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.

Zinc Finger Protein BCL11A Contributes to the Abortive Infection of Hirame novirhabdovirus (HIRRV) in B Lymphocytes of Flounder (Paralichthys olivaceus).

Hirame novirhabdovirus (HIRRV) infection is characterized by a pronounced viremia, and the high viral load is typically detected in immune-related organs and the circulatory system. In the present study, we demonstrated that HIRRV has the capacity to invade part of flounder membrane-bound IgM (mIgM+) B lymphocyte. Eight quantitative real-time PCR (qRT-PCR) standard curves involving HIRRV genomic RNA (gRNA), cRNA, and six mRNAs were established based on the strand-specific reverse transcription performed with tagged primers. It was revealed that viral RNA synthesis, especially the replication of gRNA, was inhibited in B cells, and the intracellular HIRRV even failed to produce infectious viral particles. Moreover, a range of genes with nucleic acid binding activity or related to viral infection were screened out based on the transcriptome analysis of HIRRV-infected B cells, and five molecules were further selected because of their different expression patterns in HIRRV-infected B cells and hirame natural embryo (HINAE) cells. The overexpression of these genes followed by HIRRV infection and RNA binding protein immunoprecipitation (RIP) assay revealed that the flounder B cell lymphoma/leukemia 11A (BCL11A), a highly conserved zinc finger transcription factor, is able to inhibit the proliferation of HIRRV by binding with full-length viral RNA mainly via its zinc finger domains at the C terminus. In conclusion, these data indicated that the high transcriptional activity of BCL11A in flounder mIgM+ B lymphocytes is a crucial factor for the abortive infection of HIRRV, and our findings provide new insights into the interaction between HIRRV and teleost B cells. IMPORTANCE HIRRV is a fish rhabdovirus that is considered as an important pathogen threatening the fish farming industry represented by flounder because of its high infectivity and fatality rate. To date, research toward understanding the complex pathogenic mechanism of HIRRV is still in its infancy and faces many challenges. Exploration of the relationship between HIRRV and its target cells is interesting and necessary. Here, we revealed that flounder mIgM+ B cells are capable of suppressing viral RNA synthesis and result in an unproductive infection of HIRRV. In addition, our results demonstrated that zinc finger protein BCL11A, a transcription factor in B cells, is able to suppress the replication of HIRRV. These findings increased our understanding of the underlying characteristics of HIRRV infection and revealed a novel antiviral mechanism against HIRRV based on the host restriction factor in teleost B cells, which sheds new light on the research into HIRRV control.

Isolation and characterization of hirame aquareovirus (HAqRV): A new Aquareovirus isolated from diseased hirame Paralichthys olivaceus.

We isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from Japanese flounder Paralichthys olivaceus suffering from reovirus-like infection. In electron microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The viral genome consisted of 11 segments and regions encoding 7 virion structural proteins and 5 non-structural proteins were predicted. The deduced amino acid sequences of those proteins were highly similar to those of the aquareoviruses. However, the similarity of complete genome sequence between the HAqRV and other aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences suggested that the HAqRV is not classified into the known species of Aquareovirus. Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch's postulates by experimental infection using Japanese flounder. The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese flounder at early life stages.

Pol-miR-150 regulates anti-bacterial and viral infection in Japanese flounder (Paralichthys olivaceus) via the lysosomal protein LMP2L.

MiR-150 is a microRNA (miRNA) present in a number of teleost species, but its target and regulation mechanism are unknown. Similarly, lysosome membrane protein 2-like (LMP2L) is a gene identified in fish but with unknown function. In this study, we examined the regulation mechanism and function of flounder miR-150 (named pol-miR-150) and its target gene LMP2L (named PoLMP2L) in association with bacterial and viral infection. We found that pol-miR-150 expression was not only modulated by the bacterial pathogen Streptococcus iniae but also by the viral pathogen megalocytivirus. Pol-miR-150 targeted PoLMP2L by binding to the 3'-untranslated region (3'-UTR) of PoLMP2L and inhibited PoLMP2L expression in vitro and in vivo. PoLMP2L is a member of the CD36 superfamily of scavenger receptors and homologous to but phylogenetically distinct from lysosomal integral membrane protein type 2 (LIMP2). PoLMP2L was localized mainly in the lysosomes and expressed in multiple organs of flounder. In vivo knockdown and overexpression of PoLMP2L enhanced and suppressed, respectively, S. iniae dissemination in flounder tissues, whereas in vivo knockdown and overexpression of pol-miR-150 produced the opposite effects on S. iniae dissemination. In addition, pol-miR-150 knockdown also significantly inhibited the replication of megalocytivirus. The results of this study revealed the regulation mechanism and immune functions of fish miR-150 and LMP2L, and indicated that LMP2L and miR-150 play an important role in the antimicrobial immunity of fish.

