Betanodavirus會引發多種魚類的神經壞死。其中會對龍膽石斑魚 (dragon grouper; Epinephelus lanceolatus) 造成病毒性神經壞死病 (viral Nervous Necrosis；VNN)，在幼魚死亡率高達80 ~ 100%，其常見的病症為不正常游動和體色變黑。在本篇中以大腸桿菌 (E. coli) 來大量表現龍膽石斑神經壞死病毒外殼蛋白的似病毒顆粒 (virus-like particles, VLPs)，本研究以磷酸鹽緩衝溶液(Phosphate Buffer)、三羥基氨基甲烷緩衝溶液 (Tris buffer) 純化似病毒顆粒，純化後以NTCB 和 CNBr等藥劑處理。藉由穿透式電子顯微鏡 (JEOL JEM-2100) 觀察與X-ray觀察似病毒顆粒之型態改變與研究龍膽石斑神經壞死病毒殼層蛋白之結構。並探討甘油、抗體、金屬離子對於結晶之影響。最後，利用各種3D預測軟件預測DGNNV的結構，分析和比較不同神經壞死病毒之間的親緣關係。

Nodaviridae is divided into Alphanodavirus and Betanodavirus. Alphanodavirus and Betanodavirus infect insects and fish, respectively. The infection of Betanodavirus have been demonstrated the cause of nerve necrosis in several fishes. For instance, the nervous necrosis in dragon grouper (Epinephelus lanceolatus) was infected by dragon grouper nevous necrosis virus (DGNNV) that is a member of genus Betanodavirus. The infection of DGNNV was shown that it resulted in abnormal movement and darkness in body color, with mortality as high as 100%. The capsid proteins of the DGNNV assembled into virus-like particles (VLPs) and that were expressed in Escherichia coli. In this study, VLPs were purified in phosphate buffer and Tris buffer. The VLPs of DGNNV were further treated with NTCB、CNBr. The structure information of the Shell-Domain Structure of Capsid Protein from Dragon Grouper was observed and determined by electron microscope (JEOL JEM-2100) as well as X-ray diffraction. In addition, how the effects of glycerol, antibodies and metal ions on crystallization were also investigated. Finally, a variety of 3D prediction softwares were used to predict of the structure of DGNNV and then the genetic relationship among nevous necrosis viruses was analyized and compared.

摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
壹、前言 1
一、台灣石斑魚現今概況： 1
二、Betanodavirus 的發現 3
三、魚類神經壞死病毒之傳播、感染 4
四、Betanodavirus增殖 7
五、檢測Betanodavrius的方法 8
六、 Nodavirus 相關基因體之介紹 10
七、 似病毒顆粒(Virus-like particles, VLPs) 11
八、 X-ray蛋白質晶體學 17
九、蛋白質結構預測 19
十、蛋白質結構預測 20
十一、 研究動機 21
貳、材料與方法 22
一、質體 22
1. 使用的質體 22
2. pDA8質體的獲得 22
3. DNA 的電泳分析 23
4.限制性內切酶之分析 (Restriction endonuclease analysis) 24
5、純化膠體中之DNA 24
6、DNA接合反應( ligation ) 25
7.勝任細胞 (Competent cell) 的製備 25
8. 質體的置換 26
9.菌體保存與活化、繼代流程 26
二、DGNNV VLPs 樣品的純化與分析 27
1. DGNNV VLPs 的外殼蛋白之表現 27
2. DGNNV VLPs 的純化-菌體打破-蔗糖梯度離心法 28
3. 使用Ni-NTA spin column 純化 6xHis- tagged proteins 29
4. 蛋白質定量 (Lowry method) 30
5. 蛋白質電泳 31
6. DGNNV VLPs蛋白質交聯處理 32
7.DGNNV VLPs蛋白質交聯處理後純化 33
8. 蛋白酶剪切位點和化學剪切位點試驗(Peptidecutter) 33
9. 電子顯微鏡觀察樣品的製作 34
10. 電子顯微鏡的觀察 34
11. 多株抗體與VLP結合試驗 35
三、VLP的結晶 35
1. 懸滴蒸氣擴散法結晶(hanging drop vapor diffusion method) 35
2. 坐滴蒸氣擴散法結晶(sitting drop vapor diffusion method) 36
3.蛋白質結晶的檢測 36
4. 晶體的挑選 37
5. 抗凍結晶條件修改 38
6. 蛋白質結晶前交聯處理 38
7. 蛋白質晶體結晶後的交聯 38
8. 蛋白質晶體結晶前、後的金屬離子添加 39
9.蛋白質晶體結晶前後金屬離子添加條件修改 39
四、蛋白質晶體的繞射實驗 39
1. 蛋白質的晶體上機 39
2.X-ray的開啟 40
3.X-ray軟體的操作 40
4. X-ray的關閉、晶體的卸除 41
五、結構比對、預測 41
