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Due to its wide host range and frequently causing significant mortality, infectious spleen and kidney necrosis virus (ISKNV) has caused huge economic losses on mandarin fish in China. There are no commercial drugs to cure ISKNV, and vaccines are regarded as one of the most effective measures to control virus infection. Because of the weak immunogenicity of antigen protein, subunit vaccines may require multiple booster immunizations, adjuvants or carriers to ensure strong and long‐term protective immunity. In this work, based on MCP (main capsid protein) subunit vaccine, we used single‐walled carbon nanotubes (SWCNTs) as carrier and mannose (M) as targeting factors to construct SWCNTs‐M‐MCP vaccine. After injection immunization, rapid and strong antibody titres were detected in SWCNTs‐M‐MCP groups which were significantly higher than that of MCP groups and SWCNTs‐MCP groups at the same immune doses. The antibody titre of the highest immune group (5 μg SWCNTs‐M‐MCP) reached more than 2.5 times that of the control group at 21 days postvaccination. Similar results were found in enzyme activities and immune‐related genes expression. All enzyme activities and immune‐related genes expression of 5 μg SWCNTs‐M‐MCP group at 21 days postvaccination were significantly higher than those of all dosages MCP groups and SWCNTs‐MCP groups. This study showed that SWCNTs and mannose can significantly improve the immune responses of vaccines and may provide an effective strategy against ISKNV. [ABSTRACT FROM AUTHOR]

Immersion vaccination of single‐walled carbon nanotubes loaded with mannose‐modified glycoprotein (SWCNTs‐MG) vaccine has been proved to be effective in preventing spring viraemia of carp virus (SVCV). Immunization procedure has immense consequence on the immune effect of the immersion vaccine. However, immunization procedure optimization for SWCNTs‐MG vaccine against SVCV has not been reported. In this study, accordingly, a full‐factor experiment was designed to optimize the immunization procedure of SWCNTs‐MG vaccine by three aspects of vaccine dose (30 mg/L, 40 mg/L and 50 mg/L), immunization density (8 fish L−1, 24 fish L−1 and 48 fish L−1) and immunization time (6, 12 and 24 hr). Furthermore, we used the immunization group (A1B2C1, 30 mg/L, 24 fish L−1 and 6 hr) in the previous study as a positive control (PC) to evaluate the immunization effect optimized conditions from the expression of immune‐related genes and relative percentage survival (RPS). At 28 days post‐vaccination (DPV), common carps were intraperitoneal injected SVCV challenged test indicated that the A1B2C2 group (30 mg/L, 24 fish L−1, 12 hr) displayed superiority of protective efficacy compare with other groups and the RPS with 77.9%, which was 15.6% higher than the PC group of RPS with 62.3%. Moreover, the expression of immune‐related genes such as IL‐10, CD4 and MHC‐II was also significantly higher than PC group. The specific experimental flow chart is shown in Figure 1. Conclusively, these results demonstrated that vaccine dose, immunization density and immunization time are 30 mg/L, 24 fish L−1 and 12 hr, which is the more appropriate immunization programme with juvenile carp for SWCNTs‐MG vaccine. This study provides a profitable reference for improving the immune efficiency of aquatic immersion vaccine. 1 FIGURE: The experiment process and timeline chart of the present research [ABSTRACT FROM AUTHOR]

Carbon nanotubes (CNTs) are widely used in industrial and commercial applications, but few studies systematically evaluate their developmental toxicity on aquatic organism. Using rare minnow ( Gobiocypris rarus) at early life stages as experimental models, developmental toxicity of functionalized single-walled CNTs (SWCNTs) was investigated following exposure to 0-320 mg/L for 144 h. Results revealed that significantly increased in mortality and malformation was only observed after hatching. Decreased body length, heart rate and swimming speed provide a concentration-dependent manner on larvae; values of 144 h LC50 and EC50 were 140.8 and 109.8 mg/L, respectively. Antioxidant enzyme activities (superoxide dismutase, catalase and glutathione S-transferase) and antioxidant enzyme related mRNA expressions were significant changed; cell apoptosis activities (caspase-3, -8, -9) and cell apoptosis related mRNA expressions were significant up-regulated; reactive oxygen species and DNA damage were significantly induced when the concentration of SWCNTs above 100 mg/L. Fluorescence and electron microscopy sliced observation show that SWCNTs were well dispersed in larvae within 0.5 h, eventually cleared from the larvae at 144 h. This is the first study to define uptake kinetics and to focus on behavioral consequences, physiological changes and mRNA expression following SWCNTs exposure in the early life stages of fish. The results obtained in the present study demonstrated that functionalized SWCNTs have the potential to affect aquatic life when released into the aquatic environment and reached high concentration. In the increasing economical context of SWCNTs, complementary studies must be undertaken, especially including mechanistic and environmental investigations. [ABSTRACT FROM AUTHOR]

