: Among the 44 patients, 16 were males, the onset age was (7.2±3.9) years. Twenty-four (55%) children presented with 1 to 11 times of episodes of syncope, and 18 cases of whom induced by physical activity. Syncope group had a larger proportion of New York Heart Association (NYHA) heart function class Ⅲ-Ⅳ (67% (16/24) vs. 25% (5/20), χ2=7.59, P=0.006), higher brain natriuretic peptide (BNP) value ((251±39) vs. (61±40) pg/L, t=-2.18, P=0.035), higher pulmonary-to-aorta diameter ratio (1.6±0.4 vs. 1.4±0.2, t=-2.25, P=0.030) and larger pulmonary vascular resistance index ((22±11) vs. (16±7) WU/m2, t=-2.13, P=0.039) compared with non-syncope group. The proportion of patent foramen ovale (4% (1/24) vs. 45% (9/20), χ2=10.36, P=0.001), left ventricular ejection fraction (LVEF) ((68±5)% vs. (72±8)%, t=2.23, P=0.031) and the positive rate of acute pulmonary vascular expansion test (8% (2/24) vs. 35% (7/20), χ2=4.77, P=0.029) of syncope group were significantly lower than those of non-syncope group. Multiple Logistic regression analysis showed that NYHA heart function Ⅲ-Ⅳ (OR=6.787, 95%CI 1.445-31.880), pulmonary vascular resistance index (OR=1.247, 95%CI 1.020-1.525) and BNP (OR=1.036, 95%CI 1.007-1.066) were independent risk factors for syncope. The patent foramen ovale (OR=0.010, 95%CI 0.000-0.586) was a protective factor for syncope. Conclusions: NYHA cardiac function grade, pulmonary vascular resistance index and BNP are independent risk factors for syncope. Patent foramen ovale is a protective factor for syncope. Exercise is the main inducement of syncope in children with IPAH.

Molecular cloning and characterization of CD63 in common carp infected with koi herpesvirus.

CD63 is a member of the four-transmembrane-domain protein superfamily and is the first characterized tetraspanin protein. In the present study, we cloned the common carp (Cyprinus Carpio) CD63 (ccCD63) sequence and found that the ccCD63 ORF contained 711 bp and encoded a protein of 236 amino acids. Homology analysis revealed that the complete ccCD63 sequence had 84.08% amino acid similarity to CD63 of Sinocyclocheilus anshuiensis. Subcellular localization analysis revealed that ccCD63 was localized in the cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis indicated that ccCD63 was expressed in the gill, intestine, liver, spleen, brain and kidney, with higher expression in spleen and brain tissues than in the other examined tissues. After koi herpesvirus (KHV) infection, these tissues exhibited various expression levels of ccCD63. The expression level was the lowest in the liver and highest in the brain; the expression level in the brain was 8.7-fold higher than that in the liver. Furthermore, knockdown of ccCD63 promoted KHV infection. Moreover, ccCD63 was correlated with the regulation of RIG-I/MAVS/TRAF3/TBK1/IRF3 and may be involved in the antiviral response through the RIG-I viral recognition signalling pathway in a TRAF3/TBK1-dependent manner. Taken together, our results suggested that ccCD63 upregulated the interaction of KHV with the host immune system and suppressed the dissemination of KHV.

Establishment and characterization of a new marine fish cell line derived from red-spotted grouper Epinephelus akaara.

A new marine fish cell line, EAGL, derived from the liver of red-spotted grouper Epinephelus akaara was established and characterized. The cells multiplied well in minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS) at temperatures between 25 and 30° C. The growth rate of this cell line increased as the proportion of FBS increased from 5 to 20% at 25° C, with maximum growth at the concentration of 15 or 20% FBS. Morphologically, the cells were epithelial-like and the presence of pancytokeratin confirmed their epithelial origin. Chromosome analysis revealed that the modal chromosome number was 48. The susceptibility of the cell line to four fish viruses was tested. Significant cytopathic effect (CPE) was only observed in Singapore grouper iridovirus (SGIV)-infected cells, and the virus replication was further confirmed by immunofluorescence, electron microscopy and real-time reverse-transcription (RT)-PCR assay. When the cells were transfected with pEGFP-N3 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies.