1. 龍膽石斑神經壞死病毒 (DGNNV) 與不同的病毒外殼蛋白之親緣關係樹 (phylogenetic tree) 42
2. DGNNV與不同之病毒外殼蛋白的二級結構預測、比對 42
3. DGNNV與不同之病毒外殼蛋白的三級結構預測、比對 43
參、結果 44
一、pDA8質體之確認 44
二. DGNNV VLPs似病毒顆粒之獲得 44
三純化後DGNNV VLPs以固化劑反應 45
四.蛋白質純化後蛋白酶剪切位點和化學剪切位點試驗 46
五.晶體條件的抗凍處理微調 47
六.蛋白質晶體的確認 47
七.不同之金屬離子回溶純化的VLP之影響 48
八.純化之 DGNNV VLPs與多株抗體結合試驗 49
九. DGNNV VLPs結晶同時固化處理 49
十. DGNNV VLPs晶體化學剪切位點反應 50
十一.結晶後添加金屬離子對DGNNV VLPs晶體的影響 51
十二. Ni-NTA spin column 純化DHN220 53
十三.多種病毒之外殼蛋白結構的比對、分析 53
十四.ptacD338蛋白質純化 54
十五.ptacN208殼層蛋白質之純化、分析 55
肆、討論 56
一交聯劑之固化後純化處理 56
二抗體與VLP反應在不同Detector上的差異 56
三結晶條件中加入甘油對DGNNV VLPs結晶的影響 56
四化學剪切位點實驗對VLP的影響 57
五ptacD338、ptacN208蛋白質之純化方式調整 57
伍、參考文獻 59
陸、圖表 68
柒、附錄 106
表目錄
Table 1. X-ray diffraction for DGNNV VLPs under the crystallization conditions with PEG concentrations of weight-by-weight*. 68
Table 2. Crystallization conditions with glycerol concentrations of volume-by-volume 69
Table S 1 The list of restriction enzyme 106
圖目錄
Fig 1 Micrographs of DGNNV VLPs treated with different cross .linking reagents. 70
Fig 2 The Purified DGNNV VLPs were treated with NTCB and CNBr. 72
Fig 3 Micrographs of DGNNV VLPs after treated with NTCB and CNBr. 73
Fig 4 Crystalls from EP2G conditions were diffracted in NSRRC 13B1. 74
Fig.5 Crystalls from AS3G conditions with were diffracted in NSRRC 13B1 . 75
Fig 6 Effect of glycerol on the X-ray diffraction of DGNNV VLP crystals. 76
Fig 7 The effects of different glycerol concentrations on the crystal of DGNNV VLPs were compared in the crystallization conditions of EP2G. 78
Fig 8 DGNNV VLP crystal stability test and staining by coomassie blue test. 79
Fig 9 The effect of Co, Mn, Ca, Sr and ethanol on DGNNV VLPs. 80
Fig 10 Crystals of DGNNV VLPs condition of Co, Mn, Ca, Sr on EP2G crystallization. 81
Fig 11 Crystals of DGNNV VLPs with 10 mM CoCl2 to suspension condition of EP2G crystallization. 82
Fig 12 Crystals of DGNNV VLPs with 10 mM MnCl2 to suspension condition of EP2G crystallization. 83
Fig 13 Different Dilution of antibody with VLPs that were purified in 25 mM Sodium phosphate pH 6.8. 84
Fig 14 Crystals of Anti-DGNNV VLPs from EP2G conditions were diffracted in NSRRC 13B1. 85
Fig 15 Effect of crosslink on the X-ray diffraction of DGNNV VLP crystals. 86
Fig 16 Crystals of DGNNV VLPs from EP2G conditions were treated with NTCB. 88
Fig 17 Crystals of DGNNV VLPs from EP2G conditions were treated with CNBr. 89
Fig 18 Effect of NaCl, NaI, CsCl on the X-ray diffraction of DGNNV VLP crystals by EP2G condition. 90
Fig 19 Effects of CaCl2, CoSO4, CuCl2, and NiCl2 on the X-ray diffraction of DGNNV VLP crystals by EP2G condition. 92
Fig 20 Effect of CuSO4, HgI2, Mg(NO3)2, SrCl2 on the X-ray diffraction of DGNNV VLP crystals by EP2G condition. 94
Fig 21 Effect of BaCl2, CoCl2, MnCl2 on the X-ray diffraction of DGNNV VLP crystals by EP2G condition. 96
Fig 22 Effect of CoCl2, MnCl2 on the X-ray diffraction of DGNNV VLP crystals by AS3G condition. 97