Immersion vaccination is considered as the most effective method for juvenile fish in preventing viral disease, due to its convenience for mass vaccination and stress-free administration. However, immune responses following immersion vaccination are generally less robust and of shorter duration than those induced through intraperitoneal injection. Herein, to improve the efficacy of the immersion vaccine, we constructed a targeted single-walled carbon nanotubes-based immersion vaccine delivery system (CNTs-M-VP7), the surface of which are modified with mannose to allow antigen-presenting cells' (APCs) targeting. The targeting ability of CNTs-M-VP7 was confirmed in vivo and in vitro. Critically, this immersion CNTs-M-VP7 vaccine could cross into the fish body through mucosal tissues (skin, gill, and intestine), and then present to immune-related tissues. Moreover, CNTs-M-VP7 could significantly induce the maturation and presenting process of APCs, which would then trigger robust immune responses. Altogether, this study demonstrates that the single-walled carbon nanotubes (SWCNTs)-based targeted nanovaccine delivery system shows the potential to be an effective prophylactic against fish viral disease. [ABSTRACT FROM AUTHOR]

Background: Single-walled carbon nanotubes (SWCNTs) have many potential applications in various fields. Especially, the unique physicochemical properties make them as the prime candidates for applications in biomedical fields. However, biocompatibility of SWCNTs has been a major concern for their applications. In the study, biocompatibility of oxidized SWCNTs (O-SWCNTs) was assessed using Saccharomyces cerevisiae (S. cerevisiae) as a model organism. Results: Cell proliferation and viability were significantly changed after exposure to O-SWCNTs (188.2 and 376.4 mg/L) for 24 h. O-SWCNTs were internalized in cells and distributed in cytoplasm, vesicles, lysosomes and cell nucleus. The average O-SWCNTs contents in S. cerevisiae were ranged from 0.18 to 4.82 mg/g during the exposure from 0 to 24 h, and the maximum content was reached at 18 h after exposure. Both penetration and endocytosis were involved in the internalization of O-SWCNTs in S. cerevisiae, and endocytosis was the main pathway. Cellular structures and morphology were changed after exposure to O-SWCNTs, such as undulating appearance at the membrane, shrinking of the cytosol, increased numbers of lipid droplets and disruption of vacuoles. ROS and antioxidant enzymes activities were observably changed following exposure. For the treatment at 376.4 mg/L, 20.8% of the total cells was undergone apoptosis. Decrease of mitochondrial transmembrane potential and leakage of cytochrome c from mitochondria were observed after exposure. Moreover, expression levels of apoptosis-related genes were significantly increased. Conclusions: O-SWCNTs can internalize in S. cerevisiae cells via direct penetration and endocytosis, and distribute in cytoplasm, vesicles, lysosomes and cell nucleus. Besides, O-SWCNTs (188.2 and 376.4 mg/L) can induce apoptosis in S. cerevisiae cells, and oxidative stress is involved in activation of the mitochondria-dependent apoptotic pathway. [ABSTRACT FROM AUTHOR]

To reduce the largemouth bass ulcer syndrome (LBUSV) aquatic economic losses, it must take effective preventive measures and coping strategies should be urgently investigated. In this research, the effects of a functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in largemouth bass (Micropterus Salmoides) against LBUSV were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to largemouth bass against LBUSV. We found more than 10 times increase in serum antibody levels, enzyme activities and immune-related genes (IL-6, IL-8, IFN-γ, IgM and TNF-α) expression, in the SWCNTs-pcDNA- MCP immunized groups compared with PBS group and the pure SWCNTs group. The survival rates for control group (PBS), pure SWCNTs groups (40 mg L−1), four pcDNA- MCP groups (5 mg L−1, 10 mg L−1, 20 mg L−1 and 40 mg L−1) and four SWCNTs-pcDNA- MCP groups (5 mg L−1, 10 mg L−1, 20 mg L−1 and 40 mg L−1) were 0%, 0%, 0%, 2.77%, 11.11%, 19.44%, 27.78%, 38.89%, 52.78% and 61.11%, respectively. Our results demonstrate that the SWCNTs-DNA vaccine can be used as a new method against LBUSV showing protection following challenge with LBUSV. • SWCNTs was used as an efficient DNA vaccine vector against LBUSV infection. • Immersed DNA vaccine confers strong immunity against LBUSV. • SWCNTs enhance the immunological effect of DNA vaccines. [ABSTRACT FROM AUTHOR]

