Characterization of a Rhabdovirus Isolated from the Snakehead

Home , Snakehead rhabdovirus, Viral replication

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 from the diseased fish, the etiologic agent has yet to be established. During the viral examination, two birnaviruses (Hedrick et al., 1986, Wattanavijarn et al., 1988) and two rhabdoviruses (Frerichs et al., 1986, Wattanavijarn et al., 1986) were isolated. The rhabdovirus 2 isolated by Wattanavijarn et al. (1986)was not completely characterized or compared to the other fish rhabdoviruses. The purpose of this study was as follows: (1) To study the biological and biochemical characteristics of the snakehead rhabdovirus, including analysis of the viral structural proteins. It was first necessary to determine the appropriate methods for propagation and assay of the virus. The studies involved the evaluation of cell lines, incubation temperatures, and viral purification techniques. A detailed evaluation of the biochemical characteristics of the viruswas included. (2) A comparison of the snakehead rhabdovirus structural proteins and its serological characteristics to the major fish rhabdoviruses, including two other rhabdoviruses isolated from snakehead. Once the snakehead rhabdoviral proteins were identified, the structural proteins of the known fish rhabdoviruseswere compared and their antigenic relatedness determined. (3). To develop improved methods fordiagnosis and viral identification using monoclonal antibody technology. Previously developed polyclonal antisera were unsatisfactory for diagnosis because of low titers and the toxic effect of thesesera on cultured fish cells.Moreover the rabbit polyclonal antisera produced extensive non-specific binding in fluorescent antibody tests, providinga high level of background florescence. Therefore, a battery of monoclonal antibodies was developed.These monoclonal antibodies were characterized and evaluated for usefulness in immunodiagnosis. 3

CHAPTER II

Review of Literature

Since 1980, an epizootic disease characterized bya severe ulcerative dermal necrosis has been observed ina wide variety of freshwater and estuarine fishes, both wild and pond-cultured, throughout the Indo-Pacific region.The origin of the disease is unknown, but it has been suggested by several investigatorsthat it was first encountered in Australia in 1972 (Boonyaratpalin,1989a; Frerichs et al., 1989, Tonguthai, 1985) whenan outbreak of a disease known as "red spot" occurred in sea mullet and other estuarineand freshwater fish (McKenzie and Hall, 1976). The ulcerative diseasecondition also occurred in 1981 in both freshwater and estuarine species inthe Sepik River system in Papua New Guinea (Coates, 1984). During 1979 in Malaysia, a serious necrotizing ulcerative condition was observed in several fish species in freshwaterrice fields. These included snakehead (Ophicephalus striatus), catfish(Clarias macrocephalus), and gorami (Trichogester pectoralis, Trichogester trichopterus).A severe epizootic recurred in 1983 causing high mortalities of fish cultured in paddy fields and submergedcages in Kedah (near the border with Thailand) (Boonyaratpalin, 1989a,Frerichs et al., 1989). In Indonesia, a major mortality took place during 1981 in snakehead and catfish (Clarias batrachus).These fish, from both central Java and Kalimantan, displayed the ulcerative condition (Frerichs et al., 1989).The most serious outbreak of the ulcerative 4

disease began in southern Thailand in late 1981 (Boonyaratpalin, 1989a). Since that time, it has been reported in estuarine and freshwater fishat numerous locations throughout the country. In 1985, the range of the disease was extended to the Lao People's Democratic Republic (PDR), Kampuchea and Burma (Boonyaratpalin, 1989a, Frerichs et al., 1989) and was observed in Luzon, Phillippines, in 1986 (Frerichs et al., 1989). In late 1987 and early 1988, the ulcerative diseasewas reported in Sri Lanka and India(Boonyaratpalin, 1988, 1989c) and was present in Nepal in early 1989 (Boonyaratpalin, 1989b).