Naturally occurring substitution in one amino acid in VHSV phosphoprotein enhances viral virulence in flounder.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Naturally occurring VHSV strains vary greatly in virulence. Until now, little has been known about genetic alterations that affect the virulence of VHSV in flounder. We recently reported the full-genome sequences of 18 VHSV strains. In this study, we determined the virulence of these 18 VHSV strains in flounder and then the assessed relationships between differences in the amino acid sequences of the 18 VHSV strains and their virulence to flounder. We identified one amino acid substitution in the phosphoprotein (P) (Pro55-to-Leu substitution in the P protein; PP55L) that is specific to highly virulent strains. This PP55L substitution was maintained stably after 30 cell passages. To investigate the effects of the PP55L substitution on VHSV virulence in flounder, we generated a recombinant VHSV carrying PP55L (rVHSV-P) from rVHSV carrying P55 in the P protein (rVHSV-wild). The rVHSV-P produced high level of viral RNA in cells and showed increased growth in cultured cells and virulence in flounder compared to the rVHSV-wild. In addition, rVHSV-P significantly inhibited the induction of the IFN1 gene in both cells and fish at 6 h post-infection. An RNA-seq analysis confirmed that rVHSV-P infection blocked the induction of several IFN-related genes in virus-infected cells at 6 h post-infection compared to rVHSV-wild. Ectopic expression of PP55L protein resulted in a decrease in IFN induction and an increase in viral RNA synthesis in rVHSV-wild-infected cells. Taken together, our results are the first to identify that the P55L substitution in the P protein enhances VHSV virulence in flounder. The data from this study add to the knowledge of VHSV virulence in flounder and could benefit VHSV surveillance efforts and the generation of a VHSV vaccine.

RNA-seq transcriptome analysis in flounder cells to compare innate immune responses to low- and high-virulence viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Viral growth and virulence rely on the ability to inhibit the cellular innate immune response. In this study, we investigated differences in the modulation of innate immune responses of HINAE flounder cells infected with low- and high-virulence VHSV strains at a multiplicity of infection of 1 for 12 h and 24 h and performed RNA sequencing (RNA-seq)-based transcriptome analysis. A total of 193 and 170 innate immune response genes were differentially expressed by the two VHSV strains at 12 and 24 h postinfection (hpi), respectively. Of these, 73 and 77 genes showed more than a twofold change in their expression at 12 and 24 hpi, respectively. Of the genes with more than twofold changes, 22 and 11 genes showed high-virulence VHSV specificity at 12 and 24 hpi, respectively. In particular, IL-16 levels were more than two time higher and CCL20a.3, CCR6b, CCL36.1, Casp8L2, CCR7, and Trim46 levels were more than two times lower in high-virulence-VHSV-infected cells than in low-virulence-VHSV-infected cells at both 12 and 24 hpi. Quantitative PCR (qRT-PCR) confirmed the changes in expression of the ten mRNAs with the most significantly altered expression. This is the first study describing the genome-wide analysis of the innate immune response in VHSV-infected flounder cells, and we have identified innate immune response genes that are specific to a high-virulence VHSV strain. The data from this study can contribute to a greater understanding of the molecular basis of VHSV virulence in flounder.

Bacillus subtilis Inhibits Viral Hemorrhagic Septicemia Virus Infection in Olive Flounder (Paralichthys olivaceus) Intestinal Epithelial Cells.