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-β, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines. • SWCNTs enhance the immune protective effect of bath subunit vaccine. • SWCNTs-MCP enhances the immunological activity against TGIV infection. • SWCNTs-MCP provides a good protection for pearl gentian grouper against TGIV. [ABSTRACT FROM AUTHOR]

Largemouth bass ulcerative syndrome virus (LBUSV) is an important virus induce the mortality of largemouth bass (Micropterus Salmoides). In this study, we reported a single-walled carbon nanotubes (SWCNTs) containing LBUSV major capsid protein (MCP) subunit vaccine (SWCNTs-MCP) which was evaluated for its protective effect on largemouth bass by immersion immunization. We found an elevation in serum antibody levels, enzyme activities, complement C3 content and immune-related genes (IgM, TGF-β, IL-1β, IL-8, TNF-α and CD4) expression, in the SWCNTs-MCP immunized groups compared with the pure MCP group. The survival rates for control group, pure MCP protein groups (40 mg L−1) and four SWCNTs-MCP groups (5 mg L−1, 10 mg L−1, 20 mg L−1 and 40 mg L−1) were 0%, 27.78%, 30.56%, 50.00%, 66.67% and 80.56%, respectively. The results suggests that with the assistance of SWCNTs, the immune protection of the SWCNTs-MCP group (40 mg L−1) increased up 52.78%–80.1% compared with pure MCP group (40 mg L−1). Our results demonstrate that the single-walled carbon nanotube subunit vaccine can be used as a new immunization method against LBUSV showing protection following challenge with LBUSV. Taken together, our results demonstrate that the single-walled carbon nanotube subunit vaccine can be used as a new method against LBUSV and have a high immune protection during the largemouth bass farm. • Largemouth bass immersed with SWCNTs-MCP confers strong immunity against LBUSV. • SWCNTs enhance the immunological effect of bath vaccines. • SWCNTs based immersion vaccine have prospects for application in aquatic vaccine. [ABSTRACT FROM AUTHOR]

As a high mortality disease, Infectious spleen and kidney necrosis virus (ISKNV) can cause massive economic damage on mandarin fish farming industry in China, which seriously hindered the development of mandarin fish farming industry. In this research, SWCNTs (single-walled carbon nanotubes) as a candidate for DNA vaccine carrier was vaccinated by immersion (1, 2, 5, 10, 20 mg/L) in juvenile mandarin fish. In muscle, spleen and kidney tissues, the results showed that transcription and expression of MCP gene can be detected in pcDNA-MCP and SWCNTs-pcDNA-MCP groups after bath immunization. The immune response (immune-related genes expression, serum antibody production, enzyme activities and C3 content) was significantly enhanced in fish which vaccinated with SWCNTs-pcDNA-MCP in comparison with those vaccinated with pcDNA-MCP alone. After 14 d challenge, the RPS (relative percentage survival) can be enhanced which using SWCNTs as a carrier in SWCNTs-pcDNA-MCP (82.4%) group at 20 mg/L (the highest vaccine dose) than the naked pcDNA-MCP (54.2%) group. This study reveals that functionalized SWCNTs could be a promising immersion DNA vaccine carrier in aquaculture. • SWCNTs as a promising vehicle can enhance immune effect of DNA vaccine. • Mandarin fish immersed with SWCNTs-pcDNA-MCP confers strong immunity against ISKNV. • SWCNTs based immersion DNA vaccine have prospects for application to aquatic vaccine. [ABSTRACT FROM AUTHOR]