Clinical signs The disease usually occurs during the cooler and drier months. In all affected fish, initial lesions are characterized by theappearance of single, or multiple, irregularly-circumscribed, focal areas of acute dermatitis with hyperemia (Frerichs et al., 1989).Subsequent skin erosion results in the formation of ulcerative lesions on the body surface. Large, deep ulcers are commonly found all over the body, eventually resulting in death. In certain species, such as snakehead and eel (Fluta alba), the head, particularly on the maxilla and mandible, is recognized as the principal site of the lesion.Some infected fish with minor infections demonstrate only petechial hemorrages (Boonyaratpalin, 1989). The ulcerative skin lesion of the affected fish is followed by necrosis of the underlying tissue, commonly associated with bacterial and fungal infection. The internal organs normally show only minimal 5

pathological changes. However, insome cases, especially with severely ulcerated snakehead, the pectoralor ventral fins are completely destroyed and the internal organs eventually showpathology. Boonyaratpalin (1989) reported that, in the advancedstage of ulceration, the internal organs showeda low incidence of septicemia, but an exudate was often found in the peritoneal and pericardialcavities. Little evidence of petechial hemorrhagewas noted on the surface of the liver and intestine. Although mortalitywas high in susceptible species such as snakehead, recovery with development of scar tissue and anatomical deformities was observed in less susceptible species.

Susceptible species Numerous species of both freshwater and estuarine fishare reported to be susceptible to the disease. No ulcerative diseasehas been observed in true marine fish (Frerichs et al., 1989). Tonguthai(1985) reported at least 80 species of fishwere affected with the ulcerative disease during outbreaks, which occurred in Australia, Papua New Guinea, Thailand, and Burma, between 1972 and 1985. In southeast Asia, Boonyaratpalin (1989) reported the cultured snakehead (Ophicephalus sp.), walking catfish (Clarias sp.),skin gurami (Trichogaster pectoralis), mrigala (Cirrhina mrigala), rohu (Labeo rohita), catla (Cat la catla),common carp (Cyprinus carpio), bighead carp (Aristichthys nobilis),grass carp (Ctenopharyngodon idella), and silver carp (Hypopthalmichthys molitrix)were most frequently and severely affected by the ulcerative disease. In Thailand, 6 Hedrick et al. (1986) noted that the cultured snakehead (Ophicephalus striatus), the walking catfish (Clarias bratachus), and the sand goby (Oxyeleotris mamoratus) were the most frequently affected species.

Pathogens associated with the ulcerative disease According to Tonguthai (1985), Paratransversotremasp., was the parasitic organism most frequently found on affected fish during the disease outbreak in Australia, and it was thought to be a possible vector for a primary microbial pathogen. In southeast Asia, although various types of parasitic organisms were observed, no species was specifically identified as a vector or virulent primary pathogen (Tonguthai, 1986). Roberts (1986) found numerous fungal hyphae in the infected fish in Australia. However, he suggested that this fungus did notappear to be identical to those found in the Asian outbreak. Pittayangkula and Bodhalamik (1983) identified non-septate fungal hyphae, belonging to the genus Achlya from ulcerative lesions of snakehead in Thailand.In addition, Boonyaratpalin (1987) reported the isolation of Saprolegniasp. in advanced cases of the disease. Bacteriological examination carried out by Boonyaratpalin (1989), showed that Aeromonas hydrophila, a gram negative opportunistic bacterial pathogen, was most commonly found in association with the epizootic ulcerative disease in southeast Asia.Pseudomonas sp., another opportunistic pathogen of fish, was isolated from a few moribund fish during the study. Bacteria were most often isolated from the lesions and less frequently from internal organs. Boonyaratpalin 7 (1989) suggested that it was unlikely that these opportunistic bacterial pathogens were the cause of the ulcerative disease. Hedrick et al. (1986) isolated a birnavirus from sand goby reared in freshwater cages in Thailand and experiencing the ulcerative disease. This was the first virus to be isolated in southeast Asia and the first in association with this disease.In 1986, Frerichs and his coworkers reported the isolation of a rhabdovirus from diseased snakehead in Thailand and from snakehead and freshwater eel in Burma. Since 1986, a second rhabdovirus and birnavirus have been isolated from ulcerated snakehead in Thailand (Wattanavijarn et al., 1986, Wattanavijarn et al., 1988). None of these has been conclusively demonstrated to be a causative agent of the ulcerative disease. The rhabdoviruses isolated by Frerichs et al. (1986) and Wattanavijarn et al (1986), and the birnavirus from sand goby (Hedrick et al. 1986) have been inoculated into 0. striatus without producing disease (Boonyaratpalin, pers. comm.). No pathogens or higher parasites of importance were isolated from healthy fish during this epizootic of ulcerative disease in Thailand, although, approximately 300 normal fish were examined between 1982 and 1984. Koch's or River's postulates have never been completed with any of the microorganisms associated with this disease.Thus the etiology has not been established. 8

