Antiviral effects of umbelliferone against viral hemorrhagic septicemia virus in olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to global aquaculture, prompting ongoing efforts to identify potential drug candidates for disease prevention. Coumarin derivatives have recently emerged as a promising class of compounds effective against rhabdoviruses, which severely impact the aquaculture industry. In this study, we assessed the anti-VHSV activity of umbelliferone (7-hydroxycoumarin) in fathead minnow (FHM) cells and olive flounder Paralichthys olivaceus. Umbelliferone exhibited an EC50 of 100 µg/mL by reducing cytopathic effect, with a maximum cytotoxicity of 30.9 % at 750 µg/mL. Mechanistic analyses via a time-course plaque reduction assay revealed that direct incubation with the virus for 1 h resulted in 97.0 ± 1.8 % plaque reduction, showing excellent direct virucidal activity. Pretreatment for 4 h resulted in a 33.5 ± 7.8 % plaque reduction, which increased with longer incubation times. Cotreatment led to a 33.5 ± 2.9 % plaque reduction, suggesting interference with viral binding, whereas postinfection treatment proved less effective. Umbelliferone was prophylactically administered to the olive flounder through short-term (3 days) and long-term (14 days) medicated feeding, followed by a 4-day postinfection period. Short-term administration at 100 mg/kg body weight (bw)/day resulted in the highest relative percent survival (RPS) of 56 %, whereas long-term administration achieved a maximum RPS of 44 % at 30 mg/kg bw/day. Umbelliferone administration delayed mortality at these doses. Additionally, umbelliferone significantly inhibited the expression of the VHSV N gene during viral challenge, with no observed toxic effects in fish up to an administration dose of 30 mg/kg bw/day for 28 days. Our findings suggest that the protective mechanism of short-term administration of 100 mg umbelliferone against VHSV infection may involve the overexpression of TLR2, MDA5, STAT1, and NF-κB at 24 h postinfection (hpi). IL-8 and IFN II expression was upregulated, whereas TNF-α, IL-1ß, and IFN I expression was suppressed at 24 hpi. The upregulation of ISG15 at 48 hpi may contribute to the inhibition of VHSV replication, whereas the downregulation of Caspase 3 expression at 96 hpi suggests a possible inhibition of virus-induced apoptosis at later infection stages. Overall, umbelliferone exhibited anti-VHSV activity through multiple mechanisms, with the added advantage of convenient administration via medicated feed.

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.

Formulation of chitosan microsphere-based oral vaccine against the scuticociliate parasite Miamiensis avidus: Evaluation of its protective immune potency in olive flounder (Paralichthys olivaceus).

Miamiensis avidus is a parasitic pathogen that causes scuticociliatosis, a severe and often lethal marine infection that affects marine fishes worldwide, including olive flounder (Paralichthys olivaceus) in Korea. This parasite infects all size groups of flounder year-round, causing recurring mortalities and huge economic losses to the Korean flounder industry each year. However, few efforts have been made to implement effective remedial measures to control this parasite. Therefore, our study sought to develop a chitosan microsphere (MS)-encapsulated inactivated vaccine (IMa + chitosan) for oral delivery (adsorbed in feed) to flounder fingerlings and assess its protective efficacy at different modalities via three in vivo experimental trials. Immunisation trial-1 was conducted to determine the effective concentration of chitosan. Our findings indicated that an IMa + chitosan 0.05 % vaccine formulation was safe and effective in providing moderate protection [46.67%-53.3 % relative percent survival (RPS)] against M. avidus intraperitoneal (IP) injection challenge at two weeks post-vaccination (wpv) compared to the IMa + chitosan 0.01 % and IMa + chitosan 0.005 % vaccines (0%-13.3 % RPS) irrespective of the antigen doses. In trial-2, the IMa + chitosan 0.05 % vaccine elicited similar protective immunity (30.8%-57.1 % RPS) in olive flounder against M. avidus at varying antigen doses (high: 2.38 × 106 cells/fish; low: 1.5 × 105 cells/fish), immunisation periods (2 and 5 wpv), and challenge modes (IP injection and immersion). Furthermore, experimental trial-3 validated the use of chitosan MS as an IMa antigen carrier to improve survivability (41.7 % RPS) in the host by significantly (p < 0.05) upregulating specific anti-M. avidus antibody titres in the fish sera and mucus of the group immunised with IMa-containing chitosan MS. In contrast, non-specific immunomodulatory effects (16.7 % RPS and enhanced mucosal antibody titres) were observed in the group treated with chitosan MS without IMa. Therefore, our findings suggested that oral administration of chitosan MS (0.05 %)-encapsulated IMa vaccine is a promising immunisation strategy against M. avidus that can protect the IMa antigen from digestive degradation, facilitates its targeted delivery to the host immune organs, and helps in orchestrating protective immune induction in olive flounder, thus controlling parasite infection.

