Effect of rock bream iridovirus (RBIV) contained tissue intake on rock bream (Oplegnathus fasciatus) mortality and blood cell distribution.

Rock bream (Oplegnathus fasciatus) is one of the highly priced cultured marine fish in Korea. Rock bream iridovirus (RBIV) outbreaks in aquaculture farms may involve environmental factors, co-infection with other pathogenic microorganisms and grounded (raw) fish feed. This study evaluated the effects of RBIV-containing tissue intake on mortality and oral transmission in rock bream. Virus-containing tissues administered to rock bream [50 mg (1.53 × 108/major capsid protein, MCP gene copies) to 2400 mg (7.34 × 109)] held at 23 °C lead to 100% mortality by 27 days post administration. Interestingly, the mortality rates were not viral dose- or concentration dependent. Further, high MCP gene copy numbers were observed in the gill, liver, intestine, stomach, spleen, heart, kidney, brain and muscle tissues (viral load range of 3.03 × 106 to 4.01 × 107/mg, average viral load 1.70 × 107/mg) of dead rock bream. Moreover, a high viral load was detected in the intestine and stomach, where the virus was directly administered. This indicated that the intake of RBIV-containing tissue feed weakens the intestinal mucosal immunity and increases viral load in the intestine. Moreover, the levels of complete blood cell count (CBC) indicators, such as red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) significantly decreased from 15 dpi with red blood cell distribution width (RDW), and white blood cells (lymphocyte, monocyte and granulocyte) significantly increased from the initial to later stage of infection. These results highlight the significance of blood-mediated indicators against RBIV infection in rock bream. We demonstrate the existence of an oral transmission route for RBIV in rock bream. Our findings indicate that pathogen-containing feed is an important risk factor for disease outbreaks in rock bream.

Efficacy of saponin-based inactivated rock bream iridovirus (RBIV) vaccine in rock bream (Oplegnathus fasciatus).

Rock bream iridovirus (RBIV) causes severe mortality in rock bream (Oplegnathus fasciatus) for last two decades. In view of this constant threat of RBIV to the rock bream industry, we conducted the present study with the aim to develop a safe and efficient remedial measure against the virus. In this study, we evaluated the safety and potentiality of squalene, aluminium hydroxide and saponin adjuvants, singly or in combinations, which can be used for developing an efficient inactivated (IV) vaccine to protect rock bream from RBIV infection. The evaluation results demonstrated that saponin (Sa) has the required potential in enacting the antiviral immune response in the host and in providing protection against virus mediated lethality, without causing any adverted side-effects. The study further, showed that a single primary dose of Sa-adjuvanted IV vaccine can confer moderate protections in short (60.04% relative percent mortality (RPS) at 4 wpv) and medium (53.38% RPS at 8 wpv) term post RBIV challenge; whereas, the same vaccine when administered in a prime-boost strategy, it resulted enhanced 93.34% RPS post virus challenge at 4 and 8 wpv. The moderate to high survivability demonstrated by the Sa-adjuvanted IV vaccine, was substantiated by the significant (p < 0.05) upregulation of IL-1ß, Mx and PKR gene transcript. All surviving fish from the Sa-adjuvanted IV vaccine groups were strongly protected from re-infection with RBIV (1.1 × 107) at 70 days post infection (dpi). In conclusion, it can be inferred that, Sa-adjuvanted IV RBIV vaccine can be an efficient control measure to protect the rock bream aquaculture industry against the lethal RBIV virus.

Co-Expression Network Analysis of Spleen Transcriptome in Rock Bream (Oplegnathus fasciatus) Naturally Infected with Rock Bream Iridovirus (RBIV).

