Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Betanodavirus that causes vacuolating encephalopathy and retinopathy and viral nervous necrosis. A method to purify MGNNV was proposed. The cellular components of SSN-1 reprent few viral receptors for Betanodavirus. Several cellular virus binding proteins (VBPs) were detected by employing the technique of immobilizing virus on nitrocellulose. The least VBPs were found in SSN-1 cell lysate that was treated with proteinase K. The approaches used receptor antagonists to identify the cell receptor. The antagonists are able to block the viral binding and thus potentially directly against the receptor. The results implied that the receptor of serotonin 5-HT1A or £\2 adrenergic may act as the receptor of MGNNV.

The mortality of grouper larvae and juveniles infected by nervous necrosis virus (DGNNV) was near 100%. Vacuoles were found in photoreceptor layer, outer nuclear layer and inner nuclear layer of retina and optic tectum of mesencephalon for the dragon grouper that was infected by DGNNV, after stained with haematoxylin and eosin (H&E). Recombinantly expressed in E. coli, virus-like particles (VLPs) were used for antibody preparation. By indirect fluorescence antibody test (IFAT) with the mouse anti-VLP serum, DGNNV was detected in retina inner nuclear layer and mesencephalon optic tectum. At 96 hours post infection of DGNNV with intraocular injection, vacuoles were observed, with H&E staining, in zebrafish retina photoreceptor layer and mesencephalon optic tectum. In IFAT test, DGNNV was also detected in outer nuclear layer and optic tectum of zebrafish. This study showed antibody stimulated by the recombinant VLPs was sutible for DGNNV detection in fish nervous tissues.

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.

The groupers are vital fish in Taiwan, the market of grouper fry over 300 million dollars. While grouper nervous necrosis virus (NNV) has caused mass mortality, especially 100% in larvae and juveniles, which economically impacts on culture of marine fish. The vaccination is one of the best methods to against viral diseases. The dragon grouper (Epinephelus lanceolatus), malabar grouper (E. malabaricus) and brown-marbled grouper (E. fuscoguttatus) were injected with different dosages and injection frequencies of virus-like particles (VLPs) of DGNNV, which is by the first claimed. The anti-sera of vaccinated fish were analyzed with eight kinds of immunology methods, among which antigen-capture ELISA was the best choice for qualitative and quantitative assays. The signal of antibodies in the vaccinated fish was detected in all groupers in one week after primary immunization, and the antibody titers increased markedly in one month. In dragon grouper, fish was injected with 10 £gg of VLPs, the antibody titer reached 1.05. To given booster injection once, antibody titers were raised to 35.7%. In malabar grouper, after injected twice with 50 £gg of VLPs, the antibody titer raised 33.3% than inoculation once in six weeks. After brown-marbled grouper was injected with 450 £gg of VLPs, the high antibody titer reached to 1.57 at five weeks post primary immunization. Specific antibodies still can be detected after seven months. In the in vitro assay with MGNNV of 103.5 TCID50/mL, neutralizing antibody titer of control fish were all lower than 1:50. The neutralizing antibody titer of anti-serum of dragon grouper was detected at 1:200 at one week, and raised to 1:1600 at four weeks and 1:6400 at eleven weeks after primary vaccination. In malabar grouper and brown-marbled grouper, the neutralizing antibody titers were 1:3200 and 1:400, respectively, in one month. The antibody titer can not increased by Freund¡¦s complete adjuvant. The fish produced high antibody titer and high protection by immunization with VLPs.

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.