Effect of TRAF6-knockout on gene expression and lncRNA expression in Epithelioma papulosum cyprini (EPC) cells.

TRAF6 is a key immune gene that plays a significant role in toll-like receptor signal transduction and activates downstream immune genes involved in antiviral immunity in fish. To explore the role of TRAF6 in Epithelioma papulosum cyprini (EPC) cells, we knocked out the TRAF6 gene using the Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) technique and then analyzed the transcriptomes of the knockout cells. In this study, we identified that 232 transcripts were differentially expressed in naive cells. Using the pipeline, we identified 381 novel lncRNAs in EPC cells, 23 of which were differentially expressed. Gene Ontology enrichment analysis demonstrated that differentially expressed genes (DEG) are implicated in various immune processes, such as neutrophil chemotaxis and mitogen-activated protein kinase binding. In addition, the KEGG pathway analysis revealed enrichment in immune-related pathways (Interleukin-17 signaling pathway, cytokine-cytokine receptor interaction, and TNF signaling pathway). Furthermore, the target genes of the differentially expressed lncRNAs were implicated in the negative regulation of interleukin-6 and tumor necrosis factor production. These results indicate that lncRNAs and protein-coding genes participate in the regulation of immune and metabolic processes in fish.

Antiviral effect of miR-155 in Epithelioma papulosum cyprini (EPC) cells against viral hemorrhagic septicemia virus (VHSV) infection.

MicroRNAs (miRNAs) are small non-coding RNAs known to play a significant role in the regulation of gene expression in various living organisms including fish. MiR-155 is known to enhance immunity in cells and several reports have demonstrated the antiviral properties of miR-155 in mammals. In this study, we investigated the antiviral role of miR-155 in Epithelioma papulosum cyprini (EPC) cells with viral hemorrhagic septicemia virus (VHSV) infection. EPC cells were transfected with miR-155 mimic and then infected with VHSV at different MOIs (0.01 and 0.001). The cytopathogenic effect (CPE) was observed at 0, 24, 48, and 72 h post infection (h.p.i). CPE progression appeared at 48 h.p.i in mock groups (VHSV only infected groups) and the VHSV infection group transfected with miR-155 inhibitors. On the other hand, the groups transfected with the miR-155 mimic did not show any CPE formation after infection with VHSV. The supernatant was collected at 24, 48 and 72 h.p.i., and the viral titers were measured by plaque assay. The viral titers increased at 48 and 72 h.p.i in groups infected only with VHSV. In contrast, the groups transfected with miR-155 did not show any increase in the virus titer and had a similar titer to 0 h.p.i. Furthermore, the real-time RT-PCR of immune gene expression showed upregulation of Mx1 and ISG15 at 0, 24, and 48 h.p.i in groups transfected with miR-155, while the genes were upregulated at 48 h.p.i in groups infected only with VHSV. Based on these results, miR-155 can induce the overexpression of type I interferon-related immune genes in EPCs and inhibit the viral replication of VHSV. Therefore, these results suggest that miR-155 could possess an antiviral effect against VHSV.

Isolation and Characterization of a Salt Inducible Promoter from Chlorella vulgaris PKVL7422.

Chlorella is a eukaryotic organism that can be used as an industrial host to produce recombinant proteins. In this study, a salt-inducible promoter (SIP) was isolated from the freshwater species Chlorella vulgaris PKVL7422 from the screening of genes that were upregulated after salt treatment. Several cis-acting elements, including stress response elements, were identified in the isolated SIP. Moreover, the Gaussia luciferase gene was cloned after the SIP and transformed into C. vulgaris to test the inducibility of this promoter. Reexamination of transcriptome of C. vulgaris revealed that genes involved in the synthesis of methyl jasmonic acid (MeJA), gibberellin (GA), and abscisic acid (ABA) were upregulated when C. vulgaris was treated with salt. Furthermore, the expression level of recombinant luciferase increased when the transformed C. vulgaris was treated with salt and MeJA, GA, and ABA. This study represents the first report of the C. vulgaris SIP and highlights how transformed microalgae could be used for robust expression of recombinant proteins.

