魚類之虹彩病毒感染症歷年來造成臺灣養殖漁業經濟上嚴重損失。已証實受感染之魚種包括石斑魚 (grouper; Epinephelus hybrid)、金目鱸 (giant seaperch; Lates calcarifer)、加州鱸 (largemouth bass; Micropterum salmoides)、龍膽石斑 (king grouper; Epinephelus lanceolatus)、金錢魚 (spotted butter fish; Scatophagus argus)、紅衫 (yellow-wax pomfrat; Trachinotus blochii)、黃錫鯛 (goldlined seabream Sparus sarba)、老鼠斑 (humpback grouper; Chromileptes altivelis)，銀紋笛鯛 (Mangrove red snapper; Lutjanus argentimaculatus)等魚種。研究中開發並使用引子對CY15n-F/CY15n-R及巢式聚合酶連鎖反應引子RY16-F/RY16-R分別增幅虹彩病毒DNA中1,339 bp及305 bp之片段。但對淋巴囊腫病毒、Largemouth bass virus、及正常之石斑魚、金目鱸及加州鱸等DNA均無任何產物產生。使用這套技術，在含有正常魚DNA(100 ng/μl)之樣本中可偵測到50 fg之含有CY15 DNA片段的質體DNA，或是僅需0.05 fg感染魚DNA即可產生陽性結果。比對虹彩病毒核酸中之CY15片段、ATPase、預測之major capsid protein及部份之DNA Polymerase 基因之核苷酸序列，顯示發生於臺灣之魚類虹彩病毒屬於虹彩病毒科(Iridoviruidae)中之Megalocytivirus，並與引起其他魚類大量死亡之Ranavirus 相異。根據CY15片段的序列，虹彩病毒於本區域中分成兩個基因型，分別為CY630型，即TGIV (Taiwan grouper iridovirus )及CY113型，兩型間比對核苷酸序列其相同度為91%。在光學顯微鏡下觀察受虹彩病毒感染之魚隻，可以發現其體內組織中出現巨大細胞。受感染之石斑魚其巨大細胞主要出現於脾、前腎、及後腎等。鰓部之巨大細胞由其他器官轉移所致。金目鱸之巨大細胞除出現在上述臟器外，有時大量出現於消化道及心臟組織中。受虹彩病毒感染之石斑魚體內之巨大細胞，可依其染色特性分為嗜鹼性及嗜酸性兩種。於原位雜交實驗(in situ hybridization)及超顯微結構研究中，發現病毒僅存於嗜鹼性巨大細胞中。嗜鹼性巨大細胞不僅細胞之體積較正常細胞大許多，其細胞核亦變大；然嗜酸性巨大細胞之核並未隨體積增大而變大。病毒在增殖之初期僅在嗜鹼性巨大細胞之細胞核內出現，到了中期之後在整個細胞均有分佈。由膠體碳粒吞噬實驗、酸性磷酸酶反應、及巨大細胞之超顯微結構特性判斷，病毒所感染之細胞應屬於巨噬細胞或單核球細胞。在電子顯微鏡下觀察，病毒之capsid在viromatrix組裝，virogenic stroma 可呈環狀或圓盤狀。細胞內成熟之病毒之顆粒 (mature virus particles)大小約為120-130 nm (side-side) 到160-170 nm (apex-apex)，具有外膜及內膜，高電子密度之核衣約60-110 nm，在膜與核衣間的電子低密度區約 20-50 nm。病毒可在被感染細胞中不同區域出現，其一為寄主細胞仍具有細胞核，病毒顆粒出現於細胞質之lysosome-like vesicle；其二為出現於viromatrix 中，此時細胞核已不存在或僅留下部份之核膜。

Iridovirus infections have led to serious economic loss in the aquaculture industry in Kaohsiung County as well as the whole Southern Taiwan region. Identified susceptible host species in this region includes hybrid grouper (Epinephelus hybrid), giant seaperch (Lates calcarifer), largemouth bass (Micropterum salmoides), king grouper (Epinephelus lanceolatus), spotted butter fish (Scatophagus argus), yellow-wax pomfrat (Trachinotus blochii), goldlined seabream (Sparus sarba), humpback grouper (Chromileptes altivelis), Mangrove red snapper (Lutjanus argentimaculatus). In this study, a diagnostic PCR primer pair CY15n-F/CY15nR, and its nested primer pair RY16-F/RY16-R, were designed and applied to amplify virus-specific products of 1339 bp and 305 bp, respectively. This primer set did not amplify products from lymphocystis disease virus, largemouth bass virus, or healthy control fish DNA. This sensitive technique