Rock bream iridovirus (RBIV) is a notorious agent that causes high mortality in aquaculture of rock bream (Oplegnathus fasciatus). Despite severity of this virus, no transcriptomic studies on RBIV-infected rock bream that can provide fundamental information on protective mechanism against the virus have been reported so far. This study aimed to investigate physiological mechanisms between host and RBIV through transcriptomic changes in the spleen based on RNA-seq. Depending on infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as control (0C), heavy infected fish at week 0 (0H), heavy mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and light infected fish at week 3 (3L). We explored clusters from 35,861 genes with Fragments Per Kilo-base of exon per Million mapped fragments (FPKM) values of 0.01 or more through signed co-expression network analysis using WGCNA package. Nine of 22 modules were highly correlated with viral infection (|gene significance (GS) vs. module membership (MM) |> 0.5, p-value < 0.05). Expression patterns in selected modules were divided into two: heavy infected (0H and 0MH) and control and light-infected groups (0C, 3C, and 3L). In functional analysis, genes in two positive modules (5448 unigenes) were enriched in cell cycle, DNA replication, transcription, and translation, and increased glycolysis activity. Seven negative modules (3517 unigenes) built in this study showed significant decreases in the expression of genes in lymphocyte-mediated immune system, antigen presentation, and platelet activation, whereas there was significant increased expression of endogenous apoptosis-related genes. These changes lead to RBIV proliferation and failure of host defense, and suggests the importance of blood cells such as thrombocytes and B cells in rock bream in RBIV infection. Interestingly, a hub gene, pre-mRNA processing factor 19 (PRPF19) showing high connectivity (kME), and expression of this gene using qRT-PCR was increased in rock bream blood cells shortly after RBIV was added. It might be a potential biomarker for diagnosis and vaccine studies in rock bream against RBIV. This transcriptome approach and our findings provide new insight into the understanding of global rock bream-RBIV interactions including immune and pathogenesis mechanisms.

Cloning and expressional analysis of secretory and membrane-bound IgM in rock bream (Oplegnathus fasciatus) under megalocytivirus infection and vaccination.

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future.

Protective immunity against rock bream iridovirus (RBIV) infection and TLR3-mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus) following poly (I:C) administration.

In this study, we evaluated the potential of poly (I:C) to induce antiviral status for protecting rock bream from RBIV infection. Rock bream injected with poly (I:C) at 2 days before infection (1.1 × 104) at 20 °C had significantly higher protection with RPS 13.4% and 33.4% at 100 and 200 µg/fish, respectively, through 100 days post infection (dpi). The addition of boost immunization with poly (I:C) at before/post infection at 20 °C clearly enhanced the level of protection showing 33.4% and 60.0% at 100 and 200 µg/fish, respectively. To investigate the development of a protective immune response, rock bream were re-infected with RBIV (1.1 × 107) at 200 dpi. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. Poly (I:C) induced TLR3 and Mx responses were observed at several sampling time points in the spleen, kidney and blood. Moreover, significantly high expression levels of IRF3 (2.9- and 3.1-fold at 1 d and 2 days post administration (dpa), respectively), ISG15 and PKR expression (5.4- and 10.2-fold at 2 dpa, respectively) were observed in the blood, but the expression levels were low in the spleen and kidney after poly (I:C) administration. Our results showed the induction of antiviral immune responses and indicate the possibility of developing long term preventive measures against RBIV using poly (I:C).

Innate immune responses against rock bream iridovirus (RBIV) infection in rock bream (Oplegnathus fasciatus) following poly (I:C) administration.

Poly (I:C) showed promise as an immunoprotective agents in rock bream against rock bream iridovirus (RBIV) infection. In this study, we evaluated the time-dependent virus replication pattern and antiviral immune responses in RBIV-infected rock bream with and without poly (I:C) administration. In the poly (I:C)+virus-injected group, virus copy numbers were more than 18.9-, 24.0- and 479.2-fold lower than in the virus only injected group at 4 (4.73 × 104 and 8.95 × 105/µl, respectively), 7 (3.67 × 105 and 8.81 × 106/µl, respectively) and 10 days post infection (dpi) (1.26 × 105 and 6.02 × 107/µl, respectively). Moreover, significantly high expression levels of TLR3 (8.6- and 7.7-fold, at 4 and 7 dpi, respectively) and IL1ß (3.6-fold at 2 dpi) were observed in the poly (I:C)+virus-injected group, but the expression levels were not significantly in the virus-injected group. However, IL8 and TNFα expression levels showed no statistical significance in both groups. Mx, ISG15 and PKR were significantly highly expressed from 4 to 10 dpi in the virus-injected group. Nevertheless, in the poly (I:C)+virus-injected group, Mx and ISG15 expression were significantly expressed from 2 dpi. In summary, poly (I:C) administration in rock bream induces TLR3, IL1ß, Mx and ISG15-mediated immune responses, which could be a critical factor for inhibition of virus replication.

