Generation of a recombinant viral hemorrhagic septicemia virus (VHSV) expressing olive flounder (Paralichthys olivaceus) interferon-gamma and its effects on type I interferon response and virulence

摘要

Rhabdoviruses including viral hemorrhagic septicemia virus (VHSV) are highly susceptible to type I interferon (IFN) responses, and IFN-gamma that is belonging to the type II IFN has been known to enhance type I IFN responses in mammals. In this study, we generated a recombinant VHSV that can express olive flounder IFN-gamma (rVHSV-A-IFN gamma) using reverse genetics technology, and analyzed the effect of rVHSV-A-IFN gamma infection on type I IFN response in Epithelioma papulosum cyprini (EPC) cells. Furthermore, the virulence of rVHSV-A-IFN gamma was evaluated by infection to olive flounder (Paralichthys olivaceus). Using a recombinant VHSV full genome vector in which the olive flounder IFN-gamma ORF was inserted between N and P genes, rVHSV-A-IFN gamma was successfully rescued, and the recombinant virus was grown well in EPC cells. On the other hand, the growth of rVHSV-A-IFN gamma rescued from EPC cells was severely retarded when infected into hirame natural embryo (HINAE) cells that were originated from olive flounder. These results indicate that the EPC cell's IFN-gamma receptor could not bind to olive flounder IFN-gamma, but the species-specific binding of IFN-gamma in HINAE cells induced antiviral responses. The expression of Mx1 gene in EPC cells infected with rVHSV-A-IFN gamma was not greatly different from cells infected with rVHSV-Arfp (a recombinant VHSV harboring red fluorescent protein gene between N and P genes of the genome), however, in HINAE cells, rVHSV-A-IFN gamma infection induced distinctively higher Mx1 gene expression compared to other recombinant viruses. These results suggest that olive flounder IFN-gamma produced from rVHSV-A-IFN gamma effectively enhanced type I IFN response in HINAE cells. In the present study, the lowest mortality of olive flounder fingerlings was recorded in the group of fish challenged with rVHSV-A-IFN gamma, suggesting that the recombinant VHSV was attenuated by production of IFN-gamma by itself. However, although rVHSV-A-IFN gamma induced significantly lower mortality, the mortality still reached to 40%. Therefore, to be safely used in the aquaculture farms as prophylactic vaccines or immunostmulators, further manipulations that can guarantee safety are needed. (C) 2017 Elsevier Ltd. All rights reserved.