can detect the presence of 50 fg plasmid with viral DNA insert in the presence of 100 ng/μl host DNA, or 0.05 fg DNA from infected fish. Comparing sequences of CY15 fragment, ATPase gene, predicted major capsid protein and partial DNA polymerase genes among iridoviruses, it is suggested that the viruses found in this area should be classified as the Megalocytivirus of Iridoviridae. These viruses are clearly different from the Ranavirus, another fish-pathogenic iridovirus. Those iridoviruses can be classified into two genotypes: the CY630 type, which is the Taiwan grouper iridovirus; and the CY113 type, which is similar to red seabream iridovirus (RSIV). The identity in CY15 fragment sequences is about 91%. Microscopically, enlarged cells can be found in organs of infected fishes. They appear in the spleen, head kidney, and trunk kidney in infected groupers. The enlarged cells may be relocated from other organs. In giant seaperch, the enlarged cells appeared in the above mentioned organs, and also in the digestive tract and the heart. Two kinds of the enlarged cells in grouper can be distinguished by their H&E staining properties: the basophilic and the eosinophilic enlarged cells. The result from in situ hybridization and electron microscopy suggest that the viruses only appear in the basophilic enlarged cells. Both nucleus and cell volume increase in basophilic enlarged cells, while only the cell volume increases in the eosinophilic enlarged cells. The viruses appeared first in the nuclei of the basophilic enlarged cells, after the mid-phase of the infection they distributed into the whole cells. Judging from the results of phagocytosis, acid phosphatase activity and the ultrastructure of infected cells, it is suggested that this target cell is macrophage or monocyte. The viral capsid is assembled in the viromatrix, and the virogenic stroma can be either ring-shped or disc-shaped. The diameter of mature virus is 120-130 nm from side to side, or 160-170 nm from apex to apex. The electron-lucent space between the capsid and the envelope is about 20-50 nm. The virus particles can be found in (1) lysosome-like vesicles in the cytoplasm, if the host cell still has its nucleus; or (2) the viromatrix, When the host nucleus is dissolved or only has some vestige nuclear membrane left.

中文摘要 1
英文摘要 3
前言 5
文獻探討 6
研究目的 13
材料與方法
一、 養殖場魚隻感染虹彩病毒(iridovirus)之組織病理學研究
(一)、光學顯微鏡學研究 14
(二)、電子顯微鏡學研究 15

二、 石斑魚虹彩病毒診斷技術之研發
(一)、健康魚篩選 17
(二)、病毒之純化 17
(三)、病魚之篩選及病毒之增殖 18
