Characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus

Betanodaviruses are causative agents of neurological disorders in several s pecies of fish. We cloned and sequenced the RNA2 segment of two grouper vir uses isolated from Epinephelus malabaricus (malabaricus grouper nervous nec rosis Virus, MGNNV) and Epinephelus lanceolatus (dragon grouper nervous nec rosis virus, DGNNV). The sequences of the two RNAs were 99% identical and c omparison with previously sequenced RNA2 segments of fish nodaviruses strip ed jack nervous necrosis virus, Atlantic halibut virus, sea bass encephalit is virus, and greasy grouper nervous necrosis virus (GGNNV) revealed that M GNNV and DGNNV were most closely related to GGNNV No correlation of sequenc e with geographical habitat was detected. The MGNNV coat protein, the gene product of RNA2, was expressed in Sf21 cells with a recombinant baculovirus system and virus-like particles (VLPs) spontaneously formed. Two types of VLPs were observed: a slower sedimenting particle was RNase-sensitive and s tain-permeable, while the faster sedimenting particle survived RNase treatm ent and was not stain-permeable. An image reconstruction of the latter, obt ained with electron cryomicroscopy data, revealed a morphology consistent w ith T = 3 quasi-symmetry but with features significantly different from ins ect nodavirus structures at the same resolution. This assembly system allow s the first biophysical comparisons of fish and insect nodavirus structure, assembly, and stability. (C) 2001 Academic Press.