Vaccine Molecule Design Based on Phage Display and Computational Modeling against Rhabdovirus.

Rhabdoviruses with rich species lead a variety of high lethality and rapid transmission diseases to plants and animals around the globe. Vaccination is one of the most effective approaches to prevent and control virus disease. However, the key antigenic epitopes of glycoprotein being used for vaccine development are unclear. In this study, fish-derived Abs are employed for a Micropterus salmoides rhabdovirus (MSRV) vaccine design by phage display and bioinformatics analysis. We constructed an anti-MSRV phage Ab library to screen Abs for glycoprotein segment 2 (G2) (G129-266). Four M13-phage-displayed Abs (Ab-5, Ab-7, Ab-8 and Ab-30) exhibited strong specificity to target Ag, and Ab-7 had the highest affinity with MSRV. Ab-7 (300 µg/ml) significantly increased grass carp ovary cell viability to 83.40% and significantly decreased the titer of MSRV. Molecular docking results showed that the key region of Ag-Ab interaction was located in 10ESQEFTTLTSH20 of G2. G2Ser11 and G2Gln12 were replaced with alanine, respectively, and molecular docking results showed that the Ag-Ab was nonbinding (ΔG > 0). Then, the peptide vaccine KLH-G210-20 was immunized to M. salmoides via i.p. injection. ELISA result showed that the serum Ab potency level increased significantly (p < 0.01). More importantly, the challenge test demonstrated that the peptide vaccine elicited robust protection against MSRV invasion, and the relative percentage survival reached 62.07%. Overall, this study proposed an approach for screening key epitope by combining phage display technology and bioinformatics tools to provide a reliable theoretical reference for the prevention and control of viral diseases.

Exploring the protective role of Bacillus velezensis BV1704-Y in zebrafish health and disease resistance against Aeromonas hydrophila infection.

Bacillus genus, particularly Bacillus velezensis, is increasingly considered as viable alternatives to antibiotics in aquaculture due to their safety and probiotic potential. However, the specific mechanisms through which probiotic B. velezensis confers protection against Aeromonas hydrophila infection in fish remain poorly understood. This study delved into the multifaceted impacts of B. velezensis BV1704-Y on diverse facets of zebrafish health, including gut barrier function, immune response, oxidative stress, gut environment, microbiome composition, and disease resistance. Our findings demonstrate that supplementation with B. velezensis BV1704-Y significantly alleviated symptoms and reduced mortality in zebrafish infected with A. hydrophila. Furthermore, a notable reduction in the expression of pivotal immune-related genes, such as IL-1ß, IL6, and TNF-α, was evident in the gut and head kidney of zebrafish upon infection. Moreover, B. velezensis BV1704-Y supplementation resulted in elevated activity levels of essential antioxidant enzymes, including SOD, CAT, and GSH, in gut tissue. Notably, B. velezensis BV1704-Y positively modulated the structure and function of the intestinal microbiome, potentially enhancing immune response and resilience in zebrafish. Specifically, supplementation with B. velezensis BV1704-Y promoted the relative abundance of beneficial bacteria, such as Cetobacterium, which showed a noteworthy negative correlation with the expression of pro-inflammatory genes and a positive correlation with gut barrier-related genes. Altogether, our study suggests that B. velezensis BV1704-Y holds promise as an effective probiotic for protecting zebrafish against A. hydrophila infection, offering potential benefits for the aquaculture industry.

A nanocarrier immersion vaccine encoding surface immunogenic protein confers cross-immunoprotection against Streptococcus agalactiae and Streptococcus iniae infection in tilapia.

Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.

Catechol compounds as dual-targeting agents for fish protection against Ichthyophthirius multifiliis infections.

