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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. [ABSTRACT FROM AUTHOR]

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-1β , 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. • A recombinant surface display vaccine prevented grass carp hemorrhagic disease. • Grass carp immersed in a vaccine with surface display of vp7-Mah elicited robust immunity against GCRV. • Surface display of the vp7-Mah improved the invasion of immersed vaccine. • Surface display-based immersion vaccines have prospects for application in aquaculture. [ABSTRACT FROM AUTHOR]

Our previous studies have verified that moroxydine hydrochloride (Mor) could inhibit replication of grass carp reovirus (GCRV) and suppress apoptosis of Ctenopharyngodon idella kidney cells, but be lack of information whether exists on antiviral activity in vivo. The paper was undertaken to explore the antiviral response of Mor against GCRV in grass carp and investigate the safety of drug for aquatic organisms. The results showed that injection treatment of Mor could more effectively inhibit GCRV replication than immersion administration. All the RNA systheses of vp3 and vp6 on day 7 in head kidney, gill, hepatopancreas and dorsal muscle in the Mor injection group were lesser than 0.07‐fold than that of in control group. And the GCRV‐inducing grass carp mortality was effectively controlled within 7 days post Mor injection therapy. Additionally, the reduction of superoxide dismutase activity, total antioxidant capacity and catalase activity in serum was effectively controlled by Mor. Moreover, drug safety assessment results showed that 500 mg/L of Mor was safe to C. idella, Bacillus subtilis, Chlorella vulgaris and Tetrahymena thermophila, which was far higher than the therapeutic concentration. The present study proved Mor as harmless formulations or products had potential value in the control of GCRV in aquaculture, with the advantage of super in vivo antiviral activity and environmental safety. [ABSTRACT FROM AUTHOR]

Grass carp ( Ctenopharyngodon idella ) hemorrhagic disease, caused by grass carp reovirus (GCRV), has been a serious problem in grass carp aquaculture for several decades. Characterization of the primary host factors associated with host-virus interaction is critical for understanding how a virus infects its host cell and these host factors can be antiviral targets. This study aimed to screen host factors that interacted with GCRV in the C. idella kidney (CIK) cells and used them as antiviral targets. Twelve proteins were identified by virus overlay protein binding assay and LC-MS-MS. Among these twelve proteins, Heat Shock Protein 70 (HSP70) was outstanding. Results of flow cytometry and immunofluorescence assay indicated that HSP70 was on the cell membrane. HSP70 was expressed at low levels preceding GCRV infection, but its expression was induced upon GCRV infection. Inhibition of HSP70’s function by inhibitors (VER155008 and pifithrin-μ) maintained HSP70 on the cell surface in infected cells, however GCRV quantity was decreased in the CIK cells (compared with the control group, the maximum inhibition rate of the treatment group was close to 85%), suggesting that fully functional HSP70 was required for GCRV infection. Moreover, GCRV showed a dose dependent reduction by inhibiting the entry stage of the viral life cycle following treated with VER155008 and pifithrin-μ. VER + PIF (1:1) were used at 15 μM and the expression of GCRV-VP6 downregulated nearly to 90%, which revealed that HSP70 played an important role in GCRV entering into CIK cells. This work speculated that HSP70 might be a host factor in the process of GCRV infecting CIK cells, therefore, it might be a potential antiviral target for GCRV infection. [ABSTRACT FROM AUTHOR]

Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV-infected grass carp ( Ctenopharyngodon idella ) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non-toxic concentration in vivo . Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that magnolol facilitated the expression of apoptosis-inhibiting gene bcl-2 , while suppressed the expression of apoptosis-promoting gene bax in GCRV-infected cells. Besides, RT-qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway and toll-like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV-induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small-molecule drugs possess antiviral activities, and lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry. [ABSTRACT FROM AUTHOR]