Fig 23 The Purified DHN220 by Ni-NTA column 98
Fig. 24 The phylogenetic trees of DGNNV, ETNNV, SJNNV, TPNNV, Le Blanc, Santeuil and MrNV. 99
Fig.25 Multiple alignment of amino acid sequences of the TPNNV, SJNNV, Santeuil, MrNV, Le Blanc, ETNNV, DGNNV coat proteins sequences. 100
Fig 26.Alignment of TPNNV, SJNNV, Santeuil, MrNV, Le Blanc, ETNNV, DGNNV coat protein sequences by different protein structure prediction system. 102
Fig 27 The ptacD338 protein purified by 25 mM Na-Phosphate pH6.8 103
Fig 28 The ptacN208 protein purified by 25 mM Na-Phosphate pH6.8 104
Fig 29 The ptacT338 and ptacN208 protein purified by Ni-NTA colum 105
Fig S1 The Map of pDA8 plasmid. 107
Fig S2 Sonicator、French Press efficiency of the break. 108
Fig S3 Different buffers for purification and crystallization of DGNNV VLPs. 109
Fig S4 Optimal crystallization conditions with PEG concentrations of weight-by-weight. 110
Fig. S5 Crystals of DGNNV VLPs under LS3 crystallization VLP purified in 10 mM Tris-HCl pH 8. 111
Fig. S6 Crystals of DGNNV VLPs condition of AN2 crystallization VLP purified in 10 mM HEPES pH 8. 112
Fig. S7 Crystals of DGNNV VLPs condition of AS3 crystallizationVLP purified in 10 mM Tris-HCl pH 8.0. 113
Fig.S8 Crystals of DGNNV VLPs condition of EP2 crystallization VLP purified in 25 mM Sodium phosphate pH 6.8. 114
Fig.S9 The phylogenetic tree of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro at different capsid protein. 115
Fig S10 Multiple alignment of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro of the DGNNV coat proteins sequences. 116
Fig S11 Multiple alignment of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro of the ETNNV coat proteins sequences. 117
Fig S12 Multiple alignment of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro of the Le Blanc coat proteins sequences. 118
Fig S13 Multiple alignment of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro of the MrNV coat proteins sequences. 119
Fig S14 Multiple alignment of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro of the Santeuil coat proteins sequences. 120
Fig S15 Multiple alignment of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro of the SJNNV coat proteins sequences. 121
Fig S16 Multiple alignment of APSSP, CFSSP, I-TASSER, Jpred, JUFO, porter, PSIpred, SSpro of the DGNNV coat proteins sequences. 122
Fig S17 Phylogenetic tree of DGNNV, ETNNV, SJNNV, TPNNV, Le Blanc, Santeuil and MrNV by different protein structure prediction sever. 123
S18 pDA8 124
S19 Linker 127
S 20 DN220 127
附錄B、結晶用stock準確配置 130
附錄C. 131

呂明偉。2003。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所，博士論文。
王俊雄。2010。外殼蛋白之 N 端與雙硫鍵影響石斑神經壞死病毒顆粒組裝及熱穩定性之研究。國立中山大學海洋生物科技暨資源系，博士論文。
Arimoto, M., Mushiake, K., Mizuta, Y., Nakai, T., Muroge, K., & Furusawa, I. (1992). Detection of striped jack nervous necrosis virus (SJNNV) by enzyme-linked immunosorbent assay (ELISA). Fish Pathology, 27(4), 191-195.
