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

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]

Micropterus Salmoides rhabdovirus (MSRV), as a common aquatic animal virus, can cause lethal and epidemic diseases in the cultivation of largemouth bass. In this study, we reported a kind of immersion single-walled carbon nanotubes-loaded subunit vaccine which composited by glycoprotein (G) of MSRV, and evaluated its protective effect on largemouth bass. The results showed that a stronger immune response including serum antibody levels, enzyme activities (superoxide dismutase, acid phosphatase, alkaline phosphatase and total antioxidant capacity), complement C3 content and immune-related genes (IgM , TGF-β , IL-1β , IL-8 , TNF-α , CD4) expression can be induced obviously with single-walled carbon nanotubes-glycoprotein (SWCNTs-G) groups compared with G groups when largemouth bass were vaccinated. After bath immunization with G or SWCNTs-G for 28 days, fish were challenged with a lethal dose of MSRV. The survival rates for control group (PBS), SWCNTs group (40 mg L−1), pure G protein groups (40 mg L−1) and three SWCNTs-G groups (5 mg L−1, 10 mg L−1 and 40 mg L−1) were 0%, 0%, 39.5%, 36.7%, 43.6%and 70.1%, respectively. Importantly, with the assistance of SWCNTs, the immune protective rate of the SWCNTs-G group (40 mg L−1) increased by approximately 30.6%. This study suggested that SWCNTs-G is a promising immersion subunit vaccine candidate against the death caused by MSRV. • Largemouth bass immersed with SWCNTs-G confers strong immunity against MSRV. • SWCNTs as a promising vehicle can enhance immune effect of subunit vaccine. • SWCNTs based immersion vaccine have prospects for application in aquatic vaccine. [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]

Spring viremia of carp virus (SVCV) causes devastating disease in aquaculture, resulting in significant economic impact. To develop an effective means against SVCV infection, a Lactococcus lactis (L.lactis) based subunit vaccine (pNZ-UGA) was developed based on surface displaying of SVCV glycoprotein using anchoring motif of the cA (C terminus of the peptidoglyvsn-binding) domains of AcmA, a major autolysin from L.lactis. The surface expression of SVCV glycoprotein was verified by indirect immunofluorescence assay. The efficacy of the constructed vaccine was further evaluated in common carp. The results showed that the higher levels of specific IgM could be detected in fish vaccinated with pNZ-UGA, compared with that in PBS and L.lactis groups. Immune-related genes including TNF-α , IL-6b , IL-1β , Cxcr 1 , Cxca , IFNg2b , I-IFN , and IgM expression in pNZ-UGA group were strongly up-regulated, revealing that robust innate immune response was induced. Notably, the lowest cumulative mortality (13.46%) was observed in fish vaccinated with pNZ-UGA vaccine after SVCV challenge, whereas the cumulative mortality were 100.00% and 92.31% in PBS and L.lactis groups, respectively. This study suggests the potential use of the recombinant L.lactis with surface displaying antigen proteins as effective vaccines against SVCV and other fish virus infection. • SVCV glycoprotein was displayed on the surface of Lactobacillus lactis. • Immunization by injection with pNZ-UGA strongly induced immune response in common carp. • Immunization with pNZ-UGA enhance disease resistance against SVCV. [ABSTRACT FROM AUTHOR]

Glycoprotein (G) is the most common gene used in SVCV vaccine constructions. To identify the major immunogenicity determinant region of SVCV G gene, herein we truncated G gene to 4 parts (G-1 , G-2 , G-3 and G-4). Bioinformatics and the enzyme linked immunosorbent assay (ELISA) were used to identify the antigenicity of these 4 truncated G proteins. Immunological assays (serum antibody production, enzyme activity, immune genes expression and challenge test) were carried out to further identify the immunogenicity of the screened G protein in common carp. Moreover, to further verify the immune response of the screened G protein-based subunit vaccine, its protective effects on common carp against SVCV infection using single-walled carbon nanotubes (SWCNTs) as a carrier were evaluated. Results showed that G-3 protein could induce higher antibody titer than other truncated G proteins. Furthermore, carps vaccinated with G-3 and G (positive control) showed significant enhancement of immune response (serum antibody production, enzyme activity and immune related genes expression) when compared with control groups. Meanwhile, as a promising vaccine carrier, SWCNTs could significantly enhance the immune effect of naked subunit vaccine (G-3 and G). Notably, after SVCV challenge, there was no significant difference in immune protection between G-3 and G, nor between SWCNTs-G-3 and SWCNTs-G. These results so far suggest G-3 might be the potential antigen epitope of SVCV. This study lays a foundation for developing vaccine and immunodiagnostic techniques. • SVCV antigenic sites might lies in G-3 (751–1143 bp). • The truncated G protein (G-3) could induce similar immune response to G protein. • Common carp vaccinated with SWCNTs-G-3 confers strong immunity against SVCV. [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]

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]