CpG ODN 1668 induce innate and adaptive immune responses in rock bream (Oplegnathus fasciatus) against rock bream iridovirus (RBIV) infection.

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream in Korea. CpG ODN 1668 showed promise as immunoprotective agents against RBIV infection in rock bream. In this study, we assessed innate/adaptive-related gene expression patterns in RBIV-infected rock bream with and without CpG ODN 1668 administration to determine important immune defense related factors that may affect fish survival. In the CpG ODN 1668+virus-injected group, virus copies were more than 7.4- to 790591-fold lower than in the virus-injected group at 4 d (8.79 × 104 and 6.58 × 105/µl, respectively), 7 d (5.30 × 102 and 2.29 × 107/µl, respectively) and 10 dpi (7.79 × 101 and 6.16 × 107/µl, respectively). Furthermore, in the CpG ODN 1668+virus-injected group, significantly higher levels of MyD88 (6 h, 1 d, 4 d and 7 dpi), IL1ß (1 d, 2 d and 7 dpi) and perforin/granzyme (1 dpi) expression were observed, whereas these genes were not significantly expressed in the virus-injected group at that time points. Mx, ISG15 and PKR were significantly highly expressed at 4 d and 7 dpi and reduced when low viral loads at 10 dpi in the CpG ODN 1668+virus-injected group. Conversely, in the virus-injected group, Mx, ISG15 and PKR expression were significantly higher than the control group until 10 dpi. However, MHC class I, CD8, Fas, Fas ligand and caspases (3, 8 and 9) expression levels showed no statistically significant differences between virus- and CpG ODN 1668+virus-injected group. In summary, CpG ODN 1668 administration in fish induces innate immune response or cell death pathway, which could be a major contributing factor to effective fish control over viral transcription on 4 d to 10 dpi. Expression of MyD88, IL1ß, perforin and granzyme-related immune gene response is critical factor for inhibition of RBIV replication.

Rock bream iridovirus (RBIV) replication in rock bream (Oplegnathus fasciatus) exposed for different time periods to susceptible water temperatures.

Rock bream iridovirus (RBIV) is a member of the Megalocytivirus genus that causes severe mortality to rock bream. Water temperature is known to affect the immune system and susceptibility of fish to RBIV infection. In this study, we evaluated the time dependent virus replication pattern and time required to completely eliminate virus from the rock bream body against RBIV infection at different water temperature conditions. The rock bream was exposed to the virus and held at 7 (group A1), 4 (group A2) and 2 days (group A3) at 23 °C before the water temperature was reduced to 17 °C. A total of 28% mortality was observed 24-35 days post infection (dpi) in only the 7 day exposure group at 23 °C. In all 23 °C exposure groups, virus replication peaked at 20 to 22 dpi (106-107/µl). In recovery stages (30-100 dpi), the virus copy number was gradually reduced, from 106 to 101 with faster decreases in the shorter exposure period group at 23 °C. When the water temperature was increased in surviving fish from 17 to 26 °C at 70 dpi, they did not show any mortality or signs of disease and had low virus copy numbers (below 102/µl). Thus, fish need at least 50 days from peaked RBIV levels (approximately 20-25 dpi) to inhibit the virus. This indicates that maintaining the fish at low water temperature (17 °C) for 70 days is sufficient to eradicate RBIV from fish body. Thus, RBIV could be eliminated slowly from the fish body and the virus may be completely eliminated under the threshold of causing mortality.

Gene expression regulation of the TLR9 and MyD88-dependent pathways in rock bream against rock bream iridovirus (RBIV) infection.

Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus, causes severe mass mortalities in rock bream in Korea. To date, the innate immune defense mechanisms of rock bream against RBIV is unclear. In this study, we assessed the expression levels of genes related to TLR9 and MyD88-dependent pathways in RBIV-infected rock bream in high, low or no mortality conditions. In the high mortality group (100% mortality at 15 days post infection (dpi)), high levels of TLR9 and MyD88 expressions (6.4- and 2.4-fold, respectively) were observed at 8 d and then reduced (0.6- and 0.1-fold, respectively) with heavy viral loads at 10 dpi (2.21 × 107/µl). Moreover, TRAF6, IRF5, IL1ß, IL8, IL12 and TNFα expression levels showed no statistical significance until 10 dpi. Conversely, in the low mortality group (28% expected mortality at 35 dpi), TLR9, MyD88 and TRAF6 expression levels were significantly higher than those in the control group at several sampling points until 30 dpi. Higher levels of IRF5, IL1ß, IL8, IL12 and TNFα expression were also observed, however, these were not significantly different from those of the control group. In the no mortality group (0% mortality at 40 dpi), significantly higher levels of MyD88 (2 d, 4 d and 40 dpi), TRAF6 (2 dpi), IL1ß (4 dpi) and IL8 (2 d and 4 dpi) expression were observed. In summary, RBIV-infected rock bream induces innate immune response, which could be a major contributing factor to effective fish control over viral transcription. MyD88, TRAF6, IL1ß and IL8-related immune responses were activated in fish survivor condition (low or no mortality group). This is a critical factor for RBIV disease recovery; however, these immune responses did not efficiently respond in fish dead condition (high mortality group).

Low pathogenicity of flounder iridovirus (FLIV) and the absence of cross-protection between FLIV and rock bream iridovirus.

The genus Megalocytivirus is known to infect a wide range of cultured marine fish. In this study, we examined the pathogenicity of FLIV (Megalocytivirus from olive flounder, genotype III) and RBIV (Megalocytivirus from rock bream, genotype I) to their homologous and heterologous host species. Olive flounder (7.5 ± 1.3 cm) injected with FLIV [major capsid protein (MCP) gene copies, 6.8 × 103 -6.5 × 106 /fish] at 24 °C did not die until 90 days post-infection (dpi). The average virus replication in the spleen peaked (1.27 × 106 /fish) at 20 dpi. Rock bream (6.5 ± 1.5 cm) injected with FLIV (8.8 × 105 and 6.5 × 106 /fish of MCP copies) showed no mortality until 50 dpi. The rock bream that survived after FLIV infection were rechallenged with RBIV at 50 dpi had 100% mortality, showing that there is no cross-protection between FLIV and RBIV. Temperature shifting (26 °C and 20 °C at 12 h intervals) did not cause FLIV-specific mortality into olive flounder, but higher virus copies were observed in the fish exposed to higher stocking density. This study demonstrates that FLIV and RBIV have different antigenic and pathogenic characteristics and that FLIV has low pathogenicity to olive flounder.

Effects of water temperature on mortality in Megalocytivirus-infected rock bream Oplegnathus fasciatus (Temminck et Schlegel) and development of protective immunity.

Rock bream iridovirus (RBIV) causes huge losses, especially in rock bream Oplegnathus fasciatus. Rock bream injected with RBIV and held at 29, 26, 23 or 20 °C had 100% mortality. Conversely, all infected fish held at 17 °C survived even after the temperature was progressively increased to 26 °C at 100 dpi. Rock bream exposed to virus and held for 2, 4 and 7 days at 23/26 °C before the temperature was reduced to 17 °C had mortality rates of 26.6/73.2%, 66.6/100% and 93.4/100%, respectively, through 100 dpi. When surviving fish had the water temperature increased from 17 to 26 °C at 100 dpi, they did not exhibit signs of disease and had low virus copy numbers (below 10(3)). To investigate the development of a protective immune, rock bream were infected with RBIV and held at 23 °C before shifting the water temperature to 17 °C at 4 dpi. All injected fish survived until 120 dpi. While 100% of the previously unexposed fish died, 80.2% of the previously infected fish survived. When the survivors were rechallenged again at 160 dpi, no further mortality occurred. The high survival rate of fish following rechallenge with RBIV indicates that protective immunity was established in the surviving rock bream.

