Reverse genetics를 이용한 재조합 Infectious Hematopoietic Necrosis Virus (IHNV)의 제작 및 재조합 single-cycle virus를 기반으로 하는 백신의 방어효과

Abstract

Infectious hematopoietic necrosis virus (IHNV) is a member of the genus Novirhadovirus in the family Rhabdoviridae. IHNV is an economically important pathogen causing clinical disease and mortality in a wide variety of salmonid species, including the main salmonid species produced in aquaculture. Viral hemorrhagic septicemia virus (VHSV) is also a member of the genus Novirhadovirus in the family Rhabdoviridae that has been grouped into four major genotypes (I, Ⅱ, Ⅲ, and Ⅳ), based on the sequences of N, G, and NV genes. Susceptible host species are different according to genotypes, and VHSV Ia has been causing mortality in rainbow trout. Reverse genetic technology has been used to generate recombinant viruses from the viral genome. In this study, we produced recombinant IHNV and recombinant single-cycle virus using reverse genetics and confirmed the protective efficacy of recombinant single-cycle virus-based vaccines. Recombinant IHNV-wild having the same sequence of wild-type IHNV, and rIHNV A-eGFP having the enhanced green fluorescence protein (eGFP) gene between the N and P gene were produced. In addition, we rescued single-cycle viruses that possess the transmembrane (TM) deleted glycoprotein gene, which brought defects in the production of infective viral particles and made the viruses replicate in cells only when glycoprotein is trans-supplied. To produce IHNV G△TM single-cycle virus, an EPC cell line expressing IHNV G protein was constructed, and the virus was rescued. Furthermore, we generated rVHSV Ⅳa △G expressing VHSV Ia G△TM and IHNV G△TM using EPC cells expressing VHSV Ⅳa G protein. Immunization experiments were conducted in rainbow trout to confirm the protective efficacy of the single-cycle viruses. Fish were intramuscularly (i.m.) injected with the IHNV G△TM single-cycle virus, and at 3 weeks post-immunization, a challenge test was performed with wild-type IHNV. Results showed that 100% mortality in the control group and 40% mortality in IHNV G△TM-injected group. In another immunization experiment, rainbow trout were i.m. injected with the rVHSV Ⅳa △G(VHSV Ia G△TM) △NV(IHNV G△TM), and at 3 weeks post-immunization, fish were challenged with VHSV Ⅳa, VHSV Ia, and IHNV. In the VHSV Ⅳa challenge, both control and vaccination groups showed low mortality. In the VHSV Ia and IHNV challenges, the control group showed high mortalities, while the vaccinated fish showed no or low mortalities. These results indicate that single-cycle novirhabdoviruses can be used as effective vaccines against IHNV and VHSV infection in rainbow trout.