Viral hemorrhagic septicemia virus (VHSV) is a highly pathogenic virus that infects a wide range of host fish species causing high economic losses in aquaculture. Epithelial cells in mucosal organs are target sites for VHSV entry into fish. To protect fish against VHSV infection, there is a need to develop antiviral compounds able to prevent establishment of infection at portals of virus entry into fish. Bacillus subtilis is a probiotic with excellent antiviral properties, of which one of its secretions, surfactin, has been shown to inhibit viral infections in mammals. Herein, we demonstrate its ability to prevent VHSV infection in olive flounder (Paralichthys olivaceus) intestinal epithelial cells (IECs) and infection in internal organs. Our findings show inhibition of VHSV infection in IECs by B. subtilis and surfactin. In addition, our findings showed inhibition of VHSV in Epithelioma Papulosum Cyprini (EPC) cells inoculated with intestinal homogenates from the fish pretreated with B. subtilis by oral exposure, while the untreated fish had cytopathic effects (CPE) caused by VHSV infection in the intestines at 48 h after the VHSV challenge. At 96 h post-challenge, samples from the untreated fish had CPE from head kidney and spleen homogenates and no CPE were observed in the intestinal homogenates, while the B. subtilis-pretreated fish had no CPE in all organs. These findings demonstrate that inhibition of VHSV infection at portals of virus entry in the intestines culminated in prevention of infection in internal organs. In summary, our results show that B. subtilis has the potential to prevent VHSV infection in fish and that its use as a probiotic in aquaculture has the potential to serve as an antiviral therapeutic agent against different viral infections.

Potentiality to natural immunization inducement against VHS in olive flounder by live VHSV immersion vaccination at temperature controlled culture condition.

Viral hemorrhagic septicemia virus (VHSV) is the etiological agent of viral hemorrhagic septicemia (VHS), one of the most severe viral diseases affecting cultured olive flounder (Paralichthys olivaceus) in Far East Asia. VHS occurs during the winter or spring season when the water temperature is low (9-15 °C). In our previous study found that VHSV infection had controlled by using water temperature (above 17 °C). By using water temperature, we demonstrated optimal live VHSV immersion vaccine treatment concentration, also live VHSV immersion vaccine treatment method. We confirmed that the effective VHSV immersion treatment was 105.5 TCID50/mL at 17 °C. It was no need pretreatment before live VHSV immersion vaccination. The VHSV titer of vaccinated fish organs was under the estimated limit (<1.8 log TCID50/mL) within 3 days in 105.5 TCID50/mL live VHSV immersion at 17 °C. High survival rates were observed in live VHSV immersion with 105.5 and 107.5 TCID50/mL at 17 °C and then infected VHSV at 10 °C. VHSV specific antibody was not detected from in the surviving flounder under VHSV infection after immersion treatment with live VHSV. In addition, the potentiality of natural immunization against VHS in olive flounder was suggested by live VHSV immersion vaccine at temperature controlled fish culture condition.

Lymphocystis Disease Virus (Iridoviridae) Enters Flounder (Paralichthys olivaceus) Gill Cells via a Caveolae-Mediated Endocytosis Mechanism Facilitated by Viral Receptors.

In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder (Paralichthys olivaceus) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.

Different immune responses of flounder (Paralichthys olivaceus) towards the full-length and N-terminal or C-terminal portion of hirame novirhabdovirus glycoprotein.