Aquaculture is one of the fastest growing sectors in global food production, recognized as a significant contributor to poverty alleviation, food security, and income generation. However, the frequent occurrence of diseases caused by pathogen infections result in reduced yields and economic losses, posing a substantial constraint to the sustainable development of aquaculture. Here, our study identified that four catechol compounds, quercetin, luteolin, caffeic acid, and chlorogenic acid, exhibited potent antiparasitic effects against Ichthyophthirius multifiliis in both, in vitro and in vivo. The parasite is recognized as one of the most pathogenic to fish worldwide. Using a combination of in silico methods, the dipeptidyl peptidase (DPP) was identified as a critical target for catechol compounds. The two hydroxyl radicals of the catechol group were essential for its binding to and interacting with the DPP protein. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that catechol compounds disrupt pathways associated with the metabolism and growth of I. multifiliis, thereby exerting antiparasitic effects. Furthermore, these compounds attenuated the expression of proinflammatory cytokines in vivo in fish and promoted macrophage polarization toward M2 phenotype by inhibiting the STAT1 signaling pathway. The dual activity of catechol compounds, acting as both direct antiparasitic and anti-inflammatory agents in fish, offers a promising therapeutic approach for combating I. multifiliis infections in aquaculture.

Natural product honokiol exhibits antiviral effects against Micropterus salmoides rhabdovirus (MSRV) both in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is a formidable pathogen, presenting a grave menace to juvenile largemouth bass. This viral infection frequently leads to epidemic outbreaks, resulting in substantial economic losses within the aquaculture industry. Unfortunately, at present, there are no commercially available vaccines or pharmaceutical treatments to combat this threat. In order to address the urgent need for therapeutic strategy to resist MSRV infection, the antiviral activity of natural product honokiol against MSRV was explored in this study. Firstly, cellular morphology was directly observed in an inverted microscope when treated with honokiol after MSRV infection. The results clarified that honokiol significantly lessened cytopathic effect (CPE) induced by MSRV and protected the integrity of GCO cells. Furthermore, the viral nucleic acid expression (G gene) was detected by reverse transcription real-time quantitative PCR (RT-qPCR) and the results indicated that honokiol significantly decreased the viral loads of MSRV in a concentration-dependent manner, and honokiol showed a high antiviral activity with IC50 of 2.92 µM. Besides, honokiol significantly decreased the viral titre and suppressed apoptosis caused by MSRV. Mechanistically, honokiol primarily inhibited the initial replication of MSRV and discharge of progeny virus to exert anti-MSRV activity. More importantly, in vivo experiments suggested that honokiol (40 mg/kg) expressed a fine antiviral activity against MSRV when administrated with intraperitoneal injection, which led to a notable 40% improvement in the survival rate among infected largemouth bass. In addition, it also resulted in significant reduction in the viral nucleic acid expression within liver, spleen and kidney at 2, 4 and 6 days following infection. What is more, 100 mg/kg honokiol with oral administration also showed certain antiviral efficacy in MSRV-infected largemouth bass via improving the survival rate by 10.0%, and decreasing significantly the viral nucleic acid expression in liver, spleen and kidney of largemouth bass on day 2. In summary, natural product honokiol is a good candidate to resist MSRV infection and has promising application prospects in aquaculture.

Impact of Surface Oxidation on the Morphology of Uranium Dioxide Nanoparticles.

The undeniable importance of nanoparticles has led to vast efforts, in many fields of science, to understand their chemical and physical properties. In this paper, the morphology dependence of f-element nanoparticles is correlated to the oxygen environment and the type and coverage of capping ligands. This dependence was evaluated by first-principles calculations of the surface energies of different crystallographic planes (001, 110, and 111) as a function of the relative oxygen chemical potential and under the influence of different ligands. Uranium dioxide nanoparticles were the focus of this study due to their high sensitivity to oxidation compared to thorium dioxide nanoparticles, a homoleptic material but insensitive to oxidation. To fully explain the experimental observations of uranium dioxide nanocrystals, theoretical modeling shows that the consideration of surfaces with different oxidation conditions is necessary. It is shown that, for materials with low oxidation potential, such as uranium dioxide, the oxygen environment and capping ligand concentration are competing factors in determining the nanoparticle morphology.

4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin: The first inhibitor of fish rhabdovirus glycoprotein.