ZNF295-AS1 inhibits autophagy via the ZNF295-AS1/miR-508-5p/ATG7 axis in AS.

OBJECTIVE: As a result of gene-environment interactions, the incidence of atherosclerosis (AS) is rapidly increasing worldwide. Autophagy in endothelial cells is a key process of AS and is difficult to control when it becomes excessive in the end stage of AS. MATERIALS AND METHODS: In this study, we found increased expression levels of ZNF295-AS1 in the serum of AS patients, as well as in ox-LDL-treated HUVECs. The autophagy level was also upregulated in both samples. We demonstrated that ZNF295-AS1 may interact directly with miR-508-5p to act as a miR-508-5p sponge. The negative relationship between ZNF295-AS1 and miR-508-5p indicated that ZNF295-AS1 may be an upstream suppressor of miR-508-5p. RESULTS: ATG7 plays a critical role in autophagy and was predicted to be a target of miR-508-5p. Therefore, we overexpressed miR-508-5p, which reduced the expression level of ATG7, enhanced cell proliferation and prevented autophagy. These data indicated that the ZNF295-AS1/miR-508-5p/ATG7 axis may participate in autophagy regulation in ox-LDL-treated HUVECs. The subsequent rescue experiments revealed the specificity of the ZNF295-AS1/miR-508-5p/ATG7 axis in the contribution of ZNF295-AS1 to autophagy. CONCLUSIONS: Overall, our findings demonstrate a novel mechanism by which ZNF295-AS1 silencing regulates ATG7 reduction and inhibits autophagy, which may delay the progression of AS. The ZNF295-AS1/miR-508-5p/ATG7 axis may be of therapeutic significance in AS.

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 degrees 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.

Development and characterization of a new tropical marine fish cell line from grouper, Epinephelus coioides susceptible to iridovirus and nodavirus.

The development and characterization of a new tropical marine fish cell line (GS), derived from the spleen of orange spotted grouper, Epinephelus coioides is described. The GS cells grow well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum, and have been subcultured more than 200 times. The optimal growth temperature was 27 degrees C. The GS cell culture consisted of mostly fibroblastic cells. The modal diploid chromosome number was 48. GS cell cultures showed advanced cytopathic effects after infection with a pathogenic grouper iridovirus (Singapore grouper iridovirus, SGIV) or with a grouper nodavirus (Epinephelus tauvina nervous necrosis virus, ETNNV). Analysis by transmission electron microscopy showed a large number of SGIV and ETNNV particles in the cytoplasm of virus-infected cells, respectively, indicative of high sensitivity to these two viruses. Immunofluorescence microscopy showed that iridovirus-infected GS cells reacted strongly with monoclonal antibody against the grouper iridovirus. It is suggested that the GS cell line has good potential as a diagnostic tool for isolation and propagation of iridovirus and nodavirus. When the GS cells were transfected with pEGFP vector DNA, significant fluorescent signals were observed suggesting that the GS cell line can be used as a useful tool for transgenic and genetic manipulation studies.

Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina.

The morphogenesis and the ultrastructure of a marine fish iridovirus isolated from diseased grouper, Epinephelus tauvina were studied by electron microscopy. The virus was grown on a marine fish cell line (GP) at 25 degrees C. After appearance of advanced cytopathic effect (CPE), various morphogenetic stages of virus amplification, maturation and assembly were detected in the cytoplasm of virus-infected cells. The matured nucleocapsids were probably formed by insertion of electron-dense core material into a partly forming empty capsid just before completely sealed. The nucleocapsids were located at the assembly sites as pseudocrystalline arrays or scattered individually. In the late phase of infection, the nucleocapsids were enveloped and released by budding from the plasma membrane. The budding virus particles could directly enter neighbouring cells by endocytosis to start the next round infection. Ultrastructure of the grouper iridovirus was studied using the methods of enzymatic digestions and detergent degradations. The purified iridovirus particles showed a three-layered membrane including an external lipoprotein envelope, an inner periodic protein capsid and a lipid-containing membrane. The regular array of surface capsid subunits was observed after degradation with detergent.

