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.

Antiviral, antioxidant, and anti-inflammatory activities of rhein against white spot syndrome virus infection in red swamp crayfish (Procambarus clarkii).

IMPORTANCE: Aquaculture is essential for ensuring global food security by providing a significant source of animal protein. However, the spread of the white spot syndrome virus (WSSV) has resulted in considerable economic losses in crustacean industries. In this study, we evaluated the antiviral activity of rhein, the primary bioactive component of Rheum palmatum L., against WSSV infection, and many pathological aspects of WSSV were also described for the first time. Our mechanistic studies indicated that rhein effectively arrested the replication of WSSV in crayfish by modulating innate immunity to inhibit viral gene transcription. Furthermore, we observed that rhein attenuated WSSV-induced oxidative and inflammatory stresses by regulating the expression of antioxidant and anti-inflammatory-related genes while enhancing innate immunity by reducing total protein levels and increasing phosphatase activity. Our findings suggest that rhein holds great promise as a potent antiviral agent for the prevention and treatment of WSSV in aquaculture.

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.

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.

In vitro and in vivo inhibition of a novel arctigenin derivative on aquatic rhabdovirus.

Spring viraemia of carp virus (SVCV) poses a serious threat to aquaculture industry due to the lack of approved antiviral treatments. Therefore, a novel arctigenin derivative, 4-(2-methylimidazole) octanoxy-arctigenin (MON), was synthesized to assess the antiviral activity against SVCV in vitro and in vivo. The results indicated MON decreased the SVCV glycoprotein (G) gene expression in vitro by a maximum inhibitory rate of > 99% at 3.5 µM. Furthermore, MON showed the protective effect on epithelioma papulosum cyprinid (EPC) cells and considerably decreased the cytopathic effect (CPE). More importantly, MON inhibited SVCV G gene expression levels in vitro at the half-maximal activity (IC50) of 0.18 µM at 48 h. For in vivo studies, MON demonstrated anti-SVCV activity by enhancing the survival rate of zebrafish (Danio rerio) after infection via pelvic fin base injection. These results tended to be consistent with MON decreasing the SVCV titer of infected zebrafish. During this time, viral loads of the spleen and kidney have declined in SVSV-infected zebrafish. Based on the histopathological assay, MON exhibited the high protective effect in the spleen and kidney of SVCV-infected fish. Combined, MON is on track to become a novel agent to address SVCV infection in aquaculture.

Proteome interrogation using gold nanoprobes to identify targets of arctigenin in fish parasites.

Gold nanoparticles (GNPs) are one of the most widely used nanomaterials in various fields. Especially, the unique chemical and physical properties make them as the promising candidates in drug target identification, unfortunately, little is known about their application in parasites. In this paper, GNPs were employed as new solid support to identify drug targets of natural bioactive compound arctigenin (ARG) against fish monogenean parasite Gyrodactylus kobayashi. Before target identification, GNPs with ARG on the surface showed the ability to enter the live parasites even the nucleus or mitochondria, which made the bound compounds capable of contacting directly with target proteins located anywhere of the parasites. At the same time, chemically modified compound remained the anthelminthic efficacy against G. kobayashii. The above results both provide assurance on the reliability of using GNPs for drug target-binding specificity. Subsequently, by interrogating the cellular proteome in parasite lysate, myosin-2 and UNC-89 were identified as the potential direct target proteins of ARG in G. kobayashii. Moreover, results of RNA-seq transcriptomics and iTRAQ proteomics indicated that myosin-2 expressions were down-regulated after ARG bath treatment both in transcript and protein levels, but for UNC-89, only in mRNA level. Myosin-2 is an important structural muscle protein expressed in helminth tegument and its identification as our target will enable further inhibitor optimization towards future drug discovery. Furthermore, our findings demonstrate the power of GNPs to be readily applied to other parasite drugs of unknown targets, facilitating more broadly therapeutic drug design in any pathogen or disease model.

Application of antigen presenting cell-targeted nanovaccine delivery system in rhabdovirus disease prophylactics using fish as a model organism.