(四)、健康魚及虹彩病毒感染魚之genomic DNA抽取 18
(五)、隨機增殖多形性聚合酶連鎖反應RAPD PCR 19
(六)、偵測虹彩病毒PCR之建立及靈敏度測試 21
(七)、參考前人研究之引子 23
(八)、以組織病理切片或PCR偵測虹彩病毒靈敏性之比
較 23

三、巨大細胞特性之研究
(一)、原位雜交法(in situ hybridization) 25
(二)、人工感染後各時段之光學、原位雜交及超微結構之
變化研究 26
(三)、巨大細胞之吞食作用(phagocytosis) 測試 26
(四)、巨大細胞中酸性磷酸酶特性之研究 27
(五)、電子顯微鏡學之酵素組織化學處理(酸性磷酸
酶染色) 27

四、本區虹彩病毒之種類
(一)、CY15核酸片段比對 29
(二)、ATPase gene基因之核苷酸序列比對 30
(三)、ATPase gene之胺基酸序列比對 30
(四)、推測之major capsid protein (MCP) gene 之核酸序列
之比對 31
(五)、部份DNA polymerase gene及RNA polymerase gene
部份核苷酸序列之比對 32
(六)、1F/2R PCR amplicon 及IRB5 PCR am plicon之核苷
酸序列比對 34

五、流行病學調查 35

結果
一、 養殖場魚隻自然感染虹彩病毒(iridovirus)之組織病理學研究
(一)、石斑魚虹彩病毒感染症 36
(二)、金目鱸虹彩病毒感染症 38

二、石斑魚虹彩病毒診斷技術之研發
(一)、健康魚篩選 40
(二)、病毒之純化 40
(三)、病魚之篩選及病毒之增殖 40
(四)、健康魚及虹彩病毒感染魚之genomic DNA抽取 40
(五)、隨機增殖多形性聚合酶連鎖反應RAPD PCR 41
(六)、偵測虹彩病毒PCR之建立及靈敏性測試 41
(七)、參考前人研究之引子 42
(八)、使用組織病理切片與PCR偵測虹彩病毒靈敏性之
比較 43

三、巨大細胞特性之研究
(一)、人工實驗感染魚之組織病理學研究 44
(二)、超顯微病理觀察 46
(三)、巨大細胞之吞噬能力 (phagocytotic ability ) 測試 46
(四)、巨大細胞酸性磷酸酶特性之研究 47


四、本區虹彩病毒之分類
(一)、CY15 核酸片段之DNA序列比較虹彩病毒間之親
緣關係 48
(二)、ATPase基因之DNA序列比較之親緣關係 48
(三)、ATPase gene之胺基酸序列比較之親緣關係 48
(四)、推測之MCP基因之DNA序列比較之親緣關係 48
(五) 、部份DNA polymerase、RNA polymerase 基因、
1F/2R及IBR5之核苷酸片段序列之比較。 48
五、流行病學調查 50
討論 51
結論 60
參考文獻 61
圖及說明 71
表及說明 120
附錄 126

陳建初。 1986，水質分析。 九大圖書公司. 基隆。
陳家全、李家維、楊瑞森. 1991。 生物電子顯微鏡學，行政院國家科學委員會精密儀器發展中心。 新竹。pp1-131。
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系。 博士論文。
顧倩君。 2002。 台灣虹彩病毒防治對策之相關研究. 國立海洋大學水產養殖系。 碩士論文。
Ahne, W., H. J. Schlotfeldt, and I. Thomsen. 1989. Fish viruses: isolation of an icosahedral cytoplasmic deoxyribovirus from sheatfish (Silurus glanis). Journal of Veterinary Medicine B 36: 333-336.
Anderson, I. G., H. C. Prior, B. J. Rodwell, and O. G. Harris. 1993. Iridovirus-like virions in imported dwarf gourami (Colisa lalia) with systemic amoebiasis. Australian Veterinary Journal 70: 66-67.
Appy, R.G., D.B.Burt, and T. J. Morris. 1976. Viral nature of piscine erythrocytic necrosis (PEN) in the blood of Atlantic cod (Gadus morhua). Journal of Fishery Research Board Canada 33: 1380-1385.
Armstrong, R. D., and H. W. Ferguson. 1989. Systemic viral disease of the chromide cichlid Etroplus maculatus. Diseases of Aquatic Organisms 7: 155-157.
Bellett, A. J. D., and R. B. Inman. 1967. Some properties of deoxyribonucleic acid preparations from Chilo sericesthis and Tipula iridescent viruses. Journal of Molucular Biology 25: 425-432.
Berry, E. S., T. B. Shea, and J. Gablike. 1983. Two iridovirus isolates from Carassius auratus (L). Journal of Fish Diseases 6: 501-510.