Gene expression of pro- and anti-apoptotic proteins in rock bream (Oplegnathus fasciatus) infected with megalocytivirus (family Iridoviridae).

Viruses belonging to the genus Megalocytivirus cause diseases in marine fishes primarily in East and Southeast Asian countries. Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus, causes severe mass mortalities in rock beam (Oplegnathus fasciatus) in Korea. In this study, we assessed apoptosis-related gene expression patterns in Megalocytivirus-infected rock bream in high mortality and low mortality conditions to determine important apoptosis-related factors, which may affect fish survival/or death. In the high mortality group (100% mortality at 15 dpi), significantly high levels of perforin, granzyme, Fas ligand and caspase 9 expression (5.6-, 10.2-, 13.4- and 4.2-fold, respectively) were observed in the kidney at 8 dpi. Basal expression levels of Fas and caspase 3 were observed at 8 d (1.5-/0.7-fold) and 10 dpi (1.3-/0.6-fold), accompanied by heavy viral loads (8.12 × 10(6)-2.21 × 10(7)/µl). Inhibitor of apoptosis 1 (IAP1) was highly expressed (3.5- to 4.8-fold) at 1 d and 4 dpi; however, IAP1 was reduced when fish died at 8 d and 10 dpi (1.7- to 2.0-fold), which was not significantly different from that of the control group. A similar expression pattern was observed in the low mortality group (18% expected mortality at 30 dpi), which was characterised by a delayed lower magnitude of expression with lower viral loads than the high mortality group. Perforin, granzyme and Fas ligand expression was significantly higher in the low mortality group than in the control group at several sampling points until 30 dpi. Fas and caspases 8, 9 and 3 expression levels showed no statistical significance until 30 dpi. In the low mortality group, significantly higher IAP1 expression compared with the control was observed at 10 d (2.2-fold), 20 d (3.6-fold) and 22 dpi (2.0-fold). In summary, perforin- and granzyme-related apoptosis initiation signals were activated; however, the Fas-induced apoptosis pathway did not efficiently respond. Upregulated IAP1 in RBIV-infected rock bream, which was reported for the first time in this study, exhibited inhibited apoptotic responses in RBIV-infected fish. Although it remains unclear whether apoptosis inhibition aids or impedes fish survival, our data clearly show that the apoptotic response is inhibited in RBIV-infected rock bream.

Abnormalities in red blood cell production and pathogenesis of anemia in the progression of rock bream iridovirus (RBIV).

Rock bream iridovirus (RBIV), belonging to Megalocytivirus, causes severe mortality in rock bream. Almost all deaths associated with RBIV are accompanied by splenic enlargement and anemia. Although red blood cells (RBCs) are involved in the immune response against viral infections, their involvement in rock bream has not yet been studied in terms of the immune response against RBIV. In this study, the viral replication patterns, blood characteristics and anemia-related factors were evaluated in rock bream post RBIV infection. The virus-infected RBCs of rock bream demonstrated similarities in the expression levels of hemoglobins (HGB) (α and ß), cytokine-dependent hematopoietic cell linker (CLNK) and hematopoietic transcription factor GATA (GATA), with significantly decreasing levels from 4 days post infection (dpi) to 17 (dpi), when the viral replication was at its peak. This suggests that the expression of blood-related genes is inadequate for HGB synthesis and RBC production, thereby causing anemia leading to death. Moreover, the levels of complete blood cell count (CBC) indicators, such as RBCs, HGB and hematocrit (HCT), significantly decreased from 10 to 17 dpi. This phenomenon suggests that blood-related gene expression and/or RBC-, HGB- and HCT-related levels are critical factors in RBIV-induced anemia and disease progression. These results highlight the significance of blood-mediated immune responses against RBIV infection in rock bream. Understanding blood-related gene levels to identify blood-related immune response interactions in rock bream will be useful for development of future strategies in controlling RBIV diseases in rock bream.

