臺灣博碩士論文加值系統

魚類神經壞死病毒(Betanodavirus)可以感染多種魚類的仔雉魚且致死率極高，造成養殖業極大的損失。本研究以龍膽石斑神經壞死病毒(DGNNV)的似病毒顆粒(VLPs)做為材料，分析病毒顆粒組裝(assembly)與崩解(dissociation)的特性。以螯合劑、還原劑或是氧化劑進行試驗，結果證實VLPs的組裝只需要鈣離子的參與。利用點突變來分析外殼蛋白序列可能與鈣離子結合的功能域 (motif) DxxDxD，發現其中第133個位置的天門冬氨酸(Aspartic acid, D)對VLPs的影響最大，一旦突變為不帶電的天門冬醯胺(Asparagine, N)，外殼蛋白就無法組裝成VLPs，另外兩個Aspartic acid位置的突變(D130N及D135N)則可以觀察到不規則型態的蛋白質或是似病毒顆粒兩種現象。進一步測試各種VLPs對熱處理的穩定度，結果發現D135N VLPs對溫度的耐受性稍高於野生型VLPs (85℃)，然而D130N VLPs只要超過60℃就會崩解。上述結果推知DxxDxD功能域中三個Aspartic acid對於VLPs的形成與穩定度都十分重要，對鈣離子配位而言，DxxD可能比DxD提供了更穩定的配體(ligand)。

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°C, 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°C. 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.

摘要…………………………………………………………………………..............i
Abstract………………………………………………………………………………ii
目錄…………………………………………………………………………………iii
表目錄……………………………………………………………………………….v
圖目錄……………………………………………………………………….…....vi

第一章、序論………………………………………………………………………..1
第二章、文獻回顧…………………………………………………………………..5
第一節、Betanodavirus宿主、病毒株命名與病徵…………………………..5
第二節、Nodavirus基因序列分析……………………………………………8
第三節、RNA聚合酶研究……………………………………………...……10
第四節、外殼蛋白特性分析……………………………………………........13
第五節、細胞增殖、感染途徑與內化機制…………………………………17
第六節、似病毒顆粒………………………………………………………....20
第三章、病毒顆粒崩解與再組裝的機制………………………………………....27
第一節、前言…………………………………………………………............27
第二節、材料方法………………………………………………………........31
2.1 似病毒顆粒純化及分析…………………………………............31
2.1.1 大腸桿菌表現病毒外殼蛋白………………………...........31
2.1.2 似病毒顆粒純化……………………………………….......32
2.1.3 連續式分光光度計分析…………………………………...32
2.1.4 螯合劑、核醣核酸酵素、氧化劑以及還原劑對似病毒顆粒
影響……………………………………………...................33
2.1.5 蛋白質電泳…………………………………………….......34
2.1.6 蛋白質定量...........................................................................35
2.1.7 Anti-VLP 抗血清的製備與純化……………………….....35
2.1.8 西方墨點法…………………………………………….......36
2.1.9 圓雙色光譜測定………………………...............................37
2.2 細胞培養與病毒增殖……………………………………………37
2.2.1 無菌操作基本技術………………………………………...37
2.2.2 細胞繼代……………………………………………...........38
2.2.3 細胞保存……………………………………………...........38
2.2.4 冷凍細胞活化……………………………………………...39
2.2.5 病毒增殖……………………………………………...........40
2.2.6 病毒純化……………………………………………...........40

2.3 電子顯微鏡樣品觀察…………………………………………....41
2.3.2 樣品負染色…………………………………………….......41
2.3.3 蛋白質樣品觀察……………………………………….......41
第三節、結果………………………………………………………...............42
3.1 DGNNV VLPs純化…………………………………..................42
3.2 螯合劑對似病毒顆粒的影響………………………....................43
3.3 核醣核酸酶對似病毒顆粒的影響………………………............45
3.4 雙硫鍵對似病毒顆粒的影響………………………....................45

3.5 鈣離子參與似病毒顆粒的重組裝………………………............47
3.6 二價離子對VLP重組裝的影響………………………...............48

第四節、討論………………………………………………………...............51
第四章、神經壞死病毒顆粒鈣離子模體的分析……………….......................56
第一節、前言...................................................................................................56
第二節、材料方法...........................................................................................59