Bloch,B., and J.L. Larsen. 1993. An iridovirus-like agent associlated with systemic infectin in cultured turbot Scophthalmus maximus fry in Denmark. Disease of Aquatic Organisms. 15:235-240.
Bloch, B., S. Mellegaard, and E. Nielsen. 1986. Adenovirus –like particles associated with epithelia hyperplasia in dab, Limanda Limanda. Journal of Fish Diseases 9: 281-295.
Braunwald, J., H. Nonnenmacher, and F. Tripier-Darcy. 1985. Ultrastructural and biochemical study of Frog Virus 3 uptake by BHK-21 cells. Journal of General Virology 66: 283-293.
Brookes, S. M., A. D. Hyatt, T. Wise, and R. M. E. Parkhouse. 1998. Intracellular virus DNA distribution and the acquisition of the nucleoprotein core during African swine fever virus particle assembly: Ultrastructural in situ hybridization and DNase-gold labeling. Virology 249: 175-188.
Buocias, D., J. Maruniak, and J. Pendland. 1987. Characterization of an iridovirus from the southern mole cricket, Scapteriscus vicinus. Journal of Invertebrate Pathology 50: 238-245.
Carvalho, Z.G., A. P. Alves De Matos, and C. Rodrigues-Pousada. 1988. Association of African swine fever virus with the cytoskeleton. Virus Research 11:175-192.
Chang, P. S., C. F. Lo, Y. C. Wang, and G. H. Koh. 1996. Identification of white spot syndrome associated baculovirus (WSBV) target organs in shrimp, Penaeid monodom, by in situ hybridization. Diseases of Aquatic Organisms 7: 131-139.
Chao, C. B., and V. F. Pang. 1997. An outbreak of an iridovirus-like infection in cultured grouper (Epinephelus spp.) in Taiwan. Journal of Chinese Society of Veterinarian 23: 411-422.
Chao, C. B., S. C. Yang, H. Y. Tsai, C. Y. Chen, C. S. Lin, and H. T. Huang. 2002. A nested PCR for the detection of Grouper Iridovirus in Taiwan (TGIV) in cultured hybrid grouper, giant seaperch and largemouth bass. Journal of Aquatic Animal Health 14:104-113.
Chi, S. C., C. F. Lo, G. H. Kou, P. S. Chang, S. E. Peng, and S. N. Chen. 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: 185-193.
Chi, S. C. 1997. The investigation of viral disease among cultured groupers in southern Taiwan. Reports on Fish Disease Research XVIII.COA Fisheries Series 61: 59-69.
Chou, H. Y., C. C. Hsu, and T. Y. Peng. 1998. Isolation and characterization of a pathogenic iridovirus from cultured grouper (Epinephelus sp) in Taiwan. Fish Pathology 33: 201-206.
Chua, F. H. C., M. L. Ng., K. L. Ng., I. J. Loo, and J. Y. Wee. 1994. Investigation of outbreak of a novel disease, 'Sleepy Grouper Disease', affecting the brown spotted grouper, Epinephelus tauvina Forskal. Journal of Fish Diseases 17: 417-427.
Crawford, T.B., H. Li, and D. O’toole 1999. Diagnosis of malignant catarrahal fever by PCR using formalin-fixed paraffin- embedded tissues. Journal of Veterinary Diagostic Investigation. 11: 11-116.
Crissan,D. and J.C. Mattson 1993. retrospective DNA analysis using fixed tissue specimens (review). DNA and Cell Biology. 12: 455-464.
Davidson, A. J. 1992. Channel catfish virus: a new type of herpesvirus. Virology 186: 379-396.
Day, M. F., and E. H. Mercer. 1964. Properties of an iridescent virus from the beetle, Sericesthis pruinosa. Australian.Journal of Biological Science 17: 892-904.
Devauchelle, G.. 1977. Ultrastructural characterization of an iridovirus from the marine worm Nereis diversicolor (O. F. Muller) architecture of the virion and virus morphogenesis. Virology 81: 237-246.