Protective immunity induced by ankyrin repeat-containing protein-based DNA vaccine against rock bream iridovirus (RBIV) in rock bream (Oplegnathus fasciatus).

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) and remains an unsolved problem in Korea aquaculture industry. In this study, we assessed the potential of ankyrin repeat (ANK)-containing proteins to induce protective immunity in RBIV-infected rock bream. Rock bream administered with ankyrin repeat-containing protein-based DNA vaccine (200 ng/fish) exhibited significant protection against at 4 and 8 weeks post vaccination to infected with 6.7 × 105 RBIV at 23°C; relative percent survival (RPS) of 60.04% and 40.1%, respectively. Furthermore, survivors from the first infection were strongly protected from RBIV (1.1 × 107) re-infection at 70 days post infection, as 100% RPS was observed and without clinical signs of RBIV diseases. Moreover, TLR3 (9.5-fold), TLR9 (5.2-fold), MyD88 (15.9-fold), Mx (55.5-fold), ISG15 (19.0-fold), PKR (24.2-fold), MHC class I (5.1-fold), perforin (6.5-fold), Fas (6.4-fold), Fas ligand (7.1-fold), caspase8 (5.0-fold), caspase9 (12.5-fold), and caspase3 (6.3-fold) responses were significantly elevated in the muscle (vaccine injection site) of ANK-based DNA vaccinated fish at 7 days post vaccination. However, inflammatory cytokines (IL1ß, IL8, and TNFα) were not enhanced in the vaccinated rock bream. Moreover, ANK gene may be a good candidate to detect RBIV infection or in revealing specific information to elucidate the pathogenic mechanisms underlying RBIV infection. In summary, ANK-based DNA vaccination in rock bream induced TLR- and IFN-mediated or apoptosis-related immune responses and suggest efficient preventive measures against RBIV.

Functional characterization of a novel tumor necrosis factor gene (TNF-New) in rock bream (Oplegnathus fasciatus).

The novel tumor necrosis factor (TNF-New or TNFN) gene has been identified only in teleost such as zebrafish, medaka (Oryzias latipes), fugu (Takifugu rubripes), and rainbow trout (Oncorhynchus mykiss). In this study, a putative TNFN gene in rock bream (named RB-TNFN) was cloned and its functional expression in the immune system was analyzed. Although it was previously reported to share a high degree of homology with mammalian lymphotoxin (LT)-ß, in silico analysis revealed that RB-TNFN differed slightly from mammalian LT-ß in its genomic structure, phylogenetic relationship, and predicted protein tertiary structure, whereas the genomic location of TNFN (immediately behind TNF-α) was the same as that of LT-ß. In healthy rock bream, RB-TNFN gene expression was the highest in the liver and the lowest in the head kidney. In vitro, it was significantly upregulated in head kidney cells following polyinosinic:polycytidylic acid, concanavalin A, phytohemagglutinin, or calcium ionophore (CI) stimulation and in spleen cells by lipopolysaccharide (LPS), CI, and rock bream iridovirus (RBIV). In vivo, it was upregulated in the spleen, liver, and gut on day 1 and in the blood on day 3 following LPS injection, and in the blood, head kidney, and liver following RBIV vaccination. Post-RBIV infection, the vaccinated group showed a significantly higher TNFN gene expression in the head kidney and blood than the unvaccinated group. Treatment with recombinant TNFN protein (RB-rTNFN) resulted in significantly upregulated interleukin-1ß expression in the head kidney, spleen, blood, liver, and peritoneal cells. It also enhanced IL-8 gene expression in the head kidney, blood, and peritoneal cells, and interferon γ gene expression in the gut and gills on day 1. TNFN and cyclo-oxygenase-2 gene expression was upregulated in peritoneal cells on day 3. Flow cytometry analysis revealed a significant increase in the peritoneal lymphocyte population after the intraperitoneal (i.p.) injection of RB-rTNFN. These results suggest that RB-TNFN mediated innate and adaptive immunity in rock bream.

Proteome profile of spleen in rock bream (Oplegnathus fasciatus) naturally infected with rock bream iridovirus (RBIV).