2.1 質體抽取…………………………………....................................59
2.2 氨基酸定點突變............................................................................59
2.3 勝任細胞製作................................................................................60
2.4 細胞轉型........................................................................................61

2.5 似病毒顆粒 RNA 抽取................................................................61
2.6 RNA 電泳......................................................................................61

2.7 突變株似病毒顆粒純化及分析....................................................62
第三節、結果...................................................................................................63

3.1 病毒外殼蛋白鈣離子模體序列的分析....................................63

3.2 DxxDxD模體點突變分析.............................................................64

3.3 突變株的似病毒顆粒對溫度穩定度............................................68
3.4 螯合劑對突變株似病毒顆粒的影響............................................69
第四節、討論………………………………………………………...............71

第五章、結論...........................................................................................................79
參考文獻...................................................................................................................81

圖表.........................................................................................................................101
附錄.........................................................................................................................152

附錄A. Nodavirus序列整理與比對.............................................................153
附錄B. 突變株序列.......................................................................................158

附錄D. 實驗步驟...........................................................................................170
附錄D. 參考文獻圖表...................................................................................178
附錄D. 個人著作...........................................................................................182

表目錄
Table 1. Alignment of calcium-binding motifs in viral capsid proteins.................102
Table 2. The list of primers.....................................................................................105


圖目錄
Fig. 1. Flowchart of sediment analysis of VLP.......………………………….......106
Fig. 2. Purification and anylisis of DGNNV VLPs...…………………………….107
Fig. 3. Electron micrograph of VLPs that assembled in E. coli expressing the
wild-type capsid protein of DGNNV……………………………………...108
Fig. 4. Sedimentation profiles of DGNNV and VLPs treated with chelating agents
..............................................................................................109
Fig. 5. The SDS-PAGE analysis of untreated and EGTA-dissociated VLPs..........111
Fig. 6. Electron micrographs of wild-type and EGTA-dissociated VLPs................112
Fig. 7. Sedimentation profile of DGNNV VLPs treated with RNase......................113
Fig. 8. Sedimentation profiles of DGNNV VLPs treated with reducing agents......114
Fig. 9. Sedimentation profile of DGNNV VLPs treated with diamide..................115
Fig. 10. Sedimentation profile and SDS-PAGE analysis of reassembled VLPs......116
Fig. 11. Electron micrographs of calcium-reassembled VLPs................................117
Fig. 12. Dissociation and reassembly of DGNNV VLPs........................................118
Fig. 13. Immunoblotting analysis of EGTA-dissociated VLPs...............................119
Fig. 14. Circular dichroism spectropolarimeter analyses for DGNNV VLPs.........120
Fig. 15. VLPs reassembled by alkaline metal ions..................................................121
Fig. 16. Electron micrographs of VLPs reassembled by alkaline metal ions..........122
Fig. 17. VLPs reassembled by transition metal ions...............................................123
Fig. 18. Electron micrographs of VLPs reassembled by transition metal ions.......124
Fig. 19. The calcium-binding motif of TBSV.........................................................125
Fig. 20. Alignment of DGNNV and TBSV coat protein sequences.......................126
Fig. 21. The calcium-binding motif of BBV...........................................................127
Fig. 22. Multiple alignment of amino acid sequences of the betanodavirus coat
proteins.......................................................................................................128
Fig. 23. Sedimentation profile and SDS-PAGE analysis of D130N VLPs.............131
Fig. 24. Electron micrographs of D130N VLPs......................................................132
Fig. 25. Sedimentation profile and SDS-PAGE analysis of D133N VLPs.............133
Fig. 26. Electron micrographs of D133N VLPs......................................................134
Fig. 27. Sedimentation profile and SDS-PAGE analysis of D135N VLPs.............135
Fig. 28. Electron micrographs of D135N VLPs......................................................136
Fig. 29. Sedimentation profiles and SDS-PAGE analysis of mutant VLPs............137
Fig. 30. RNA analysis of wild-type and mutant VLPs............................................138
Fig. 31. Sedimentation profiles of thermal stability of wild-type VLPs.................139
Fig. 32. Electron micrographs of thermal stability of wild-type VLPs...................140
Fig. 33. Sedimentation profiles of thermal stability of D130N VLPs.....................143
Fig. 34. Electron micrographs of thermal stability of D130N mutant.....................144
Fig. 35. Sedimentation profiles of thermal stability of D135N VLPs.....................145
Fig. 36. Electron micrographs of thermal stability of D135N mutant.....................146
Fig. 37. Disassembly of D130N and D135N VLPs.................................................149
Fig. 38. Hypothetical scheme: dual mode of calcium-ligand interactions for virion
formation...................................................................................................150