Characteristics of the Family Rhabdoviridae Rhabdoviruses are important pathogens ofa wide variety of plants and animals, including fish and humans. Currently,more than 60 viruses isolated from animals and plants are classified in the Family Rhabdoviridae based on morphology (Emerson, 1985). All rhabdoviruses are elongated and are either bullet-shaped or bacilliform. The size and shape of the rhabdoviruses are relatively uniform. Those from animals possess a bullet-shaped morphology, 60 to 85nm in width and 180 to 230 nm in length, and the plant rhabdovirusesare usually longer and bacilliform in shape (Bishop and Smith, 1977). In addition, most rhabdoviruses generate defective interfering (DI) particles, shorter truncated particles which have less RNA than is present in complete virions. These DI particles are unable to replicate but interfere with replication of complete virus (Emerson, 1985). Rhabdovirusesconsistof twostructuralunits,the ribonucleocapsid and the envelope. The genome of the rhabdovirus is single-stranded RNA of approximately 4 to 5 x 106 daltons. The genomic RNA is of the negative sense (i.e., complementary to mRNA). One of the unique features of the negative-strand RNA genome of a rhabdovirus is that it serves as the template for both transcription (mRNA synthesis) and replication (genomic RNA synthesis) (Banerjee, 1987). Because this virus contains its own polymerase, transcription begins as soon as the viral envelope is removed. The products of standard virus transcription, both in vivo and in vitro, consist of five monocistronic mRNAs anda leader RNA (Emerson, 1985). The five mRNAs are translated into the five viral proteins.Nomenclature for the five structural proteins are: 9 the viral polymerase (L), glycoprotein (G), nucleoprotein (N), nucleocapsid-associated phosphoprotein or nonstructural protein (NS), and the matrix protein (M) (Wagner et al., 1972). The two best described rhabdoviruses are the animal pathogen, vesicular stomatitis virus (VSV) and the human pathogen, rabies virus. These two prototypes can be differentiated based on their structural proteins: VSV, the prototype virus of the Vesiculovirus genus, has one matrix protein and an additional nonstructural (NS) protein (Wagner, 1975). Rabies virus has two matrix proteins, the M1 and M2 which place it in the genus Lyssavirus (Cos lett et al., 1980). The M1 protein of rabies virus is associated with nucleocapsid proteins and is identified as a phosphoprotein. Therefore, the Ml protein of rabies virus is analogous to the NS protein of VSV (Wunner et al., 1985).

Fish Rhabdoviruses There are at least 11 fish rhabdoviruses that have been isolated and successfully grown in fishcell lines (Wolf, 1988). The characteristics of the fish rhabdoviruses described below have been compared using all available information (Table 1).

1. Infectious Hematopoietic Necrosis Virus Serious epizootics were reported among sockeye salmon (Oncorhynchus nerka) fry in the Pacific Northwest, USA, in late 1940s and 1950s (Rucker et al., 1953). Wingfield et al. (1969) isolated a virus from hatchery-reared sockeye salmon in Oregon, USA and named this 10 virus Oregon Sockeye Virus (OSV). During this same period, another virus was isolated from chinook salmon (Oncorhynchus tshawytscha) cultured in a hatchery on the Sacramento River in California, USA. This virus was called Sacramento River Chinook Virus (SRCV). Wingfield and Chen (1970) suggested this virus was similar to OSV. Amend et al. (1969) isolated a virus from infected rainbow trout (Oncorhynchus mykis) and sockeye salmon that caused extensive necrosis of the blood-forming tissue of the kidney and of the spleen. This isolate was named infectious hematopoietic necrosis virus (IHNV). Amend and Chambers (1970) compared morphology, cellline susceptibility, and pathogenicity of OSV, SRCV, and IHNV. They found that OSV and SRCV had similar characteristics to those of IHNV and suggested the name IHNV be applied to all strains.Using a cross- neutralization test, McCain et al. (1971) compared the three rhabdoviral isolates and found that IHNV and OSV were indistinguishable and SRCV was closely related. McAllister and Wagner (1975) compared five structural proteins of IHNV and Egtved virus (a rhabdovirus isolated from brown trout in Europe) and found that these proteins were similar to those of rabies virus. This conclusion was confirmed by Hill et al. (1975) and Lenoir and de Kinkelin (1975). Kurath and Leong (1985) discovered that IHNV, in fact, had six polypeptides: L, G, N, M1, M2, and a non-virion (NV) protein.The NV protein is present only in infected cells but not in mature virions.By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Leong et al. (1981) distinguished strains of IHNV into four groups based on the differences in the molecular 11 weights of the N and G proteins.Hsu et al. (1986) used the same technique to compare 71 different IHNV isolates andgroup them into five types (four specific and one less defined). They suggestedthat a particular viral isolate was more specific to geographicarea than to host.

