Myung-Hwa Jung

Inactivated vaccine against viral hemorrhagic septicemia (VHS) emulsified with squalene and aluminum hydroxide adjuvant provides long term protection in olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia (VHS) in olive flounder (Paralichthys olivaceus) remains an unsolved health problem in Korean aquaculture. Vaccination plays a significant role in modern aquaculture, and the duration of protection provided is of vital importance. Here, we have demonstrated the efficacy, duration of protection and safety of an inactivated vaccine emulsified with squalene (5%) and aluminum hydroxide (0.5%). The inactivated VHS vaccine provided a moderate protection of 37% and 47% relative percent survival (RPS) at 4 and 10 weeks post vaccination (wpv). Addition of squalene and aluminum hydroxide into inactivated VHS vaccine clearly enhanced the level of protection showing 58% and 83% RPS at 4 and 10 wpv, respectively, indicating the need for adjuvants to enhance the efficacy. The vaccinated fish showed significant protection at 3, 6, 12, 18, 24, and 40 wpv (except week 57) than non-vaccinated fish to an intraperitoneal challenge of 10(7.1)TCID₅₀/fish at 15 °C, with RPS of 60%, 64%, 71%, 55%, 52% and 50% (45% at 57 week), respectively, covering the duration of natural outbreak. Fish challenged at 18 wpv at 6 °C showed 56% RPS and protection at a low temperature. The antibody titer was high at 3 wpv with an OD of 1.08 ± 0.13, but decreased gradually and was undetectable by 24 wpv. The vaccine formulation was safe without injection site reactions, adhesions, or pigmentation observed at 6, 12, 18, or 24 wpv. Inflammatory reactions were observed in the spleen intestine at 6 and 12 wpv but were similar as control by 24 wpv. These results confirm that this vaccine is efficient and safe for olive flounder and could offer an appropriate strategy to prevent VHS without causing side effects.

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.

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

Abstract 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 &mgr;g/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 &mgr;g/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). HighlightsTreatment with poly (I:C) at before/post infection had significantly higher protection.Poly (I:C) provided significant protection for 200 days.TLR3 and Mx were highly expressed in poly (I:C) administered rock bream.IRF3, ISG15 and PKR induced immune responses were high activated in blood.

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).

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

ABSTRACT 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/&mgr;l). Moreover, TRAF6, IRF5, IL1&bgr;, IL8, IL12 and TNF&agr; 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, IL1&bgr;, IL8, IL12 and TNF&agr; 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), IL1&bgr; (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, IL1&bgr; 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). HighlightsRBIV copy number displayed a correlation with fish mortality.MyD88, TRAF6 and pro‐inflammatory cytokines related immune responses were not enhanced in the high mortality group.MyD88 and TRAF6 were highly expressed in the low mortality group.MyD88, TRAF6, IL1&bgr; and IL8 induced immune responses were high activated in the no mortality group.

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

Abstract 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/&mgr;l, respectively), 7 (3.67 × 105 and 8.81 × 106/&mgr;l, respectively) and 10 days post infection (dpi) (1.26 × 105 and 6.02 × 107/&mgr;l, respectively). Moreover, significantly high expression levels of TLR3 (8.6‐ and 7.7‐fold, at 4 and 7 dpi, respectively) and IL1&bgr; (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 TNF&agr; 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, IL1&bgr;, Mx and ISG15‐mediated immune responses, which could be a critical factor for inhibition of virus replication. HighlightsPoly (I:C) inhibited virus replication in spleen, kidney, liver and blood of rock bream.TLR3 and IL1&bgr; were highly expressed in the poly (I:C)+virus‐injected group.Mx and ISG15 were significantly expressed earlier (from 2 dpi) in the poly (I:C)+virus‐injected group than in the virus‐injected group (from 4 dpi).