Aranguren R, Tafalla C, Novoa B, Figueras A (2002) Nodavirus replication in a turbot cell line. J Fish Dis 25: 361-366

Arimoto M, Mushiake K, Mizuta Y, Muroga K, Furusawa I (1992) Detection of striped jack nervous necrosis virus (SJNNV) by enzyme-linked immunosorbent assay (ELISA). GyoByo Kenkyu 27: 191-195

Athanassopoulou F, Billinis C, Prapas T (2004) Important disease conditions of newly cultured species in intensive freshwater farms in Greece: first incidence of nodavirus infection in Acipenser sp. Dis Aquat Orqan 60: 247-252

Baker TS, Drak J, Bina M (1988) Reconstruction of the three-dimensional structure of simian virus 40 and visualization of the chromatin core. Proc Natl Acad Sci U S A 85: 422-426

Ball JM, Graham DY, Opekun AR, Gilger MA, Guerrero RA, Estes MK (1999) Recombinant Norwalk virus-like particles given orally to volunteers: phase I study. Gastroenterology 117: 40-48

Ball LA (1995) Requirements for the self-directed replication of flock house virus RNA. J Virol 69: 2722

Bancroft JB (1970) The self-assembly of spherical plant viruses. Advan Virus Res 16: 99-134
Benson EM, Clarkson J, Law M, Marshall P, Kelleher AD, Smith D E, Patou G, Stewart, GJ, Cooper DA, French RA (1999) Therapeutic vaccination with p24-VLP and zidovudine augments HIV-specific cytotoxic T lymphocyte activity in asymptomatic HIV-infected individuals. AIDS Res Hum Retroviruses 15: 105-113

Beynon R, Bond JS (2001) Proteolytic enzymes, a practical approach. 2nd edn. Oxford Univ Press, UK, pp 322

Bhuvaneshwari M, Subramanya HS, Gopinath K, Savithri HS, Nayudu MV, Murthy MR (1995) Structure of sesbania mosaic virus at 3 Å resolution. Structure 3: 1021-1030

Bloch B, Gravningen K, Larsen JL (1991) Encephalomyelitis among turbot associated with a picornavirus-like agent. Dis Aquat Org 10: 65-70

Boonyaratplin S, Supamattaya K, Kasornachandra J, Hoffman RW (1996) Picorna-like virus associated with mortality and a spongiosus encephalopathy in grouper Epinephelus malabaricus. Dis Aquat Org 26: 75-80

Bouamr F, Garnier L, Rayne F, Verna A, Rebeyrotte N, Cerutti M, Mamoun RZ (2000) Differential Budding Efficiencies of Human T-Cell Leukemia Virus Type I (HTLV-I) Gag and Gag-Pro Polyproteins from Insect and Mammalian Cells. Virology 278: 597-609

Brady JN, Kendall JD, Consigli RA (1979) In vitro reassembly of infectious polyoma virions. J Virol 32: 640-647

Brady JN, Winston VD, Consigli RA (1977) Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J Virol 23: 717-724

Brady JN, Winston VD, Consigli RA (1978) Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N’-tetraacetic acid and dithiothreitol. J Virol 27: 193-204

Bräutigam S, Snezhkov E, Bishop DH (1993) Formation of poliovirus-like particles by recombinant baculoviruses expressing the individual VP0, VP3, and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein. Virology 192: 512-524

Breuil G, Bonami JR, Pepin JF, Pichot Y (1991) Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea bass (Dicentrarchus labrax) larvae and juveniles. Aquaculture 97: 109-116