Eaton, B.T., A. D. Hyatt, and S. H. Hengstberger. 1991. Epizootic harmatopoietic necrosis virus purification and classification. Journal of Fish Diseases 14: 157-169.
Essbauer S., and W. Ahne. 2002. The epizootic haematopoietic necrosis virus (Iridoviridae) induces apoptosis in vitro. Journal of Veterinary Medicine B 49: 25-30.
Fijan, N., Z. Matasin, Z. Petrinex, I. Valpotic, and O. Zwillenberg. 1991. Isolation of an iridovirus-like agent from the green frog (Rana esculenta L). Veterinary Archives Zagreb 3:151-158.
Frank, T.S., S.M. Svoboda-Newman, and E.D.His. 1996. comparison of methods for extracting DNA from formali-fixed paraffin sections fro nonisotopic PCR. Diagnostic Molecular Pathology. 5(3):220-224.
Goorha, R. 1982. Frog virus 3 replication occurs in two stages. Journal of Virology 43: 519-528.
Goorha, R. 1995. Virus taxonomy: the classification and nomenclature of viruses. In Murphy F. A., C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo, and M. D. Summers, editors. The 6th Report of the International Committee on Taxonomy of Viruses. Springer-Verlag. Vienna. Pp95-99.
Gould, A. R., A. D. Hyatt, S. H. Hengstberger, R. J. Whittington, and B. E. H. Coupar. 1995. A polymerase chain reaction (PCR) to detect epizootic haematopoietic necrosis virus and Bohle iridovirus. Diseases of Aquatic Organisms 22: 211-215.
Grizzle, J.M., I. Altinok, and A.D. Noyes. 2003. PCR method for detection of largemouth bass virus. Diseases of Aquatic Organisms 54: 29-23.
He, J. G., Z. Li, M. Deng, H. H. He, S. P. Weng, H. X. Wang, S. Y. Zhou, Q. X. Long, Z. X. Wang, and S. M. Chen. 2002. Sequence analysis of the complete genome of an iridovirus isolated from the tiger frog. Virology 292: 185-197.
He, J. G., M. Deng, S. P. Weng, Z. Li., S.Y. Zhou, X. Q. Long, X. Z. Wang, and S. M. Chen. 2001. Complete genome analysis of the mandarin fish infectious spleen and kidney necrosis iridovirus. Virology 291: 126-139.
He, J. G., K. Zeng, S. P. Weng, and S. M. Chan. 2002. Experimental transmission, pathogenicity and physical –chemical properties of infectious spleen and kidney necrosis virus (ISKNV). Aquaculture 204:11-24.
He, J. G., S. P. Wang, K. Zeng, Z. J. Huang, and S. M. Chan. 2000. Systemic disease caused by an iridovirus-like agent in cultured mandarinfish, Siniperca chuatsi (Basilewsky), in China. Journal of Fish Diseases 23: 219-222.
Hedrick, R. P., and T. S. McDowell. 1995. Properties of iridoviruses from ornamental fish. Veterinary Research 26: 423-427.
Hedrick, R. P., T. S. McDowell, W. Ahne, C. Torhy, and P. DeKinkelin. 1992. Properties of three iridovirus-like agents associated with systemic infections of fish. Diseases of Aquatic Organisms 13: 203-209.
Hengstberger, S. G., A. D. Hyatt, R. Speare, and B. H. Coupar. 1993. Comparison of epizootic haematopoietic necrosis and Bohle iridoviruses, recently isolated Australian iridoviruses. Diseases of Aquatic Organisms 15: 93-107.
Hetrick, F. M., and R. P. Hedrick. 1993. New virus described in finfish from 1998-1992. Annual Reviews of Fish Diseases 3: 187-207.
Hyatt, A. D., A. R. Gould, Z. Zupanovic, A. A. Cunningham, S. Hengstberger, R. J. Whittington, J. Kattenbelt, and B. E. H. Coupar. 2000. Comparative studies of piscine and amphibian iridoviruses. Archives of Virology 145: 301-331.
