Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 12 Jan 2019 06:48:52 Journal of General Virology (2001), 82, 2653–2662. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodaviruses Tokinori Iwamoto, 1 † Kazuyuki Mise, 2 Koh-ichiro Mori, 3 Misao Arimoto, 3 Toshihiro Nakai 1 and Tetsuro Okuno 2 1 Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima, 739-8528, Japan 2 Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 3 Kamiura Station, Japan Sea-Farming Association, Oita 879-2602, Japan A system has been established to produce infectious RNA transcripts for Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses, which infect fish. An enzymological analysis suggested that both RNA1 and RNA2 of SJNNV have a 5 cap. Both RNAs were largely resistant to 3 polyadenylation and ligation, suggesting the presence of an interfering 3 structure, while a small quantity of viral RNAs were polyadenylated in vitro. The complete 5 and 3 non- coding sequences of both segments were determined using the rapid amplification of cDNA ends method. Based on the terminal sequences obtained, RT–PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promoter, were constructed. These plasmids were cleaved at a unique restriction site just downstream of the 3 terminus of each SJNNV sequence and were transcribed in vitro into RNA with a cap structure analogue. A mixture of the transcripts was transfected into the fish cell line E-11. Using indirect immunofluorescence staining with anti-SJNNV serum, fluorescence was observed specifically in these transfected cells ; this culture supernatant exhibited pathogenicity to striped jack larvae. Northern blot analysis of E-11 cells infected with the recombinant virus or SJNNV showed small RNA (ca. 04 kb) that was newly synthesized and corresponded to the 3-terminal region of RNA1. Finally, the complete nucleotide sequences of these functional cDNAs (RNA1, 3107 nt ; RNA2, 1421 nt) were determined. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed. Introduction Betanodaviruses, members of the family Nodaviridae, cause highly destructive diseases in hatchery-reared larvae and juveniles of a variety of marine fish species. Since its first description in 1990 (Glazebrook et al., 1990 ; Yoshikoshi & Inoue, 1990), the disease, named viral nervous necrosis or viral Author for correspondence : Kazuyuki Mise. Fax 81 75 753 6131. e-mail kmisekais.kyoto-u.ac.jp † Present address : Kamiura Station, Japan Sea-Farming Association, Oita 879-2602, Japan. The DDBJ accession nos of the sequences reported in this paper are AB056571 and AB056572. encephalopathy and retinopathy, has spread to 19 or more marine fish species of 10 families in the Indo-Pacific region, Mediterranean and Scandinavia (Munday & Nakai, 1997 ; Office International des Epizooties, 2000). Recently, the virus has been proven to have existed in North America (Curtis et al., 2001). Affected fish exhibit a range of neurological abnor- malities, which are characterized by vacuolization and cellular necrosis in the central nervous system and retina. Nodaviruses have a bipartite genome of positive-sense RNA, with RNA1 encoding the RNA-dependent RNA polymerase and RNA2 encoding the coat protein (CP). Both RNAs are capped, but not polyadenylated. During RNA replication, a subgenomic RNA3, which is co-terminal with RNA1 and encodes small proteins, is synthesized. The family Nodaviridae comprise two genera : Alphanodavirus and Beta- 0001-7873  2001 SGM CGFD