Infection with Grass carp reovirus (GCRV) is becoming unprecedentedly widespread in grass carp ( Ctenopharyngodon idella ) aquaculture industry, yet the management of GCRV infection still remains a challenge. Therefore, it is of importance to develop effective means against GCRV. As a delivery system of viral antigens, surface displaying of heterologous proteins on bacteria using anchoring motifs has successfully been implemented in human and veterinary vaccines research. In this study, a novel vaccine (BL21/InpN/vp7) was developed based on surface displaying a major capsid protein (vp7) of GCRV using the anchoring motif of N-terminal unique domain of ice-nucleation protein (InpN) on Escherichia coli BL21 (DE3) vaccine. Then the grass carp were immunized by surface displaying BL21/InpN/vp7 vaccine against GCRV using both intraperitoneal injection and bath immunization and their immune responses were tested. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly increased in grass carp post injection inoculation (vp7 dose ranged from 10 to 20 μg). The specific antibody levels against GCRV and the transcriptional of immune-related genes ( TNF-α , IL-1β , MHCI and IgM ) were also significantly enhanced in grass carp by injection inoculation (vp7 dose ranged from 5 to 20 μg). On the other hand, only the highest dose of bath vaccination significantly induced the production of specific antibody and up-regulated transcriptions of several immune-related genes ( IgM and MHCI ) in grass carp. The lower cumulative mortality of grass carp in vaccinated groups after GCRV challenge clearly demonstrated that surface displayed vp7 vaccine could protect fish against GCRV infection. The relative percentage survival (RPS) value in injection vaccinated group (88.89%) was much higher compared to bath group (18.89%), which was in consistent with the production of specific serum antibodies, non-specific immune response and immune related genes expression. To sum up, our results indicated the surface display of heterologous antigenic proteins on E. coli BL21 (DE3) using the anchoring motif of ice-nucleation protein may provide a promising approach to the vaccine development of aquatic animals and suggested its potential to be used as vaccine to fight against GCRV infection. [ABSTRACT FROM AUTHOR]

Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp ( Ctenopharyngodon idella ). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non-living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost-based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial ghost (DH5α-BG), delivering a major capsid protein gene ( vp 7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments -i) naked pcDNA- vp 7 (containing plasmid 1, 2.5 and 5 μg, respectively), ii) DH5α-BG/pcDNA- vp 7 (containing plasmid 1, 2.5 and 5 μg, respectively) and iii) naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune-related genes were significantly enhanced in fish immunized with DH5α-BG/pcDNA- vp 7 vaccine (DNA dose ranged from 2.5 to 5 μg). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5α-BG/pcDNA- vp 7 vaccine and the relative percentage survival reached to 90% in DH5α-BG/pcDNA- vp 7 group than that of naked pcDNA- vp 7 (42.22%) at the highest DNA dose (5 μg) after 14 days of post infection. Moreover, the level of pcDNA- vp 7 plasmid was higher in DH5α-BG/pcDNA- vp 7 groups than naked pcDNA- vp 7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection. [ABSTRACT FROM AUTHOR]

Medicinal plants have been widely used for a long history. Exploration of pharmacologically active compounds from medicinal plants present a broad prevalent of application. By examining viral mRNA expression in GCRV-infected Ctenopharyngodon idella kidney (CIK) cells treated with thirty kinds of plant extracts, we identified Magnolia officinalis Rehd et Wils. was able to preferably suppress viral replication. Further studies demonstrated that the main ingredients of magnolia bark, namely, magnolol and honokiol presented protective pharmacological function when treated GCRV-infected CIK cells with a concentration of 2.00 μg/ml and 1.25 μg/ml, respectively. Furthermore, reverse transcript quantitative polymerase chain reaction (RT-qPCR) and western blot showed that both magnolol and honokiol were efficient to restrain the replication of GCRV in CIK cells at non-toxic concentration (2.51 ± 0.51 μg/ml for magnolol, and 3.18 ± 0.61 μg/ml for honokiol). Moreover, it was found that magnolol and honokiol promoted the expression of immune-related genes. Magnolol obviously significantly increased the expression of interferon (IFN) regulatory factor ( IRF ) 7 rather than that of IRF3 in the GCRV-infected cells, leading to the activation of type I IFN ( IFN-I ). Simultaneously, magnolol drastically facilitated the expression of interleukin ( IL)-1β , but failed to induce the molecules in nuclear factor (NF)-κB pathways. Differently, honokiol strikingly motivated not only the expression of IL-1β , but also those of tumor necrosis factor α ( TNFα ) and NF-κB . Interestingly, though honokiol motivated the expression of IFN-β promoter stimulator 1 ( IPS-1 ), IRF3 and IRF7 , it failed to up-regulate the expression of IFN-I , indicating that honokiol enhanced the host innate antiviral response to GCRV infection via NF-κB pathways. Collectively, the present study revealed that magnolol and honokiol facilitated the expression of innate immune-related genes to strengthen the innate immune signaling responses to resist GCRV infection, which contributed to understanding the mechanisms by which small-molecule drugs possessed antiviral activities. In addition, these results lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry. [ABSTRACT FROM AUTHOR]

• UA effectively inhibits MSRV proliferation in vitro. • UA has a protective effect on MSRV-infected fish. • UA is a valuable agent for MSRV in the industry. Micropterus salmoides rhabdovirus (MSRV) has a high mortality rate and causes huge economic losses to the aquaculture industry. In this study, we identified that ursolic acid (UA) had antiviral efficacy against MSRV in vitro and in vivo. The results showed that UA inhibited MSRV replication in grass carp ovary (GCO) cells with a half-maximal inhibitory concentration (IC 50) of 5.55 μM, reduced viral titers and decreased cytopathic effects (CPE). Mechanistically, UA does not directly damage viral particles. On the other hand, UA inhibits MSRV replication by altering viral binding and release. Furthermore, pre- and post-treatment assays revealed that UA had preventive and therapeutic effects. For in vivo studies, UA could enhance the survival rate of MSRV-infected largemouth bass. Similarly, UA reduced the viral load of MSRV in the heart, spleen and brain at 3, 5 and 7 d post-infection. In conclusion, UA is an effective inhibitor of rhabdovirus in aquaculture. [ABSTRACT FROM AUTHOR]

To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in juvenile grass carp against Aeromonas hydrophila were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to juvenile grass carp against A. hydrophila . Moreover, SWCNTs conjugated with DNA vaccine provided significantly protective immunity compared with free DNA vaccine. Thereby, SWCNTs may be considered as a potential efficient DNA vaccine carrier to enhance the immunological activity. [ABSTRACT FROM AUTHOR]

Moroxydine hydrochloride (Mor) is known to have multi-antiviral activities against DNA and RNA viruses but very little information exists on its pharmacology. The paper was undertaken to explore the antiviral response and antiapoptotic mechanism of Mor against grass carp reovirus (GCRV) in Ctenopharyngodon idella kidney (CIK) cells. The results showed that exposing GCRV-infected cell to 6.3 μg mL −1 of Mor for 96 h avoid ca. 50% apoptosis. Meanwhile, Mor had lower cytotoxicity than ribavirin (Rib) as the value of safe concentration was threefold higher than effective concentration and the compound could ensure sufficient into and out of cells within 4 h when tested at the maximal safe concentration. Mor blocked the GCRV-induced cytopathic effects and eliminated nucleocapsids in CIK cells to keep the normal morphological structure. Moreover, the expressions of viral protein genes were significantly inhibited especially the guanylyl transferase and RNA-dependent RNA polymerase related expression. Furthermore, GCRV caused Bcl-2 down-regulation and Bax mitochondrial translocation was prevented by treatment of CIK cells with Mor. The downstream effector, caspase activity was also significantly inhibited in Mor treated cells. The potential mechanism might be that mitochondrial apoptotic signals were not activated by the intervention of Mor for targeting viral gene expression. Taken together, Mor showed high anti-GCRV activity and had been proved as a secure and promising agent in viral controlling in aquaculture industry. [ABSTRACT FROM AUTHOR]

To reduce the lethal hemorrhagic disease caused by grass carp reovirus (GCRV) and improve the production of grass carp, efficient and economic prophylactic measure against GCRV is the most pressing desired for the grass carp farming industry. In this work, a novel SWCNTs-pEGFP- vp5 DNA vaccine linked vp5 recombinant in the form of plasmid pEGFP- vp5 and ammonium-functionalized SWCNTs by a chemical modification method was prepared to enhance the efficacy of a vp5 DNA vaccine against GCRV in juvenile grass carp. After intramuscular injection (1, 2.5 and 5 μg) and bath administration (1, 10, and 20 mg/L), the ability of the different immune treatments to induce transgene expression was analyzed. The results showed that higher levels of transcription and expression of vp5 gene could be detected in muscle tissues of grass carp in SWCNTs-pEGFP- vp5 treatment groups compare with naked pEGFP- vp5 treatment groups. Moreover, antibody levels, immune-related genes, and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pEGFP- vp5 vaccine. In addition, we found that a good immune protective effect was observed in bath immunization group; which at a concentration of 20 mg/L could reach the similar relative percentage survival (approximately 100%) in injection group at a dose of 5 μg. All these results indicated that ammonium-functionalized SWCNTs could provide extensive application prospect to aquatic vaccine and might be used to vaccinate fish by intramuscular injection or bath administration method. [ABSTRACT FROM AUTHOR]

Intestinal microbiota has become an integral component of the fish, and plays a key role in host metabolism, immunity and health maintenance. However, information on the immune responses after vaccine administration in relation to the intestinal microbiota is absent in fish. The present study focused on the effect of a new recombinant Aeromonas hydrophila vaccine (Aera) by using a novel functionalized, single-walled carbon nanotubes (SWCNTs) as a delivery vehicle on the intestinal microbiota of grass carp ( Ctenopharyngodon idella ) through the bath immunization, and further explored the immunological responses in intestine, kidney and spleen. By performing deep sequencing, a total of 81,979 valid reads and 609 OTUs obtained from 4 intestine samples were analyzed. We detected 141 genuses, most of which belonged to Firmicutes, Fusobacteria and Proteobacteria. Of note, the quantity of Aeromonas in library Aera (after 6 h Aera vaccine pretreatment, fish were transferred to tanks without SWCNTs-Aera for 28 d) and Aera-GD (6 h Aera vaccine pretreated prior to the group injected by A. hydrophila ) was declined 6.5% and 14.6% compared with the control, respectively. Moreover, the expression of seven immune-related genes (IFN-I, TNF-α, CRP, IL-8, IgM, MHC I and CD8α) in the intestine, kidney and spleen of Aera treated fish was significantly enhanced, which indicated that a better tissue immune response in grass carp was induced by the SWCNTs-Aera vaccine. Therefore, a new recombinant SWCNT-Aera vaccine may represent potentially efficient and immunological role in grass carp intestine to resist A. hydrophila infection. [ABSTRACT FROM AUTHOR]

Infectious diseases of viral origin cause major aquatic production losses in different parts of the world. Because of formidable barriers for gastrointestinal tract, skin and cell, large amounts of antiviral drugs have limited therapeutic effect. In the current study, functionalized single-walled carbon nanotubes (SWCNTs) were selected as a drug carrier to carry antiviral drug for the treatment of viral diseases on fish. The results show that increasing antiviral drug (ribavirin) intake was observed by SWCNTs carrier and therapeutic dosage to kill grass carp reovirus is significantly reduced. At 12 d post infection, survival rate and infection rate were 29.7% and 50.4% for naked ribavirin treatment group exposed to the highest concentration (20 mg/L); however, survival rate of 96.6% and infection rate of 9.4% were observed in 5 mg/L ribavirin-SWCNTs treatment group. In addition, the drug detention time in different organs and tissues (blood, gill, liver, muscle, kidney and intestine) was also significantly extended (about 72 h) compared with the same dosage in naked ribavirin treatment group. Moreover, the toxicity of functionalized SWCNTs in grass carp can be minimal, and physiological markers (some antioxidant enzymes activities, apoptotic factors activities and their corresponding genes expression) remained within normal ranges following treatment. Our results indicated that drug delivery with functionalized SWCNTs can improve the antiviral effect on grass carp and has a potential application value to control fish viral diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Intestinal microorganisms of aquatic animals have a crucial impact on the host and some may have the potential to be used as probiotics. In this work, three strains of autochthonous bacteria were isolated from the intestinal tract of the healthy grass carp ( Ctenopharyngodon idellus ). The effects of single or combined use of dietary Shewanella xiamenensis A-1, S. xiamenensis A-2, and Aeromonas veronii A-7 were assessed on innate immunity and disease resistance of grass carp. The grass carp were fed 28 days by five experimental diets: i) one control diet with no probiotics, ii) three diets supplemented with 1 × 10 8 cell g − 1 of intestinal bacteria A-1 (group A1), A-2 (group A2) and A-7 (group B), and iii) mixed diet (group MIX) comprising of bacteria A 1, A-2 and A 7 dosed at 1 × 10 8 cell g − 1 , at a ratio of 4:2:1, which was set according to their quantity ratio in our study. At the end of the 28-day feeding experiment, the survival of grass carp against Aeromonas hydrophila was challenged for 14 days. Results showed that responses of several non-specific immune parameters of groups fed probiotics were enhanced; i.e. all probiotic groups led to significant enhancement of both respiratory burst, phagocytic and lysozyme activities than that of control activity. The complement C3, total serum proteins, albumin and globulin levels were also significantly improved on day 14 of feeding regime with probiotics compared to the control. Moreover, the expression of four immune-related genes (IL-8, IL-1β, lysozyme-C, and TNF-α genes) was significantly up-regulated in grass carp fed probiotic diets compared to control group on days 7, 14, and 28. The cumulative mortality of grass carp, experimentally challenged with A. hydrophila , was reduced in groups fed probiotics, i.e. A-1 = 46.67%, A-2 = 33.33%, B = 53.33% and mixed = 26.67%, respectively, than that of controls (80%) after 14 days of post-infection. Overall, the results of this study indicated the potential of using the three isolated autochthonous bacteria (single or combined) to improve immunity and disease resistance of grass carp. [ABSTRACT FROM AUTHOR]

The uses of single walled carbon nanotubes (SWCNTs) as carriers for DNA delivery have received considerable attention in cell studies. DNA vaccination of fish has been shown to elicit durable transgene expression, but no reports exist on intramuscular administration of SWCNTs-DNA vaccine electrostatic complexes which prepared through non-covalent conjugation. In this study, we injected grass carp intramuscularly with a plasmid vector containing a major capsid protein gene ( vp7 ) of grass carp reovirus as a) naked pcDNA- vp7 , b) SWCNTs-pcDNA- vp7 , c) empty plasmid vector, or phosphate buffered saline. After intramuscular administration, the ability of the different immune treatments to induce transgene expression was analyzed. The results indicated that higher levels of transcription and expression of the vp7 gene could be detected in muscle tissues of grass carp 28 days intramuscular injection in SWCNTs-pcDNA- vp7 treatment groups compare with naked pcDNA- vp7 treatment groups. Moreover, the serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, immune-related genes, antibody levels and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pcDNA- vp7 vaccine. The data in this study suggested that SWCNTs were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by intramuscular approach. [ABSTRACT FROM AUTHOR]

To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for recombinant Aeromonas hydrophila vaccine administration via bath or injection in juvenile grass carp were studied. The results showed that SWCNT as a vector for the recombinant protein aerA, augmented the production of specific antibodies, apparently stimulated the induction of immune-related genes, and induced higher level of survival rate compared with free aerA subunit vaccine. Furthermore, we compared the routes of bath and intramuscular injection immunization by SWCNTs-aerA vaccine, and found that similar antibody levels induced by SWCNTs-aerA were observed in both immunization routes. Meanwhile, a similar relative percentage survival (approximately 80%) was found in both a 40 mg/L bath immunization group, and a 20 μg injection group. The results indicate that functionalized SWCNTs could be a promising delivery vehicle to potentiate the immune response of recombinant vaccines, and might be used to vaccinate juvenile fish by bath administration method. [ABSTRACT FROM AUTHOR]

Grass carp reovirus (GCRV), the most pathogenic aquareovirus, can cause fatal hemorrhagic disease in fingerling and yearling grass carp. Vaccination by injection is by far the most effective method of combating disease. However it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Thus, an efficient and economic strategy for the prevention of GCRV infection becomes urgent. Here, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture SWCNTs-VP7 subunit vaccine with chemical modification. Different developmental stages of grass carps were immunized by VP7/SWCNTs-VP7 subunit vaccine against GCRV by intramuscular injection and bath immunization. The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP7 subunit vaccine were induced in comparison with VP7 subunit vaccine alone. Immunization doses/concentrations are significantly reduced (about 5–8 times) to prevent GCRV infection in different developmental stages of grass carp with injection or bath treatment when SWCNTs carrier was used. A good immune protective effect (relative percentage survival greater than 95%) is observed in smaller size fish (0.2 g) with SWCNTs-VP7 bath immunization. In addition, serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes and antibody levels were significantly enhanced in fish immunized with vaccine. This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach. [ABSTRACT FROM AUTHOR]

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1β, TNFα, MyD88 and IgM) in C . idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1β, TNFα, and MyD88 in C . idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L −1 ). According to in vivo insecticidal assays against Dactylogyrus intermedius , compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L −1 and safe to goldfish ( Carassius auratus ). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C . idella kidney cells and to some extent, eliminate the virus and parasitic infections. [ABSTRACT FROM AUTHOR]

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of crude extracts, fractions, and compounds from the leaves of Macleaya cordata against I. multifiliis were investigated under in vitro conditions by bioactivity-guided isolation method. The dried ethanol extract of M. cordata was extracted successively in a separating funnel with petroleum ether, ethyl acetate, chloroform and n-butanol. Among them, only the chloroform extract showed promising activity and therefore, was subjected to further separation and purification using various chromatographic techniques. Four compounds were isolated from chloroform extract, but only one compound showed potent activity. The structure of the active compound was elucidated as sanguinarine by hydrogen, carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. In vitro antiparasitic efficacy tests exhibited that sanguinarine was 100% effective against I. multifiliis at a concentration of 0.7 mg l, with LC and LC values of 0.437 and 0.853 mg l after 4 h of exposure. In vivo antiparasitic efficacy tests showed that the number of I. multifiliis on the gills in the treatment group (in 0.9 mg l sanguinarine) was reduced by 96.8%, in comparison to untreated group at 25°C for 48 h. Mortality of fish did not occur in the treatment group during the trail, although 40% of untreated fish died. Our results indicate that the studied plant extracts, as well as sanguinarine might be potential sources of new antiparasitic drug for the control of I. multifiliis. [ABSTRACT FROM AUTHOR]

Grass carp hemorrhagic disease has widely received great attention for Chinese freshwater aquaculture due to its strong infectiousness and high fatality rate. The pathogen is grass carp reovirus (GCRV). DNA vaccination is a high efficient approach for disease control. Although some encouraging progress have been made, developing a highly-efficient DNA vaccine with genetic type II GCRV predominant antigen epitope is less successful and still require further research. However, the DNA vaccine has made it challenging to avoid the problem of easily metabolized and degraded in vivo. In an effort to overcome this challenge, we constructed the DNA vaccine using single-walled carbon nanotubes (SWCNTs) as delivery vector (SWCNTs-pcDNA- vp4-3) and immunized grass carp (1.2 ± 0.2 g) by intramuscular injection. Experiments are grouped as follows: control group, pcDNA- vp4-3 immune group (10 μg per fish) and SWCNTs-pcDNA- vp4-3 immune groups (2.5, 5 and 10 μg per fish, respectively). The immune effect of DNA vaccine is evaluated comprehensively by testing immune-related parameters (the persistence and expression of DNA vaccine, serum antibody level, immune-related physiological indexes, immune-related genes expression and relative percentage survival). The results showed that serum antibody levels of grass carp were elevated significantly after immunization with DNA vaccines. And the serum antibody levels in SWCNTs-pcDNA- vp4-3 immune groups were higher than pcDNA- vp4-3 immune group. Likewise, the immune-related physiological indexes and immune-related genes expression were elevated significantly after immunization. The IgM gene expression in the SWCNTs-pcDNA- vp4-3 immune group (5 μg per fish) was 3.14 times as much as control group. The relative percentage survival in SWCNTs-pcDNA- vp4-3 immune group (5 μg per fish) reached the peak, protected 74% of grass carp from type II GCRV. Conclusively, the vp4-3 -centric DNA vaccine can induce efficient immune response by injection. Single-walled carbon nanotubes extend the retention time of DNA vaccine in the body and improve its expression intensity. SWCNTs-loaded DNA vaccine induce stronger immune response and enhance the resistance to grass carp haemorrhagic disease. • We introduced a DNA vaccine (pcDNA-vp4-3) based on the GCRV II predominant antigen epitope VP4-3. • SWCNT-loaded DNA vaccine based on predominant antigen epitope widely applied to prevent and control grass carp hemorrhagic diseases in the future. • SWCNTs is promising suitable candidate for improving the delivery efficiency of DNA vaccine in fish. [ABSTRACT FROM AUTHOR]

