Cyprinid herpesvirus 2 (CyHV-2) infection results in huge economic losses in gibel carp (Carassius auratus gibelio) industry. In this study, we first constructed recombinant plasmids pcORF25 and pcCCL35.2 as DNA vaccine and molecular adjuvant against CyHV-2, respectively, and confirmed that both recombinant plasmids could be effectively expressed in vitro and in vivo. Then, the vaccination and infection experiments (n = 50) were set as seven groups. The survival rate (70%) in ORF25/CCL35.2 group was highest. The highest specific antibody levels were found in ORF25/CCL35.2 group in major immune tissues by qRT-PCR, and confirmed in serum by ELISA assay, antibody neutralization titer, and serum incubation-infection experiments. Three crucial innate immune indices, namely C3 content, lysozyme, and total superoxide dismutase (TSOD) activities, were highest in ORF25/CCL35.2 group in serum. pcORF25/pcCCL35.2 can effectively up-regulate mRNA expressions of some important immune genes (IL-1β, IL-2, IFN-γ2, and viperin), and significantly suppress CyHV-2 replication in head kidney and spleen tissues. The minimal tissue lesions can be seen in ORF25/CCL35.2 group in gill, spleen, and trunk kidney tissues by histopathological examination. The results indicated that the combination of DNA vaccine pcORF25 and molecular adjuvant pcCCL35.2 is an effective method against CyHV-2 infection, suggesting a feasible strategy for the control of fish viral diseases.
Grass carp reovirus (GCRV) is a severe virus that causes great losses to grass carp culture every year, and GCRV-II is the current popular and fatal strain. VP56, fibrin on the outer surface of GCRV-II, mediates cell attachment. In this study, we firstly divided the VP56 gene into four fragments to screen the optimal antigen by enzyme-linked immunosorbent assay and neutralizing antibody methods. The second fragment VP56-2 demonstrates the optimal efficiency and was employed as an antigen in the following experiments. Bacillus subtilis were used as a carrier, and VP56-2 was expressed on the surface of the spores. Then, we performed the oral immunization for grass carp and the challenge with GCRV-II. The survival rate was remarkably raised, and mRNA expressions of IgM were significantly up-regulated in spleen and head kidney tissues in the B. s-CotC-VP56-2 group. Three crucial immune indexes (complement C3, lysozyme and total superoxide dismutase) in the sera were also significantly enhanced. mRNA expressions of four important genes (TNF-α, IL-1β, IFN1 and MHC-II) were significantly strengthened. Tissue lesions were obviously attenuated by histopathological slide examination in trunk kidney and spleen tissues. Tissue viral burdens were significantly reduced post-viral challenge. These results indicated that the oral recombinant B. subtilis VP56-2 subunit vaccine is effective for controlling GCRV infection and provides a feasible strategy for the control of fish virus diseases.
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