An antiviral optimized extract from Sanguisorba officinalis L. roots using response surface methodology, and its efficacy in controlling viral hemorrhagic septicemia of olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia causes considerable economic losses for Korea's olive flounder (Paralichthys olivaceus) aquaculture farms; therefore, effective antiviral agents for controlling viral hemorrhagic septicemia virus (VHSV) infection are imperative. The present study implemented a Box-Behnken design and cytopathic reduction assay to derive an optimized extract of Sanguisorba officinalis L. roots (OE-SOR) with maximum antiviral activity against VHSV. OE-SOR prepared under optimized extraction conditions (55% ethanol concentration at 50 °C for 5 h) exhibited potent antiviral activity against VHSV, with a 50% effective 0.21 µg/mL concentration and a 340 selective index. OE-SOR also showed direct virucidal activity in the plaque reduction assay. Administering OE-SOR to olive flounder exhibited substantial efficacies against VHSV infection. Fish receiving 100 mg/kg body weight/day of OE-SOR as a preventive (40.0%; p < 0.05) or therapeutic (44.4%; p < 0.05) exhibited a higher relative survival than the untreated VHSV-infected control group (mortalities of 100% and 90%, respectively). In addition, fish fed with OE-SOR (100 mg/kg body weight/day) for two weeks conveyed a significantly higher inflammatory cytokine expression (nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB], interleukin-1 beta [IL-1ß], and tumor necrosis factor-alpha [TNF-α]) than the control group one to two days post-administration. Moreover, no hematological or histological changes were observed in olive flounder treated with OE-SOR over four weeks. Liquid chromatography-quadrupole-time of flight tandem mass spectrometry and -triple quadrupole tandem mass spectrometry analyses identified ziyuglycoside I as a prominent OE-SOR constituent and marker compound (content: 14.5%). This study verifies that OE-SOR is an effective alternative for controlling viral hemorrhagic septicemia in olive flounder farms as it exhibits efficient in vivo anti-VHSV activity and increases innate immune responses.

Complete Genome Sequence of Anguillid Herpesvirus 1 Isolated from Imported Anguilla rostrata (American Eel) from Canada.

We report the full-length genome sequence (compared to reference sequences) of a variant strain of Anguillid herpesvirus 1 (AngHV-1) isolated from imported Anguilla rostrata (American eel) from Canada. This should help to further identify such viruses in the North America.

Efficacy of Chitosan-PLGA encapsulated trivalent oral vaccine against viral haemorrhagic septicemia virus, Streptococcus parauberis, and Miamiensis avidus in olive flounder (Paralichthys olivaceus).

The present study was conducted to assess the protective efficacy of a trivalent oral vaccine containing chitosan-PLGA encapsulated inactivated viral haemorrhagic septicemia virus (VHSV), Streptococcus parauberis serotype I and Miamiensis avidus antigens, followed by its oral (incorporated in feed) administration to olive flounder (Paralichthys olivaceus) fingerlings for a period of 15-consecutive days. After 35 days of initial vaccination, three separate challenge studies were conducted at the optimal temperature of the targeted pathogens using an intraperitoneal injection route. RPS analysis revealed moderate protection in the immunized group against all the three pathogens viz., VHSV (53.30% RPS), S. parauberis serotype-I (33.30% RPS), and M. avidus (66.75% RPS), as compared to the respective non-vaccinated challenge (NVC) control group. In addition, the immunized fish demonstrated significantly (p < 0.05) higher specific antibody titres in serum and significant (p < 0.05) upregulation in the transcript levels of immune genes of Igs (IgM, IgT, pIgR), TLRs (TLR 2, TLR 7), cytokines (IL-1ß, IL-8) and complement pathway (C3) in the mucosal and systemic tissues than those of NVC control fish, suggesting orchestration of pathogen-specific host immune responses thereby favouring its combativeness against the three pathogens. The expression dynamics of IFN-γ, Mx, caspase 3 genes post VHSV challenge; IFN-γ, TLR 2, caspase 1 genes post S. parauberis serotype I challenge and CD-8α, IL-10, TNF-α genes post M. avidus challenge further substantiates the efficacy of the vaccine in stimulating antiviral, antibacterial and antiparasitic immune responses in the host resulting in their better survival. The findings from the present study reflect that the formulated trivalent oral vaccine incorporating VHSV, S. parauberis serotype I and M. avidus antigens can be a promising prophylactic strategy to prevent the associated disease outbreaks in olive flounder.