Arimoto, M., Mori, K., Nakai, T., Muroga, K., & Furusawa, I. (1993). Pathogenicity of the causative agent of viral nervous necrosis disease in striped jack, Pseudocaranx dentex (Bloch & Schneider). Journal of Fish Diseases, 16(5), 461-469.
Bernal, J. D., & Crowfoot, D. (1934). X-ray photographs of crystalline pepsin. Nature, 133(3369), 794-795.
.
Breuil, G., Bonami, J. R., Pepin, J. F., & Pichot, Y. (1991). Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea-bass (Dicentrarchus labrax) larvae and juveniles. Aquaculture, 97(2-3), 109-116.
Breuil, G., Pepin, J. F., Castric, J., Fauvel, C., & Thiery, R. (2000). Detection of serum antibodies against nodavirus in wild and farmed adult sea bass: application to the screening of broodstock in sea bass hatcheries. BULLETIN-EUROPEAN ASSOCIATION OF FISH PATHOLOGISTS, 20(3), 95-100.
Bowie, J. U., Lüthy, R., & Eisenberg, D. (1991). A method to identify protein sequences that fold into a known three-dimensional structure. Science, 164-170.
Chen, Y. M., Kuo, C. E., Chen, G. R., Kao, Y. T., Zou, J., Secombes, C. J., & Chen, T. Y. (2014). Functional analysis of an orange-spotted grouper (Epinephelus coioides) interferon gene and characterisation of its expression in response to nodavirus infection. Developmental & Comparative Immunology, 46(2), 117-128.
Caparrós-Wanderley, W., Savage, N., Hill-Perkins, M., Layton, G., Weber, J., & Davies, D. H. (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. Journal of general virology, 80(4), 1025-1033.
Chi, S. C., Lo, C. F., Kou, G. H., Chang, P. S., Peng, S. E., & Chen, S. N. (1997). Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery‐reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel). Journal of Fish Diseases, 20(3), 185-193.
Chi, S. C., Lo, B. J., & Lin, S. C. (2001). Characterization of grouper nervous necrosis virus (GNNV). Journal of Fish Diseases, 24(1), 3-13.
Comps, M., Pepin, J. F., & Bonami, J. R. (1994). Purification and characterization of two fish encephalitis viruses (FEV) infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture, 123(1), 1-10.
Comps, M., Trindade, M., & Delsert, C. L. (1996). Investigation of fish encephalitis viruses (FEV) expression in marine fishes using DIG-labelled probes. Aquaculture, 143(2), 113-121.
Cui, L. C., Guan, X. T., Liu, Z. M., Tian, C. Y., & Xu, Y. G. (2015). Recombinant lactobacillus expressing G protein of spring viremia of carp virus (SVCV) combined with ORF81 protein of koi herpesvirus (KHV): a promising way to induce protective immunity against SVCV and KHV infection in cyprinid fish via oral vaccination. Vaccine, 33(27), 3092-3099.
Dalla Valle, L., Zanella, L., Patarnello, P., Paolucci, L., Belvedere, P., & Colombo, L. (2000). Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT‐PCR plus nested PCR. Journal of Fish Diseases, 23(5), 321-327.
Dauter, Z. (2002). New approaches to high-throughput phasing. Current opinion in structural biology, 12(5), 674-678.
Dempster, T., Uglem, I., Sanchez-Jerez, P., Fernandez-Jover, D., Bayle-Sempere, J., Nilsen, R., & Bjørn, P. A. (2009). Coastal salmon farms attract large and persistent aggregations of wild fish: an ecosystem effect. Marine Ecology Progress Series, 385, 1-14.
Ellis, A. E. (1988). General principles of fish vaccination. In Fish Vaccination, pp. 1-19. Edited by A. E. Eillis. London: Academic Press.
Ellis, A. E. (2001). Innate host defense mechanisms of fish against viruses and bacteria. Developmental & Comparative Immunology, 25(8), 827-839.
Garman, E. F. (2014). Developments in x-ray crystallographic structure determination of biological macromolecules. Science, 343(6175), 1102-1108.
Glazebrook, J. S., Heasman, M. P., & Beer, S. W. (1990). Picorna‐like viral particles associated with mass mortalities in larval barramundi, Lates calcarifer Bloch. Journal of Fish Diseases, 13(3), 245-249.
Grotmol, S., Totland, G. K., Thorud, K., & Hjeltnes, B. K. (1997). Vacuolating encephalopathy and retinopathy associated with a nodavirus-like agent: a probable cause of mass mortality of cultured larval and juvenile Atlantic halibut Hippoglossus hippoglossus.
Grotmol, S., Totland, G. K., & Kryvi, H. (1997). Detection of a nodavirus-like agent in heart tissue from reared Atlantic salmon Salmo salar suffering from cardiac myopathy syndrome (CMS). Diseases of Aquatic Organisms, 29(2), 79-84.
Guardiola, F. A., Cuesta, A., Abellán, E., Meseguer, J., & Esteban, M. A. (2014). Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish. Fish & shellfish immunology, 40(1), 24-31.
Hasoon, M. F., Daud, H. M., Abdullah, A. A., Arshad, S. S., & Bejo, H. M. (2011). Development and partial characterization of new marine cell line from brain of Asian sea bass Lates calcarifer for virus isolation. In Vitro Cellular & Developmental Biology-Animal, 47(1), 16-25.
Hasoon, M. F., Daud, H. M., Arshad, S. S., & Bejo, M. H. (2011). Betanodavirus experimental infection in freshwater ornamental guppies: diagnostic histopathology and RT–PCR. J Adv Med Res, 1, 45-54.
Huang, B., Tan, C., Chang, S. F., Munday, B., Mathew, J. A., Ngoh, G. H., & Kwang, J. (2001). Detection of nodavirus in barramundi, Lates calcarifer (Bloch), using recombinant coat protein-based ELISA and RT-PCR. Journal of Fish Diseases, 24(3), 135-142.
Iwamoto, T., Mori, K. I., Arimoto, M., & Nakai, T. (1999). High permissivity of the fish cell line SSN-1 for piscine nodaviruses. Diseases of aquatic organisms, 39(1), 37-47.
Iwamoto, T., Mise, K., Takeda, A., Okinaka, Y., Mori, K. I., Arimoto, M., Okuno, T. & Nakai, T. (2005). Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2. Journal of General Virology, 86(10), 2807-2816.
Jithendran, K. P., Shekhar, M. S., Kannappan, S., & Azad, I. S. (2011). Nodavirus infection in freshwater ornamental fishes in India: diagnostic histopathology and nested RT–PCR. Asian Fish Sci, 24, 12-19.
Kiron, V. (2012). Fish immune system and its nutritional modulation for preventive health care. Animal Feed Science and Technology, 173(1), 111-133.