BACKGROUND: Rock bream iridovirus (RBIV) is one of the most dangerous pathogens that causes the highest mortality in the aquaculture of rock bream (Oplegnathus fasciatus). Even though RBIV infection leads to huge economic loss, proteome studies on RBIV-infected rock bream have not been conducted to provide information about the differential protein expression pattern by the host protection system. OBJECTIVE: The purpose of this study was to investigate the protein expression patterns in spleens of rock bream olive after infection by RBIV or mixed infection by RBIV and bacteria. METHODS: Depending on the infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as the control (0C), heavily infected fish at week 0 (0H), heavily mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and lightly infected fish at week 3 (3L). Proteins were extracted from the spleens of infected rock bream. We used 2-DE analysis with LC-MS/MS to investigate proteome changes in infected rock bream. RESULTS: The results of the LC-MS/MS analyses showed different protein expression profiles after infection. Proteins related to oxygen transport and energy generation, such as hemoglobin, beta-globin, and ATP synthase, were mostly expressed in the infected spleen. Whereas proteins involved in structure and cell movement, such as tubulin, myosin, actin binding proteins, and intermediate filament proteins, were down-regulated in the infected spleens. The protein expression profiles between infection by RBIV and mixed infection by RBIV and bacteria showed similar patterns. CONCLUSIONS: Our results indicated that infection by RBIV or mixed infection by RBIV and bacteria triggered energy generation and oxygen-transport, but cell migration and constructional changes in the spleen were extremely decreased.

Cloning and characterization of tumor necrosis factor superfamily 15 in rock bream, Oplegnathus fasciatus; phylogenetic, in silico, and expressional analysis.

Tumor necrosis factor superfamily (TNFSF)15 is a member of TNFSF which shares a high homology with other TNFSFs, especially lymphotoxin (LT)-α in teleost. In this study, we have cloned a putative TNFSF15 gene in rock bream which was highly homologous with other fish TNFSF15 and performed bioinformatic analysis to confirm the membership. The RB-TNFSF15 cDNA consists of 3192 bp (193 bp of 5'-untranslated region (UTR), 732 bp of ORF, and 2267 bp of 3'-UTR) and encodes a polypeptide of 243 amino acids containing a predicted TNF superfamily signature with 43-61% identities with fish TNFSF15. The predicted 3D structure was similar to human TNFSF15 with ß barrel structure containing 10 ß strands and 1 α helix while human LT-α and ß contain 10 ß strands and 2 α helices. Consequently, the synteny and phylogenetic analysis of fish TNFSF15 genes and structural similarity of the predicted protein to mammalian TNFSF15 implicate that they can be identified as TNFSF15. In healthy rock bream, RB-TNFSF15 gene expression level was the highest in fin and the lowest in blood. In vitro, TNFSF15 gene expression was up-regulated by lipopolysaccharide, polyinosinic:polycytidylic acid (poly I:C) and rock bream iridovirus (RBIV) in head kidney, while up-regulated by poly I:C and RBIV at later time in spleen. In vivo, RB-TNFSF15 gene expression was up-regulated in head kidney, liver and blood after vaccination with a formalin inactivated RBIV. After challenging with RBIV, RB-TNFSF15 gene expression was up-regulated in unvaccinated group at day 3 post-infection in head kidney. In gill, it was significantly up-regulated in vaccinated group at day 1 post-challenge and all groups at day 7, indicating that RB-TNFSF may play a key role in mucosal immunity during viral infection. Since the regulation mechanism of TNFSF15 gene expression in fish has not yet been elucidated, the present study will help to understand the roles of TNFSF15 in fish immune system.

DNA vaccine encoding myristoylated membrane protein (MMP) of rock bream iridovirus (RBIV) induces protective immunity in rock bream (Oplegnathus fasciatus).