Saponin and chitosan-based oral vaccine against viral haemorrhagic septicaemia virus (VHSV) provides protective immunity in olive flounder (Paralichthys olivaceus).

Production losses of olive flounder (Paralichthys olivaceus) have increased owing to viral haemorrhagic septicaemia virus (VHSV) infection. In this study, we determined safe concentrations of orally administered saponin and chitosan by analysing serum enzyme (AST/ALT) levels as biochemical markers of hepatic injury. Furthermore, we demonstrated the efficacy, duration of protection, and safety of saponin and chitosan-based vaccines with inactivated VHSV (IV). Oral administration of saponin, chitosan, and their combination did not induce fish mortality at all tested concentrations (0.29, 1.45, and 2.9 mg/g of fish body weight/day) 10 days after administration. However, AST level was high at a dose >0.29 mg/g of fish body weight/day. Both saponin and chitosan were found to be safe and acceptable for vaccination studies at a dose of 0.29 mg/g of fish body weight/day. Administration of IV alone did not induce protection at 2 and 4 weeks post vaccination (wpv). Olive flounders administered saponin + IV and chitosan + IV vaccines had higher immunity against VHSV with relative percentage survival (RPS) of 12.5-7.5% and 0-20.1%, respectively; however, additional immunisation with combination of saponin + chitosan + IV clearly enhanced the protection with RPS values of 10-15%, 26.7%, 42.9%, and 37.5% at 4, 8, 12, and 20 wpv, respectively. Although the RPS value of oral immunisation was not comparable to that of injectable vaccines, the manufacturing process is simple and oral administration causes less stress to juvenile fish. To investigate the development of a protective immune response, olive flounder were re-challenged with VHSV (107.8 TCID50/fish) at 70 days postinfection; 100% of the previously unexposed fish died, whereas 80-100% of the previously immunised fish survived. Our results showed the possibility of developing preventive measures against VHSV using saponin and chitosan-based oral vaccines with inactivated virus.

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.

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.

Potential Efficacy of Chitosan-Poly (Lactide-Co-Glycolide)-Encapsulated Trivalent Immersion Vaccine in Olive Flounder (Paralichthys olivaceus) Against Viral Hemorrhagic Septicemia Virus, Streptococcus parauberis Serotype I, and Miamiensis avidus (Scuticociliate).

Olive flounder (Paralichthys olivaceus) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae, have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis, and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus, respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly (p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant (p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder.

Comparative study on antigen persistence and immunoprotective efficacy of intramuscular and intraperitoneal injections of squalene - aluminium hydroxide (Sq + Al) adjuvanted viral hemorrhagic septicaemia virus vaccine in olive flounder (Paralichthys olivaceus).

The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and relish the raw muscles as 'sashimi'. For sustaining high production, easy-to-deliver and efficient vaccination strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality. We previously developed a squalene-aluminium hydroxide (Sq + Al) adjuvanted VHSV vaccine that conferred moderate to high protection in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a comparative analysis of vaccine efficacy and serum antibody response. The clinical and histological observation of the IM and IP groups showed that our vaccine remained persistence at the injection sites for 10-17 weeks post vaccination (wpv), without causing any adverse effects to the fish. The relative percentage of survival were 100% and 71.4% for the IP group and 88.9% and 92.3% for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule - IM vaccination for small-sized fish and IP vaccination for table-sized fish.

