넙치 interferon-γ 발현 재조합 viral hemorrhagic septicemia virus (VHSV) 제작 및 특성

Abstract

Viral hemorrhagic septicemia virus (VHSV) has been a major cause of mass mortality in farmed olive flounder (Paralichthys olivaceus) in Korea. As there are no effective commercial control measures against VHSV disease, the development of effective vaccines or therapeutics are urgently needed. Interferon-gamma (IFN-γ) is a key factor for the activation of not only adaptive immunity but also innate immunity. In this study, we hypothesized that production of olive flounder IFN-γ by a recombinant VHSV would induce robust antiviral responses. To test our hypothesis, a new recombinant VHSV (rVHSV-A-IFNG) was generated by the insertion of olive flounder IFN-γ gene between nucleocapsid (N) and phosphoprotein (P) genes. The replication ability, the induction of immune-related genes expression in host cells, and in vivo virulence to olive flounder fingerlings of rVHSV-A-IFNG were analyzed and compared with previously constructed recombinant viruses, rVHSV-wild and rVHSV-A-RFP that contains red fluorescent gene ORF between N and P genes in the genome. The replication ability of each recombinant VHSV was analyzed through the plaque assay and the real-time PCR to enumerate the viral copy number (VCN). In hirame natural embryo (HINAE) cells, VCN and infectious particles of rVHSV-A-IFNG were significantly decreased when compared to other recombinant viruses. However, in epithelioma papulosum cyprini (EPC) cells, plaque forming units of rVHSV-A-IFNG were similar to rVHSV-A-RFP and lower than rVHSV-wild. When HINAE cells were infected with each recombinant virus at MOI 0.001, cells infected with rVHSV-A-IFNG showed significantly higher up-regulation of Mx, IsG15, IRF3, and IFN-γ than other recombinant VHSVs from 36 h post-infection. In the in vivo experiments, the group of olive flounder fingerlings infected with rVHSV-A-IFNG showed the highest increase of Mx and IsG15 gene expression, and showed the lowest mortality rates, suggesting that although rVHSV-A-IFNG was not completely safe at the present used doses, it still has the potential to be used as an antiviral agent and attenuated vaccines. Further studies to increase safety and to broaden the applicability are needed.