Glycoprotein is an important immunogenic protein of Hirame novirhabdovirus (HIRRV). In this study, the full-length and N-/C-terminal portions of glycoprotein were recombinantly expressed (rG, rGn and rGc protein), and the induced immune responses were investigated in flounder (Paralichthys olivaceus) model. The results showed that compared to PBS control, rG, rGn and rGc proteins and inactivated HIRRV suspension (iVS) could all stimulate significant increases of flounder CD4-1+, CD4-2+ T lymphocytes and surface IgM positive (sIgM+) B lymphocytes in peripheral blood, spleen and head kidney (p < 0.05). However, no significant differences of the percentages of CD4-1+ or CD4-2+ T lymphocytes were observed among three protein vaccination groups (p > 0.05). iVS could induce the highest mean levels of CD4+ T lymphocytes in peripheral blood and spleen. For sIgM+ B lymphocytes, the average peak percentages in rG and rGc groups were higher than rGn group. Moreover, significant increases of specific serum IgM against HIRRV or rG protein were observed in iVS, rG, rGn and rGc groups, but rG group exhibited the highest mean level. Furthermore, rG protein induced the highest titer of neutralizing antibodies against HIRRV, followed by iVS. Meanwhile, the challenge test showed that the relative percent survival (RPS) of rG, rGn, rGc and iVS groups were 75.0%, 35.7%, 53.6% and 60.7%, respectively. These results revealed that the full-length G protein would be a more effective subunit vaccine candidate against HIRRV infection.

Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus.

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

Genome based quantification of VHSV in multiple organs of infected olive flounder (Paralichthys olivaceus) using real-time PCR.

BACKGROUND: Viral hemorrhagic septicemia (VHS) is a serious viral disease that infects the olive flounder in South Korea. The Korean aquaculture industry experienced an economic loss caused by the high infectivity and mortality. OBJECTIVE: This study aimed to evaluate the infection density of VHSV in various organs of the olive flounder including spleen, liver, kidney, stomach, esophagus, intestine, gill, muscle, heart, and brain. Olive flounders were collected from a local fish farm and injected subcutaneously with 106 PFU/fish. METHODS: Each 15 fish were sampled at 0, 3, and 7 days post challenge (dpc), respectively, to perform quantitative analysis of VHSV using SYBR-green based real-time PCR in various tissues including spleen, liver, head-kidney, body-kidney, muscle, esophagus, stomach, intestine, gill, and brain. RESULTS: Organs infected with VHSV were obtained after 3 and 7 days. Each organs were examined for viral infection using real-time PCR. The data obtained from this experiment revealed copy numbers higher than 10 copies per 100 ng cDNA in the spleen (15.26 ± 3.11 copies/100 ng of cDNA), muscle (11.24 ± 2.25 copies), and gill (14.23 ± 6.26 copies), but lower in liver, head-kidney, body-kidney, esophagus, brain and stomach. CONCLUSION: The present study, together with previous data, demonstrated that the gill, spleen, and muscle are the major target organs of VHSV in olive flounder. Therefore, central monitoring of spleen, gill and muscle should be considered and might be necessary if anti-VHSV treatment is to be successful in infected olive flounder.

Involvement of CD4-1 T cells in the cellular immune response of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and nervous necrosis virus (NNV) infection.

The occurrence of CD4 helper T cells has already been established for a number of teleost species, though, it has not been possible to analyze these responses at a cellular level due to a large lack of appropriate monoclonal antibodies (mAbs). In the present study, we produced a mAb against olive flounder (Paralichthys olivaceus) CD4-1 lymphocyte to investigate the functional activity of the cells to improve our understanding of the T cell response in this species. This mAb is specifically able to detect CD4-1 lymphocytes in olive flounder proved by immunofluorescence staining and RT-PCR analysis. In flow cytometry analysis, the number of CD4-1-positive lymphocytes was observed to gradually increase from 3 days post infection (dpi) and then reach peak at 7 dpi against two viruses challenge. As a conclusion, both the basic properties of CD4-1 T cells and its response to viral infections in olive flounder are very similar to the helper T cells in terrestrial animals.

Characterization of the Long Terminal Repeat of the Endogenous Retrovirus-derived microRNAs in the Olive Flounder.

Endogenous retroviruses (ERVs) have been identified at different copy numbers in various organisms. The long terminal repeat (LTR) element of an ERV has the capacity to exert regulatory influence as both a promoter and enhancer of cellular genes. Here, we describe olive flounder (OF)-ERV9, derived from chromosome 9 of the olive flounder. OF-ERV9-LTR provide binding sites for various transcription factors and showed enhancer activity. The OF-ERV9-LTR demonstrates high sequence similarity with the 3' untranslated region (UTR) of various genes that also contain seed sequences (TGTTTTG) that bind the LTR-derived microRNA(miRNA), OF-miRNA-307. Additionally, OF-miRNA-307 collaborates with transcription factors located in OF-ERV9-LTR to regulate gene expression. Taken together, our data facilitates a greater understanding of the molecular function of OF-ERV families and suggests that OF-miRNA-307 may act as a super-enhancer miRNA regulating gene activity.

