Many fish undergo betanodavirus infection. To study the infection process of dragon grouper nervous necrosis virus (DGNNV), native virus and E. coli-produced virus-like particles (VLPs) were used to analyze the binding and internalization in SSN-1 cells. The binding of DGNNV and VLPs to SSN-1 cells was demonstrated using Western blotting and indirect enzyme-linked immunosorbent assay (ELISA). As estimated by ELISA, the DGNNV particles bound SSN-1 cells in a dose-dependent manner up to 8 ¡Ñ 104 particles per cell. The binding of VLPs was sensitive to neuraminidase and tunicamycin, suggesting that cell-surface sialic acid is involved in binding. The recombinant VLPs block attachment of native virus to the surface of cultured fish nerve cells, blocking infection by the native virus. It is suggesting that the outer shell of DGNNV VLPs is structurally indistinguishable from native virus. The penetration of DGNNV into cells, which was monitored by electron microscopy, appeared mainly to occur via the spherical pit and membrane ruffling pathways. Occasionally, a spherical pit was engulfed by membrane ruffling so as to form a large figure 8-shaped vesicle with an open connection. Our observations suggest that DGNNV utilizes both micro- and macro-pinocytosis pathways to enter SSN-1 cells. Both of nucleotide and amino acid sequences of MGNNV protein A were comparison with all of Nodaviridae members, revealed that MGNNV were most closely related to RGNNV. No correlation of sequences of betanodavirus with geographical habitat was detected. All thirteen nodavirus protein A amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. By Phyre web server identification, the BVDV RdRp as the best template for fold recognition of the RdRp domain of MGNNV and allowed the construction of a congruent 3D model.

Abstract Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Piscinodavirus (Betanodavirus) that causes vacuolating encephalopathy and retinopathy or viral nervous necrosis. MGNNV propagated in SSN-1 cells that were derived from fry tissue of striped snakehead fish, Channa striatus. SSN-1 was highly permissive to MGNNV infection and production, in which the TCID50 per ml increased from the magnitude of 10 4 to 10 8 after 5 passages. The thermal stability analysis unveiled that the optimal activity of MGNNV can be resumed at the temperature from frozen to less than 25 ¢J. Oreochromis mossambica was highly susceptible to MGNNV only when injecting the virus into the vitreous body of fish eye, via which the virus could propagate in the fish brain. Employing the technique of immobilizing virus on itrocellulose, several cellular MGNNV-binding proteins were detected. Two MGNNV-binding proteins of 100 and 56 kDa were found in SSN-1 cell lysate; four proteins of 60, 43, 36, and 30 kDa in rotifer lysate; four proteins of 62, 50, 20 and 25 kDa on the membrane of grouper fertilized eggs. In different organ lysates of Epinephelus malabaricus, two MGNNV-binding proteins of 80 and 43 kDa were found in brain; four MGNNV-binding proteins of 60, 45, 43 and 23 kDa were found in eye; two MGNNV-binding proteins of 80, 18 kDa were found in liver; four MGNNV-binding proteins of 60, 45, 43 and 30 kDa were found in pancreas; two MGNNV-binding proteins of 45 and 24 kDa were found in spleen; one MGNNV-binding proteins of 80 kDa were found in kidney; six MGNNV-binding proteins of 100, 95, 80, 48, 45 and 30 kDa were found in ovary.