Spring viremia of carp virus (SVCV) has caused mass mortality in cyprinids, with case fatality rates of young fish up to 90%, resulting in enormous economic losses in the aquaculture industry. Immersion vaccination is considered as the most effective method for juvenile fish to combating disease, due to its convenience for mass vaccination and stress-free administration. However, immune responses following immersion vaccination are generally less robust and of shorter duration as those induced through intraperitoneal injection. Herein, to enhance the efficient of immersion vaccine, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture immersion DNA vaccine system (SWCNTs-pEGFP-M) with chemical modification. Results showed that SWCNTs-pEGFP-M could enter into fish body via immersion administration and express antigen proteins in fish kidney and spleen. Moreover, stronger and longer duration immune responses (including serum antibody production and immune genes expression) can be induced in fish vaccinated with SWCNTs-pEGFP-M in comparison with those vaccinated with pEGFP-M alone. Notably, SWCNTs can increase the immune protective effect of naked DNA vaccine by ca. 23.8%. Altogether, this study demonstrates that SWCNTs as a promising DNA vaccine carrier might be used to vaccinate large-scale juvenile fish by bath administration approach, which can provide an outlook for future vaccination strategies against SVCV. • SWCNTs-pEGFP-M encoding the SVCV matrix protein were constructed. • SWCNTs as a promising vehicle can enhance immune effect of DNA vaccine. • Common carps immersed with SWCNTs-pEGFP-M confers duration immunity against SVCV. • SWCNTs based immersion vaccine have prospects for application to aquatic vaccine. [ABSTRACT FROM AUTHOR]

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which lead to significant economic loss on mandarin fish in China. There is no effective drug or vaccine against this fatal disease at present. Meanwhile, many drugs and vaccines had no effect in many cases account of several impenetrable barriers (cell, skin and gastrointestinal tract). Here we reported an immersion subunit vaccine system (SWCNTs-MCP) encoding MCP gene of ISKNV based on single-walled carbon nanotubes (SWCNTs). To evaluate its efficacy against ISKNV, we found a stronger and longer duration immune response (serum antibody production, enzyme activities and immune-related genes expression) can be induced in fish vaccinated with SWCNTs-MCP in comparison with those vaccinated with MCP alone. Importantly, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 23.8%. Thereby, this study demonstrates that SWCNTs as a promising carrier for subunit vaccine might be used to vaccinate large-scale juvenile mandarin fish by bath administration approach. • SWCNTs-MCP encoding the ISKNV MCP gene were constructed. • ►SWCNTs as a promising vehicle can enhance immune effect of subunit vaccine. • Mandarin fish immersed with SWCNTs-MCP confers strong immunity against ISKNV. • SWCNTs based immersion vaccine have prospects for application to aquatic vaccine. [ABSTRACT FROM AUTHOR]

Highlights • SWCNTs can be used as carriers for subunit vaccine. • SWCNTs-subunit vaccine can enhance the immunological activity against SVCV infection. • SWCNTs based subunit vaccine have prospects for application to aquatic vaccine. Abstract As a successful prevention strategy for controlling the highly contagious and pathogenic disease of spring viremia of carp (SVC), DNA vaccines reported in recent years could trigger protective responses against SVC with the means of injection. However, there remains many concerns and uncertainties related to DNA vaccination as well as injection is labor intensive, costly and not suitable to vaccinate large numbers of fish. Therefore, more efficient and safe prophylactic measures should be urgently investigated. In this research, single-walled carbon nanotubes (SWCNTs) as the candidate SWCNTs-pET32a- G subunit vaccine carrier were 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 were analyzed. The results showed that SWCNTs-pET32a- G could enter fish body after immersion for 10 h, furthermore, compared to control groups, antibody levels, the non-specific immune parameters (complement activity, superoxide dismutase activity and alkaline phosphatase activity), and immune-related genes (especially the TNF-α and IFNg2b) in vaccinated groups were significantly enhanced in fish immunized with SWCNTs-subunit vaccine. In addition, as a promising carrier, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 16% in bath immunization group and by ca. 23% in injection group. This study suggests that SWCNTs-vaccine may represent a potentially efficient immersion vaccine against viral pathogens of fish in the future. [ABSTRACT FROM AUTHOR]

To elicit the immune protective of vaccine against the highly contagious and pathogenic disease caused by spring viremia of carp virus (SVCV), a novel functionalized single-walled carbon nanotubes (SWCNTs) were applied as a delivery vehicle for DNA vaccine. In this study, we report a SWCNTs-DNA vaccine encoding matrix protein of SVCV which, when injected in the muscle at a dose of 10 μg SWCNTs-pcDNA- M vaccine, confers up to 51.3% protection against intraperitoneal challenge with SVCV. In addition, SWCNTs as a promising vehicle can enhance about 17.5% of the immune protective effect in SWCNTs-pcDNA- M vaccinated common carp compared with fish injected with naked pcDNA- M DNA vaccine. In addition, serum antibody production, none specific immunity parameters (complement activity, superoxide dismutase activity (SOD), acid phosphatase activity (ACP) and alkaline phosphatase activity (AKP)) and immune-related genes were used to verify the enhancement immune response induced in SWCNTs-pcDNA- M vaccinated fish, herein all these mentioned immune activities were significantly enhanced after immunization. Thereby, it is revealed that the M gene of SVCV could be used as an antigen for DNA vaccine constructs, and SWCNTs could be a candidate DNA vaccine carrier to enhance the immunological response against fish disease. [ABSTRACT FROM AUTHOR]

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. [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]

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]

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]

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]

Grouper is an important cultured fish in China. However, a large number of larval and juvenile groupers dead from the infection of nervous necrosis virus every year, which calls for an effective prevention and control measure. In this study, recombinant major capsid protein (MCP) of nervous necrosis virus (NNV) was produced through a prokaryotic expression vector as a subunit vaccine. We processed single walled carbon nanotube (SWCNTs) by conc H 2 SO 4 and conc. HNO 3 to make it a carrier for the vaccine. The fish were inoculated by immersion immunization for 6 h and three concentrations included 5, 10, 20 mg/L were set. Antibody levels, immunization related enzyme activities of acid phosphatase, superoxide dismutase, and alkaline phosphatase in serum as well as the transcription of immune genes (IgM, Mx1, IL1-β, TNFα, CD4, CD8. MHC I, and MHC II) were tested. According to the results, the data of vaccine with the carrier had a significant increase compared with that without. After the virus challenge test, the vaccines carried by SWCNTs with concentrate of 5, 10, 20 mg/L of provided the relative survival rates of 28.57%, 50.62%, 70.76% respectively, while 16.67%, 25.93%, 33.33% were provided by the vaccines without SWCNTs. Besides, the body length and weight of a fish were measured every week, the data showed that there was no significant difference among the groups. The research showed the immersion subunit vaccine loaded by SWCNTs can protect pearl gentian grouper from NNV. • Major capsid protein (MCP) of nervous necrosis virus (NNV) induces effective immune response. • SWCNTs enhance the immunological activity by immersion. • SWCNTs-MCP provides a good protection for pearl gentian grouper against nervous necrosis virus. [ABSTRACT FROM AUTHOR]

Infectious diseases of viral origin cause major aquatic production losses in different parts of the world. There are lots of limited therapeutic effects for large amounts of antiviral drugs because of several impenetrable barriers (cell, skin, and gastrointestinal tract). In this study, we selected moroxydine hydrochloride (Mor) for the treatment of infectious spleen and kidney necrosis virus (ISKNV) and choose single-walled carbon nanotubes (SWCNTs) as drug carrier by immersion bath on mandarin fish infected with ISKNV. The results showed that increasing Mor intake was observed by SWCNTs carrier and therapeutic dosage to kill ISKNV is significantly reduced. After 4 h treatment, the concentration of Mor was 103.48 μg/g in 40 mg/L Mor group, and 182.35 μg/g in 40 mg/L Mor-SWCNTs groups. Comparing with the same dosage in Mor group, the detention time of Mor was extended more than 48 h in Mor-SWCNTs group. After 11 d treatment, cumulative mortality was 11.51% and infection rate was 3.81% in 40 mg/L Mor-SWCNTs group. In the same dosage of Mor group, cumulative mortality was 43.34% and infection rate was 22.67%. Moreover, enzyme activities, complement (C3) content and immune-related genes expression were significantly increased in Mor and Mor-SWCNTs groups than control group, herein all these mentioned activities reached the highest level at 3 days post-treatment. Our results indicated that drug delivery with functionalized SWCNTs can improve the antiviral effect on mandarin fish and has a potential application value to control fish viral diseases in aquaculture. • Our study indicated that Mor delivery with functionalized SWCNTs can improve the antiviral effect on mandarin fish. • Comparing with the same dosage in Mor group, the detention time of Mor was extended more than 48 h in Mor-SWCNTs group. • Our study showed that Mor-SWCNTs can be a plausible candidate for preventing and controlling ISKNV in farmed mandarin fish. [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]

