Hirame Natural Embryo Cells Research Articles

Expression characteristics of non-virion protein of Hirame novirhabdovirus and its transfection induced response in hirame natural embryo cells

The non-virion (NV) protein is the signature of genus Novirhabdovirus, which has been of considerable concern due to its potential role in viral pathogenicity. However, its expression characteristics and induced immune response remain limited. In the present work, it was demonstrated that Hirame novirhabdovirus (HIRRV) NV protein was only detected in the viral infected hirame natural embryo (HINAE) cells, but absent in the purified virions. Results showed that the transcription of NV gene could be stably detected in HIRRV-infected HINAE cells at 12 h post infection (hpi) and then reached the peak at 72 hpi. A similar expression trend of NV gene was also found in HIRRV-infected flounders. Subcellular localization analysis further exhibited that HIRRV-NV protein was predominantly localized in the cytoplasm. To elucidate the biological function of HIRRV-NV protein, NV eukaryotic plasmid was transfected into HINAE cells for RNA-seq. Compared to empty plasmid group, some key genes in RLR signaling pathway were significantly downregulated in NV-overexpressed HINAE cells, indicating that RLR signaling pathway was inhibited by HIRRV-NV protein. The interferon-associated genes were also significantly suppressed upon transfection of NV gene. This research would improve our understanding of expression characteristics and biological function of NV protein during HIRRV infection process.

Optimization of green LED light intensity for accelerating wound healing in olive flounder Paralichthys olivaceus (Temminck et Schlegel)

Recent studies showed that green wavelength light-emitting diodes (LEDs) were effective for wound healing in various animal models, including olive flounder. However, determining the optimal light intensity for phototherapy is very important for accelerating wound healing and safety. Therefore, the purpose of this study was to investigate the optimal intensity of green LEDs (520 nm) for accelerating wound healing using olive flounder Paralichthys olivaceus. In vitro studies, a Paralichthys olivaceus-derived embryo cell line (Hirame natural embryo cells; HINAE) was used to verify the proliferative effects of various intensities (20, 40, 80, and 120 μmol·m−2·s−1) of green LED (520 nm) light in 24-h light for seven days. In vivo studies, artificial wounds (4 mm in diameter) were made in flounders using a biopsy punch, and the wounds were exposed to green LED (520 nm) light at 20, 40, and 60 μmol·m−2·s−1, as well as ambient light as a control, in 12-h light/ dark cycles for 28 days. The results showed that 520 nm LED treatment at 40 μmol·m−2·s−1 significantly stimulated HINAE cell proliferation relative to ambient light (3 ± 1 μmol·m−2·s−1) and the 20, 80 and 120 μmol·m−2·s−1 LED intensity groups. The relative wound healing rate of fish exposed to 40 μmol·m−2·s−1 of green LED light was significantly higher than that of the other groups and showed a high expression of wound healing-related genes such as matrix metalloproteinase (mmp)-9, mmp-13, epidermal growth factor (egf), and vascular endothelial growth factor (vegf)-a. After the injuries, the survival rate of fish exposed to 520 nm LED light at an intensity of 60 μmol·m−2·s−1 was markedly lower (82%) than those of the control and 20 and 40 μmol·m−2·s−1-treated groups (over 95%). Stress-related factors such as catalase and superoxide dismutase (SOD) in the fish wound areas and serum glucose levels were significantly increased in the 60 μmol·m−2·s−1 exposure group, showing that prolonged exposure to strong light intensity could negatively affect physiology and wound healing. Therefore, the results showed that 40 μmol·m−2·s−1 intensity green LED (520 nm) light was the most effective light condition for accelerating wound healing in olive flounder.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Protective Effects of Nargenicin A1 against Tacrolimus-Induced Oxidative Stress in Hirame Natural Embryo Cells.

Tacrolimus is widely used as an immunosuppressant to reduce the risk of rejection after organ transplantation, but its cytotoxicity is problematic. Nargenicin A1 is an antibiotic extracted from Nocardia argentinensis and is known to have antioxidant activity, though its mode of action is unknown. The present study was undertaken to evaluate the protective effects of nargenicin A1 on DNA damage and apoptosis induced by tacrolimus in hirame natural embryo (HINAE) cells. We found that reduced HINAE cell survival by tacrolimus was due to the induction of DNA damage and apoptosis, both of which were prevented by co-treating nargenicin A1 or N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, with tacrolimus. In addition, apoptosis induction by tacrolimus was accompanied by increases in ROS generation and decreases in adenosine triphosphate (ATP) levels caused by mitochondrial dysfunction, and these changes were significantly attenuated in the presence of nargenicin A1, which further indicated tacrolimus-induced apoptosis involved an oxidative stress-associated mechanism. Furthermore, nargenicin A1 suppressed tacrolimus-induced B-cell lymphoma-2 (Bcl-2) down-regulation, Bax up-regulation, and caspase-3 activation. Collectively, these results demonstrate that nargenicin A1 protects HINAE cells against tacrolimus-induced DNA damage and apoptosis, at least in part, by scavenging ROS and thus suppressing the mitochondrial-dependent apoptotic pathway.