Nanoencapsulation of inactivated-viral vaccine using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in olive flounder (Paralichthys olivaceus) against viral haemorrhagic septicaemia virus (VHSV) infection.

Viral haemorrhagic septicaemia virus (VHSV), a (-) ssRNA virus belonging to the genus Novirhabdovirus of rhabdoviridae family, is the aetiological agent of viral haemorrhagic septicaemia (VHS) disease which causes huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. Previously, we developed an inactivated vaccine viz., formalin-inactivated VHSV mixed with squalene as adjuvant which was effective in conferring protective immunity (58-76% relative percentage survival) against VHSV but the mode of administration was intraperitoneal injection which is not feasible for small sized fingerling fish. To overcome this limitation, we presently focused on replacing the injection route of vaccine delivery by oral and immersion routes. In this context, we encapsulated the inactivated VHSV vaccine with chitosan nanoparticles (CNPs-IV) by water-in-oil (W/O) emulsification method. After encapsulation, two sets of in vivo vaccination trials were conducted viz., preliminary trial-I and final trial-II. In preliminary trial-I, olive flounder fingerlings (10.5 ±â€¯1.7 g) were vaccinated with CNPs-IV by different delivery strategies involving oral and immersion routes (single/booster dose) followed by challenge with VHSV (1 × 106 TCID50 virus/fish) to evaluate an effective method amongst different applied delivery strategies. Subsequently, a final trial-II was conducted to better understand the immune mechanism behind the efficacy of the employed delivery strategy and also to further improvise the delivery mechanism with prime-boost (primary immersion and oral boosting) combination in order to improve the transient anti-VHSV response in the host. Evaluation of RPS analysis in trial-I revealed higher RPS of 46.7% and 53.3% in the CNPs-IV (immersion) and CNPs-IV (immersion/immersion) groups, respectively compared to 0% RPS in the CNPs-IV (oral) group and 20% RPS in the CNPs-IV (oral/oral) group when calculated against 100% cumulative mortality percentage in the NVC (non-vaccinated challenged) control group, whereas, in the trial-II, RPS of 60% and 66.6% were obtained for CNPs-IV (immersion/immersion) and CNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, CNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to control, in both the systemic (kidney) and mucosal (skin and intestine) immune compartments of the host post immunization as well as post challenge. To conclude, mucosal immunization with CNPs-IV vaccine can orchestrate an effective immunization strategy in organizing a coordinative immune response against VHSV in olive flounder thereby exhibiting higher protective efficacy to the host with minimum stress.

The Megalocytivirus RBIV Induces Apoptosis and MHC Class I Presentation in Rock Bream (Oplegnathus fasciatus) Red Blood Cells.

Rock bream iridovirus (RBIV) causes severe mass mortality in Korean rock bream (Oplegnathus fasciatus) populations. To date, immune defense mechanisms of rock bream against RBIV are unclear. While red blood cells (RBCs) are known to be involved in the immune response against viral infections, the participation of rock bream RBCs in the immune response against RBIV has not been studied yet. In this study, we examined induction of the immune response in rock bream RBCs after RBIV infection. Each fish was injected with RBIV, and virus copy number in RBCs gradually increased from 4 days post-infection (dpi), peaking at 10 dpi. A total of 318 proteins were significantly regulated in RBCs from RBIV-infected individuals, 183 proteins were upregulated and 135 proteins were downregulated. Differentially upregulated proteins included those involved in cellular amino acid metabolic processes, cellular detoxification, snRNP assembly, and the spliceosome. Remarkably, the MHC class I-related protein pathway was upregulated during RBIV infection. Simultaneously, the regulation of apoptosis-related proteins, including caspase-6 (CASP6), caspase-9 (CASP9), Fas cell surface death receptor (FAS), desmoplakin (DSP), and p21 (RAC1)-activated kinase 2 (PAK2) changed with RBIV infection. Interestingly, the expression of genes within the ISG15 antiviral mechanism-related pathway, including filamin B (FLNB), interferon regulatory factor 3 (IRF3), nucleoporin 35 (NUP35), tripartite motif-containing 25 (TRIM25), and karyopherin subunit alpha 3 (KPNA3) were downregulated in RBCs from RBIV-infected individuals. Overall, these findings contribute to the understanding of RBIV pathogenesis and host interaction.