Caparros-Wanderley W, Savage N, Hill-Perkins M, Layton G, Weber J, Davies DH (1999) Intratype sequence variation among clinical isolates of the human papillomavirus type 6 L1 ORF: clustering of mutations and identification of a frequent amino acid sequence variant. J Gen Virol 80: 1025-1033
Caspar D (1963) Assembly and stability of the tobacco mosaic virus particle. Advan Protein Chem 18: 37–121

Chang S, Ngoh GH, Kueh LFS, Qin QW, Chen CL, Lam TJ, Sin YM (2001) Development of a Tropical Marine Fish Cell-Line from Asian Sea-Bass (Lates-Calcarifer) for Virus Isolation. Aquaculture 192: 133-145

Chen PL, Wang M, Ou CW, Lii CK, Chen LS, Chang D (2001) Disulfide bonds stabilize JC virus capsid-like structure by protecting calcium ions from chelation. FEBS Lett 500: 109-113

Chi SC, Hu WW, Lo BJ (1999) Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV). J Fish Dis 22: 173-182

Conner ME, Zarley CD, Hu B, Parsons S, Drabinski D, Greiner S, Smith R, Jiang B, Corsaro B, Barniak V, Madore HP, Crawford S, Estes MK (1996) Virus-like particles as a rotavirus subunit vaccine. J Infect Dis 174 Suppl 1: S88-S92

Crawford SE, Labbe M, Cohen J, Burroughs MH, Zhou YJ, Estes M K (1994) Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. J Virol 68: 5945-5952

Dawson RMC (1986) Data for Biochemical Research. 3rd edn. Oxford Univ Press, New York, pp 404-405

Delos SE, Montross L, Moreland RB, Garcea RL (1993) Expression of the polyomavirus VP2 and VP3 proteins in insect cells: coexpression with the major capsid protein VP1 alters VP2/VP3 subcellular localization. Virology 194: 393–398

Delsert C, Morin N, Comps M (1997a) A fish encephalitis virus that differs from other nodaviruses by its capsid protein processing. Arch Virol 142: 2359-2371

Delsert C, Morin N, Comps M (1997b) Fish nodavirus lytic cycle and semipermissive expression in mammalian and fish cell cultures. J Virol 71: 5673-5677

Deml L, Kratochwil G, Osterrieder N, Knuchel R, Wolf H, Wagner R (1997) Increased incorporation of chimeric human immunodeficiency virus type 1 gp120 proteins into Pr55gag virus-like particles by an Epstein-Barr virus gp220/350-derived transmembrane domain. Virology 235: 10-25

Dong XF, Natarajan P, Tihova M, Johnson JE, Schneemann A (1998) Particle polymorphism caused by deletion of a peptide molecular switch in a quasi-equivalent virus. J Virol 72: 6024-6033

Dudev T, Lim C (2003) Principles governing Mg, Ca, and Zn binding and selectivity in proteins. Chem Rev 103: 773-788

Dudev M, Wang J, Dudev T, Lim C (2003) First-second shell interactions in metal binding sites in proteins: a PDB survey and DFT/CDM calculations. J Am Chem Soc 125: 3168-3180

Fernandez FM, Conner ME, Parwani AV (1998) Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cowsimmunized with recombinant SA11 rotavirus core-like particle (CLP) or virus-like particle (VLP) vaccines. Vaccine 16: 507-516

Forstova J, Krauzewicz N, Wallace S, Street AJ, Dilworth SM, Beard S, Griffin BE (1993) Cooperation of structural proteins during late events in the life cycle of polyomavirus. J Virol 67: 1405-1413

French TJ, Roy P (1990) Synthesis of blue-tongue virus (BTV) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Virol 64: 1530-1536

Frerichs GN, Rodger HD, Peric Z (1996) Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax. J Gen Virol 77: 2067-2071

Gallagher TM, Friesen PD, Rueckert RR (1983) Autonomous replication and expression of RNA1 of black beetle virus. J Virol 46: 481-489

Gallagher TM, Rueckert RR (1988) Assembly-dependent maturation cleavage in provirions of a small icosahedral insect ribovirus. J Virol 62: 3399-3406

Gheysen D, Jacobs E, de Foresta F, Thiriart C, Francotte M, Thines D, De Wilde M (1989) Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell 59: 103-112