Inouye, K., K. Yamano, Y. Maeno, K. Nakajima, M. Matsuoka, Y. Wada, and M. Sorimachi. 1992. Iridovirus infection of cultured red sea bream, Pagrus major. Fish Pathology 27: 19-27.
Jensen, N. J., and B. Bloch. 1980. Adenovirus-like particle associated with epidermal hyperplasia in cod, Gadus morthus. Nordic Veterinary Medicine 32: 173-175.
Jung S.J. and M.J. Oh. 2000. Iridovirus-like infection associated with high mortalities of striped beakperch , Oplegnathus fasciatus (Temminck et Schlegel), in southern coastal areas of the Korean peninsula. Journal of Fish Diseasees. 23:223-226.
Kallio, P., Syrjanen, A.Tervahauta. and K. Syrjanen. 1991. A simple method for isolation of DNA from formalin-fix paraffin-embedded samples for PCR. Jorunal of Virological Methods. 35:39-47.
Kattenbelt, J. A. A. D. Hyatt, and A. R. Gould. 2000. Recovery of ranavirus dsDNA from formalin-fixed archival material. Diseases of Aquatic Organisms 39: 151-154.
Kawakami, H., and K. Nakajima. 2002. Cultured fish species affected by red sea bream iridoviral disease from 1996 to 2000. Fish pathology 37: 45-47.
Kelley, D.C. and M.A. Atkinson 1975. Frog Virus 3 replication :electron microscope observations on the terminal stages of infection in chronically infected cell cultures. Journal of General Virology 28:391-407
Kelly, D. C., M. D. Ayres, T. Lescott, J. S. Robertson, and G. M. Happ. 1979. A small iridescent virus (type 29) isolated from Tenebrio molitor: A comparison of its proteins and antigens with six other iridescent viruses. J. General Virology 42: 95-105.
Kim, Y.J., S.J. Jung, T.J. Choi, H.R.Kim, K.V. Rajendran, and M.J. Oh. 2002. PCR amplification and sequence analysis of irido-like virus infecting fish in Korea. Journal of Fish Disease.25:121-124.
Kurita, J., K. Nakajima, I. Hirono, and T. Aoki. 2000. Decision of complete genome DNA sequence of red sea bream iridovirus genome. Abstract of 2000 spring meeting of Japanese Society of Fish Pathology, p. 7 (in Japanese).
Kurita, J., K. Nakajima, I. Hirono, and T. Aoki. 1998. Polymerase chain reaction (PCR) amplification of DNA of red sea bream iridovirus (RSIV). Fish Pathology 33: 17-23.
Lai,Y. S., S. Murali, H. Y. Ju, M. F. Wu, I. C. Guo, S. C. Chen, K. Gang, and C. Y. Chang. 2000. Two iridovirus-susceptible cell lines established from kidney and liver, Epinephelus awoara (Temminck& Schlegel), and partial characterization of grouper iridovirus. Journal of Fish Diseases 23: 379-388.
Langdon, J. S., J. D. Humphrey, L. M. Williams, A. D. Hyatt, and H. A. Westbury. 1986. First virus isolation from Australian fish: an iridovirus-like pathogen from redfin perch, Perca fluviatilis L. Journal of Fish Diseases 9: 263-268.
Langdon, J. S., and J. D. Humphrey. 1987. Epizootic hematopoietic necrosis: a new viral disease in redfin perch Perca fluviatilis L. in Australia. Journal of Fish Diseases 10: 289-297.
Langdon, S., J. D. Humphrey, and L. M. Williams. 1988. Outbreaks of an EHNV-like iridovirus in cultured rainbow trout, Salmo gairdneri Richardson, in Australia. Journal of Fish Diseases 11: 93-96.
Li, P. –C., H. T. Huang, and J. T. Liang. 2001. Neurophysiological effects of recurrent laryngeal and thoracic vagus nerves on mediatingthe neurogenic inflammation of the trachea, bronchi, and esophagus of rat. Autonomic Neuroscience Basic Clinics 88: 142-150.
