[논문]Protection of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus (IHNV) by immunization with G gene's cytoplasmic and transmembrane region-deleted single-cycle IHNV

Jae Young, Kim; Jun Soung, Kwak; Hyoung Jun, Kim; Ki Hong, Kim

doi:10.7847/jfp.2022.35.2.157

Protection of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus (IHNV) by immunization with G gene's cytoplasmic and transmembrane region-deleted single-cycle IHNV 원문보기

韓國魚病學會誌= Journal of fish pathology, v.35 no.2, 2022년, pp.157 - 165

Jae Young, Kim (Department of Aquatic Life Medicine, Pukyong National University) , Jun Soung, Kwak (Centre for Integrative Genetics (CIGENE), Faculty of Biosciences, Norwegian University of Life Sciences) , Hyoung Jun, Kim (OIE Reference Laboratory for VHS, National Institute of Fisheries Science) , Ki Hong, Kim (Department of Aquatic Life Medicine, Pukyong National University)

Abstract ▼ AI-Helper

Single-cycle viruses generated by reverse genetic technology are replication-incompetent viruses due to the elimination of gene(s) essential for viral replication, which provides a way to overcome the safety problem in attenuated viruses. Infectious hematopoietic necrosis virus (IHNV) is a major pathogen causing severe damage in cultured salmonid species. In the present study, we generated a single-cycle IHNV lacking the transmembrane and cytoplasmic domain in the G gene (rIHNV-GΔTM) and evaluated the prophylactic potential of rIHNV-GΔTM in rainbow trout (Oncorhynchus mykiss). To produce rIHNV-GΔTM, IHNV G protein-expressing Epithelioma papulosum cyprini (EPC) cells were established. However, as the efficiency of rIHNV-GΔTM production in EPC cell clones was not high, fish were immunized with a low-tittered single-cycle virus (1.5 × 102 PFU/fish). Despite the low dose, the single-cycle IHNV induced significant protection in rainbow trout against IHNV infection, suggesting high immunogenicity of rIHNV-GΔTM. No significant difference in serum ELISA titers against IHNV between the rIHNV-GΔTM immunized group and the control group suggests that the immunized dose of rIHNV-GΔTM might be too low to induce significant humoral adaptive immune responses in rainbow trout. The involvement of adaptive cellular immunity or innate immunity in the present significantly higher protection by the immunization with rIHNV-GΔTM should be further investigated to know the protection mechanism.

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표/그림 (4)

표 Table 1. Primers used in this study
그림 Fig. 1. Vector maps for the production of (A) a recombinant IHNV-wild (rIHNV-wild), (B) a single-cycle recombinant IHNV (rIHNV-GΔTM), and (C) IHNV G gene-expressing cells. F, fragment; gRNA, guide RNA; HDV ribozyme, Hepatitis delta virus ribozyme, copGFP, Green Fluorescent Protein from the copepod Pontellina plumata; T2A, WPRE,- Woodchuck hepatitis virus posttranscriptional regulatory element.
그림 Fig. 2. (A) The RT-qPCR-based expression level of IHNV G gene in EPC cells that were transfected with pIHNV-G-T2A-copGFP and phiC31integrase vector, selected using G-418, then sorted into one cell clones. (B) Plaque assay of rIHNV-GΔTM in IHNV G gene-expressing EPC cells. (C) Verification of G gene's transmembrane and cytoplasmic region deletion in the genome of rIHNV-GΔTM by RT-PCR. lane 1, N gene; lane 2, G gene (the reverse primer matching for the cytoplasmic region of G gene); lane 3, L gene. In wild-type IHNV, all three genes were successfully amplified, but the G gene was not amplified in rIHNV-GΔTM. Marker (M), 1 kb ladder.
그림 Fig. 3. Kaplan-Meier curve of survival of rainbow trout. Fish were immunized with 1.5 × 10² PFU/fish of rIHNV-GΔTM or L-15 medium alone. At 3 weeks post-immunization, fish were intramuscularly challenged with 2.0 × 10⁶ PFU/fish of wild-type IHNV.

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Protection of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus (IHNV) by immunization with G gene's cytoplasmic and transmembrane region-deleted single-cycle IHNV 원문보기

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Protection of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus (IHNV) by immunization with G gene's cytoplasmic and transmembrane region-deleted single-cycle IHNV 원문보기

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