Generation of a recombinant viral hemorrhagic septicemia virus (VHSV) expressing olive flounder (Paralichthys olivaceus) interferon-γ and its effects on type I interferon response and virulence

Rhabdoviruses including viral hemorrhagic septicemia virus (VHSV) are highly susceptible to type I interferon (IFN) responses, and IFN-γ that is belonging to the type II IFN has been known to enhance type I IFN responses in mammals. In this study, we generated a recombinant VHSV that can express olive flounder IFN-γ (rVHSV-A-IFNγ) using reverse genetics technology, and analyzed the effect of rVHSV-A-IFNγ infection on type I IFN response in Epithelioma papulosum cyprini (EPC) cells. Furthermore, the virulence of rVHSV-A-IFNγ was evaluated by infection to olive flounder (Paralichthys olivaceus). Using a recombinant VHSV full genome vector in which the olive flounder IFN-γ ORF was inserted between N and P genes, rVHSV-A-IFNγ was successfully rescued, and the recombinant virus was grown well in EPC cells. On the other hand, the growth of rVHSV-A-IFNγ rescued from EPC cells was severely retarded when infected into hirame natural embryo (HINAE) cells that were originated from olive flounder. These results indicate that the EPC cell's IFN-γ receptor could not bind to olive flounder IFN-γ, but the species-specific binding of IFN-γ in HINAE cells induced antiviral responses. The expression of Mx1 gene in EPC cells infected with rVHSV-A-IFNγ was not greatly different from cells infected with rVHSV-Arfp (a recombinant VHSV harboring red fluorescent protein gene between N and P genes of the genome), however, in HINAE cells, rVHSV-A-IFNγ infection induced distinctively higher Mx1 gene expression compared to other recombinant viruses. These results suggest that olive flounder IFN-γ produced from rVHSV-A-IFNγ effectively enhanced type I IFN response in HINAE cells. In the present study, the lowest mortality of olive flounder fingerlings was recorded in the group of fish challenged with rVHSV-A-IFNγ, suggesting that the recombinant VHSV was attenuated by production of IFN-γ by itself. However, although rVHSV-A-IFNγ induced significantly lower mortality, the mortality still reached to 40%. Therefore, to be safely used in the aquaculture farms as prophylactic vaccines or immunostmulators, further manipulations that can guarantee safety are needed.

Relationship between Expression of Cellular Receptor-27.8 kDa and Lymphocystis Disease Virus (LCDV) Infection.

The 27.8kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells.

RNA Aptamers Inhibit the Growth of the Fish Pathogen Viral Hemorrhagic Septicemia Virus (VHSV)

Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

Characterization of the flounder IL-6 promoter and its regulation by the p65 NF-κB subunit

Interleukin (IL)-6 plays important roles in the regulation of the immune response and inflammation in many cell types and its induction by bacterial endotoxins or cytokines is regulated at the transcriptional level. The present study demonstrates the isolation and characterization of the flounder IL-6 promoter sequence and its transcriptional regulation in olive flounder (hirame) natural embryo (HINAE) cells. The promoter region (−400 to +79 bp from the transcription initiation site) of the flounder IL-6 gene contains putative cis-regulatory elements for CCAAT-enhancer-binding proteins (C/EBP; −346 to −355 bp and −166 to −160 bp), cAMP response element binding (CREB; −81 to −85 bp), the activator protein 1 (AP-1; −56 to −62 bp), and NF-κB (−39 to −48 bp). Transfection of p65 stimulated the PoIL-6-luc-WT reporter, but not the PoIL-6-luc-κB mt reporter, and treatment with LPS augmented p65-stimulated reporter activity in HINAE cells. In contrast, transfection of C/EBPβ or c-Jun failed to induce synergistic effects in the LPS-driven PoIL-6-luc-WT reporter activity. These results give us new insight into the regulation of flounder IL-6 transcription by the p65 NF-κB subunit.