Graft-versus-host reaction (GVHR) in clonal amago salmon, Oncorhynchus rhodurus.

The graft-versus-host reaction (GVHR) was demonstrated in a salmonid model system of clonal diploid and triploid amago salmon. Triploid operculum grafts on clonal diploid evoked an acute rejection within 12 days. Grafts exchanged among triploid amago salmon exhibited prolonged survival for 18 days. In contrast, diploid grafts on triploid, and allografts among clonal diploid amago salmon were accepted. A typical GVHR was induced in triploid recipients by intraperitonal injection of head kidney cells from sensitised diploid donors. The clinical signs of graft-versus-host disease (GVHD) were observed in the recipients after 1 week of cell injection as a loss of appetite and appearance of solid faeces, followed by haemorrhage, local swelling of ventral skin and an enlarged spleen. Three of six fish died within 1 month. Water temperature and frequency of sensitisation are critical to induce GVHR. Diploid donors had to be sensitised three times at 20 degrees C to induce the typical GVHR. GVHR was most effectively induced by head kidney cells, followed by peripheral blood leucocytes (PBL) and spleen cells. Ploidy analysis by flow cytometry revealed that the donor head kidney cells greatly increased in the recipient liver, head kidney and spleen, and reached the peak after 9 days of donor cell injection. The results in the present study are quite similar to the findings in ginbuna and ginbuna-gold fish hybrid system, suggesting the presence of T cells in salmonid as well as cyprinid fish.

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.

The pathology of systemic iridoviral disease in fish.

Iridoviruses have been associated with severe disease and economic loss in farmed food fish and ornamental fish, with mortality often reported to reach 50% or more. In the present study, three tropical marine food fish species and four tropical freshwater ornamental fish species with systemic iridovirus infections were examined histopathologically and ultrastructurally. Light microscopy consistently revealed pale to intensely basophilic hypertrophied virus-infected cells in spleen, kidney and intestine from all seven species. Ultrastructural examination showed changes in the vascular endothelium overlying hypertrophied virus-infected cells suggestive of pressure necrosis. Viral isolation was improved by the use of fibroblastic cell lines. This, together with the sub-endothelial location of infected cells in all infected species examined, suggests that systemic iridoviruses are mesotheliotropic.

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China.

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Tumor necrosis factor alpha (TNFalpha)-like factor produced by macrophages in rainbow trout, Oncorhynchus mykiss.

The presence of a Tumor Necrosis Factor alpha (TNFalpha)-like molecule has been suggested in fish by biological assays and biological and antigenic cross-reactivities with human TNFalpha. In the present study, whether rainbow trout macrophages produce TNFalpha was examined. Murine recombinant TNFalpha (m-rTNFalpha) was used as the standard mammalian TNFalpha. The supernatants were harvested from trout macrophage culture stimulated with lipopolysaccharide (LPS) and then passed through a Polymyxin B column to remove LPS. Results show that trout macrophage culture supernatants exhibit TNF-like activities. The supernatants significantly enhanced neutrophil migration and macrophage respiratory burst activity as assessed by NBT reduction test. The supernatants were also highly cytotoxic to murine L929 cells, which are known to be sensitive to mammalian TNFalpha. The biological activities of TNF in the trout macrophage culture supernatant was determined as 2.6 U ml(-1) in the presence of actinomycin D. This indicates biological cross-reactivity of trout TNFalpha-like factor on mammalian cells. Moreover, these activities were inhibited by a rabbit anti-mTNFalpha antibody. These results suggest that rainbow trout macrophages produce a TNFalpha-like factor that is similar to the mammalian TNFalpha in functions.