Induction of apoptosis in SSN-1cells by Snakehead Fish Vesiculovirus (SHVV) via Matrix protein dependent intrinsic pathway.

An increasing important area in immunology is the process cell death mechanism, enabling the immune system triggered thru extrinsic or intrinsic signals to effectively remove unwanted or virus infected cells called apoptosis. A recently isolated infectious Snakehead fish vesiculovirus (SHVV), comprising negative strand RNA and encoded viral matrix (M) proteins, is responsible for causing cytopathic effects in infected fish cells. However, the mechanism by which viral M protein mediates apoptosis has not been elucidated. Therefore, in the present experiments, it was investigated the regulatory potential of apoptosis signals during SHVV infection. By employing the model of SHVV infection in SSN-1 cells, the accelerated apoptosis pathway involves an intrinsic pathway requiring the activation of caspase-9 but not caspase-3 or -8. In the groups of infection (SHVV) or treatment (hydrogen peroxide) were induced apoptotic morphological changes and indicated the activation of the main caspases, i.e.; executioner caspase-3, initiators caspase-8 and caspase-9 using colorimetric assays. Turning to the role of viral M protein when it was overexpressed in SSN-1 cells, it was indicated that the viral M gene alone has the ability to induce apoptosis. To elucidate the mechanism of apoptosis in SSN-1 cells, the activation inhibitors of main caspases were used showing that inhibiting of caspase-3 or caspase-8 activation did not seize induction of apoptosis in virus-infected SSN-1 cells. However, the inhibiting of caspase-9 activation reduced significantly the apoptosis initiation process and sharply the expression of viral M gene, suggesting that SHVV plays a major role in the early induction of apoptosis by caspase-9. Interestingly, there were also differences in the mitochondrial membrane potential after the apoptotic induction of caspases, which confirm that caspase-9 is primarily responsible for the cleavage of caspases during apoptosis. Taken together, these findings can therefore be assumed that viral M protein induces apoptosis via the intrinsic apoptotic pathway in SHVV infecting SSN-1 cells.

Explorations of the optimal method for isolating oocytes from zebrafish (Danio rerio) ovary.

Obtaining oocytes from the adult female zebrafish (Danio rerio) ovary has enormous importance in the studies of developmental biology, toxicology, and genetics. It is vital to establish a simple and effective approach to ensure the quantity and quality of oocytes, which will enable the success of follow-up experimental investigation finally. Usually, oocytes are separated with mechanical or enzymatic methods, however, little studies have been done with concerns about the comparative effects. The present study separated zebrafish oocytes of Stage III with five frequently used methods, including stripping, pipetting, hyaluronidase (1.6 mg/ml), collagenase (0.4 mg/ml), and trypsin (0.1%). The cell viability, oxidative stress, mitogen-activated protein kinase (MAPK) protein phosphorylation, and apoptosis levels were selected as main biomarkers to evaluate the oocytes health status. The results showed that both trypsin and hyaluronidase isolation significantly upregulated germinal vesicle breakdown (GVBD) rates and downregulated p38 MAPK activity simultaneously. GVBD rates and survival rates were decreased notably in oocytes separated by the collagenase method. Above results indicate that zebrafish oocytes in vitro are sensitive to enzymatic treatments and the enzymatic isolation is not the suitable mean for collecting zebrafish oocytes although it is time-saving. The mechanical strategy of pipetting remarkably increased the reactive oxygen species and malondialdehyde level in isolated oocytes. Interestingly, oocytes separated with stripping show less physiological and biochemical damages. Therefore, stripping isolation is comparatively recommended as the optimum method for separating and collecting numerous intact and healthy zebrafish oocytes in vitro for the subsequent developmental research.

Autophagy induced by snakehead fish vesiculovirus inhibited its replication in SSN-1 cell line.

Autophagy plays an important role in host protection against pathogen infection through activating innate and adaptive immunity. In the present study, we observed that the infection of snakehead fish vesiculovirus (SHVV) could induce apparent autophagy in striped snakehead fish cell line (SSN-1), including clear double-membrane vesicles, fluorescent punctate pattern of microtubule-associated protein 1 light chain 3B (SSN-LC3B) and the conversion of SSN-LC3B-Ⅰ to SSN-LC3B-Ⅱ. Furthermore, we verified that autophagy inhibited the replication of SHVV by assessing mRNA and protein level of nucleoprotein as well as virus titer in the supernatant. These results will shed a new light on the prevention of the infection of SHVV.

Identification and Characterization of MicroRNAs in Snakehead Fish Cell Line upon Snakehead Fish Vesiculovirus Infection.