2. Viral Hemorrhagic Septicemia Virus The Egtved virus, designated strain F1,was isolated from the North American rainbow trout whichwere introduced into Europe beginning in 1879 (Jensen, 1965). By electron microscopy, Zwillenberget al. (1965) demonstrated that Egtved viruswas a rhabdovirus.Like IHNV, Egtved virus, also known as viral hemorrhagic septicemia virus (VHSV), contains five structural proteins similar to those of rabies virus (Hill et al., 1975, Lenior and de Kinkelin, 1975, McAllister and Wagner, 1975).For many years, the geographic distribution of VHSVwas restricted to Europe.In 1988, the first VHSV isolations in North America were obtained from chinook (Hopper, 1989) and coho salmon (Oncorhynchus. kisutch) in North America (Brunson et al., 1989). The two north American VHSV isolates were characterized by Winton et al. (1989) who found them to contain identical protein patterns, with five structural proteins that matched those of the Fl serotype of VHSV. In addition, the north American isolates were neutralized by antiserum against VHSV Fl.Winton et al. (1989) concluded thata close biochemical and antigenic relationship exists between the two isolates and the Fl serotype of VHSV. 12

3. Spring Viremia of Carp Virus In Europe, the most serious cause of loss among cyprinid fish is the disease known as infectious dropsy.Fijan et al. (1971) isolated a rhabdovirus from common carp (Cyprinus carpio) experiencing this disease. They named this virus, Rhabdovirus carpio, and the disease, spring viremia of carp. The biochemical and serological characteristics of this virus have been studied (de Kinkelin and LeBerre ,1974, Hill et al., 1975). In 1974, Bachmann and Ahne (1974) characterized a rhabdovirus from a disease known as swim bladder inflammation (SBI) in common carp, and this virus proved to be indistinguishable from R. carpio. Hill et al. (1975) showed that R. carpio contains five structural proteins which resemble those of VSV.

4. Pike Fry Rhabdovirus Isolation of the pike fry rhabdovirus (PFR), from diseased pike (Esox lucius) in northern Europe in 1973, was reported by Bootsama and Vorstenbosch (1973) and de Kinkelin et al. (1973). At that time, two different diseases known as "red disease" and "hydrocephalus" occurred in pike.Later they were shown to be caused by the same rhabdovirus (Bootsma and Vorstenbosch, 1973). The characterization of PFR revealed that this virus contained four structural proteins similar to those of VSV (de Kinkelin et al., 1974).This finding has been confirmed by other investigators, Clerx et al. (1975), Hill et al. (1975), and Roy et al. (1975). Ahne et al. (1982) reported the isolation of PFR from tench (Tinca tinca) and white beam, (Blicca bjoerkna) in Germany. 13

5. Atlantic Cod Ulcus Syndrome The Atlantic cod ulcus syndrome was recognized as a chronic or subacute pathogenic skin condition of Atlantic cod (Gadus morhua) in Denmark since the 1940s, and the disease was thought to be caused by vibrios (Larsen and Jensen, 1977). However, in 1979, Jensen et al. (1979) isolated two viruses, a rhabdovirus and an iridovirus, from affected fish. Jensen and Larsen (1982) showed transmission of the viral infection by cohabitation of healthy and affected fish.They also injected intracardially culture-grown iridovirus and rhabdovirus into separate groups of experimental fish. They found that 19% of the fish injected with iridovirus developed lesions and fish injected with rhabdovirus remained healthy.They suggested that the iridovirus may be the etiologic agent as the in vitro grown rhabdovirus alone did not induce disease.

6. Rio Grande Cichlid Rhabdovirus In 1980, Malsberger and Lautenslager isolated a rhabdovirus from diseased Rio Grande perch (Cichlasoma cyanoguttatum). The virus was named Rio Grande perch rhabdovirus and had characteristics unique among fish rhabdoviruses.The Rio Grande perch rhabdovirus exhibits a bacilliform morphology rather than the bullet-shape typical of other animal rhabdoviruses.This virus later proved to be an acute pathogen for the original host species and Cichlasoma nigrofasciatum. 14

7. Perch Rhabdovirus In France, a second perch rhabdovirus, causing disease of the central nervous system in Perca fluviatilis,was reported by Dorson et al. (1984). The virus showed a typical bullet-shapeas observed by electron microscopy. No serological relatedness was observed between this virus and the salmonid rhabdoviruses (IHNV and VHSV),or the eel virus American (EVA). Other characters have not yet been reported.