Low, J. 1874. Fauna and flora of Norfolk part 4 Fishes. Transection of Norfolk Norwich Nature Society 21: 56.
Liu, J.H. 1975. Cytodiagnosis. In S. G. Yu, editor, Laboratory Medicine-Clinical Pathology. Wen Jong Press, Ltd. Taiwan. Pp. 117-148.
Luna, L.G. 1976. Mamual of histologic staining methods of the Armed forces institute of Pathology New York.pp1-77.
Mao, J., J. Wang, G. D. Chinchar, and V. G. Chinchar. 1999. Molecular characterization of a ranavirus isolated from largemouth bass Micropterus salmoides. Diseases of Aquatic Organisms 37: 107-114.
Mao, J., R. P. Hedrick, and V. G. Chinchar. 1997. Molecular characterization, sequence analysis, and taxonomic position of newly isolated fish iridoviruses. Virology 229: 212-220.
Mathieson, W. B., and P. E.Lee.1981. Cytology and autoradiography of Tipula iridescent virus infection of insect suspension cell cultures. Journal of Ultrastructural Research 74: 59-68.
McGrogan, D.G. V.E. Ostland, P.J. Byrne, and H.W.Ferguson. 1998. Systemic disease involving an iridovirus-like agent in cultured tilapia, Oreochomis Niloticus L. –a case repot. Journal of Fish Disease. 21: 149-152.
McMillan, N. A. J., S. Davison, and J. Kalmakoff. 1990. Comparison of the genomes of two sympatric iridescent viruses (type 9 and 16). Archives of Virology 114: 277-284.
Miyata, M., K. Matsuno, S. J. Jung, Y. Danayadol, and T. Miyazaki. 1997. Genetic similarity of iridoviruses from Japan and Thailand. Journal of Fish Diseases 20: 127-134.
Mohan, D., K.B.C. Appa Rao, A. Dixit, S. Ali. 1995. Assessment of amplicons in the DNA from boiled tissue by PCR and AP-PCR amplification. Gene Analysis Techniques. 12: 57-62.
Moody, N. J. G., and L. Owens. 1994. Experimental demonstration of the pathogenicity of a frog virus, Bohle iridovirus, for a fish species, baramundi Lates calcarifer. Diseases of Aquatic Organisms 18: 95-102.
Mori, K., T. K. Nakai, M. Muroga, K. Arimoto, K. Mushiake, and I. Turusawa. 1992 Properties of new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx) with nervous necrosis. Virology 187, 368-371.
Murali, S., M. F. Wu, I. C. Guo, S. C. Chen, H. W. Yang, and C. Y. Chang. 2002 molecular characterization and pathogenicity of a grouper iridovirus (GIV) isolated from yellow grouper, Epinephelus awoara (Temminck & Schlegel). Jorunal of Fish Diseases 25: 91-100.
Nishizawa, T., K. I. Mori, T. Nakai, I. Furusawa, and K. Muroga. 1994. Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV). Diseases of Aquatic Organisms 18: 103-107.
Oshima, S.I., J.I. Hata, C. Segawa, N. Hirasawa. And S. Yamashita. 1996. A method for direct DNA amplification of uncharacterized DNA virus and for development of a viral polymerase chain reactio assay :Application to the red sea bream iridovurus. Analytical Biochemistry. 242: 15-19.
Oshima, S.I., J. I. Hata, N. Hirasawa, T. Ohtaka, I. Hirono, T. Aoki, and S. Yamashita. 1998. Rapid diagnosis of red sea bream iridovirus infection using the polymerase chain reaction. Diseases of Aquatic Organisms 32: 87-90.
Pozet, F., M. Morand, A. Moussa, C. Torhy, and P. de Kinkelin. 1992. Isolation and preliminary characterization of a pathogenic icosahedral deoxyribovirus from the catfish Ictalurus melas. Diseases of Aquatic Organisms 14: 35-42.
