Abstract
The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.
Highlights
Grass carp (Ctenopharyngodon idella) is an economically important freshwater fish, occupying a vital position in aquaculture of China [1]
Hemorrhagic disease caused by grass carp reovirus (GCRV) poses a serious threat to the grass carp cultivation industry [2], resulting in a greater than 80% mortality rate and significant economic losses [3]
Epidemiological analyses have shown that the three genotypes exist simultaneously, but GCRV II accounts for the major pandemic of grass carp hemorrhagic disease in China [6]
Summary
Grass carp (Ctenopharyngodon idella) is an economically important freshwater fish, occupying a vital position in aquaculture of China [1]. Xue et al (2013) showed that the silkworm pupae powder containing baculovirus-expressed VP6 protein administered orally with feed induced BacFish-vp specific antibody in grass carp [10]. We generated a novel recombinant baculovirus BmNPV-VP35-VP4 expressing the recombinant VP35-VP4 protein (rVP35-VP4) of GCRV II and used it to infect silkworm pupae to prepare a lyophilized powder for oral immunity to grass carp. We determined the immune response and further evaluated the protective effects of the recombinant VP35-VP4 protein against GCRV infection in grass carp.
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