Oral immunization with cell-free self-assembly virus-like particles against orange-spotted grouper nervous necrosis virus in grouper larvae， Epinephelus coioides.

Nervous necrosis virus (NNV) infection causes viral nervous necrosis， inflicting serious economic losses in marine fish cultivation. Vaccination is the most effective choice for controlling and preventing viral infection. Virus-like particles (VLPs) are considered a novel vaccine platform because they are not infectious and they induce neutralizing antibodies efficiently. In the present study， we investigated the effect of the recombinant orange-spotted grouper NNV (OSGNNV) capsid proteins produced in Escherichia coli and cell-free self-assembled into VLPs on protective immune responses in orange-spotted grouper following immersion， intramuscular injection and oral immunization. We found the OSGNNV VLPs elicited neutralizing antibody with high efficacy， and provided the fish with full protection against OSGNNV challenge. In addition， the cell-free self-assembled OSGNNV VLPs did not contain residual host cell components and was safer compared with the intracellular assembled VLPs. Thus， oral vaccination is a more convenient and preferred route for fish vaccination. Our results show that the fish fed four times with a diet supplemented with 50-200?μg/g OSGNNV VLPs at 7-day intervals have sufficient protection. These findings demonstrate that cell-free self-assembled OSGNNV VLPs have potential as oral vaccines in grouper.