Spring viraemia of carp virus (SVCV), a highly pathogenic rhabdovirus, could cause spring viraemia of carp (SVC) with up to 90% lethality. Like other rhabdoviruses, the entry of SVCV into susceptible cells was mediated by a single envelope glycoprotein G. Specific inhibitors targeting the glycoprotein were the most effective means to alleviate the epidemic. The programs including SWISS-MODEL, I-TASSER, Phyre2 and AlphaFold2 were used to build a three-dimensional structural model of glycoprotein. The structural comparison between SVCV-G and homology protein VSV-G revealed that the SVCV glycoprotein ectodomain (residues 19 to 466) folded into four distinct domains. Based on the potential small molecule binding sites on glycoprotein surfaces, virtual screening of the anti-SVCV drug libraries was performed using Autodock software and 4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin (MOA) with a high binding affinity was identified. The solubility enhancer tags including trigger factor and maltose binding protein were fused with the ectodomain of glycoprotein, and the target protein with a purity of about 90% was successfully obtained. The interaction confirmation tests revealed that the fluorescence intensity of a characteristic peak induced by the endogenous chromophores in glycoprotein was decreased with the addition of MOA, indicating changes in the microenvironment of glycoprotein. Moreover, the interaction could cause a slight conformational change in glycoprotein, as shown by the content of ß-turn, ß-folding, and random coil of protein all increased with the decrease of α-helix content after the addition of MOA compound. These results demonstrated that MOA could act as a novel drug against fish rhabdovirus via direct targeting of glycoprotein.

Effects of dietary Cetobacterium somerae on the intestinal health, immune parameters and resistance against Nocardia seriolae of largemouth bass, Micropterus salmoides.

Largemouth bass (Micropterus salmoides), one of the most important freshwater commercial fish species has been widely cultivated in China. In recent years, the nocardiosis caused by Nocardia seriolae has greatly damaged the M. salmoides industry and there is no effective treatment at present. Currently, Cetobacterium somerae, the predominant bacteria in the gut of many freshwater fishes has been reported to be associated with fish health. However, whether the native C. somerae could protect the host from N. seriolae is unclear. In this study, M. salmoides were fed with three different diets, including control diet (CD), low C. somerae diet (106 CFU/g as LD) and high C. somerae diet (108 CFU/g as HD). After 8-week feeding, growth performance, gut health index, serum enzyme activities and the expression of inflammation-related genes were tested. Results showed that the LD and HD diets had no adverse effects on the growth performance. Moreover, dietary HD enhanced gut barrier and reduced intestinal ROS and ORP, as well as increased serum enzyme activities including ACP, AKP, SOD and LZM compared to the CD group. In addition, the HD diet significantly up-regulated the expression of TNF-α, IL8, IL-1ß and IL15, while down-regulating the expression of TGF-ß1 and IL10 in kidney. Moreover, the expression of antibacterial genes was significantly increased in HD group after being challenged by N. seriolae. And the fish fed HD diet exhibited higher survival rate (57.5%) than that in CD (37.5%) and LD groups (42.5%). To summarize, our study demonstrates that dietary HD can enhance gut health, improve immune response and strengthen pathogen resistance, suggesting that C. somerae is a potential probiotic for defending against N. seriolae infection in M. salmoides.

Recombinant surface display vaccine enhances the immersion immune effect against grass carp reovirus in grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is subject to a hemorrhagic disease caused by grass carp reovirus (GCRV), which can lead to mass mortality in grass carp culture, causing significant economic loss. Vaccination is the most promising strategy for the prevention of infectious diseases. Immersion vaccination is considered the most effective disease prevention method for juvenile fish because it can be implemented on many fish at once and administered without causing stress. However, immune responses by immersion vaccination are markedly less robust due to the skin barrier and insufficient antigen uptake. The display of heterologous proteins on the cell surface has been explored as a delivery system for viral antigens in veterinary and human vaccine studies. To improve the efficacy of the immersion vaccine, the major capsid protein (VP7) of GCRV was co-displayed with Aeromonas hydrophila outer membrane protein a (OmpA) and major adhesion protein (Mah) on the outer membrane surface of nonpathogenic Escherichia coli BL21 using the anchoring motif of ice-nucleation protein (Inp). The immune responses and protection efficiency against GCRV infection via both the injection and immersion routes were evaluated. The results indicated that the activities of anti-oxidant enzymes (ACP, AKP, SOD and T-AOC), as well as the expression of immune-related genes (TNF-α, IL-1ß, MHCI and IgM) and specific VP7 antibody levels, were strongly increased in the grass carp from 7 to 21 days post-injection inoculation in a dose dependent manner. The cumulative mortality rates of injection-vaccinated groups were much lower than those of the control group after the GCRV challenge, and the relative percent survival (RPS) was greater than 80 %. Vitally, the surface co-display of vp7-Mah protein conferred marked protection to grass carp against GCRV infection after immersion administration (RPS >50 %); this was consistent with the production of high level of specific serum antibodies, non-specific immune responses, and the expression of immune-related genes. Moreover, the invasion analysis further showed that surface co-display of the vp7-Mah protein indeed significantly improved the invasion of E. coli BL21 (DE3) in vitro. Altogether, this study demonstrated that surface display GCRV core antigen vaccine system accompanied by invasion component from aquatic pathogenic microorganism is an effective prophylactic against GCRV viral diseases via the immersion administration approach.

Adamantoyl chloride inhibited replication of the largemouth bass virus via enhanced immunity and inhibition of apoptosis.

The largemouth bass virus (LMBV) is a commonly encountered pathogen in aquaculture and presents significant challenges to development of the largemouth bass industry due to the lack of effective treatment methods. Here, the inhibitory potential and underlying mechanisms of adamantoyl chloride (AdCl) against LMBV were assessed both in vitro and in vivo. The results showed that AdCl (IC50 = 72.35 µM) significantly inhibited replication of LMBV in epithelioma papulosum cyprini (EPC) cells. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and cytopathic effect (CPE) assays confirmed that AdCl inhibited replication of LMBV in EPC cells and significantly reduced the CPE effect, respectively. As a potential mechanism, AdCl inhibited apoptosis as determined by fluorescence and transmission electron microscopy. The results of flow cytometry showed that the apoptosis rate was decreased by 69 % in the AdCl-treated group as compared to the LMBV-infected group. Additionally, AdCl inhibited viral release. In vivo, the survival rate was 16.2 % higher in the AdCl-treated group as compared to the LMBV-infected group (26.9 % vs. 10.7 %, respectively). Additionally, the results of quantitative reverse transcription polymerase chain reaction (RT-qPCR) showed that AdCl significantly reduced the viral load of the fish liver, spleen, and kidneys at 3, 6, and 9 days postinfection. In addition, RT-qPCR analysis found that AdCl upregulated expression of immune-related genes to suppress replication of LMBV. Collectively, these results confirmed the anti-LMBV activities of AdCl for use in the aquaculture industry.

In Situ High-Temperature Raman Spectroscopy of UCl3: A Combined Experimental and Theoretical Study.

Uranium trichloride (UCl3) has received growing interest for its use in uranium-fueled molten salt reactors and in the pyrochemical processing of used fuel. In this paper, we report for the first time the experimentally determined Raman spectra of UCl3, at both ambient condition and in situ high temperatures up to 871 K. The frequencies of five of the Raman-active vibrational modes (vi) of UCl3 exhibit a negative temperature derivative ((∂νi/∂T)P) with increasing temperature. This red-shift behavior is likely due to the elongation of U-Cl bonds. The average isobaric mode Grüneisen parameter (γiP = 0.91 ± 0.02) of UCl3 was determined through use of the coefficient of thermal expansion published in Vogel et al. (2021) and the (∂νi/∂T)P values determined in this study. These results are in general agreement with those calculated here by density functional theory (DFT+U). Finally, a comparison of the ambient band positions of UCl3 to those of isostructural lanthanide (La-Eu) and actinide chlorides (Am-Cf) has been made.

The oral protective efficacy of magnolol against Aeromonas hydrophila and A. veronii infection via enhancing anti-inflammatory ability in goldfish (Carassius auratus).

Aeromonas hydrophila and A. veronii are widespread and important critical pathogenic bacteria in the aquaculture industry and cause severe economic damage. At present, magnolol has been proved to be a broad-spectrum antibacterial activity, such as A. hydrophila, Staphylococcus aureus and Streptococcus mutans. In order to explore the cause of in vivo disease resistance of magnolol and promote its safe application in aquaculture, the pathological detection and changes in immune indicators of fish after feeding with magnolol were conducted in this paper. Results showed that the diets supplemented with magnolol (3 g magnolol/kg commercial feed) significantly increase the expression level of anti-inflammatory cytokines (IL-10, TGF-ß and IL-4) in the liver of goldfish (p < .05). Additionally, the expression levels of proinflammatory cytokines (IL-1ß, IL-8 and IFN-γ) did not increase significantly. Subsequently, this study investigated the resistance of goldfish to A. hydrophila and A. veronii infection after feeding with magnolol. The results showed that the survival rates of treatment groups fed 3 g magnolol/kg commercial feed daily increased by 23.1% and 38.5% after 10 days post A. hydrophila and A. veronii (p = .0351) infection, respectively. Meanwhile, growth performance (body weight and length), major internal organs (liver, spleen, kidney and intestine) and the serum biochemistry indicators (ATL and AST) all exhibited no significant adverse effects after the goldfish fed with magnolol for 30 days. TP showed an increasing concentration in the treatment group (p < .05). Results of the mRNA expression of stress response indicated that the expression level of cyp1a and hsp70 was significantly down-regulated after a 30-day treatment (p < .05), and the two genes recovered to the similar level as the control group after a commercial feed diet. In brief, the diets supplemented with magnolol protected the host from the excessive immune response caused by A. hydrophila and A. veronii via enhancing its anti-inflammatory capacity and had no adverse effects with feeding.

Inhibition of the largemouth bass virus replication by piperine demonstrates potential application in aquaculture.

Largemouth bass virus (LMBV) is a systemic viral pathogen that can cause high mortality rates in cultivated largemouth bass. However, no treatment is currently approved. Therapeutic strategies against LMBV infection are urgently needed. In this study, we investigated the antiviral activity of piperine against LMBV in vitro and in vivo. In vitro antiviral activity assay showed that 210.28 µM piperine significantly decreased LMBV major capsid protein (MCP) gene expression in epithelioma papulosum cyprinid (EPC) cells by a maximum inhibitory rate of >95%. Piperine treatment inhibited LMBV replication in a dose-dependent manner, with the half-maximal activity (IC50 ) of 34.61 µM. Moreover, piperine significantly decreased the viral titers and cytopathic effects (CPE), contributing to the protection of infected cells. With regard to the steps of piperine affecting the life cycle of viruses, piperine had a direct inactivating effect on LMBV. During the virus adsorption phase, piperine prevented the adsorption of LMBV to EPC cells. Furthermore, piperine played an antiviral role mainly in the later stages of viral infection (4-8 h). To further evaluate the antiviral activity of piperine against LMBV in vivo, largemouth bass as a model organism was carried out in relevant experiments. Intraperitoneal injection of piperine (25 mg/kg) effectively improved the survival rate of LMBV-infected largemouth bass by 20%. In addition, RT-qPCR results of viral replication in liver, spleen, kidney, gill and swim bladder tissues showed that piperine significantly inhibited LMBV replication in vivo, thus protecting largemouth bass from LMBV-induced death. Together, our results suggested that piperine is a therapeutic and preventative agent against LMBV infection.

Nocardia seriolae mediates liver granulomatous chronic inflammation in Micropterus salmoides through pyroptosis.

Granulomatous diseases caused by Nocardia seriously endanger the health of cultured fish. These bacteria are widely distributed, but prevention and treatment methods are very limited. Chronic granulomatous inflammation is an important pathological feature of Nocardia infection. However, the molecular mechanisms of granuloma formation and chronic inflammation are still unclear. Constructing a granuloma infection model of Nocardia is the key to exploring the pathogenesis of the disease. In this study, we established a granuloma model in the liver of largemouth bass (Micropterus salmoides) and assessed the infection process of Nocardia seriolae at different concentrations by analysing relevant pathological features. By measuring the expression of pro-inflammatory cytokines, transcription factors and a pyroptosis-related protein, we revealed the close relationship between pyroptosis and chronic inflammation of granulomas. We further analysed the immunofluorescence results and the expression of pyroptosis-related protein of macrophage infected by N. seriolae and found that N. seriolae infection induced macrophage pyroptosis in vitro. These results were proved by flow cytometry analysis of infection experiment in vivo. Our results indicated that the pyroptosis effect may be the key to inducing chronic inflammation in the fish liver and further mediating granuloma formation. In this study, we explored the molecular mechanism underlying chronic inflammation of granulomas and developed research ideas for understanding the occurrence and development of granulomatous diseases in fish.

Screening the potential part of the G protein antigen is an achievable strategy to improve the immune effect of DNA vaccine against MSRV infection.