Kokou, F., Sarropoulou, E., Cotou, E., Kentouri, M., Alexis, M., & Rigos, G. (2017). Effects of graded dietary levels of soy protein concentrate supplemented with methionine and phosphate on the immune and antioxidant responses of gilthead sea bream (Sparus aurata L.). Fish & shellfish immunology, 64, 111-121.
Koppang, E. O., Thomas, G. A., Ronningen, K. & Press, C. McL. (1998). Expression of insulin growth factor-1 in gastrointestinal tract of Atlantic salmon (Salmo salar L.). Fish Physiology and Biochemistry 18, 167-175.
Laurent, S., Vautherot, J. F., Madelaine, M. F., Le Gall, G., & Rasschaert, D. (1994). Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection. Journal of virology, 68(10), 6794-6798.
Lengyel, J., Hnath, E., Storms, M., & Wohlfarth, T. (2014). Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR. Journal of structural and functional genomics, 15(3), 117-124.
Lorenzen, N. (1999). Recombinant vaccines: experimental and applied aspects. Fish & Shellfish Immunology, 9(4), 361-365.
Lu, M. W., & Lin, C. S. (2003). Involvement of the terminus of grouper betanodavirus capsid protein in virus-like particle assembly. Archives of virology, 148(2), 345-355.
Lu, M. W., Chao, Y. M., Guo, T. C., Santi, N., Evensen, Ø., Kasani, S. K., Hong JR & Wu, J. L. (2008). The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model. Molecular immunology, 45(4), 1146-1152.
Lua, L. H., Connors, N. K., Sainsbury, F., Chuan, Y. P., Wibowo, N., & Middelberg, A. P. (2014). Bioengineering virus‐like particles as vaccines. Biotechnology and bioengineering, 111(3), 425-440.
Luo, Y. C., Wang, C. H., Wu, Y. M., Liu, W., Lu, M. W., & Lin, C. S. (2014). Crystallization and X-ray diffraction of virus-like particles from a piscine betanodavirus. Acta Crystallographica Section F: Structural Biology Communications, 70(8), 1080-1086.
Magnadóttir, B. (2006). Innate immunity of fish (overview). Fish & shellfish immunology, 20(2), 137-151.
McLoughlin, M. F., & Graham, D. A. (2007). Alphavirus infections in salmonids–a review. Journal of Fish Diseases, 30(9), 511-531.
Mori, K. I., Nakai, T., Muroga, K., Arimoto, M., Mushiake, K., & Furusawa, I. (1992). Properties of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology, 187(1), 368-371.
Munday, B. L., Langdon, J. S., Hyatt, A., & Humphrey, J. D. (1992). Mass mortality associated with a viral-induced vacuolating encephalopathy and retinopathy of larval and juvenile barramundi, Lates calcarifer Bloch. Aquaculture, 103(3-4), 197-211.
Munday, B. L., Kwang, J., & Moody, N. (2002). Betanodavirus infections of teleost fish: a review. Journal of Fish Diseases, 25(3), 127-142.
Dadar, M., Dhama, K., Vakharia, V. N., Hoseinifar, S. H., Karthik, K., Tiwari, R., ... & Joshi, S. K. (2017). Advances in Aquaculture Vaccines Against Fish Pathogens: Global Status and Current Trends. Reviews in Fisheries Science & Aquaculture, 25(3), 184-217.Nishi, S., Yamashita, H., Kawato, Y., & Nakai, T. (2016). Cell culture isolation of piscine nodavirus (betanodavirus) in fish-rearing seawater. Applied and environmental microbiology, 82(8), 2537-2544.
Nishizawa, T., Furuhashi, M., Nagai, T., Nakai, T., & Muroga, K. (1997). Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Applied and Environmental Microbiology, 63(4), 1633-1636.
Panzarin, V., Fusaro, A., Monne, I., Cappellozza, E., Patarnello, P., Bovo, G., ... & Cattoli, G. (2012). Molecular epidemiology and evolutionary dynamics of betanodavirus in southern Europe. Infection, Genetics and Evolution, 12(1), 63-70.