PLGA encapsulated inactivated-viral vaccine: Formulation and evaluation of its protective efficacy against viral haemorrhagic septicaemia virus (VHSV) infection in olive flounder (Paralichthys olivaceus) vaccinated by mucosal delivery routes.

Viral haemorrhagic septicaemia virus (VHSV), an OIE listed viral pathogen, is the etiological agent of a contagious disease, causing huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. In continuation with our previous work, where injection vaccination with inactivated VHSV mixed with squalene (as adjuvant) conferred higher protective immunity to olive flounder, the present study focused on replacing the injection route of vaccine delivery by immersion/oral route to overcome the limitations of the parenteral immunization method. Here, we encapsulated the inactivated VHSV vaccine with PLGA (poly lactic-co-glycolic acid) nanoparticles (PNPs-IV) and evaluated its ability to induce protective immunity in olive flounder (12.5 ±â€¯1.5 g) by initially immunizing the fishes by immersion route followed by a booster with the same dose two weeks later with half of the fish through immersion route and other half through oral route (incorporated into fish feed). Cumulative mortalities post-challenge (1 × 106 TCID50 virus/fish) with virulent VHSV-isolate, were lower in vaccinated fish and RPS of 60% and 73.3% were obtained for PNPs-IV (immersion/immersion) and PNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, PNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to controls, in both the systemic (kidney) and mucosal (skin and intestine) immune compartment of the host post immunization as well as post challenge. Thus it can be inferred that the adopted immunization strategy efficiently protected and transported the inactivated viral antigen to target immune organs and positively stimulated the protective immune response against VHSV in olive flounder.

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.).

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country-wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin-inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin-killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate-buffered saline or vaccine formulated with Montanide by intra-peritoneal (i.p.) injection and challenged by intra-muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post-vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un-adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.

Comparison of antibacterial activity and phenolic constituents of bark, lignum, leaves and fruit of Rhus verniciflua.

Rhus verniciflua is commonly known as a lacquer tree in Korea. The bark of R. verniciflua has been used as an immunostimulator in traditional medicine, but also causes allergic dermatitis due to urushiol derivatives. For the development of active natural resources with less toxicity, the antibacterial activity of various parts of R. verniciflua such as bark, lignum, leaves and fruit, together with chemical composition, were investigated. Among the various parts of R. verniciflua, lignum showed the most potent antibacterial activity against fish pathogenic bacteria such as Edwardsiella tarda, Vibrio anguillarum and Streptococcus iniae. Measurement of total phenolic content and flavonoid content clearly showed a high content of phenolic and flavonoids in lignum among the various parts of R. verniciflua. Further analysis showed a close correlation between antibacterial activity and phenolic content. In addition, methyl gallate and fustin, the major constituents of bark and lignum, showed antibacterial activity, which suggested phenolic constituents as active constituents. The content of urushiols, however, was highest in bark, but there was a trace amount in lignum. LC-MS-MS and PCA analysis showed good discrimination with the difference of phenolic composition in various parts of R. verniciflua. Taken together, phenolic compounds are responsible for the antibacterial activity of R. verniciflua. The lignum of R. verniciflua contains high content of phenolic compounds with less urushiols, which suggests efficient antibacterial activity with less toxicity. Therefore, the lignum of R. verniciflua is suggested as a good source for antibacterial material to use against fish bacterial diseases.

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.

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus.

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30kDa; serotype II, 38kDa; serotype III, 34kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores-<51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.

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

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 µg/g/day of Ext was 31.25% and 12.50%, respectively. In trial II, the RPS for 1000, 500 and 50 µg/g/day of Ext was 31.57%, 0% and 0%, respectively. In trial III, the RPS after 1 and 2 weeks (1000 µg/g/day) of exposure to Ext was 26.31% and 31.57%, respectively. Oral administration of Ext (1000 µg/g/day) significantly induced inflammatory cytokine responses (IL-1ß, IL-6 and IFN-γ) at 1 and 2 days post-oral administration (dpa). Additionally, IFN-α/ß (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.

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.