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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]

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]