Parra, F., Boga, J. A., Marin, M. S., & Casais, R. (1993). The amino terminal sequence of VP60 from rabbit hemorrhagic disease virus supports its putative subgenomic origin. Virus research, 27(3), 219-228.
Pfister, H., Gissmann, L., & Zur Hausen, H. (1977). Partial characterization of the proteins of human papilloma viruses (HPV) 1–3. Virology, 83(1), 131-137.
Plummer, E. M., & Manchester, M. (2011). Viral nanoparticles and virus‐like particles: platforms for contemporary vaccine design. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 3(2), 174-196.
Press, C. M., & Evensen, Ø. (1999). The morphology of the immune system in teleost fishes. Fish & Shellfish Immunology, 9(4), 309-318.
Roldão, A., Mellado, M. C. M., Castilho, L. R., Carrondo, M. J., & Alves, P. M. (2010). Virus-like particles in vaccine development. Expert review of vaccines, 9(10), 1149-1176.
Schneemann, A., Dasgupta, R., Johnson, J. E., & Rueckert, R. R. (1993). Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus. Journal of virology, 67(5), 2756-2763.
Secombes, C. J. (1996). The nonspecific immune system: Cellular defences In: The fish immune system: Organism, pathogens, environment (eds. Iwama, G. and Nakanish, T.): pp. 63-103.
Skliris, G. P., Krondiris, J. V., Sideris, D. C., Shinn, A. P., Starkey, W. G., & Richards, R. H. (2001). Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia. Virus research, 75(1), 59-67.
Smith, D. M., Simon, J. K., & Baker Jr, J. R. (2013). Applications of nanotechnology for immunology. Nature Reviews Immunology, 13(8), 592-605.
Sommerset, I., & Nerland, A. H. (2004). Complete sequence of RNA1 and subgenomic RNA3 of Atlantic halibut nodavirus (AHNV).
Stuart, D. I., & Abrescia, N. G. (2013). From lows to highs: using low-resolution models to phase X-ray data. Acta Crystallographica Section D: Biological Crystallography, 69(11), 2257-2265.
Su, Y., Xu, H., Ma, H., Feng, J., Wen, W., & Guo, Z. (2015). Dynamic distribution and tissue tropism of nervous necrosis virus in juvenile pompano (Trachinotus ovatus) during early stages of infection. Aquaculture, 440, 25-31.
Svergun, D. I., Richard, S., Koch, M. H. J., Sayers, Z., Kuprin, S., & Zaccai, G. (1998). Protein hydration in solution: experimental observation by x-ray and neutron scattering. Proceedings of the National Academy of Sciences, 95(5), 2267-2272.
Tang, L., Lin, C. S., Krishna, N. K., 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. Journal of virology, 76(12), 6370-6375.
Tort, L., Balasch, J. C., & Mackenzie, S. (2003). Fish immune system. A crossroads between innate and adaptive responses. Inmunología, 22(3), 277-286.
Valero, Y., Arizcun, M., Esteban, M. Á., Bandín, I., Olveira, J. G., Patel, S., Cuesta, S. & Chaves-Pozo, E. (2015). Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions. PloS one, 10(12), e0145131.
Veesler, D., Kearney, B. M., & Johnson, J. E. (2015). Integration of X-ray crystallography and electron cryo-microscopy in the analysis of virus structure and function. Crystallography Reviews, 22(2), 102-127.
Walker, P. J., & Winton, J. R. (2010). Emerging viral diseases of fish and shrimp. Veterinary research, 41(6), 51.
Yoshikoshi, K., & Inoue, K. (1990). Viral nervous necrosis in hatchery‐reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel). Journal of Fish Diseases, 13(1), 69-77.
Zhou, H., & Zhou, Y. (2005). Fold recognition by combining sequence profiles derived from evolution and from depth‐dependent structural alignment of fragments. Proteins: Structure, Function, and Bioinformatics, 58(2), 321-328.