Glazebrook JS, Heasman MP, De Beer SW (1990) Picorna-like viral particles associated with mass mortalities in larval barramundi, Lates calcarifer (Bloch). J Fish Dis 13: 245-249

Griffiths JC, Harris SJ, Layton GT, Berrie EL, French TJ, Burns N R, Adams SE, Kingsman AJ (1993) Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion. J Virol 67: 3191-3198

Grotmol S, Totland GK, Thorud K, Hjeltnes BK (1997) Vacuoloating encephalopathy andretinopathy associated with a nodavirus-like agent: a probable cause of mass mortality ofcultured larval and juvenile Atlantic halibut Hippoglossus hippoglossus. Dis Aquat Org 29: 85-97

Guo YX, Chan SW, Kwang J (2004) Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal. J Virol 78: 6498-6508

Haffar OK, Smithgall MD, Moran PA, Travis BM, Zarling JM, Hu S L (1991) HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles. Virology 183: 487-495

Hegde A, Teh HC, Lam TJ, Sin YM (2003) Nodavirus infection in freshwater ornamental fish, guppy, Poicelia reticulata--comparative characterization and pathogenicity studies. Arch Virol 148: 575-586

Hosur MV, Schmidt T, Tucker RC, Johnson JE, Gallagher TM, Selling BH, Rueckert RR (1987) Structure of an insect virus at 3.0 Å resolution. Proteins 2: 167-176

Hu YC, Hsu JT, Huang JH, Ho MS, Ho YC (2003) Formation of enterovirus-like particle aggregates by recombinant baculoviruses co-expressing P1 and 3CD in insect cells. Biotechnol Lett 25: 919-925

Ikenaga T, Tatecho Y, Nakai T, Uematsu K (2002) Betanodavirus as a novel transneuronal tracer for fish. Neurosci Lett 331: 55-59

Iwamoto T, Mise K, Takeda A, Okinaka Y, Mori K, Arimoto M, Okuno T, Nakai T (2005) Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2. J Gen Virol 86: 2807-2816

Iwamoto T, Mori K, Arimoto M, Nakai T (1999) High permissivity of the fish cell line SSN-1 for piscine nodaviruses. Dis Aquat Organ 39: 37-47

Iwamoto T, Nakai T, Mori K, Arimoto M, Furusawa I (2000) Cloning of the fish cell line SSN-1 for piscine nodaviruses. Dis Aquat Organ 43: 81-89

Ishizu KI, Watanabe H, Han SI, Kanesashi SN, Hoque M, Yajima H, Kataoka K, Handa H (2001) Roles of disulfide linkage and calcium ion-mediated interactions in assembly and disassembly of virus-like particles composed of simian virus 40 VP1 capsid protein. J Virol 75: 61-72
Jiang XI, Wang M, Graham DY, Estes MK (1992) Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. J Virol 66: 6527-6532

Johansen R, Sommerset IT, Korsnes K, Hjortaas MJ, Nilsen F, Nerland AH, Dannevig BH (2004) Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.). J Fish Dis 27: 591-601

Kanesashi SN, Ishizu K, Kawano MA, Han SI, Tomita S, Watanabe H, Kataoka K, Handa H (2003) Simian virus 40 VP1 capsid protein forms polymorphic assemblies in vitro. J Gel Virol 84: 1899-1905

Kima Y, Nielsen PR, Hodgins D, Changa KO, Saif LJ (2002) Lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (VLPs) of two serotypes or inactivated bovine rotavirus vaccines. Vaccine 20: 1248-1258

Kosukegawa A, Arisaka F, Takayama M, Yajima H, Kaidow A, Handa H (1996) Purification and characterization of viruslike particles and pentamers produced by the expression of SV40 capsid proteins in insect cells. Biochim Biophys Acta 1290: 37-45

Kratz PA, Böttcher B, Nassal M (1999) Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci U S A 96: 1915-1920

Krondiris JV, Sideris DC (2002) Intramolecular disulfide bonding is essential for betanodavirus coat protein conformation. J Gen Virol 83: 2211-2214

Labbe M, Charpilienne A, Crawford SC, Estes MK, Cohen J (1991) Expression of rotavirus VP2 produces empty core-like particles. J Virol 65: 2946-52

