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 Str iped jack nervous necrosis virus (SJNNV), the type species of the betanodav iruses, which infect fish. An enzymological analysis suggested that both RN A1 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 vitr o. The complete 5' and 3' noncoding sequences of both segments were determi ned using the rapid amplification of cDNA ends method. Based on the termina l sequences obtained, RT-PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promote r, were constructed. These plasmids were cleaved at a unique restriction si te just downstream of the 3' terminus of each SJNNV sequence and were trans cribed in vitro into RNA with a cap structure analogue. A mixture of the tr anscripts was transfected into the fish cell line E-11. Using indirect immu nofluorescence staining with anti-SJNNV serum, fluorescence was observed sp ecifically in these transfected cells; this culture supernatant exhibited p athogenicity to striped jack larvae. Northern blot analysis of E-11 cells i nfected with the recombinant virus or SJNNV showed small RNA (ca. 0.4 kb) t hat was newly synthesized and corresponded to the 3'-terminal region of RNA 1. Finally, the complete nucleotide sequences of these functional cDNAs (RN A1, 3107 nt; RNA2, 1421 nt) were determined. This is the first report of be tanodavirus cDNA clones from which infectious genomic RNAs can be transcrib ed.