BACKGROUND: Targeted delivery of virus-associated antigens to professional antigen-presenting cells (APCs) is considered as an efficient strategy to enhance the pyrophytic effect of vaccines against rhabdovirus disease. MATERIALS AND METHODS: In this study, we constructed a targeted carbon nanotubes-based vaccine deliver system (SWCNTs-MG) which can recognize the signature receptor (mannose) of APCs. An environmentally and economically important disease called spring viremia of carp (SVC) was studied as a model to evaluate the feasibility of single-walled carbon nanotubes (SWCNTs) conjugated with mannosylated antigen for rhabdovirus prevention. RESULTS: Results showed that SWCNTs-MG could cross into fish body and present to internal immune-related tissues through gill, muscle and intestine within 6 h immersed vaccination. With further modification of mannose moiety, the obtained nanovaccine showed enhanced uptake by carp macrophages and immune-related tissues, which would then trigger strong immune responses against spring viremia of carp virus (SVCV) infection. Moreover, the survival rate of fish vaccinated with SWCNTs-MG (30 mg/L) was 63.5% after SVCV infection, whereas it was 0% for the control group. CONCLUSION: This study not only provide a theoretical basis and research template for the application of targeted nanovaccine system in aquatic animals, but also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.

Ursolic acid from Prunella vulgaris L. efficiently inhibits IHNV infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a fish viral pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. However, anti-IHNV drugs currently are scarce. For the purpose of seeking out anti-IHNV drugs, the anti-IHNV activities of 32 medicinal plants were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Prunella vulgaris L. (PVL) showed the strongest inhibition on IHNV replication with an inhibitory percentage of 99.3% at the concentration 100 mg/L. Further studies demonstrated that ursolic acid (UA), a major constituent of PVL, also showed a highly effective anti-IHNV activity. The half-maximal inhibitory concentration (IC50) at 72 h of UA on IHNV was 8.0 µM. Besides, UA could significantly decrease cytopathic effect (CPE) and the viral titer induced by IHNV in EPC cells. More importantly, UA also showed a strong anti-IHNV activity in vivo, as indicated by increasing the survival rate of rainbow trout and inhibiting viral gene expression. Intraperitoneal injection of UA increased the relative percentage of survival of rainbow trout by 18.9% and inhibited IHNV glycoprotein mRNA expression by > 90.0% in the spleen at the 1st-day post-infection. Altogether, UA was expected to be a therapeutic agent against IHNV infection in aquaculture.

Synthesis and antiviral activity of a new arctigenin derivative against IHNV in vitro and in vivo.

Viral diseases in aquaculture were challenging because there are few preventative measures and/or treatments. Our previous study indicated that imidazole arctigenin derivatives possessed antiviral activities against infectious hematopoietic necrosis virus (IHNV). Based on the structure-activity relationship in that study, a new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), was designed, synthesized and its anti-IHNV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC50), we found that EOA (IC50 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole arctigenin derivatives in our previous study. Besides, EOA could significantly decrease cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum cyprinid (EPC) cells. In addition, EOA significantly inhibited apoptosis induced by IHNV in EPC cells. Further data verified that EOA inhibited IHNV replication in rainbow trout, with reducing 32.0% mortality of IHNV-infected fish. The results suggested that EOA was more stable with a prolonged inhibitory half-life in the early stage of virus infection (1-4 days). Consistent with above results, EOA repressed IHNV glycoprotein gene expression in virus sensitive tissues (kidney and spleen) in the early stage of virus infection. Moreover, histopathological evaluation showed that tissues from the spleen and kidney of fish infected with IHNV exhibited pathological changes. But there were no lesions in any of the tissues from the control group and EOA-treaten group. In accordance with the histopathological assay, EOA could elicited anti-inflammation response in non-viral infected rainbow trout by down-regulating the expression of cytokine genes (IL-8, IL-12p40, and TNF-α). Altogether, EOA was expected to be a therapeutic agent against IHNV infection in the field of aquaculture.

Synthesis and antiviral activity of coumarin derivatives against infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of juvenile salmonid species. However, robust anti-IHNV drugs currently are extremely scarce. For the purpose of seeking out anti-IHNV drugs, here a total of 24 coumarin derivatives are designed, synthesized and evaluated for their anti-viral activities. By comparing the half maximal inhibitory concentrations (IC50) of the 12 screened candidate drugs in epithelioma papulosum cyprini (EPC) cells infected with IHNV, the imidazole coumarin derivative C4 is selected for additional validation studies, with an IC50 of 2.53 µM at 72 h on IHNV glycoprotein. Further experiments revealed that C4 could significantly inhibit apoptosis and cellular morphological damage induced by IHNV. On account of these findings, derivative C4 could be a viable way of controlling IHNV and considered as a promising lead compound for the development of commercial drugs.

