2종 외래단백질 발현 재조합 viral hemorrhagic septicemia virus(VHSV) 생산 및 dexamethasone이 바이러스 증식에 미치는 영향

Abstract

Viral diseases caused by viral hemorrhagic septicemia virus (VHSV) have seriously damaged on the aquaculture of olive flounder in Korea. The objectives of this study were ⅰ) generation of two-foreign-proteins-express ing recombinant VHSV(rVHSV-RFP-eGFP) using reverse genetics technology and evaluation of the potential of the recombinant VHSV as a prophylactic trivalent live attenuated vaccine and ⅱ) Effect of dexamethasone treatment on typeⅠ IFN response of Epthelioma papulosum cyprini (EPC) cells and replication of the recombinant VHSV.

Previously, it was indicated that insertion of a foreign gene between N and P gene(rVHSV-A-RFP) or P and M gene (rVHSV-B-RFP) of VHSV genome would be advantageous for development of VHSV-based viral-vectored vaccines. In this study, a two-foreign-protein-expressing recombinant VHSV (rVHSV-RFP-eGFP) was rescued by insertion of a RFP and eGFP between N & P and between P & M genes of VHSV genome, respectively. Successful generation of the rVHSV-RFP-eGFP was confirmed by RT-PCR, plaque analysis, and RFP or eGFP fluorescence. The replication cycle and final titers of the rVHSV-RFP-eGFP were distinctly lower than those of the wild-type VHSV, and slightly lower than those of the recombinant wild-type VHSV, rVHSV-△NV-eGFP, rVHSV-A-RFP and rVHSV-B-RFP. Pathogenesis of the rVHSV-RFP-eGFP in olive flounder was different from that of the wild-type VHSV by causing a slowly progressing mortalities.

Thus, the second part of the study has a purpose to increase the virus titer of the recombinant VHSVs. Type I interferons (IFNs) play an important role in infection and replication of VHSV. In this study, we speculated that suppression of type I IFN response of host cells before viral infection would lead to increase of viral titers the NV gene-knockout recombinant VHSV (rVHSV-ΔNV-eGFP) and wild-type VHSV. Type I IFN response measured by Mx gene expression in EPC cells stimulated with poly I:C or infected with wild-type VHSV or rVHSV-ΔNV- EGFP was significantly suppressed by dexamethasone exposure. However, in spite of significant suppression of type I IFN response, the titer of both wild-type VHSV and rVHSV-ΔNV-eGFP was not increased but decreased by dexamethasone exposure. These results suggest that dexametha sone not only down-regulated type I interferon responses but also affected certain factors that are necessary for VHSV replication in EPC cells. Recently, down-regulation of heat shock protein90(HSP90) by glucocorticoids and essential role of HSP90 on the polymerases of RNA viruses have been reported. In this study, dexamethasone treatment down-regulated HSP90 expression in EPC cells that were stimulated with poly I:C. Furthermore, cells treated with a HSP90 inhibitor, geldanamycin, showed clearly decreased titers of VHSVs, indicating that HSP90 is an essential host component for VHSV replication, and HSP90 inhibition might be one of the causes of the present viral titer reduction by dexamethasone. In conclusion, dexamethasone was not appropriate for enhancing replication efficiency of wild-type VHSV and rVHSV-△NV-eGFP in EPC cells despite having strong anti-type I IFN activity. However, we demonstrated for the first time that a HSP90 inhibitor geldanamycin suppressed replication of VHSV in EPC cells, which suggests that targeting host HSP90 can be a way to control VHSV infection in fish.