Lai YS, Chiu HC, Murali S, Guo IC, Chen SC, Fang K, Chang CY (2001) In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus awoara (Temminck and Schlegel). J Fish Dis 24: 237-244

Latham T, Galarza JM (2001) Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. J Virol 75: 6154-6165

Laurent S Vautherot JF, Madelaine MF, Le Gall G, Rasschaert D (1994) Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection. J Virol 68: 6794-6798

Li H, Li WX, Ding SW (2002) Induction and suppression of RNA silencing by an animal virus. Science 296: 1319-1321

Li PP, Naknanishi A, Tran MA, Ishizu K, Kawano M, Phillips M, Handa H, Liddington RC, Kasamatsu H (2003) Importance of Vp1 calcium-binding residues in assembly, cell entry, and nuclear entry of simian virus 40. J Virol 77: 7527-7538

Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T (1997) Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol 71: 7207-7213

Lin CS, Lu MW, Tang L, Liu W, Chao CB, Lin CJ, Krishna NK, Johnson JE, Schneemann A (2001) Characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus. Virology 290: 50-58

Lu MW, Lin CS (2003) Involvement of the terminus of grouper betanodavirus capsid protein in virus-like particle assembly. Arch Virol 148: 345-355

Lu MW, Liu W, Lin CS (2003) Infection competition against grouper nervous necrosis virus by virus-like particles produced in Escherichia coli. J Gen Virol 84: 1577-1582

Marshall D, Schneemann A (2001) Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein. Virology 285: 165-175

Mena I, Vivo A, Pérez E, Portela A (1996) Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids. J Virol 70: 5016-5024

Mézeth KB, Nylund S, Henriksen H, Patel S, Nerland AH, Szilvay AM (2007) RNA-dependent RNA polymerase from Atlantic halibut nodavirus contains two signals for localization to the mitochondria. Virus Res 130: 43-52

Montefiori DC, Safrit JT, Lydy SL, Barry AP, Bilska M, Vo HTT, Klein M, Tartaglia J, Robinson HL, Rovinski B (2001) Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques. J Virol 75: 5879-5890

Mori K, Nakai T, Muroga K, Arimoto M, Mushiake K, Frusawa I (1992) Properties of a new virus belonging to Nodaviridae found in larval striped jack Pseudocaranx dentex with virus nervous necrosis. Virology 187: 368-371

Mori K, Nakai Muroga, TK, Arimoto M, Mushiake K, Furusawa I (1992) Properties of new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187: 368-371

Munday BL, Kwang J, Moody N (2002) Betanodavirus infections of teleost fish: a review. J Fish Dis 25: 127-142

Munday BL, Nakai T (1997) Special topic review: nodaviruses as pathogens in larval and juvenile marine finfish. World J Microbiol Biotechnol 13: 375-381

Muriaux D, Mirro J, Harvin D, Rein A (2001) RNA is a structural element in retrovirus particles. Proc Natl Acad Sci U S A 98:5246-5251

Muroga K (1995) Viral and bacterial diseases in larval and juvenile marine fish and shellfish: a review. Fish Pathol 30: 71-85

Nakai T, Mori T, Nishizawa T, Muroga K (1995) Viral nervous necrosis of larval and juvenile marine fish. Proc International Symposium on Biotechnology Applications in Aquaculture. Asian Fish Soc (Spec Publ) 10: 147-152

Ng SC, Bina M (1981) Disulfide bonds protect the encapsidated chromosomes of Simian virus 40. FEBS Lett 130: 47-49

Nguyen HD, Nakai T, Muroga K (1996) Progression of striped jack nervous necrosis virus (SJNNV) infection in naturally and experimentally infected striped jack Pseudocaranx dentex larvae. Dis Aquat Org 24: 99-105

Nishizawa T, Furuhashi M, Nagai T, Nakai T, Muroga K (1997) Genomic classfication of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Appl Environ Microbiol 63: 1633-1636

Nishizawa T, Mori K, Furuhashi M, Nakai T, Furusawa I, Muroga K (1995) Comparsion of the coat protein genes of five fish nodaviruses, the causative agent of viral nervous necrosis in marine fish. J Gen Virol 76: 1563-1569

Noad R, Roy P (2003) Virus-like particles as immunogens. Trends Microbiol 11: 438-444