8. Eel Rhabdovirus Two different rhabdoviruses have been isolated by Sano (1976) and Sano et al. (1977) from cultured eels imported into Japan from Europe and North America.Because the first virus was isolated froman American eel (A. rostrata), the virus was designated EVA (Sano, 1976). The second eel rhabdovirus was obtained froman eel (A. anguilla) imported from Europe and was named EVEX (Sano, 1976). Both viruses had bullet-shaped morphology; although, the EVA particleswere shorter than those of EVEX. Physicochemical characteristics, serology, and polypeptides of the two eel rhabdoviruseswere compared by Hill et al. (1980).They were antigenically related, but neutralization tests showed they had no relationship to the other fish rhabdoviruses. Therefore, he proposed these two rhabdoviruses be named Rhabdovirus anguilla, with EVA and EVEX being referred to as strains A and X, respectively. Two other European eel rhabdoviruses came from France, and were designated B12 and C30 (Castric and Chastel, 1980). Only the C30 isolate was serologically related to EVA and EVEX. Castric et al. (1984) 15 demonstrated that EVA, EVEX, and C30 had structural proteins similar to those of VSV and considered them to be members of the Vesiculovirus genus. The B12 isolate exhibited five polypeptides and resembled the Lyssavirus subgroup.

9. Hirame Rhabdovirus The hirame rhabdovirus (HRV) was isolated from diseased hirame (Paralichthys olivaceus) and ayu (Plecoglossus altivelis) and was identified and characterized by Kimura et al. (1986). The isolation of HRV from the other fish such as black seabream, Milio macrocephalus, and mebaru, Sebastes inermis, have been reported (Sano and Fukuda, 1987, Yoshimizu et al., 1987).Like IHNV and VHSV, HRV contained five structural proteins, similar to rabies virus (Kimura et al., 1989). In western blot analysis, Nishizawa et al. (1991) reported the structural proteins, G, N, and M2, of HRV shared antigenic determinants with IHNV and VHSV.Even though Rivers' postulates have not been fulfilled, HRV infection can be induced in rainbow trout at 12°C by intraperitoneal inoculation.

10. Ulcerative Disease Rhabdovirus and Snakehead Rhabdovirus In southeast Asia, two fish rhabdoviruses have been isolated and successfully grown in fish cell culture. One is the ulcerative disease rhabdovirus (UDRV) isolated by Frerichs et al. (1986) from affected snakehead in both Thailand and Burma. The second is the snakehead virus, obtained from diseased snakehead in Thailand by Wattanavijarn et al. (1986). Biochemical and biological characters of these two viruses 16 have been reported (Frerichs et al., 1988, Frerichset al., 1989, Ahne et al., 1988). No cross-neutralizationwas observed between UDRV and the other fish rhabdovirus (Frerichs et al., 1988). Ahneet al. (1988) reported that the rhabdovirus isolated by Wattanavijarn showedno serological relatedness to any other fish rhabdovirus. Although thesetwo viruses have been individually described,no detailed comparative studies have been conducted.

Serology Serological techniques have been developed for the detection, identification, and diagnosis of rhabdoviral infections, bothin tissue culture and in infected host species.In general, rabbits are used for producing antisera against viral agents, althoughgoats and sheep are sometimes employed. Sartorelli et al. (1966) suggested thatascitic fluid from immunized mice might be another possiblesource for viral antibodies. Serum neutralization tests are commonly used in identification of viral agents (Rovozzo and Bruke, 1973). The testsmeasure the loss of viral infectivity resulting from binding of antibody moleculesto the surface proteins of viruses, preventing attachment,or entry into, susceptible cells (Van Regenmortel, 1981).Using cross serum- neutralization tests, McCain et al. (1971) showed that IHNV and OSV were indistinguishable while SRCV was closely related to IHNV. McAllister et al. (1974) used polyclonal rabbit antisera to determine that IHNV was serologically different from other fish rhabdoviruses. Table ILL Comparison of the characteristics among the known fish rhabdoviruses