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

ABSTRACT 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/&mgr;l). 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/&mgr;l). 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. HighlightsShifting water temperature inhibited RBIV replication in rock bream.Fish mortality and virus copy number were highly dependent on period of exposure days at susceptible water temperatures.Virus completely eliminated under the threshold of causing mortality at the late stage.Fish need at least 50 days from peaked virus level to avoid re‐activation of the virus.

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.

Recent Advances in Application of Nanoparticles in Fish Vaccine Delivery

ABSTRACT There is a constant need for the development of efficient vaccines and delivery systems to prevent and control the emerging and re-emerging infectious diseases in aquaculture. There are innumerable infectious diseases for which the development of efficient vaccines has been difficult to achieve. The failure is mainly due to the inability to design vaccines evoking appropriate immune responses. The use of nanoparticles has provided a tremendous opportunity to design vaccine delivery systems that are efficient in targeted delivery, providing stability to antigens, and act as efficient adjuvants. Many of the nanoparticles are able to enter the antigen presenting cells by different pathways and induce appropriate immune responses to the antigen. A number of different nanoparticles are used in fish vaccine delivery, which includes biodegradable polymers, nanoliposomes, carbon nanotubes, calcium phosphate, and immunostimulating complexes (ISCOMs), among which poly (lactic-co-glycolic acid) and chitosan are the most studied form of nanoparticles to date. Hence, the use and application of other forms of nanoparticles need to be explored. This review provides an overview of the use of different nanoparticle systems for the delivery of fish vaccines and compares the potential of these delivery systems for the development of new vaccines against different fish pathogens.

Efficacy of algal Ecklonia cava extract against viral hemorrhagic septicemia virus (VHSV).

ABSTRACT The inhibition efficacy of an extract from Ecklonia cava (E. cava) was studied to determine whether the extract and compounds exhibited inhibitory activity against VHSV in the fathead minnow (FHM) cell line and following oral administration to the olive flounder. Based on its low toxicity and effective concentration, the E. cava extract (Ext) and compounds (eckol and phlorofucofuroeckol A) were selected for further analysis. In the plaque reduction assay, simultaneous co‐exposure of VHSV to Ext, eckol and phlorofucofuroeckol A showed a higher level of inhibition than the pre‐ and post‐exposure groups. The antiviral activity in the FHM cell line was time‐dependent and increased with the exposure time with the virus and Ext or the compounds. In the in vivo experiments, different Ext concentrations were orally administered to the olive flounder. In trial I, the relative percent survival (RPS) following oral administration of 500 and 50 &mgr;g/g/day of Ext was 31.25% and 12.50%, respectively. In trial II, the RPS for 1000, 500 and 50 &mgr;g/g/day of Ext was 31.57%, 0% and 0%, respectively. In trial III, the RPS after 1 and 2 weeks (1000 &mgr;g/g/day) of exposure to Ext was 26.31% and 31.57%, respectively. Oral administration of Ext (1000 &mgr;g/g/day) significantly induced inflammatory cytokine responses (IL‐1&bgr;, IL‐6 and IFN‐&ggr;) at 1 and 2 days post‐oral administration (dpa). Additionally, IFN‐&agr;/&bgr; (7–12 dpa), ISG15 (2, 7 and 10 dpa) and Mx (7–12 dpa) were significantly activated in the olive flounder. In conclusion, we demonstrated an inhibitory ability of the E. cava extract and compounds against VHSV in the FHM cell line. Moreover, oral administration of the E. cava extract to the olive flounder enhanced antiviral immune responses and the efficacy of protection against VHSV, resulting in an anti‐viral status in the olive flounder. HighlightsE. cava (extract and compounds) exhibited high inhibitory activity against VHSV in vitro.Feeding of the E. cava extract (1000 &mgr;g/g/day) resulted in higher protection against VHSV.IL‐1&bgr;, IL‐6 and IFN‐&ggr; were highly expressed by oral administration of the E. cava extract.IFN‐&agr;/&bgr;, ISG15 and Mx were activated by E. cava extract in the olive flounder.