Effects of Non-Virion Gene Expression Level and Viral Genome Length on the Replication and Pathogenicity of Viral Hemorrhagic Septicemia Virus.

Fish novirhabdoviruses, including viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus (HIRRV), and infectious hematopoietic necrosis virus (IHNV), harbor a unique non-virion (NV) gene that is crucial for efficient replication and pathogenicity. The effective levels and the function of the N-terminal region of the NV protein, however, remain poorly understood. In the present study, several recombinant VHSVs, which completely lack (rVHSV-ΔNV) or harbor an additional (rVHSV-dNV) NV gene, were generated using reverse genetics. To confirm the function of the N-terminal region of the NV protein, recombinant VHSVs with the NV gene that gradually mutated from the start codon (ATG) to the stop codon (TGA), expressed as N-terminally truncated NV proteins (rVHSV-NV1, -NV2, and -NV3), were generated. CPE progression and viral growth analyses showed that epithelioma papulosum cyprini (EPC) cells infected with rVHSV-ΔNV or rVHSV-NV3-which did not express NV protein-rarely showed CPE and viral replication as opposed to EPC cells infected with rVHSV-wild. Interestingly, regardless of the presence of two NV genes in the rVHSV-dNV genome, EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP (control) failed to induce CPE and viral replication. In EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP, which harbored a longer VHSV genome than the wild-type, Mx gene expression levels, which were detected by luciferase activity assay, were particularly high; Mx gene expression levels were higher in EPC cells infected with rVHSV-ΔNV, -NV2, or -NV3 than in those infected with rVHSV-wild or rVHSV-NV1. The total amount of NV transcript produced in EPC cells infected with rVHSV-wild was much higher than that in EPC cells infected with rVHSV-dNV. However, the expression levels of the NV gene per viral particle were significantly higher in EPC cells infected with rVHSV-dNV than in cells infected with rVHSV-wild. These results suggest that the NV protein is an essential component in the inhibition of host type-I interferon (IFN) and the induction of viral replication. Most importantly, viral genome length might affect viral replication efficiency to a greater extent than does NV gene expression. In in vivo pathogenicity experiments, the cumulative mortality rates of olive flounder fingerlings infected with rVHSV-dNV or rVHSV-wild were similar (60-70%), while those of fingerlings infected with rVHSV-A-EGFP were lower. Moreover, the virulence of rVHSV-ΔNV and rVHSV, both harboring a truncated NV gene (rVHSV-NV1, -NV2, and -NV3), was completely attenuated in the olive flounder. These results suggest that viral pathogenicity is affected by the viral replication rate and NV gene expression. In conclusion, the genome length and NV gene (particularly the N-terminal region) expression of VHSVs are closely associated with viral replication in host type-I IFN response and the viral pathogenicity.

pH Changes Have a Profound Effect on Gene Expression, Hydrolytic Enzyme Production, and Dimorphism in Saccharomycopsis fibuligera.

Saccharomycopsis fibuligera is an amylolytic yeast that plays an important role within nuruk (a traditional Korean fermentation starter) used for the production of makgeolli (Korean rice wine), which is characterized by high acidity. However, the effect of pH change (neutral to acidic) on the yeast cell to hyphal transition and carbohydrate-hydrolyzing enzyme activities for S. fibuligera has not been investigated yet. In this study, S. fibuligera strains were cultured under the different pH conditions, and the effect on the enzyme production and gene expression were investigated. An acidic pH induced a hyphal transition from yeast cell of S. fibuligera KPH12 and the hybrid strain KJJ81. In addition, both strains showed a gradual decrease in the ability to degrade starch and cellulose as the pH went down. Furthermore, a transcriptome analysis demonstrated that the pH decline caused global expression changes in genes, which were classified into five clusters. Among the differentially expressed genes (DEGs) under acidic pH, the downregulated genes were involved in protein synthesis, carbon metabolism, and RIM101 and cAMP-PKA signaling transduction pathways for the yeast-hyphal transition. A decrease in pH induced a dimorphic lifestyle switch from yeast cell formation to hyphal growth in S. fibuligera and caused a decrease in carbohydrate hydrolyzing enzyme production, as well as marked changes in the expression of genes related to enzyme production and pH adaptation. This study will help to elucidate the mechanism of adaptation of S. fibuligera to acidification that occur during the fermentation process of makgeolli using nuruk.