Viruses shape size (nm) temperature of propagated Reaction to Selectedreferences Or incubation °C celllines heat low pH lipid halogenated Diseases solvent pyrimidine

IHNV bullet 160-180x70-90 13-18 CHSE-214, RTG-2, EPC, FHM + + + Wingfield etal.1969 STE-137

VHSV bullet 1 8 5x6 5 10-15 BF-2, CHSE-214, EPC, FHM, + + + de Kinkelin and Scherrer 1970; PG, RTG-2 Zwillenberg e al. 1965

HRV bullet 180-220x80 15-20 FHM, EPC, RTG-2, BF-2, YNK, + + + Kimura etal. 1986

SVCV bullet 90-180x60-90 20-22 BB, BF-2, EPC, FHM, RTG-2 + + + de Kinkelin etal.1973 de Kinkelin and Le Berre 1974

PFRV bullet 115-135x80 21-28 RTG-2, BB, FHM, EPC + + + de Kinkelin et al.1974

EVA bullet 100-160x75-80 15 RTG-2, EPC, FHM, BF-2, EK-1 + + + Hill et al. 1980; Sano 1976; EO-2 Sano et al. 1977 cod ulcus syndrome bullet 115-195x50-80 15 PS nd nd nd nd Jensen et al.1979

Rio Grande perch rhabdovirusbacilliform 180x50 23 FHM, BF-2, RTG-2 nd nd + Malsberger and Lautenslager 1980 Perch rhabdovirusbullet 20 Oxl 0 0 14 RTG-2 + nd nd nd Dorson et al. 1980

UDRV bullet 110-130x75-85 25-30 BF-2, SSN-1,AS, RSN, CP + nd nd nd Frerichs etal.1986; Frerichs etal.1989 Snakehead isolation bullet 1 7 Ox6 0 15-28 CLC, SH + + + Ahne et al. 1988 nd = no data + = sensitive = resistant 18 Hill et al. (1975) used serum neutralization tests to determine the serological relatedness among fish rhabdoviruses (IHNV, VHSV, SVCV, SBIV, and PFR). They explained that the SVC and SBI viruses were indistinguishable; whereas, IHNV showed no serological relationship to the other four viruses although slightcross reactions were noted.Using serum neutralization tests, Clerx et al. (1978) demonstrated that PFR was monotypic.They also suggested that neutralization accomplished with rabbit antiserum was complement- dependent; whereas, neutralization with antiserum prepared in pike was not. Hill et al. (1980) demonstrated that EVA and EVX viruses were antigenically closely related, but were serologically unrelated toany other fish rhabdovirus by cross-neutralization tests. Kimura et al. (1986) proved that 11 isolates of HRV were antigenically identical when reacted with anti-HRV rabbit antiserum using plaque neutralization tests, but, in cross-neutralization tests, this virus was distinguishable from IHNV, VHSV, SVCV, PFR, EVA, and EVX. Frerichs et al. (1989) determined that the six isolates of UDRV were identical but differed from the other seven fish rhabdoviruses (VHSV, IHNV, EVX, EVA, SVCV, PFR, PRV) by serum neutralization tests. Although the neutralization test is routinely used in identification of fish rhabdoviruses, it is a process taking several days to complete. Other immunological techniques have been developed in recent years for rapid diagnosis, detection, and identification of fish rhabdoviruses both in vitro and in vivo. Faisal and Ahne (1984) compared the sensitivity of the immunoperoxidase and fluorescent antibody techniques (FAT) for detecting SVCV in infected tissues. They suggested that the techniques 19 were equivalent in sensitivity, and both techniques were useful in studying the location of SVCV in cells of infected organs. Dixon andHill(1984)developedan enzyme-linked immunosorbent assay (ELISA) for rapid identification of IHNV and the two eel rhabdoviruses, EVA and EVX, in cell cultures or directly from tissues of infected fish. Although the ELISA was successfully developed for rapid identification of IHNV and the eel rhabdoviruses, this techniques was ineffective in detection of VHSV and SVCV. They thought that the antisera against these two viruses, VHSV and SVCV, had low titers. Jorgensen et al. (1991) used three different techniques, plaque neutralization tests, immunofluorescence, and ELISA, to detect anti-IHNV and anti-VHSV antibodies in cultured rainbow trout surviving disease outbreaks. They reported that immunofluorescence was the most sensitive method for detecting anti-IHNV antibodies, but the ELISA was best in detecting anti-VHSV antibodies. They suggested that these three serological methods are useful for IHNV and VHSV epidemiology, even though cross-reactivity between the two viruses was observed in sera with high titers of viral antibodies. Hsu and Leong (1985) described methods for characterizing different strains of IHNV by using solid phase direct binding assays with 125lodinated Protein A or with immunoperoxidase staining. They suggested that both immunological methods were highly specific and sensitive to viral protein. Schultz et al. (1985) successfully produced a monoclonal antibody (MAb) against IHNV. Although the MAb showed little neutralizing activity (a titer of 1:50 against 50 TCID50 of IHNV), it reacted with IHNV 20 by ELISA but did not recognize IPNVor any of three serotypes of VHSV. Winton et al. (1988), using three different neutralizing MAbs, classified 12 isolates of IHNV into four groups. Their results supported the idea that grouping of IHNV isolates correlated with geographicareas rather than with host species. LaPatra et al. (1990) succesfully developedan FAT for detection of IHNV in infected tissues using either MAbor polyclonal antibody against IHNV. They suggested that the indirect FAT utilizing MAb is sensitive and rapid for detection and study of the pathogenesis of IHNV infection. Ristow and de Avila (1991) produced MAbs to the G and N proteins of several isolates of IHNV and used them in viral typing.In their study, 17 isolates of IHNV from various locations, years, and different fish stocks were analyzed by SDS-PAGE, serum neutralization, and indirect FAT (IFAT). Using SDS-PAGE they found minor variations in the N proteins of these viral isolates.In serum neutralization assays, 17 isolates were reacted with each of two different MAbs produced against the G protein, 3GH127B and 3GH92A. They divided these isolates into two groups: one neutralized by both MAbs and the other would not.In an IFAT, they found 12 (7 MAbs produced against the N protein and 5 MAbs against the G protein) of the 27 MAbs tested gave positive results to 17 IHNV isolates, suggesting that antigenic variation existed among isolates. Lorenzen et al. 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CHAPTER III

