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.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0123106-180812 |
| Date | 23 January 2006 |
| Creators | Liu, Wang-ta |
| Contributors | none, none, Chan-Shing Lin, none, Chi-Hsin Hsu, none, Yi-Ren Hong |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0123106-180812 |
| Rights | withheld, Copyright information available at source archive |
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