Antiviral activity of anisomycin against spring viraemia of carp virus in epithelioma papulosum cyprini cells and zebrafish.

Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) is an acute and highly lethal viral disease of cyprinid fish. However, effective therapy for SVC is still scarce until now. Here we evaluated the inhibition of anisomycin (Ani), a metabolite produced by Streptomyces griseolus, on the replication of SVCV in vitro and in vivo. Our results demonstrated that Ani could suppress SVCV replication with the maximum inhibitory rate > 95% in epithelioma papulosum cyprini (EPC) cells. And the half maximal inhibitory concentrations (IC50) of Ani on SVCV glycoprotein (G), nucleoprotein (N) and phosphoprotein mRNA expressions were 21.79, 13.13 and 12.24 nM, respectively. Besides, Ani decreased SVCV-induced cytopathic effects and nucleus damages. As expected, Ani also showed a strong anti-SVCV activity in vivo, as indicated by inhibiting viral gene expression and increasing the survival rate of zebrafish. Intraperitoneal injection of Ani increased the survival rate of zebrafish by 30% and markedly inhibited the expressions of G and N mRNA by > 60% in kidney and spleen at day 1 and day 4 post-infection. Results so far suggest that Ani as a powerful agent against SVCV can be applied to the control of SVC in aquaculture.

Evaluation on the antiviral activity of ribavirin against infectious hematopoietic necrosis virus in epithelioma papulosum cyprini cells.

Infectious hematopoietic necrosis virus (IHNV) causes high mortality in several economically important salmonid fishes, but there is no approved therapy up to now. To address the urgent need for therapeutics to combat IHNV infection, we investigate the anti-IHNV activities of 14 common antiviral agents using epithelioma papulosum cyprini (EPC) cells in this study. Among these agents, ribavirin shows the highest inhibition on IHNV replication, with an inhibitory percentage of 99.88%. And the 72 h half maximal inhibitory concentrations (IC50) of ribavirin on IHNV glycoprotein is 0.40 mg/L. In addition, ribavirin significantly inhibits apoptosis and cellular morphological damage induced by IHNV. Mechanistically, ribavirin could damage the viral particle of IHNV. Moreover, ribavirin could be used for prevention of IHNV infection. Therefore, ribavirin is considered to develop as a promising agent to treat IHNV infection.

Synthesis of arctigenin derivatives against infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. Herein, a series of arctigenin derivatives are synthesized to evaluate their antiviral activity against IHNV. The results indicate that the length of linker and imidazole substituent groups play an important role in decreasing IHNV replication. In this study, the arctigenin-imidazole hybrid derivative 15 with an eight carbon atoms length of the linker reduces IHNV replication with an IC50 value of 1.3 µM. In addition, derivative 15 significantly inhibits apoptosis and cellular morphological damage induced by IHNV. Mechanistically, derivative 15 can not damage the viral particle directly. While time-of-addition and viral binding assays reveal that derivative 15 mainly affect the early replication of IHNV but do not interfere with IHNV adsorption. Overall, derivative 15 could be considered to develop as a promising agent to treat IHNV infection.

The developmental toxicity, bioaccumulation and distribution of oxidized single walled carbon nanotubes in Artemia salina.