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) in Korea. In this study, we investigated the potential of viral membrane protein to induce antiviral status protecting rock bream against RBIV infection. We found that fish administered with ORF008L (myristoylated membrane protein, MMP) vaccine exhibited significantly higher levels of survival compared to ORF007L (major capsid protein, MCP). Moreover, ORF008L-based DNA vaccinated fish showed significant protection at 4 and 8 weeks post vaccination (wpv) than non-vaccinated fish after infected with RBIV (6.7 × 105) at 23 °C, with relative percent survival (RPS) of 73.36% and 46.72%, respectively. All of the survivors from the first RBIV infection were strongly protected (100% RPS) from re-infected with RBIV (1.1 × 107) at 100 dpi. In addition, the MMP (ORF008L)-based DNA vaccine significantly induced the gene expression of TLR3 (14.2-fold), MyD88 (11.6-fold), Mx (84.7-fold), ISG15 (8.7-fold), PKR (25.6-fold), MHC class I (13.3-fold), Fas (6.7-fold), Fas ligand (6.7-fold), caspase9 (17.0-fold) and caspase3 (15.3-fold) at 7 days post vaccination in the muscle (vaccine injection site). Our results showed the induction of immune responses and suggest the possibility of developing preventive measures against RBIV using myristoylated membrane protein-based DNA vaccine.

Protective immunity against Megalocytivirus infection in rock bream (Oplegnathus fasciatus) following CpG ODN administration.

Rock bream iridovirus (RBIV) disease in rock bream (Oplegnathus fasciatus) remains an unsolved problem in Korea aquaculture farms. CpG ODNs are known as immunostimulant, can improve the innate immune system of fish providing resistance to diseases. In this study, we evaluated the potential of CpG ODNs to induce anti-viral status protecting rock bream from different RBIV infection conditions. We found that, when administered into rock bream, CpG ODN 1668 induces better antiviral immune responses compared to other 5 CpG ODNs (2216, 1826, 2133, 2395 and 1720). All CpG ODN 1668 administered fish (1/5µg) at 2days before infection (1.1×107) held at 26°C died even though mortality was delayed from 8days (1µg) and 4days (5µg). Similarly, CpG ODN 1668 administered (5µg) at 2days before infection (1.2×106) held at 23/20°C had 100% mortality; the mortality was delayed from 9days (23°C) and 11days (20°C). Moreover, when CpG ODN 1668 administered (1/5/10µg) at 2/4/7days before infection or virus concentration was decreased to 1.1×104 and held at 20°C had mortality rates of 20/60/30% (2days), 30/40/60% (4days) and 60/60/20% (7days), respectively, for the respective administration dose, through 100 dpi. To investigate the development of a protective immune response, survivors were re-infected with RBIV (1.1×107) at 100 and 400 dpi, respectively. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. The high survival rate of fish following re-challenge with RBIV indicates that protective immunity was established in the surviving rock bream. Our results showed the possibility of developing preventive measures against RBIV using CpG ODN 1668 by reducing RBIV replication speed (i.e. water temperature of 20°C and infection dose of 1.1×104).

Expression Analysis of Interferon-Stimulated Gene 15 in the Rock Bream Oplegnathus fasciatus against Rock Bream Iridovirus (RSIV) Challenge.

Interferon-stimulated gene 15 (ISG15) is known to interfere with viral replication and infection by limiting the viral infection of cells. Interferon-stimulated gene 15 (ISG15) interferes with viral replication and infectivity by limiting viral infection in cells. It also plays an important role in the immune response. In this study, tissue-specific expression of ISG15 in healthy rock bream samples and spatial and temporal expression analysis of rock bream ISG15 (RbISG15) were performed following rock bream iridovirus (RSIV) infection. RbISG15 expression was significantly higher in the eye, gill, intestine, kidney, liver, muscle, spleen, and stomach, but low in the brain. There were particularly high levels of expression in the liver and muscle. RbISG15 expression was also examined in several tissues and at various times following RSIV infection. ISG15 expression increased within 3 h in the whole body and decreased at 24 h after infection. In addition, temporal expression of several tissues following RSIV infection showed a similar pattern in the muscle, kidney, and spleen, increasing at 3 h and decreasing at 72 h. These results suggest that ISG15 plays an important role in the immune response of rock bream. Overall, this study characterizes the response of RbISG15 following RSIV infection.