Characterization of a Rhabdovirus Isolated from the Snakehead Fish (Ophicephalus striatus)

J. Kasornchandral, C. N. Lannan2, J. S. Rohovecl and J. L. Fryers

1Department of Microbiology, Oregon State University, Nash Hall 220, Corvallis, Oregon 97331-3804 USA

2Laboratory for Fish Disease Research, Department of Microbiology, Oregon State University, Mark 0. Hatfield Marine Science Center, Newport, Oregon 97365-5296 USA.

Submitted for publication in the Proceeding of the Second International Symposium on Viruses of Lower Vertebrates 33 Abstract

During a severe epizootic among the snakehead fish, Ophicephalusstriatus, in Thailand, a virus belonging to the Rhabdoviridae was isolated in cell culture. The viruswas bacilliform 60-70 nm in width and 180-200 nm in lenght and surrounded bya lipid envelope. The virus replicated optimally at 24-30°C in snakehead fin and carp cell lines. Exponential growth on EPC cells at 27°C began after a 4 h latent period and continued during the next 6 h.After 14 h of incubation, all of the cell-associated virus had been released. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed five virion structural proteins, the L, G, N, Mi,and M2, similar to those of the salmonid rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. The estimated molecular weights of the five snakehead rhabdoviral polypeptideswere > 150, 69, 48.5, 26.5, and 20 Kilodaltons (Kd).The 69 Kd molecular weight protein was glycosylated. The virus has been refered to as snakehead rhabdovirus. 34 Introduction

An epizootic disease characterized by a severe ulcerative necrosis was observed in both wild and pond-cultured fish throughout southeast Asia. The disease caused extensive losses among fish populations in Malaysia between 1979 and 1983 and 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). The ulcerative syndrome was first observed in southern Thailand in 1981 (Boonyaratpalin, 1989).Since that time, it has been reported in estuarine and freshwater fish at numerous locations throughout the country. Although the disease was observed in several fish species, the cultured snakehead (Ophicephalus striatus), the walking catfish (Clarias bratachus), and the sand goby (Oxyeleotis marmoratus) were the most noticeably affected (Hedrick et al., 1986). Clinical signs associated with this disease are initially characterized by raised areas of induration and erythema, followed by erosion of the skin.Large, deep, ulcerative lesions are formed on the body surface, especially on the head and jaws of diseased fish. Moribund fish collected during the outbreak in Thailand were examined for bacteria,fungi, viruses, and higher parasites. Aeromonas hydrophila, a gram-negative opportunistic bacterial pathogen, was most commonly found in association with the ulcerative diseased fish (Boonyaratpalin, 1989). Although various types of parasitic organisms were observed, no species was specifically identified as the vector or pathogen (Tonguthai, 1985). During the viral examination, two 35 birnaviruses (Hedrick et al., 1986; Wattanavijarn et al., 1988) and two rhabdoviruses (Frerichs et al., 1986, Wattanavijarn et al., 1986)were isolated. In this report, the virus isolated by Wattanavijarn et al. (1986) is characterized. 36 Materials and Methods