With the increasing production and applications of single walled carbon nanotubes (SWCNTs), concerns about the likelihood of SWCNTs being present in the aquatic environment and the subsequent effects on aquatic organisms are emerging. This work aimed to investigate the developmental toxicity, bioaccumulation and distribution of oxidized SWCNTs (O-SWCNTs) in a marine model organism, Artemia salina (A. salina). The results indicated that the hatching rates of capsulated and decapsulated cysts were decreased as the O-SWCNT concentration increased from 0 to 600 mg L-1 at 12, 18, 24 and 36 h. For instar I, II and III larvae exposure to 600 mg L-1, the mean mortality rates were 36.1%, 57.9% and 45.2%, respectively. Both the body length and swimming speed showed a concentration-dependent decrease after exposure to O-SWCNTs for 24 h. The inhibition of swimming may be caused by (1) the malformation of gills; (2) the attachment of O-SWCNTs on the gills. Reactive oxygen species (ROS) and antioxidant enzyme (catalase, superoxide dismutase and glutathione peroxidase) activities substantially increased following exposure, indicating that the toxic effects were related to oxidative stress. O-SWCNTs can be ingested, accumulated and excreted by A. salina, and distributed in the intestine, lipid vesicles and phagocytes. However, the accumulated O-SWCNTs were not completely excreted by A. salina. Uptake kinetics data showed that the O-SWCNT content increased from 1 to 48 h followed by a decrease from 48 to 72 h in the range from 0.08 to 5.7 mg g-1. The combined results so far indicate that O-SWCNTs have the potential to affect aquatic organisms when released into the marine ecosystems.

Synthesis and in vitro activities evaluation of arctigenin derivatives against spring viraemia of carp virus.

Spring viraemia of carp virus (SVCV) is a viral fish pathogen causing high mortality in several carp species and other cultivated fish. However, robust anti-SVCV drugs currently are extremely scarce. For the purpose of seeking out anti-SVCV drugs, here a total of 35 arctigenin derivatives were designed, synthesized and evaluated for their anti-viral activities. By comparing the inhibitory concentration at half-maximal activity (IC50) of the 15 screened candidate drugs (max inhibitory response surpassing 90%) in epithelioma papulosum cyprini (EPC) cells infected with SVCV, 2Q and 6 A were chosen for additional validation studies, with an IC50 of 0.077 µg/mL and 0.095 µg/mL, respectively. Further experiments revealed that 2Q and 6 A could significantly decrease SVCV-induced apoptosis and have a protective effect on cell morphology at 48 and 72 h post-infection. Moreover, the reactive oxygen species (ROS) induced upon SVCV infection could be obviously inhibited by 2Q and 6 A, while SVCV-infected cells were clearly observed. On account of these findings, 2Q and 6 A could have a promising application for the treatment of infection of SVCV and provide a considerable reference for novel antivirals in aquaculture.

Ligand induced shape transformation of thorium dioxide nanocrystals.

Nanocrystals (NCs) with size and shape dependent properties are a thriving research field. Remarkable progress has been made in the controlled synthesis of NCs of stable elements in the past two decades; however, the knowledge of the NCs of actinide compounds has been considerably limited due the difficulties in handling them both experimentally and theoretically. Actinide compounds, especially actinide oxides, play a critical role in many stages of the nuclear fuel cycle. Recently, a non-aqueous surfactant assisted approach has been developed for the synthesis of actinide oxide NCs with different morphologies, but an understanding of its control factors is still missing to date. Herein we present a comprehensive study on the low index surfaces of thorium dioxide (ThO2) and their interactions with relevant surfactant ligands using density functional calculations. A systematic picture on the thermodynamic stability of ThO2 NCs of different sizes and shapes is obtained employing empirical models based on the calculated surface energies. It is found that bare ThO2 NCs prefer the octahedral shape terminated by (111) surfaces. Oleic acid displays selective adsorption on the (110) surface, leading to the shape transformation from octahedrons to nanorods. Other ligands such as acetylacetone, oleylamine, and trioctylphosphine oxide do not modify the equilibrium shape of ThO2 NCs. This work provides atomic level insights into the anisotropic growth of ThO2 NCs that was recently observed in experiments, and thus may contribute to the controlled synthesis of actinide oxide NCs with well-defined size and shape for future applications.

Probiotic potential of Bacillus velezensis JW: Antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus.