DNA vaccines, as an effective prophylactic technology to induce both humoral and cellular immune responses, have already been widely studied to prevent and control viral and bacterial infections in aquaculture. To find a more effective and safer way to control Micropterus salmoides rhabdovirus (MSRV) infection in largemouth bass, two different DNA vaccines expressing partial (pcDNA3.1-G2) and full-length (pcDNA3.1-G) of the MSRV G protein were developed and injected intramuscularly with different doses. The immune effect was comprehensively compared and evaluated by detecting immune-related parameters including serum antibody levels, immune-related physiological indexes, immune-related gene expression and relative survival rates in this study. The results showed that compared with the pcDNA3.1-G vaccine, the pcDNA3.1-G2 vaccine induced higher serum antibody levels, a lower nonspecific immune response in serum (ACP, SOD and T-AOC activities), higher immune-related gene expression and a higher relative survival rate. Moreover, the immune effect of pcDNA3.1-G2-vaccinated fish showed gradually higher with the increasing pcDNA3.1-G2 concentration, especially in pcDNA3.1-G2 (10µg/per fish) group, the relative survival rate reached to 82.5%, which was significant higher (p < 0.05) than pcDNA3.1-G (10µg/per fish) group. This study indicated that screening the potential core part of an antigen is an achievable strategy to improve the immunogenicity and immunoprotective effect of DNA vaccine.

Amantadine, a promising therapeutic agent against viral encephalopathy and retinopathy.

Outbreaks of viral encephalopathy and retinopathy (VER) in marine and freshwater species severely devastate the aquaculture worldwide. The causative agent of VER is nervous necrosis virus (NNV), which mainly infects the early developmental stages of fish, limiting the effectiveness of vaccines. To counter this case, the anti-NNV potentials of nine drugs with broad-spectrum antiviral activity were explored using ribavirin as a positive drug. Toxicity of the selected drugs to SSN-1 cells and grouper was firstly evaluated to determine the safety concentrations. For screening in vitro, amantadine and oseltamivir phosphate can relieve the cytopathic effects and inhibit NNV replication with the 90% inhibitory concentrations (IC90 ) of 38.74 and 106.75 mg/L, respectively. Amantadine has a stronger anti-NNV activity than ribavirin at the with- and post-NNV infection stages, indicating that it is a potential therapeutic agent against VER by acting directly on NNV. Similarly, amantadine also has a strong anti-NNV activity in vivo with the IC90 of 27.91 mg/L at the 7 days post-infection, while that was 73.25 mg/L for ribavirin. Following exposure to amantadine (40 mg/L) and ribavirin (100 mg/L) for 7 days, the survival rates of NNV-infected grouper were increased to 44% and 39%, respectively. The maximum amantadine content (11.88 mg/Kg) in grouper brain was reached following exposure for 24 hr, and amantadine can be quickly excreted from fish, reducing the risk of drug residue. Results so far indicated that amantadine is a promising therapeutic agent against NNV in aquaculture, providing an effective strategy for VER control at the early developmental stages of fish.

Quinoline, with the active site of 8-hydroxyl, efficiently inhibits Micropterus salmoides rhabdovirus (MSRV) infection in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is an significant pathogen that causes high mortality and related economic losses in bass aquaculture. There is no effective or approved therapy to date. In this study, we evaluated the anti-MSRV effects of 22 quinoline derivatives in grass carp ovary (GCO) cells. Among these compounds, 8-hydroxyquinoline exhibited valid inhibition in decreasing MSRV nucleoprotein gene expression levels of 99.3% with a half-maximal inhibitory concentrations (IC50 ) value of 4.66 µM at 48 h. Moreover, 8-hydroxyquinoline significantly enhanced a protective effect in GCO cells by reducing the cytopathic effect (CPE). By comparing the anti-MSRV activity of 22 quinoline derivatives, we found that 8-hydroxyquinoline possessed the efficient active site of 8-hydroxyl and inhibited MSRV infection in vitro. For in vivo studies, 8-hydroxyquinoline via intraperitoneal injection exhibited an antiviral effect in MSRV-infected largemouth bass by substantially enhancing the survival rate by 15.0%. Importantly, the viral loads in the infected largemouth bass notably reduced in the spleen on the third days post-infection. Overall, 8-hydroxyquinoline was considered to be an efficient agent against MSRV in aquaculture.

Screening and evaluating honokiol from Magnolia officinalis against Nocardia seriolae infection in largemouth bass (Micropterus Salmoides).