Qin, Q. W, T. J. Lam, Y. M. Sin, H. Shen, S. F. Chang, G. H. Ngoh, and C. L. Chen. 2001. Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina. Journal of Virological Methods 98: 17-24.
Qin, Q. W., S. F. Chang, G. H. Ngoh-Lim, S. Gibson-Kuen, C. Shi, T. J. Lam. 2003. Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina. Diseases of Aquatic Organisms 53: 1-9.
Reno, P. W., P. H. Philippon-Fried, and B. I. Nicholson. 1978. Ultrastructural studies of piscine erythrocytic necrosis (PEN) in Atlantic herring (Clupea harengus). Journal of Fishery Research Board Canada 35: 148-154.
Robert, R. J. 2001. Fish Pathology, 3rd Ed. W. B. Saunders. London, UK. p196.
Roberts, R. J. 1989. The pathophysiology and systemic pathology of teleosts. In: Roberts R. J., editor. Fish Pathology. Bailliere Tindall. London, UK. Pp56-134.
Rodger, H. D., M. Kobs, A. Macartney, and G. N. Frerichs. 1997. Systemic iridovirus infection in freshwater angelfish, Pterophyllum scalare (Lichtenstein). Journal of Fish Diseases 20: 69-72.
Roger, B. B., L. C. Alpert, E. A. Hine, and G. J. Buffone. 1990. Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction. American Journal of Pathology 136: 57-62.
Saiki, P. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K.B. Mullis, and H.A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487-491.
Sano, M., M. Minagwa, and K. Nakajima. 2002. Multiplication of Red sea bream iridovirus (RSIV) in the experimentally infected grouper Epinephelus Malabaricus Fish Pathology 37: 163-168.
Schierwater, B., and A. Ender. 1993. Different thermostable DNA polymerase may amplify different RAPD products. Nucleic Acid Research 21: 4647-4648.
Sierra, M. A., A. Bernabe, E. Mozos, A. Mendez, and A. Jover. 1987. Ultrastructure of the liver in pigs with experimental African swine fever. Veterinary Pathology 24: 460-462.
Siwicki, A.K., F. Pozet, M. Morand ,C. Volatier, and E. Terech- Majewska. 1999. Effects of iridovirus-like agent on the cell-mediated immunity in sheatfish(Silurus glanis)- an in vitro study. Virus Research 63:115-119.
Sudthongkong, C., M. Miyata, and T. Miyazaki. 2002a. Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and drawf gourami. Diseases of Aquatic Organisms 48: 163-173.
Sudthongkong, C., M. Miyata, and T. Miyazaki. 2002b. Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan, South China Sea and Southeast Asian countries. Archives of Virology 147: 2089-2109.
Tajbakhsh, S., P. E. Lee, D. C. Watson, and V. L. Seligy. 1990. Molecular cloning characterization and expression of the Tipula iridecent virus capsid gene. Journal of Virology 64: 125-136.
Tidoma, C. A., and G. Darai. 1997. The complete sequence of lymphocystis disease virus. Virology 230: 207-216.
Watson, L.R.,J.M. Groff, R.P. Hedrick, 1998. Replication and pathogenesis of white sturgeon iridovirus(WSIV) in experimentally infected white sturgeon Acipenser transmontanus juveniles and sturgeon cell lines. Diseases of Aquatic Organisms 32.173-184.
Walker, R. 1962. Fine structure of lymphocstis virus in fish. Virology 18: 503-508.
Ward, V. K., and J. Kalmakoff. 1987. Physical mappping of the DNA of the genome of insect iridescent virus type 9 from Wiseana spp. larvae. Virology 160: 507-510.
Williama, T. 1994. Comparative studies of iridoviruses: Further support for a new classification. Virus Research 33: 99-121.
Willians, K.G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers and useful as genetic marker. Nucleic Acid Research 18: 6531-6535.
Wolf, K. 1988. Fish Viruses and Fish Viral Diseases. Cornell University Press. Ithaca, New York. USA. Pp268-291.