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which leads to a significant economic loss on mandarin fish in China. At present, there is no effective drug against this fatal disease because of several impenetrable barriers (cell, skin and gastrointestinal tract). In this study, we selected ganciclovir for the treatment of ISKNV and choose single-walled carbon nanotubes (SWCNTs) as ganciclovir carrier by immersion bath on mandarin fish infected with ISKNV. After 7 d post-infection, cumulative mortality was 14.75% and infection rate was 26.55% in naked ganciclovir group (40 mg/L). Meanwhile, cumulative mortality of 32.50% and infection rate of 15.30% were observed in G-SWCNTs group (20 mg/L). In the control group, cumulative mortality was 88.75% and the infection rate was 100%. At 6 h post-infection, the concentration of ganciclovir was detected to be 34.58 μg/g in naked ganciclovir group (20 mg/L). And in G-SWCNTs groups (10 mg/L) at 6 h, the concentration of ganciclovir was 39.35 μg/g. Besides, the time detention of ganciclovir in G-SWCNTs group was significantly extended compared with the naked ganciclovir group in the same dosage. Moreover, enzyme activities, complement (C3) content and immune-related genes expression were used to verify the enhancement effect induced in G-SWCNTs groups, herein all these mentioned activities reached the highest level at 3 days post-treatment. The results showed that ganciclovir-SWCNTs can be a plausible candidate for preventing and controlling ISKNV among farmed mandarin fish. • Our study showed that ganciclovir has antiviral effect on ISKNV in mandarin fish in vivo. • Ganciclovir loaded with SWCNTs has a significant in vivo effect against ISKNV. • The ganciclovir detention time was significantly extended >24 h by using SWCNTs as drug carrier. [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]

Viral nervous necrosis (VNN) disease has caused mass mortality in cultured marine and freshwater fish worldwide, resulting in enormous economic losses in the aquaculture industry. The causative agent of VNN is betanodavirus [also known as nervous necrosis virus (NNV)], which classified as a member of the Nodaviridae family. NNV mainly infects the central nervous system (CNS) of hosts, which is protected by an elaborate barrier system, limiting the number of drugs reaching the infected sites to tackle the infections. Therefore, it is important to develop alternative drugs and smart delivery technologies for treatment the NNV infection. In the study, the anti-NNV activity of an immunomodulatory antiviral drug, isoprinosine, was checked both in vitro and in vivo. Besides, single-walled carbon nanotubes (SWCNTs) were used as a carrier to improve the anti-NNV activity. Results showed that isoprinosine could effective protect SSN-1 cell from NNV infection, and the percentage of apoptosis was significantly decreased following isoprinosine treatment. Similarly, isoprinosine also showed a strong anti-NNV activity in zebrafish, and the IC 50 (inhibitory concentration at half-maximal activity) values were 86.44 and 52.47 mg/L after exposure to isoprinosine for 3 and 5 days, respectively. The expression levels of IL-1β, IFN-γ and TNF-α were up-regulated following isoprinosine treatment, indicating that isoprinosine can improve the immune responses to against NNV infection. A smart delivery system (named as SWCNTs-I) based on SWCNTs, bovine serum albumin (BSA) and isoprinosine was constructed and characterized by transmission electron microscope, FTIR spectroscopy and UV–vis spectroscopy. SWCNTs-I was internalized inside the SSN-1 cells, and showed a systemic distribution in the zebrafish larvae. SWCNTs-I has a stronger anti-NNV activity than naked isoprinosine, and the mortality of larvae treated with SWCNTs-I was 39.3% during 7 days post infection, while that were 96.7% and 58% for the control and naked isoprinosine treatment groups, respectively. Results so far indicated that isoprinosine is a powerful anti-NNV drug, and drug delivery with SWCNTs has a potential application value to control fish viral diseases in aquaculture. • Isoprinosine shows a strong anti-NNV activity both in vitro and in vivo. • Isoprinosine improves the immune responses to against NNV infection. • A smart delivery system was constructed, named as SWCNTs-I. • SWCNTs-I can be internalized in SSN-1 cells and zebrafish larvae. • Isoprinosine delivery with SWCNTs has a positive impact on VNN therapy. [ABSTRACT FROM AUTHOR]