Juvenile olive flounder immersed in live VHSV at 17 °C and 20 °C shows resistance against VHSV infection at 10 °C.

Viral hemorrhagic septicemia (VHS) causes serious economic loss in olive flounder aquaculture industry in Korea. Water temperature is known to play a critical role in VHS disease outbreak. Here, we assessed the potential efficacy of VHSV immersion treatment in relation to resistance conferred at differential water temperatures in olive flounder. VHSV acquired resistance was compared between formalin-killed VHSV immersion treatment and live VHSV immersion treatment at three different water temperatures viz., 10 °C, 17 °C, and 20 °C. At 10 °C, cumulative mortality was around 80% in live VHSV immersed group while 30% cumulative mortality was observed in formalin-killed VHSV treated group. After 4 weeks, surviving olive flounder at 17 °C and 20 °C were challenged with VHSV at 10 °C following which the VHS outbreaks took place at host susceptible water temperature. For the pre-treated flounder at 17 °C, survival rates were 80% and 30% after challenge at 10 °C in live VHSV immersed group and formalin-killed VHSV immersed group, respectively. Whereas, the pre-treated flounder at 20 °C showed survival rate of 75% and 20% after challenge at 10 °C in live VHSV immersed group and formalin-killed VHSV immersed group, respectively. Our results propose the fact that live VHSV immersion using non-susceptible water temperature has the potential to protect olive flounder against VHSV infection. Moreover, the protective efficacy of live immersion treatment in a non-excited immune state without the use of an adjuvant combined with water temperature adjustment was investigated for the first time at 17 °C. Further studies should be targeted to explore the host-associated immune factors responsible for the protective effect and acquired resistance in olive flounder after live VHSV immersion treatment.

Kinetics of infectious virus and viral RNA copy number in the blood of olive flounder infected with viral hemorrhagic septicemia virus (VHSV).

Viral hemorrhagic septicemia (VHS) is a cold-water disease caused by viral hemorrhagic septicemia virus (VHSV) at an optimal temperature of 9 °C-15 °C. VHSV isolation and detection have been accomplished by using a number of diagnostic methods such as cell culture and qRT-PCR. Spleen and kidney have been reported as the main target organs of VHSV-infection; however, how VHSV spreads throughout the fish body has not been clearly studied. The purpose of this study was 1) to investigate viral titer and viral RNA copy number in the blood of VHSV-infected olive flounder at 10 °C and 13 °C; 2) to compare VHSV titer and viral RNA copy numbers in blood from fish exposed to the virus by two different challenges. VHSV titer at 10 °C was higher than at 13 °C in blood samples of injection challenged group. Whereas, similar titer was observed at 10 °C and 13 °C in the blood samples of the immersion challenged group. At 10 °C, copy numbers of VHSV-N gene in blood of immersion challenged group increased slightly in comparison to injection challenged group. At 13 °C, similar patterns were observed between the injection and immersion challenged groups. Also, higher titer and copy number were observed in fish blood compared to tested organs from our previous study. Our results indicate that VHSV genome existed in fish blood at earlier time points after infection, and the blood may contribute to the spread of the virus in whole fish body. In addition, VHSV diagnosis by qRT-PCR from fish blood samples, not requiring sacrificing the host fish can be valuable to collect the kinetic information of viral infection.

Generation of VHSV replicon particles carrying transmembrane and C-terminal cytoplasmic region-deleted G gene (rVHSV-GΔTM) and comparison of vaccine efficacy with G gene-deleted VHSV (rVHSV-ΔG).