MicroRNAs (miRNAs) play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection. Snakehead fish Vesiculovirus (SHVV) has caused mass mortality in snakehead fish during the past few years. To identify specific miRNAs involved in SHVV infection, we performed microRNA deep sequencing on a snakehead fish cell line (SSN-1) with or without SHVV infection. A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs, and nine novel miRNAs were identified using MiRDeep2 software. Eighteen and 143 of the 205 known miRNAs were differentially expressed at three and 24 h post-infection (poi), respectively. From the differentially-expressed miRNAs, five were randomly selected to validate their expression profiles using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and their expression profiles were consistent with the microRNA sequencing results. In addition, the target gene prediction of the SHVV genome was performed for the differentially-expressed host miRNAs, and a total of 10 and 58 differentially-expressed miRNAs were predicted to bind to the SHVV genome at three and 24 h poi, respectively. The effects of three selected miRNAs (miR-130-5p, miR-214 and miR-216b) on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting. The results showed that all three miRNAs were able to inhibit the multiplication of SHVV; whereas the mechanisms underlying the SHVV multiplication inhibited by the specific miRNAs need to be further characterized in the future.

Mannose receptor mediated phagocytosis of bacteria in macrophages of blunt snout bream (Megalobrama amblycephala) in a Ca(2+)-dependent manner.

Mannose receptor (MR) is an important pattern-recognition receptor in macrophages and plays a critical role in immune responses. It is has been reported that mammalian macrophages are able to engulf a wide range of microorganisms mediated by Ca(2+)-dependent MR binding to terminal mannose residues which are frequently found on the pathogen surfaces. However, little is known about the MR-mediated phagocytosis in macrophages of fish. In this report, the distributions of MR in the macrophage and head kidney tissue from blunt snout bream were examined using MaMR specific antibody generated in our lab. Mannan and MaMR specific antibody inhibition experiments results collectively showed that MR was involved in the GFP-expressed E. coli engulfed in the macrophages, resulting in respiratory burst, nitric oxide production as well as inflammatory cytokines secretion, and the MaMR-mediated phagocytosis was Ca(2+)-dependent. These results will shed a new light on the immune functions of teleost MRs.

Abortive infection of snakehead fish vesiculovirus in ZF4 cells was associated with the RLRs pathway activation by viral replicative intermediates.

Snakehead fish vesiculovirus (SHVV) is a negative strand RNA virus which can cause great economic losses in fish culture. To facilitate the study of SHVV-host interactions, the susceptibility of zebrafish embryonic fibroblast cell line (ZF4) to the SHVV was investigated in this report. The results showed that high amount of viral mRNAs and cRNAs were detected at the 3 h post-infection. However, the expressions of the viral mRNAs and cRNA were decreased dramatically after 6 h post-infection. In addition, the expressions of interferon (IFN) and interferon-induced GTP-binding protein Mx were all up regulated significantly at the late stage of the infection. Meanwhile, the expressions of Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated gene 5 (MDA5) were also all up-regulated significantly during the infection. Two isoforms of DrLGP2 from zebrafish were also cloned and analyzed. Interestingly, the expression of DrLGP2a but not DrLGP2b was significantly up-regulated at both mRNA and protein levels, indicating that the two DrLGP2 isoforms might play different roles during the SHVV infection. Transfection experiment showed that viral replicative intermediates were required for the activation of IFN-α expression. Taken together, the abortive infection of SHVV in ZF4 cells was associated with the activation of RLRs pathway, which was activated by viral replicative intermediates.

Accumulation and depuration of dissolved hexavalent chromium and effects on the antioxidant response in bighead carp (Aristichthys nobilis).

The present study investigated the accumulation and depuration effects of hexavalent chromium (Cr6+) in ten tissues (gills, intestines, liver, kidney, blood, heart, bladder, spleen, skin and muscle) of the bighead carp (Aristichthys nobilis). Fish were exposed to graded levels of waterborne Cr6+ (0.01, 0.1, 1 and 5 mg/L) for 4, 7 and 14 days, and subsequently transferred to Cr6+-free water for 14 days. After 14-day exposure, a dose-dependent increase of Cr6+ has been observed in most tissues. While after 14-day depuration, Cr6+ contents were significantly decreased in various tissues except in kidney and spleen where Cr6+ contents significantly increased at the group of 5 mg/L. Considering that Cr6+ highly accumulated in gills, intestines, liver and kidney, the oxidative damage of Cr6+ on the four tissues were further investigated and found that the antioxidant response to Cr6+ were organ-specific. The results in this study indicated that a 14-day period is effective for accumulation and depuration of Cr6+ in bighead carp and there was no health risk of fish muscle consumption. Additionally, the delayed efflux of Cr6+ in the fish kidney and spleen indicates that high importance should be attached to them when evaluating the toxic effects and risk assessments of Cr6+.

