The grouper is a high-value fish in Taiwan. However, nervous necrosis virus, which contains two single strand RNAs and doesn¡¦t have an envelope, causes groupers die in early stages of development. It economically impacts on the aquaculture of this marine fish. Vaccination is one of the best methods to prevent viral diseases. Virus-like particles (VLPs) were used for studying the ability and efficiency of producing antibodies against dragon grouper nervous necrosis virus. Different injection dosages and injection frequency of VLPs were performed on Epinephelus lanceolatus. The anti-sera of vaccinated fish were acquired by microvoluminal phlebotomization method and analyzed by antigen-capture dot blotting with ECL detections, which are the best choices for qualitative and quantitative assays. The signal of antibodies in the vaccinated fish was detected every week after primary immunization. The antibody signal reached 8.4x106 U in one month when the dragon groupers were injected with 10 µg of VLPs, but giving additional injection of VLPs didn¡¦t increase production of antibodies after one month. Moreover, dragon groupers that were injected with 50 µg and 100 µg of VLPs generated antibody signals up to 7.2x106 U and 6.7x106 U, respectively. The antibody signal can remain at a higher level in the presence of high dosage injection for at least three weeks. Furthermore, a polyvalent vaccine that included killed bacteria was applied in field to test dragon grouper immune response. The results not only support the data that dragon grouper boots antibody production within a short period after immunization, but also demostrates that the production of antibody by dragon grouper against virus and bacteria can be monitored by the microvoluminal phlebotomization method.

Piscine nodaviruses are members of genus Betanodavirus, which infect more than 30 species of fish and cause massive mortality in larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the dissociation and reassembly of betanodavirus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. The experiments with calcium-chelating or reducing/oxidizing reagents elicited that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. In the mutational analysis of DxxDxD that is putatively involved in the coordination of calcium ions, the results showed that the D133N mutation significantly disrupted the assembly of VLPs while D130N and D135N mutants produced heterogeneous particles with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of D135N mutant were stable at a temperature of 85¢XC, which is slightly higher than that for wild-type, whereas VLPs of D130N mutant could not tolerate the thermal effects at a temperature higher than 60¢XC. It is deduced that three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and, among them, the DxxD provides a more stable coordinate of calcium-ligand than DxD.

The groupers are vital fish in the market of over 350 million dollars, while grouper nervous necrosis virus (NNV) has caused mass mortality at about 100% in larvae and juveniles, which impacts on economic of marine cultured fish. The monoclonal antibody is one of the best methods to identify the epitopes on the 3D structure. For evaluation, the Balb/c mice were injected with DGNNV and virus-like particles (VLPs) in this study. The results showed that ascite of mAb-cells produced 1200 times higher than the cell secretion in the medium whereas our best clone hAb_VLP8 can only produced 100 times less antibody than the cell secretion. In the meantime before the monoclonal producer is established, the hAb_VLP8 could be used for ascite production to gain high antibody production.

In order to investigate the effects of C-terminus modification of Dragon Grouper Nervous Necrosis Virus capsid protein on the virus particle formation, we used E. coli expression system to express DGNNV capsid protain with different truncations at C-teminus fused with six or three histidines (His-Tag). These poly-His tagged clones, including ¡µC334-C6H, ¡µC335-C6H, ¡µC336-C6H, ¡µC337-C6H, C3H and C6H (His6 tagged at the C-teminus of wild-type capsid protein)¡Awere expressed and VLPs formation ability were examined. Wild-type and N-terminal recombination (N6H, His6 tagged at the N-teminus of wild-type capsid protein) were also used for comparison. These His-tagged VLPs can be further purified by Ni-NTA agarose, and their thermal stability of mutant VLPs were analyzed by Circular Dichroism. The Western blotting and ELISA assay were utilized to analyzed N-teminus or C-terminus was located at the surface of virus icosahedron. Once the four amino acids at the C-terminus of capsid protein were truncated (¡µC334-C6H), the mutated cpasid protein cannot assemble into VLPs. The same phenomenon was also observed in C6H. The related productions of wild-type, ¡µC335-C6H, ¡µC336-C6H, ¡µC337-C6H, C3H VLPs were about 100%, 56%, 116%, 141%, and 193%, respectively. Using Circular Dichroism to observe the thermal stability of mutant VLPs, the results revealed that the Tm of mutant VLPs were about 3oC lower than wild-type VLPs (61oC). The results of Western blotting and ELISA assay suggest that the C-termius of DGNNV capisid protein was exposed to the surface of virus structure.