Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodaviruses.
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.
Iwamoto
Tokinori
T
Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima, 739-8528, Japan1.
Mise
Kazuyuki
K
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan2.
Mori
Koh-Ichiro
KI
Kamiura Station, Japan Sea-Farming Association, Oita 879-2602, Japan3.
Arimoto
Misao
M
Kamiura Station, Japan Sea-Farming Association, Oita 879-2602, Japan3.
Nakai
Toshihiro
T
Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima, 739-8528, Japan1.
Okuno
Tetsuro
T
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan2.
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005393:Fish Diseases / Q000821:virology*; D005399:Fishes / Q000821:virology; D008969:Molecular Sequence Data; D030381:Nodaviridae / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D012327:RNA Virus Infections / Q000662:veterinary* / Q000821:virology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014774:Virulence
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral