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.

Grouper nervous necrosis viruses belong to the Betanodavirus genus in the Nodaviridae family that is a group of small, non-enveloped icosahedron viruses. More than 30 species of fish are infected by the betanodaviruses, which cause massive mortality in hatchery-reared larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the effects of N-terminus and disulfide bonds of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. Deletion of 35 residues at the N-terminus completely ruined the VLP assembly. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. Site-directed mutagenesis was used to investigate the effects of N-terminus of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus. Althought all arginine mutants could produce VLPs, the relative amounts and thermal stabilities of arginine-mutated VLPs were decrease. The VLPs from £GN25-R29A and £GN25 mutants have similar structural properties on particle formation and thermal stability. Therefore, the effects of Arg29 mutations are negligible. The relative amounts and thermal stabilities of VLPs from £GN25-R30A and £GN25-R31A mutants are lower than £GN25-R29A VLP. When 25 amino acids at N-terminus of DGNNV capsid protein were removed, Arg30 and Arg31 are important for particle formation and particel stability. Although particle could form as 12 positively charged amino acids were lost (¡µN25-R293031A), the efficiency of particles assembly were decrease to 1.2 ¡Ó 0.9% as compare to wild-type VLPs (WT-VLPs). Site-directed mutagenesis and chemical reducing reagents were used to investigate the roles of disulfide bonds in particle formation and thermal stability of grouper nervous necrosis virus. The homogeneous particles from C187A, C331A and C187A/C331A mutants are indistinguishable from the native virus and WT-VLPs in their sizes and shapes. C115A and C201A mutants could not produce VLPs. The dissociated capsomers from arginine- or cysteine-mutant VLPs all can be reassembled to icosahedrons with efficiencies as high as 100%. When VLP particles are pre-fabricated, the reducing agent cannot disrupt the VLP icosahedron structure. The thiol reduction only caused effects on the disulfide linkages inside the icosahedrons. £]-mercaptoethanol-treated WT-VLPs could not tolerate the thermal effects at a temperature higher than 70¢XC. Once the disulfide linkages in dissociated capsomers were entirely disrupted by £]-mercaptoethanol treatment, the resulting capsomers could not reassemble back to icosahedron particles.These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles. In the observation of Cryo-EM, the shapes and sizes of the N-terminus truncated particle (£GN25-VLP) are indistinct from the full-length particle (WT-VLP). The maximum diameter of DGNNV is approximately 380 Å. Like that of the insect nodaviruses, the surface morphologies of £GN25-VLP and WT-VLP are consistent with a T = 3 quasi-equivalent lattice. The protrusions (~154 to 192 Å), the inner shell of the capsid (~112 to 154 Å), and the RNA (¡Õ112 Å) were observed in the DGNNV structure. The protrusion domain is consisting of three capsid subunits, and the interactions between these subunits are different. Deletion of 25 residues at the N-terminus did not affect VLPs formation and the structure of £GN25-VLP is similar to WT-VLPs. Resolutions was calculated by Fourier shell correlation, and the resolution of WT-VLPs and £GN25-VLPs is 6.5Å and 11.8Å, respectively.

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.

Grouper in Taiwan is of high value, but nervous necrosis virus infection causes 100% mortality. Our laboratory had developed a good expression system to produce virus-like particles that induced immune functions. In this study, cells producing monoclonal antibody against the virus-like particles were used to induce BALB/c mice production of high titer ascites. The ascite from the H1 cells generated 6000-fold of antibodies higher than cell culture, using enzyme-linked immune-sorbent assay, although relatively less than the previous monoclonal cells of mX, 8000-fold. In the electron microscopy, the ascite antibody bound to various mutants of virus-like particles. Using the polymerase chain reaction to amplify fragments of IgG cDNAs, we will clone and express such cDNAs to efficiently produce the desired monoclonal antibodies.