Development of a novel candidate subunit vaccine against Grass carp reovirus Guangdong strain (GCRV-GD108)

Abstract

Grass carp reovirus Guangdong 108 strain (GCRV-GD108) was recently isolated in Guangdong province, China. M6 gene of GCRV-GD108 was speculated encoding virus major outer capsid protein VP4. Blast analysis showed that the amino acid sequence of GCRV-GD108 VP4 was homologous to the structural protein VP4 of known Aquareoviruses (27.3-32.9%). Immunogenicity prediction by DNAStar software revealed there were multiple B cell epitopes on GCRV-GD108 VP4. Prokaryotic expression vector pET32a was used to express VP4 recombinant protein (rVP4) in E. coli BL21(DE3) strain. As expected, the molecular weight of recombinant VP4 was about 87 kDa showed by SDS-PAGE result. Neutralization assay demonstrated that the rabbit polyclonal antibody of rVP4 could prevent virus infection efficiently. After 14 days immunization with the rVP4, grass carps were challenged with GCRV-GD108, the results showed that different doses of rVP4 (1 μg/g, 3 μg/g and 5 μg/g) all provided protection against virus infection (47-82%). The relative percent survival reached 82% in the group immunized with 3 μg/g of rVP4. ELISA revealed rVP4 induced high antibody titer in immunized fish. IgM expression levels in head kidney of grass carp were detected by RT-PCR, and the results showed that IgM expressed at a significantly higher level in immunization groups than in blank control, indicating the rVP4 can induce strong immune response. In conclusion, rVP4 is a candidate vaccine against GCRV-GD108.

Keywords: B cell epitopes; Neutralization assay; Outer capsid protein; Relative percent survival; VP4.