Nocardiosis caused by Nocardia seriolae is a major threat to the aquaculture industry. Given that prolonged therapy administration can lead to a growth of antibiotic resistant strains, new antibacterial agents and alternative strategies are urgently needed. In this study, 80 medicinal plants were selected for antibacterial screening to obtain potent bioactive compounds against N. seriolae infection. The methanolic extracts of Magnolia officinalis exhibited the strongest antibacterial activity against N. seriolae with the minimal inhibitory concentration (MIC) of 12.5 µg/ml. Honokiol and magnolol as the main bioactive components of M. officinalis showed higher activity with the MIC value of 3.12 and 6.25 µg/ml, respectively. Sequentially, the evaluation of antibacterial activity of honokiol in vivo showed that honokiol had good biosafety, and could significantly reduce the bacterial load of nocardia-infected largemouth bass (p < .001). Furthermore, the survival rate of nocardia-infected fish fed with 100 mg/kg honokiol was obviously improved (p < .05). Collectively, these results suggest that medicinal plants represent a promising reservoir for discovering active components against Nocardia, and honokiol has great potential to be developed as therapeutic agents to control nocardiosis in aquaculture.

Protective immunity by DNA vaccine against Micropterus salmoides rhabdovirus.

Micropterus salmoides rhabdovirus (MSRV) is one of the common pathogens in the largemouth bass industry, which can cause lethal diseases in juvenile fish and enormous economic losses. To establish effective means to prevent MSRV infection, the pcDNA3.1-G plasmid containing the MSRV glycoprotein gene was successfully constructed and intramuscularly injected into the largemouth bass to evaluate the immune responses and protective effects in our study. As the results showed, the serum antibody levels of the fish vaccinated with different doses of pcDNA3.1-G were significantly higher compared with the control groups (PBS and pcDNA3.1). Meanwhile, the immune parameters (acid phosphatase and alkaline phosphatase) were also significantly up-regulated. Several immune-related genes (IgM, IL-8, IL-12p40 and CD40) were expressed in the pcDNA3.1-G groups at higher levels than in the control groups, which indicated that strong immune responses were induced. Besides, the survival percentages of fish in the control groups (PBS and pcDNA3.1) and pcDNA3.1-G groups (2.5, 5, 10 and 20 µg/fish) at 14 days after challenge experiment with MSRV were 0%, 0%, 6.1%, 15.2%, 29.0% and 48.5% respectively. This study indicated that pcDNA3.1-G was a prospective DNA vaccine candidate against MSRV-induced mortality.

Epitope screening of the major capsid protein within grouper iridovirus of Taiwan and the immunoprotective effect with SWCNTs as the vaccine carrier.

Iridovirus can cause a mass of death in grouper, leading to huge economic loss in recent years. At present, practical vaccine is still the best way to control the outbreak of this virus. Many researches had indicated that the major capsid protein (MCP) of grouper iridovirus of Taiwan (TGIV) is an effective antigen to induce a specific immune response in grouper. However, these traditional vaccines that based on large proteins or whole organisms are faced with challenges because of the unnecessary antigenic load. Thus, in this study, we screened the dominant linear epitope within the MCP of TGIV and then, a new peptide vaccine (P2) was developed via prokaryotic expression system. Furthermore, SWCNTs was used as a vaccine carrier to enhance the immunoprotective effect. To evaluate the immunoprotective effect of this vaccine, a total of 245 fish were vaccinated with P2 (5, 10, 20 mg L-1) and SWCNTs-P2 (5, 10, 20 mg L-1) via immersion before being challenged with live TGIV at 28 days post immunization (d.p.i.). Results showed that the serum antibody titer, enzymatic activity, expression level of some immune-related genes (CC chemokine, IgM and TNF-α) and survival rate were significantly increased (SWCNTs-P2, 20 mg L-1, 100%) compared to the control group (0%). These results indicated that this peptide vaccine could effectively induce specific immune response in vaccinated groupers. Functionalized SWCNTs could serve as a carrier of the peptide vaccine to enhance the immunoprotective effect via immersion. To sum up, epitope screening might be a potential way to develop an effective vaccine nowadays, and SWCNTs might provide a practical method that can be used in large-scale vaccination, especially for juvenile fish, to fight against diseases in aquaculture industry.