Characterization of grouper nervous necrosis virus (GNNV)

Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper la rvae, Epinephelus sp., using a fish cell line GF-1. The present study descr ibes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) a fter 12 h post-infection (p.i.), although no cytopathic effect (CPE) appear ed at that time. Clear CPE developed on the third day, and complete disinte gration of the-monolayer occurred over the subsequent two days. The infecti vity of GNNV can be blocked following treatment at 60 degreesC for 1 h. GNN V was sensitive to pH 3 and pH 10-12 with a 4 log(10) drop in infectivity. Purified GNNV was analysed by SDS-PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions an d analysed. The molecular weights of genomic RNAs were 1.02 x 10(6) and 0.5 0 x 10(6) Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 se quence of two GNNV isolates with four genotypes of fish nodaviruses reveale d that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution o f GNNV in naturally infected grouper larvae was investigated by in situ hyb ridization using a diglabelled probe, which showed that GNNV was not only d etected in the brain and retina, but also in the gill, skeletal muscle, liv er, pyloric gland, intestine and blood cells in the heart.