Characterization of a Rhabdovirus Isolated from the Snakehead
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AN ABSTRACT OF THE THESIS OF Jiraporn Kasornchandra for the degree of Doctor of Philosophy in Microbiology presented on December 2. 1991 Title Characterization of a Rhabdovirus Isolated from the Snakehead Fish (Ophicephalus striatus) Redacted for Privacy Abstract approved: Dr. J. L. Fryer- A new rhabdovirus was isolated from snakehead fish (Ophicephalus striatus) during an outbreak of ulcerative disease in Thailand in 1985.The virus was named snakehead rhabdovirus (SHRV), and it replicated optimally at 24-30°C in snakehead fin (SHF) and epithelioma papulosum cyprini (EPC) cell lines.The cytopathic effect produced by this virus in both cell lines began with infected cells becoming rounded, followed by detachment and then lysis. Exponential growth of SHRV on EPC cells at 27°C began after a 4 h latent period and reached a maximum plateau at 12 h post infection. The rhabdoviral particle exhibited a bacilliform morphology in thin section. It was 60-70 nm wide, 180-200 nm long and surrounded by a lipid envelope. SHRV consisted of a single-stranded RNA genome. The virus was sensitive to chloroform, pH 3, and heat (56°C), but stable to freeze-thaw. Electron microscopy and biochemical studies confirmed SHRV to be a member of the family Rhabdoviridae. SHRV consisted of five structural proteins (L, G, N, M1,and M2) similar to rabies virus, the prototype rhabdovirus of the Lyssavirus genus, with molecular weights of >150, 69, 48.5, 26.5, and 20 Kilodaltons. The serological comparison of SHRV with the other fish rhabdoviruses showed that SHRV was unique. Fifteen monoclonal antibodies (MAbs) were produced against SHRV and characterized by immunofluorescence, neutralization tests, and an immunoprecipitation assay. Eight of the MAbs, recognized the viral glycoprotein in an immunoprecipitation assay; of these, three had neutrailizing activity. Twelve MAbs showed good binding activity by immunofluorescence.Five of these recognized the G protein, five recognized the N protein, and two recognized the M1 protein of SHRV. In an indirect fluorescence assay, the MAbs gave varied staining patterns depending upon the viral structural proteins recognized. Characterization of a Rhabdovirus Isolated from the SnakeheadFish (Ophicephalus striatus) by Jiraporn Kasornchandra A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed December 2, 1991 Commencement June, 1992 APPROVED: 7-. Redacted for Privacy Profer of MicrobioCI4y in charge of Major Redacted for Privacy ChairmaW, DepartmentgMicrobiology Redacted for Privacy Dean of Grad School (I Date of thesis presentation December 2, 1991 Typed by Jiraporn Kasornchandra for Jiraporn Kasornchandra ACKNOWLEDGEMENTS I would like to dedicate this thesis tomy family- my father Pol, my mother, Chaleourat; my sister, Chintana; my brothers, Vichai and Thongchai for their love, guidance, and constantsupport, without whom I would not have had a chance to accomplish thislongtime survival in the USA or finishing this goal. Also to Drs. Sitdhi andMali Boonyaratpalin for their encouragement, guidance, andsupport over the years of my study. I would like to express my deep appreciation tomy major professor, Dr. J. L. Fryer, for the opportunity to work in the FishDisease Laboratory and for his support, encouragement, guidance, andpatience during my three and a half yearsas his graduate student. Because of him everything was made possible. To Dr. J. S. Rohovec for his assistance and encouragement during the progess of my thesis work especially with this manuscript. To Cathy Lannan,"my favorite MOM", for her love, patience, guidance, encouragement, and great support which made allmy study and manuscripts possible. To Dr. H. M. Engelking for his assistance and guidance in viral purification and gel electrophoresis. And a special thanks to my closest friends, Jerri Bartholomew, Susan Gutenberger, John Kaufman, Carol Shanks, Harriet Lorz, Margo Whipple, and Marcia House for their friendship, advice, and support. And I would like to thank the members of the fish disease group both on campus and at HMSC, Dr. Tony Amandi, Dr. Rich Holt, Leslie Smith, Craig Banner, Dr. Jim Bertolini, Dr. BobOlson, Dr. Paul Reno, and Prudy-Caswell Reno, for their friendshipand warm hospitality. I would like to thank Micheal for proofreading thismanuscript. I do appreciate it. To Dr. Wattanavijarn for providing the SHRV; Dr. Frerichsfor providing the UDRV-BP and UDRV-19; Dr. Kimura forproviding the HRV and PFR, and to Dr. Winton for providing theanti-VHSV antiserum. To Sheri Shenk and special thanks to Julie Duimstra fortheir excellent work in the electron microscopy. This work is a result of research sponsored bygrants from the Oregon State University Sea Grant College Program,supported by NOAA Office of Sea Grant, Department of Commerce,under grant No. NA 89AA-D-SG 108 (project No. R/FSD-14) and USAIDproject grant No. 7-276. CONTRIBUTION OF AUTHORS J. S. Rohovec and J. L. Fryer served as advisors to this project and appear in Chapter III and IV.J. L. Fyer is also a co-author on Chapter V. C. N. Lannan assisted in cell cultures and the rhabdoviral assays in Chapter III, serum neutralization tests in Chapter IV, monoclonal antibody production and fluorescent antibody tests in Chapter V.In Chapter IV, H. M. Engelking assisted in viral purification and structural protein analysis. P. Caswell-Reno provided assistance in the monoclonal antibody production in Chapter V. TABLE OF CONTENTS Chapter Page I. Introduction 1 II. Review of Literature 3 Literature Cited 21 Characterization of a Rhabdovirus Isolated from the Snakehead Fish (Ophicephalus striatus) 32 Abstract 33 Introduction 34 Materials and Methods 36 Results 43 Discussion 59 Acknowledgements 61 Literature Cited 62 IV. Characteristics of Three Rhabdoviruses Isolated from Snakehead Fish (Ophicephalus striatus) 66 Abstract 67 Introduction 69 Materials and Methods 70 Results 76 Discussion 87 Acknowledgements 90 Literature Cited 91 V. Development and Characterization of Monoclonal Antibodies against Snakehead Rhabdovirus 95 Abstract 96 Introduction 97 Chapter Page V. (Continued) Materials and Methods 98 Results 104 Discussion 111 Acknowledgements 113 Literature Cited 114 SUMMARY AND CONCLUSIONS 118 BIBLIOGRAPHY 120 LIST OF FIGURES CHAPTER III. Page Replication of snakehead rhabdovirus at selected temperatures, MOI = 0.1. 47 111.2 Replication of snakehead rhabdovirus in selected cell lines at 27°C, MOI = 1.0. 49 111.3. One-step growth curve of snakehead rhabdovirus in EPC cells incubated at 27°C, MOI = 1.0. 51 111.4. Thin sections of EPC cells infected with snakehead rhabdovirus. 53 111.5. Electrophoretic profile of three fish rhabdoviruses. 55 111.6 Enzyme immunoassay of the snakehead rhabdovirus glycoprotein. 57 CHAPTER IV. IV.1 a. Electron micrograph of an ultrathin section of EPC cells infected with snakehead rhabdovirus. 77 IV.1b. Electron micrograph of an ultrathin section of SHF cells infected with ulcerative disease rhabdovirus-BP (UDRV-BP). 77 IV.1 c. Electron micrograph of an ultrathin section of SHF cells infected with ulcerative disease rhabdovirus-1 9 (UDRV-1 9). 77 IV.2. A comparison of the viral polypeptides of snakehead rhabdovirus (SHRV) and the other eight fish rhabdoviruses in a 10% acrylamide gel. 79 CHAPTER V. Page V.1. Immunoprecipitation of snakehead rhabdovirus proteins with monoclonal antibodies recognized the G protein. 107 V.2. Immunoprecipitation of snakehead rhabdovirus recognized the N proteins. 107 V.3. Immunoprecipitation of snakehead rhabdovirus recognized the M1 proteins. 107 V.4. Mono layer cultures of EPC cells infected with snakehead rhabdovirus fixed at 10 h post infection and examined by indirect fluorescent antibody test.109 LIST OF TABLES CHAPTER II. Page II.1. Comparison of the characteristics among the known fish rhabdoviruses. 17 CHAPTER IV. IV.1. The estimated molecular weights (x103 daltons) of virion proteins of the snakehead rhabdovirus (SHRV), infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia (VHSV), and hirame rhabdovirus (HRV). 84 IV.2. The estimated values of molecular weights (x103 dalton) of virion proteins of the spring viremia of carp virus (SVCV),ulcerative disease rhabdovirus (UDRV-BP), UDRV-19, and pike fry rhabdovirus (PFR). 85 IV.3. Cross-neutralization tests for eight fish rhabdoviruses incubated with homologous and heterologous antibodies. 86 Characterization of a Rhabdovirus Isolated from the Snakehead Fish (Ophicephalus striatus) Chapter I Introduction A rhabdovirus,serologicallydifferent from other fish rhabdoviruses, was isolated from snakehead fish (Ophicephalus striatus) in Thailand during an epizootic disease characterized by a severe ulcerative necrosis.The ulcerative disease was reported in Australia in 1972 and then spread throughout the Indo-Pacific region (Tonguthai, 1985). The disease caused extensive losses among wild and pond-cultured fish in Malaysia between 1979 and 1983 and also in central Java, Indonesia in 1983 (Tonguthai, 1985). In 1985, the disease spread to the Lao People's Democratic Republic (PDR) and to Burma (Boonyaratpalin 1989). A major epizootic began in southern Thailand in late 1980 and spread throughout the country. Although the disease was observed in several fish species, the cultured snakehead, the walking catfish (Clariasbratachus), and the sand goby (Oxyeleotris marmoratus) were the most noticeably affected (Hedrick et al,1986). Although potentially pathogenic organisms were isolated and identified