Oliveira-Ferreira J, Miyahira Y, Layton GT, Savage N, Esteban M, Rodriguez D, Rodriguez JR, Nussenzweig RS, Zavala F, Myahira Y (2000) Immunogenicity of Ty-VLP bearing a CD8(+) T cell epitope of the CS protein of P. yoelii: enhanced memory response by boosting with recombinant vaccinia virus. Vaccine 18: 1863-1869

Osterrieder N, Wagner R, Brandmuller C, Schmidt P, Wolf H, Kaaden OR (1995) Protection against EHV-1 challenge infection in the murine model after vaccination with various formulations of recombinant glycoprotein gp14 (gB). Virology 208: 500-510

Paintsil J, Muller M, Picken M, Gissmann L, Zhou J (1998) Calcium is required in reassembly of bovine papillomavirus in vitro. J Gen Virol 79: 1133-1141

Parra F, Boga JA, Marin MS, Casais R (1993) The amino terminal sequence of VP60 from rabbit hemorrhagic disease virus supports its putative subgenomic origin. Virus Res 27: 219-228

Paintsil J, Müller M, Picken M, Gissmann L, Zhou J (1998) Calcium is required in reassembly of bovine papillomavirus in vitro. J Gen Virol 79: 1133-1141
Rayment I, Baker TS, Caspar DL, Murakami WT (1982) Polyoma virus capsid structure at 22.5 Å resolution. Nature 295: 110-115

Renault T, Haffner P, Baudin Laurencin F, Breuil G, Bonami JR (1991) Mass mortalities in hatchery-reared sea bass (Lates calcarifer) larval associated with the presence in the brain and retina of virus-like particles. Bull. Eur. Associa of Fish Pathol 11: 68-73

Romestand B, Bonami JR (2003) A sandwich enzyme linked immunosorbent assay (S-ELISA) for detection of MrNV in the giant freshwater prawn, Macrobrachium rosenbergii (de Man). J Fish Dis 26: 71-75

Rovinski B, Haynes JR, Cao SX, James O, Sia C, Zolla-Pazner S, Matthews TJ, Klein MH (1992) Expression and characterization of genetically engineered human immunodeficiency virus-like particles containing modified envelope glycoproteins: implications for development of a cross-protective AIDS vaccine. J Virol 66: 4003-4012

Roy P, Bishop DH, LeBlois H, Erasmus BJ (1994) Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection. Vaccine 12: 805-811

Rümenapf T, Strauss EG, Strauss JH (1994) Subgenomic mRNA of Aura alphavirus is packaged into virions. J Virol 68: 56-62

Rümenapf T, Brown DT, Strauss EG, König M, Rameriz-Mitchel R, Strauss JH (1995) Aura alphavirus subgenomic RNA is packaged into virions of two sizes. J Virol 69: 1741-1746
Salunke DM, Caspar DL, Garcea RL (1986) Self-assembly of purified polyomavirus capsid protein VP1. Cell 46: 895-904

Sapp M, Volpers C, Muller M, Streeck RE (1995) Organization of the major and minor capsid proteins in human papillomavirus type 33 virus-like particles. J Gen Virol 76: 2407-2412

Satheshkumar PS, Lokesh GL, Sangita V, Saravanan V, Vijay CS, Murthy MRN, Savithri HS (2004) Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T=3 and T=1 capsids. J Mol Biol 342: 1001-1014

Schiller JT, Hidesheim A (2000) Developing HPV virus-like particle vaccines to prevent cervical cancer: a progress report. J Clin Virol 19: 67-74
Schneemann A, Marshall D (1998) Specific encapsidation of nodavirus RNAs is mediated through the C terminus of capsid precursor protein alpha. J Virol 72: 8738-8746

Schneemann A, Dasgupta R, Johnson JE, Rueckert RR (1993) Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus. J Virol 67: 2756-2763

Skliris GP, Krondiris JV, Sideris DC, Shinn AP, Starkey WG, Richards RH (2001) Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia. Virus Res 75: 59-67

Tanaka S, Aoki H, Nakai T (1998) Pathogenicity of the nodavirus detected from diseased sevenband grouper Epinephelus septemfasciatus. Fish Pathol 33: 31-36

Tang L, Lin CS, Krishna NK, Yeager M, Schneemann A, Johnson J E (2002) Virus-like particles of a fish nodavirus display a capsid subunit domain organization different from that of insect nodaviruses. J Virol 76: 6370-6375

Tobin GJ, Li GH, Fong SE, Nagashima K, Gonda MA (1997) Chimeric HIV-1 virus-like particles containing gp120 epitopes as a result of a ribosomal frameshift elicit Gag- and SU-specific murine cytotoxic T-lymphocyte activities. Virology 236: 307-315

Totland GK, Grotmol S, Morita Y, Nishioka T, Nakai T (1999) Pathogenicity of nodavirus strains from striped jack Pseudocaranx dentex and Atlantic halibut Hippoglossus hippoglossus, studied by waterborne challenge of yolk-sac larvae of both teleost species. Dis Aquat Org 38: 169-175

Trus BL, Roden RB, Greenstone HL, Vrhel M, Schiller JT, Booy FP (1997). Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 Å resolution. Nat Struct Biol 4: 413-420
Wagner R, Deml L, Fliessbach H, Wanner G, Wolf H (1994) Assembly and extracellular release of chimeric HIV-1 Pr55gag retrovirus-like particles. Virology 200: 162-175

Wagner R, Teeuwsen VJ, Deml L, Notka F, Haaksma AG, Jhagjhoorsingh SS, Niphuis H, Wolf H, Heeney JL (1998) Cytotoxic T cells and neutralizing antibodies induced in rhesus monkeys by virus-like particle HIV vaccines in the absence of protection from SHIV infection. Virology 245: 65-74

Walter G, Deppert W (1975) Intermolecular disulfide bonds: an important structural feature of the polyoma virus capsid. Cold Spring Harb Symp Quant Biol 39: 255-257

Wizemann H, Brunn AV (1999) Purification of E. coli-expressed His-tagged hepatitis B core antigen by Ni2+ chelate affinity chromatography. J Virol Methods 77: 189-197

Wu SX, Ahlquist P, Kaesberg P (1992) Active complete in vitro replication of nodavirus RNA requires glycerophospholipid. Proc Natl Acad Sci USA 89: 11136-11140

Yoshikoshi K, Inoue K (1990) Viral nervous necrosis in hatchery-reared larval and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck Schlegel). J Fish Dis 13: 69-77

Zhang LF, Zhou J, Chen S, Cai LL, Bao QY, Zheng FY, Lu JQ, Padmanabha J, Hengst K, Malcolm K, Frazer IH (2000) HPV6b virus like particles are potent immunogens without adjuvant in man. Vaccine 18: 1051-1058

Zlotnick A, Reddy VS, Dasgupta R, Schneemann A, Ray WJ, Rueckert RR, Johnson JE (1994) Capsid assembly in a family of animal viruses primes an autoproteolytic maturation that depends on a single aspartic-acid residue. J Biol Chem 269: 13680-13684

呂明偉。(2002)。龍膽石斑神經壞死病毒外殼蛋白與其似病毒顆粒之研究。國立中山大學海洋資源研究所博士論文。

呂明毅。(1993)。石斑魚種苗生產技術之現況與展望。海水魚繁養殖大全。養魚世界雜誌社。pp 201-212。

呂明毅。(1998)。石斑魚的繁養殖。臺灣省漁業局漁業推廣叢書第049A，臺灣省漁業局，臺北，30pp

李婉琦。(2002)。石斑神經壞死病毒結合蛋白及免疫法檢測。國立中
山大學海洋資源研究所碩士論文。

柯德宏。(1993)。石斑魚類的繁養殖。海水魚繁養殖大全。養魚世界雜誌社。pp 213-220。

陳家全。(1991)。生物電子顯微鏡學。儀器科技研究中心。國家實驗研究院。pp 100-102

曾文陽。(1989)。石斑魚養殖學。前程出版社。pp 119-146。

劉旺達。(2006)。龍膽石斑神經壞死病毒入侵SSN-1細胞初期之機轉。國立中山大學海洋生物科技暨資源研究所博士論文。

羅秉貞。(1997)。南台灣養殖石斑魚病毒性神經性壞死症之研究。國立台灣大學動物學研究所碩士論文。