Hemorrhagic disease caused by grass carp reovirus (GCRV) is considered to be one of the most serious threats to grass carp, resulting in significant economic loss in grass carp culture industry. This study was aimed at optimizing the immunization program of SWCNTs-vaccine (SWCNTs-M-VP4-3) controlling infectious GCRV, determining the best immunization program for grass carp, and then providing reference for the commercial promotion and use of vaccines in aquaculture. We designed an compete experiment to optimize different parameters affecting vaccination such as immunization time, antigen concentration, and fish density when grass carp was vaccinated by bath treatment. Our results showed that the highest relative percent survival (88.33%) was found in 12 h of immunization time, 10 mg L−1 of antigen concentration, and 15 fish per liter of fish density compared to 100% mortality for control groups. And other immune responses (serum antibody production, enzyme activities, and immune genes expression) also demonstrated similar results. These results indicated that the administration protocol which induce highest immune response of the host, had the highest vaccine effect against the disease. This study lays the foundation for providing reference for the commercial promotion and use of vaccines in aquaculture. • The optimum procedure of a SWCNTs based vaccine with mannose modification were evaluated. • Fish density play an important role in response to the immune response of SWCNTs-M-VP4-3 vaccine. • Fish immersed for 12 h at the density of 15 fish L-1 with 10 mg L-1 SWCNTs-M-VP4-3 might be the optimum procedure. [ABSTRACT FROM AUTHOR]

Grass carp reovirus (GCRV), causative agent of grass carp hemorrhagic disease, causes high mortality of grass carp and results in huge economic losses in grass carp farming. Vaccination is considered the most effective way of preventing GCRV infection and losses of grass carp. In the study, recombination pcDNA3.1-vp4 and pcDNA3.1-vp4-3 plasmids were constructed and respectively transformed into E. coli DH5α. The pcDNA3.1-vp4 and pcDNA3.1-vp4-3 plasmids were used to vaccinate grass carp via intramuscular injection and we evaluated the immune responses induced by different forms of the DNA vaccine and investigated the efficacy in vaccinated fish. The results indicate that better immune responses of grass carp immunized with the pcDNA3.1-vp4 (10 μg/fish) compared with the pcDNA3.1-vp4-3 (10 μg/fish) DNA vaccine. Immune genes were expressed at higher levels in pcDNA3.1-vp4-3 treatment group compared to pcDNA3.1-vp4. The survival post vaccination and challenge of pcDNA3.1-vp4-3 vaccinated fish was 57.89% compared to 36.84% for pcDNA3.1-vp4 group and at 100% mortality in control groups. These results indicated that the segment of vp4-3 could be a plausible candidate for preventing GCRV-JX02 infection among grass carp. • Grass carp injected with pcDNA3.1-vp4 and pcDNA3.1-vp4-3 confers strong immunity against GCRV-JX02. • The DNA vaccine was manufactured by the core of dominant antigen epitope of GCRV-JX02, and its protective effects on grass carp against GCRV-JX02 infection by injection were assessment. • pcDNA3.1-vp4-3 could be a potential therapeutic vaccine against GCRV-JX02. [ABSTRACT FROM AUTHOR]

Hemorrhagic disease caused by grass carp reovirus (GCRV) is considered to be one of the most serious threats to grass carp, resulting in significant economic loss in aquaculture industry. Currently, vaccination is the most effective method to against GCRV infection and improve the production of grass carp. In the study, the antigen epitope of GCRV-II was screened, and its protective effects on grass carp against GCRV-II infection using carbon nanotubes as a carrier were assessment. The vp4 gene of GCRV-II was divided into four segments and respectively expressed in Escherichia coli. The potential antigen epitope was screened by the enzyme linked immunosorbent assay (ELISA). Afterwards, the VP4 and VP4-3 were conjugated with single-walled carbon nanotubes (SWCNTs) to vaccinate grass carp, and the protective effects were assessed by checking immune related parameters (specific serum antibodies, enzyme activities and immune related genes). The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP4 subunit vaccine were induced in comparison with naked VP4 subunit vaccine. Besides, compared to the SWCNTs-VP4 groups, the immune related parameters showed higher levels in SWCNTs-VP4-3 treatment groups. The survival of grass carp treated with SWCNTs-VP4-3 was 76.7% during 10 days post infection, while that were 58.3% and 0% for the SWCNTs-VP4 and control groups, respectively. These results indicated that SWCNTs-VP4-3 can be a plausible candidate for preventing and controlling GCRV-II among grass carp. • Grass carp immersed with SWCNTs-VP4 and SWCNTs-VP4-3 confers strong immunity against GCRV-II. • SWCNTs as a promising vehicle can enhance immune effect of subunit vaccine. • The antigen epitope of GCRV-II was screened. [ABSTRACT FROM AUTHOR]