This study evaluated the probiotic potential of B. velezensis JW through experimental and genomic analysis approaches. Strain JW showed antimicrobial activity against a broad range of fish pathogenic bacteria including Aeromonas hydrophila, Aeromonas salmonicida, Lactococcus garvieae, Streptococcus agalactiae, and Vibrio Parahemolyticus. Fish (Carassius auratus) were fed with the diets containing 0 (control), 107, and 109 cfu/g of B. velezensis JW for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that JW supplemented diets significantly increased acid phosphatase (ACP), alkaline phosphatase (AKP), and glutathione peroxidase (GSH-PX) activity. The mRNA expression of immune-related genes in the head kidney of C. auratus was measured. Among them, the interferon gamma gene (IFN- γ) and tumor necrosis factor-α (TNF-α) showed higher expression after 3 and 4 weeks of feeding (P < 0.05). The expression of interleukin-1 (IL-1) only being significantly upregulated by 109 cfu/g of JW after 1 week of feeding (P < 0.05). The upregulation of interleukin-4 (IL-4) increased over time from 1st to 4th week. The expression of interleukin-10 (IL-10) and interleukin-12 (IL-12) showed an opposite expression pattern with IL-10 significantly upregulated and IL-12 significantly downregulated by JW containing diets at 2, 3, and 4 weeks of post-feeding (P < 0.05). Moreover, fish fed with JW supplemented diets showed significantly improved survival rate after A. hydrophila infection. The analysis of the genome of JW revealed several features aiding host health and being relevant to the GIT adaptation. Four bacteriocins, three Polyketide Synthetase (PKS), and five Nonribosomal Peptide-Synthetase (NRPS) gene clusters were identified in the genome. In summary, the above results clearly proved that B. velezensis JW has the potential to be developed as a probiotic agent in aquaculture.

Targeting Heat Shock Protein 70 as an antiviral strategy against grass carp reovirus infection.

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.

Evaluation on the antiviral activity of arctigenin against spring viraemia of carp virus.

Spring viraemia of carp virus (SVCV) causes high morality in several economically important cyprinid fishes, but there is no approved therapy up to now. To address the urgent need for therapeutics to combat SVCV infection, we investigated the anti-SVCV activities of 12 natural compounds and 7 common antiviral agents using epithelioma papulosum cyprini (EPC) cells in this study. From the 19 compounds, we identified arctigenin (ARG) has the highest inhibition on SVCV replication, with maximum inhibitory percentage on SVCV > 90%. And the 48 h half maximal inhibitory concentrations (IC50) of ARG on SVCV glycoprotein and nucleoprotein were 0.29 (0.22-0.39) and 0.35 (0.29-0.41) mg/L respectively. In addition, ARG significantly reduced SVCV-induced apoptosis and recovered SVCV-activated caspase-3/8/9 activity. Further, cellular morphological damage induced by SVCV was blocked by ARG treatment. Mechanistically, ARG did not affect SVCV infectivity. Moreover, ARG could not induce reactive oxygen species (ROS) generation, which plays an antiviral role on SVCV. Interestingly, SVCV-induced autophagy which is necessary for virus replication was inhibited by ARG treatment. These results indicated that the inhibition of ARG on SVCV replication was, at least in part, via blocking SVCV-induced autophagy. Taken together, ARG has the potential to work as an agent for protecting economically important fishes against SVCV.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effect of a DNA vaccine against spring viremia of carp virus in common carp.

Spring viremia of carp virus (SVCV) is highly contagious and pathogenic to cyprinid fish, causing enormous economic losses in aquaculture. Efficient and economic prophylactic measure against is the most pressing desired for the common carp farming industry. In this research, single-walled carbon nanotubes (SWCNTs) as a candidate DNA vaccine carrier was administrated via bath (1, 5, 10, 20, 40 mg L-1) or injection (1, 4, 8, 12, 20 µg) in common carp juvenile, and the different immune treatments to induce immunoprotective effect was analyzed. The results showed that higher levels of transcription and expression of G gene could be detected in muscle, spleen and kidney tissues via bath administration or intramuscular injection in SWCNTs-pEGFP-G treatment groups compared with naked pEGFP-G treatment groups. Meanwhile, complement activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes (especially the TNF-α) and antibody levels were significantly enhanced in fish immunized with DNA vaccine combined with SWCNTs. The relative percentage survival were significantly enhanced in fish bathed with SWCNTs-pEGFP-G vaccine and the relative percentage survival reached to 57.5% in SWCNTs-pEGFP-G group than that of naked pEGFP-G (40.0%) at the highest vaccine dose (40 mg L-1) after 22 days of post infection, and fish in bath immunization group at a concentration of 40 mg L-1 could reach the similar relative percentage survival in injection group at a dose of 12 µg. This study suggest that ammonium-functionalized SWCNTs is the promising carrier for DNA vaccine and might be used to vaccinate large-scale juvenile fish by bath administration approach in aquaculture.