Establishment of reverse genetics system of betanodavirus for the efficient recovery of infectious particles.
Betanodaviruses, a member of the family Nodaviridae, have small positive-stranded bipartite RNA genomes and are the causal agent of viral nervous necrosis in marine-farmed fish. To facilitate the study of betanodavirus, infectious cDNA clones of its two genomic RNAs were generated. The full-length cDNA of the new Redspotted grouper nervous necrosis virus strain (SG2001Nag) RNA1 and RNA2 were co-transcribed by T7 RNA polymerase in baby hamster kidney cells expressing T7 RNA polymerase. The transcription of precise viral RNAs from cDNAs neither lead to viral protein synthesis nor the production of infectious particles. However, the additional two guanine residues following T7 promoter increased the transcription of viral RNAs from cDNAs, and 1.0 x 10(6)TCID(50)/ml of infectious particles was collected from the transfected cells. The ability to reproduce the entire life cycle of betanodavirus from cDNA clones by this reverse genetics system would therefore facilitate a further analysis of the mechanism of betanodavirus RNA replication, structure, and assembly. These findings may thus help in the future development of a betanodavirus vaccine.
Takizawa
Naoki
N
Center for International Collaborative Research, Nagasaki University, 1-12-4 Sakamoto-machi, Nagasaki 852-8523, Japan; Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Adachi
Kei
K
Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Kobayashi
Nobuyuki
N
Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan; Central Research Center, AVSS Corporation, 1-22 Wakaba-machi, Nagasaki 851-8137, Japan.
D000818:Animals; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005393:Fish Diseases / Q000821:virology*; D005399:Fishes; D030381:Nodaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012327:RNA Virus Infections / Q000175:diagnosis*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral