A developed subunit vaccine based on fiber protein VP56 of grass carp reovirus providing immune protection against grass carp hemorrhagic disease

Abstract

Grass carp reovirus (GCRV) is the main viral pathogen that endangers grass carp seriously. Application of vaccine has been considered to be the most effective way to prevent virus infection. VP56 is a protein encoded by gene segment 7 of grass carp reovirus, and is predicted to share homology with fiber protein of mammalian reovirus (MRV). In our study, the immunogenicity of VP56 was evaluated by neutralization test. GCRV was incubated with mouse anti-VP56 antibody, and then was injected into grass carp. Results showed that disease progress and death occurrence was hindered in the experimental group compared with the control group. For further study, the recombinant VP56 protein (rVP56) expressed by pET-32a (+) vector was purified, and was used as subunit vaccine to immunize grass carp. After each fish (15 ± 1.5 g) was injected with 30 μg purified rVP56 intraperitoneally, the immune protective efficacy of recombinant VP56 protein was assessed by a series of immune parameters. The population of red blood cells in immunized fish increased significantly after 5 d post injection (dpi), and reached a peak with (2.98 ± 0.17) × 109/ml at 7 dpi (p < 0.05). The numbers of white blood cells peaked with (8.42 ± 1.01) × 107/ml at 7 dpi (p < 0.05). Additionally, the percentage of monocytes and neutrophils rose to a peak with (9.05 ± 0.92)% and (25.93 ± 2.60)% respectively at 5 dpi (p < 0.05 or p < 0.01), whereas lymphocytes reached the highest value of (85.81 ± 2.73) % at 14 dpi (p < 0.01). Serum antibody titer in the vaccinated fish increased significantly and reached a peak at 21 dpi (p < 0.01). The mRNA expression levels of type I interferon (IFN1), major histocompatibility complex class I (MHC I), Toll-like receptor 22 (TLR22), and immunoglobulin M (IgM) were significantly up-regulated in head kidney and spleen (p < 0.05 or p < 0.01). The GCRV challenge test showed that the relative survival rate in immunized group was 71%–75%. Collectively, the results indicated that rVP56 protein can induce immune protection in grass carp, and can be consider as a candidate vaccine against GCRV infection.