A comparison of accumulation and depuration effect of dissolved hexavalent chromium (Cr6+) in head and muscle of bighead carp (Aristichthys nobilis) and assessment of the potential health risk for consumers.

In this study, bighead carp (Aristichthys nobilis) were exposed to waterborne Cr6+ of 0.01, 0.1, 1 and 5 mg/L for 14 days and subsequently transferred to clean water for another 14 days. The Cr6+ contents in some edible parts, such as dorsal muscle, ventral muscle and head were detected. The Cr6+ concentrations in the three parts were in the order of: head > ventral muscle > dorsal muscle with significant increase during exposure period and remarkable decrease when kept at clean water during depuration stage. The head contained higher fat than that of muscle and the Cr6+ levels of these parts showed significantly positive correlation with fat content, however, the Cr6+ contents in the separated fat were extremely low. The Cr6+ levels determined in tissues can tell a real story rather than the correlation coefficient. Fish head poses a higher potential health risk than muscle due to heavy metals pollution.

µEvaluation of microcystin-LR absorption using an in vivo intestine model and its effect on zebrafish intestine.

Microcystin-LR (MC-LR) is regarded as one of the most toxic microcystins (MCs) isoforms. Microcystins could cause multiple organs dysfunction, and more attention has been drawn to the toxic effects on the gastrointestinal disorder. By using ex vivo everted gut sac model in 6 fish (Carassius auratus, Megalobrama amblycephala, Hypophthalmichthys molitrix, Aristichthys nobilis, Ctenopharyngodon idellus and Cyprinus carpio) and determining the accumulation of MC-LR in zebrafish intestine, we found a dose-dependent manner in the absorption and accumulation of MC-LR. Until now, little studies have been reported concerning the gut microbiota composition caused by different MC-LR exposure. The present study is the first time characterized the phylogenetic composition and taxonomic of the bacterial communities growth in the intestines of zebrafish treated with MC-LR using 16S rRNA pyrosequencing. After 30 days of treatment with 0, 1, 5 or 20 µg/L MC-LR, the alpha and beta diversity did not generate significant differences, indicating the existence of a core microbiota. However, db-RDA analysis showed that treatment with 20 µg/L MC-LR changed the characteristics of high abundances microbiota. The expression of Oatp2b1, stress related enzyme activities in gut and their associations with gut microbiota were also determined. The identified phylotypes including Actinobacteria, Lactobacillus and some opportunistic pathogens highlight the increasing risks of pathogen invasion and recovery tendency via potential probiotics resistance in zebrafish exposed to MC-LR.

MicroRNA miR-214 Inhibits Snakehead Vesiculovirus Replication by Promoting IFN-α Expression via Targeting Host Adenosine 5'-Monophosphate-Activated Protein Kinase.

BACKGROUND: Snakehead vesiculovirus (SHVV), a new rhabdovirus isolated from diseased hybrid snakehead, has emerged as an important pathogen during the past few years in China with great economical losses in snakehead fish cultures. However, little is known about the mechanism of its pathogenicity. MicroRNAs are small noncoding RNAs that posttranscriptionally modulate gene expression and have been indicated to regulate almost all cellular processes. Our previous study has revealed that miR-214 was downregulated upon SHVV infection. RESULTS: The overexpression of miR-214 in striped snakehead (SSN-1) cells inhibited SHVV replication and promoted IFN-α expression, while miR-214 inhibitor facilitated SHVV replication and reduced IFN-α expression. These findings suggested that miR-214 negatively regulated SHVV replication probably through positively regulating IFN-α expression. Further investigation revealed that adenosine 5'-monophosphate-activated protein kinase (AMPK) was a target gene of miR-214. Knockdown of AMPK by siRNA inhibited SHVV replication and promoted IFN-α expression, suggesting that cellular AMPK positively regulated SHVV replication and negatively regulated IFN-α expression. Moreover, we found that siAMPK-mediated inhibition of SHVV replication could be partially restored by miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting AMPK. CONCLUSION: The findings of this study complemented our early study, and provide insights for the mechanism of SHVV pathogenicity. SHVV infection downregulated miR-214, and in turn, the downregulated miR-214 increased the expression of its target gene AMPK, which promoted SHVV replication via reducing IFN-α expression. It can therefore assume that cellular circumstance with low level of miR-214 is beneficial for SHVV replication and that SHVV evades host antiviral innate immunity through decreasing IFN-α expression via regulating cellular miR-214 expression.