Transcriptome analysis of gene expression in Chlorella vulgaris under salt stress.

Chlorella vulgaris is an important freshwater alga that is widely used as a food source for humans and animals. High-salinity environments can cause accumulation of lipids and proteins in this species, but the mechanism of this accumulation and the salt response remain unclear. In this work, transcriptome analysis was performed for the C. vulgaris response to salt stress (1% and 3% NaCl) applied for different times (2 h and 4 h). In total, 5232 and 9196 were differentially expressed after 1% NaCl for 2 and 4 h, and 3968 and 9035 unigenes were differentially expressed after 3% NaCl for 2 and 4 h, respectively. The number of upregulated genes after 4 h of salinity stress was greater than the number of downregulated genes, suggesting that the alteration of gene expression may be related to a mechanism of adaptation to a high-salinity environment. Furthermore, gene ontology and KEGG pathway analyses revealed that numerous biological pathways are affected by salt stress. Among the upregulated pathways, the cytoplasmic calcium signaling pathway, which is involved in the regulation of homeostasis, was highly upregulated. Genes involved in the photosystem I light-harvesting pathway were downregulated under salt stress. These results provide foundational information on the effects of salt stress on C. vulgaris metabolism and its possible mechanism of surviving high concentrations of NaCl.

Effect of miR-155 as a molecular adjuvant of DNA vaccine against VHSV in olive flounder (Paralichthys olivaceus).

Rhabdoviral G protein-based DNA vaccines have been recognized as a useful way to protect cultured fish from rhabdoviral diseases. In Korea, viral hemorrhagic septicemia virus (VHSV) genotype IVa has been the primary culprit of high mortalities of cultured olive flounder (Paralichthys olivaceus). In this study, we inserted a miR-155-expressing cassette into the VHSV's G protein-based DNA vaccine, and analyzed the effects of miR-155 on the antiviral activity and on the vaccine efficacy in olive flounder. Olive flounder fingerlings were intramuscularly (i.m.) immunized with 10 µg/fish (1st experiment) or 1 µg/fish (2nd experiment) of DNA vaccine plasmids. However, there were no significant differences in mortalities and serum neutralization titers between fish immunized with 1 µg and 10 µg plasmids/fish, suggesting that i.m. injection with 1 µg plasmids/fish would be enough to induce effective adaptive immune responses in olive flounder fingerlings. In survival rates, as fish immunized with just G protein expressing plasmids showed no or too low mortalities, the adjuvant effect of miR-155 was not discernible. Also, in the serum neutralization activities, although G gene or G gene plus miR-155 expressing DNA vaccines induced significantly higher activities than control vaccines (PBS and vacant vector), no significant differences were found between G gene alone and G gene plus miR-155 expressing DNA vaccines. In the serum virucidal activity, fish immunized with G gene plus miR-155 expressing DNA vaccine showed significantly higher activity against hirame rhabdovirus (HIRRV) at 3 days post-immunization (d.p.i.) compared to other groups, suggesting that miR-155 produced from the vector can enhance innate immune responses in olive flounder. The significantly enhanced serum virucidal activities against VHSV especially at 28 d.p.i. in the groups immunized with G gene alone and G gene plus miR-155 expressing DNA vaccines reflect the increased antibodies against G protein, which could activate the classical complement pathway and subsequent viral inactivation. As the available information on the DNA vaccines in olive flounder is not sufficient, more diverse researches on the protective efficacy of DNA vaccines are needed to make more practical use of DNA vaccines in olive flounder farms.