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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]

• 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]

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]

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]

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]