Lymphocystis disease virus (LCDV)의 major capsid protein (MCP) 유전자를 발현하는 재조합 viral hemorrhagic septicemia virus (VHSV) 제작 및 그 특성

Abstract

Viral diseases caused by viral hemorrhagic septicemia virus (VHSV) and lymphocystis disease virus (LCDV) have seriously damaged on the aquaculture of olive flounder (Paralichthys olivaceus). VHSV infections can lead to mass mortality of olive flounder at low temperature periods and LCDV disease reduces salability of fish by outstanding nodular lesions. However, there has been no effective control measure against these viral diseases yet. Recently, recombinant viruses generated through reverse genetics system have been used to uncover functions of viral genes and to develop attenuated live vaccines. Previously, a recombinant VHSV (rVHSV-ΔNV-EGFP) having the green fluorescent protein (GFP) gene instead of the NV gene had been produced using the reverse-genetics method and the high vaccine potential of the recombinant VHSV as a live attenuated vaccine was confirmed by in vivo immunization experiments. In this study, another recombinant VHSV (rVHSV-ΔNV-MCP) containing the major capsid protein (MCP) gene of LCDV instead of the NV gene was rescued by reverse genetics technology and basic characteristics of the recombinant virus were analyzed for further use as a dual vaccine against VHSV and LCDV. Epithelioma papulosum cyprini (EPC) cells expressing T7 RNA polymerase was transfected with a mixture of pVHSV-ΔNV-MCP, pCMV-N, pCMV-L, pCMV-P. Generation of rVHSV-ΔNV-MCP was confirmed by RT-PCR and rescue of infectious rVHSV-ΔNV-MCP was confirmed by observation of plaque formation. Replication efficiency of rVHSV-ΔNV-MCP was distinctly lower than that of wild-type VHSV. The cytopathic effect (CPE) induced by infection of EPC cells, hirame natural embryo (HINAE) cells or Chinook salmon embryo (CHSE-214) cells with wild-type VHSV or rVHSV-ΔNV-MCP at MOI 0.1, MOI 0.001, or MOI 0.00001 after 7 days post-infection was observed. EPC cells and HINAE cells were more sensitive than CHSE-214 cells, indicating differences in viral susceptibility according to cell types. To determine type Ⅰ interferon response of hosts, expression of Mx gene in EPC cells and in olive flounder in response to infection with wild-type VHSV or rVHSV-ΔNV-MCP was investigated using luciferase reporter system and semi-quantitative RT-PCR, respectively. The highest increase of luciferase activity was detected from supernatant of cell infected with rVHSV-ΔNV-MCP at MOI 1.0. In vivo experiment, the Mx gene expression in olive flounder injected with rVHSV-ΔNV-MCP was higher than fish injected with wild-type VHSV. These results suggest that the present rVHSV-ΔNV-MCP possesses characteristics that are needed to be used as live attenuated vaccines. Further in vivo vaccine studies are needed to apply the rVHSV-ΔNV-MCP to a combined vaccine against VHSV and LCDV infections in olive flounder.