Vaccines based on viral replicon particles would be advantageous to induce immune responses compared to inactivated viruses in that they can infect host cells (only once) and can produce viral proteins in the infected cells like live viruses. Furthermore, as viral replicon particles are replication-defective, they are safer than live attenuated viruses. Previously, we had rescued viral hemorrhagic septicemia virus (VHSV) replicon particles lacking full ORF of G gene (rVHSV-ΔG). In the present study, to enhance the immunogenicity of VHSV replicon particles, we newly generated another form of VHSV replicon particles that can produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM), and compared the protective efficacy of rVHSV-GΔTM with that of rVHSV-ΔG through immunization of olive flounder (Paralichthys olivaceus). In addition, we evaluated the safety of rVHSV-GΔTM by the analysis of effects on wild-type VHSV replication. In the vaccine experiment, olive flounder immunized with rVHSV-GΔTM showed significantly higher titers of serum neutralization activity than fish immunized with rVHSV-ΔG suggesting that the G protein that is not only spiked on the viral envelop but also secreted extracellularly can contribute to the enhancement of adaptive humoral immunity. Moreover, fish immunized with rVHSV-GΔTM showed higher survival rates than fish immunized with rVHSV-ΔG, suggesting that the amount of G protein provided to hosts is an important factor for the enhancement of vaccine efficacy against VHSV disease. In a safety aspect, rVHSV-GΔTM could not replicate in infected cells, and significantly inhibited the replication of wild-type VHSV when co-infected, suggesting that rVHSV-GΔTM would not worsen disease progression caused by wild-type VHSV infection.

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.

Infection of covert mortality nodavirus in Japanese flounder reveals host jump of the emerging alphanodavirus.

Interspecies transmission of viruses, where a pathogen crosses species barriers and jumps from its original host into a novel species, has been receiving increasing attention. Viral covert mortality disease, caused by covert mortality nodavirus (CMNV), is an emerging disease that has recently had a substantial impact on shrimp aquaculture in Southeast Asia and Latin America. While investigating the host range of CMNV, we found that this virus is also capable of infecting populations of the farmed Japanese flounder Paralichthys olivaceus, a vertebrate host. The infected fish were being raised in aquaculture facilities that were also producing marine shrimp. Through RT-nPCR, targeting the RNA-dependent RNA polymerase (RdRp) gene of CMNV, we found that 29 % of the fish sampled were positive. The amplicons were sequenced and aligned to the RdRp gene of shrimp CMNV and were found to have 98 % identity. Histopathological examination indicated that CMNV-positive fish showed vacuolation of nervous tissue in the eye and brain, as well as extensive necrosis of cardiac muscle. In situ hybridization showed positive reactions in tissues of the eye, brain, heart, liver, spleen and kidney of infected fish. Transmission electron microscopy showed the presence of CMNV-like particles in all of the above-mentioned tissues, except for brain. The novel finding of a shrimp alphanodavirus that can also infect farmed P. olivaceus indicates that this virus is capable of naturally crossing the species barrier and infecting another vertebrate. This finding will contribute to the development of efficient strategies for disease management in aquaculture.

Gene expression profiles alteration after infection of virus, bacteria, and parasite in the Olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is one of economically valuable fish species in the East Asia. In comparison with its economic importance, available genomic information of the olive flounder is very limited. The mass mortality caused by variety of pathogens (virus, bacteria and parasites) is main problem in aquaculture industry, including in olive flounder culture. In this study, we carried out transcriptome analysis using the olive flounder gill tissues after infection of three types of pathogens (Virus; Viral hemorrhagic septicemia virus, Bacteria; Streptococcus parauberis, and Parasite; Miamiensis avidus), respectively. As a result, we identified total 12,415 differentially expressed genes (DEG) from viral infection, 1,754 from bacterial infection, and 795 from parasite infection, respectively. To investigate the effects of pathogenic infection on immune response, we analyzed Gene ontology (GO) enrichment analysis with DEGs and sorted immune-related GO terms per three pathogen groups. Especially, we verified various GO terms, and genes in these terms showed down-regulated expression pattern. In addition, we identified 67 common genes (10 up-regulated and 57 down-regulated) present in three pathogen infection groups. Our goals are to provide plenty of genomic knowledge about olive flounder transcripts for further research and report genes, which were changed in their expression after specific pathogen infection.