Viruses The snakehead rhabdovirus (SHRV) was isolated from diseased snakehead in Nakorn Pathom Province, Thailand (Wattanavijarn et al., 1986). Two rhabdoviruses from fish, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) were used as reference for comparison of viral structural proteins.In addition to IHNV, the birnavirus, infectious pancreatic necrosis virus (IPNV), and the herpesvirus, channel catfish virus (CCV), were usedas controls for biochemical characterization of SHRV.

Cell lines An uncharacterized cell line from the blotched snakehead (Ophicephalus lucius) (Wattanavijarn et al., 1986) was used for primary isolation. For viral characterization, the virus was routinely propagated on Epithelioma papulosum cyprini (EPC) cells (Fijan et al., 1983). In addition, the following cell lines were tested for their susceptibility to infection by SHRV: fathead minnow (Pimephales promelas) (FHM) (Gravell and Malsburger, 1965), blue gill (Lepomis macrochirus) fry (BF-2) (Wolf and Quimby, 1966), channel catfish (Ictalurus punctatus) ovary (CCO) (Bowser and Plumb, 1980), brown bullhead (Ictalurus nebulosus) (BB) (Wolf and Quimby, 1966), snakehead (Ophicephalus striatus) fin (SHF) (Kasornchandra et al., 1988), and three cell lines originating from fin (GCF), snout (GCS-2), and swim bladder (GCSB) of the grass carp (Ctenopharyngodon idella) (Lu et al., 1990). 37 The three grass carp cell lines were grown in Medium 199 with Hanks' balanced salts (HBSS) (Sigma Chemical Co., St. Louis, MO), supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT). The SHF cell line was grown in Leibovitz's L-15 medium (Gibco, Laboratories, Grand Island, NY), also supplemented with 10% FBS. All other cell lines were propagated in Eagle's minimum essential medium with Earle's salts (MEM) (Flow Laboratories Inc., McLean, VA), supplemented with 10% FBS and 1% L-glutamine (Flow).In addition, all culture media contained 100 U penicillin and 100 streptomycin/ml (Sigma).

Titration of virus The viral titer was determined in EPC cells by end-point dilution assay (TCID 50) (Rovozzo and Burke, 1973). The cells were incubated for 7 d at 27°C. Dilution end-points were calculated by the method of Reed and Muench (1938).

Optimum temperature The replication rate of SHRV at selected temperatures was determined in EPC cells. However, replication of EPC cells is confined to temperatures < 33°C (Fijan et al., 1983); so, the SHF cell line was used to examine viral replication at 35°C. The optimum temperature for viral replication was determined as follows:the EPC cells were inoculated with SHRV at a multiplicity of infection (MOT) of 0.1.After 1 h adsorption at 25°C, the cell sheet was washed and 5 ml of MEM containing 5% FBS (MEM-5) was added to each flask. The cultures were 38 incubated at 14, 18, 21, 24, 27, 30°C, and 35°C. Each day for 7 d, a flask incubated at each temperature was removed and frozen at -70°C. At the end of the experimental period, these cultures were rapidly thawed, and the viral titer of each determined.

Cell line susceptibility and virus replication Nine fish cell lines were seeded in 96-well plates. Ten-fold serial dilutions of SHRV were inoculated into six wells of each line. The plates were incubated for 7 d at 27°C and the viral titer on each cell line computed. To investigate the ability of the nine fish cell lines to support the growth of SHRV, cultures were infected with the virus at an MOI of 1.0. When cytopathic effect (CPE) reached the completion, flasks were frozen at -70°C, rapidly thawed, and the viral titer determined on EPC cells.If no CPE was produced after 14 d, a blind pass procedure was used. After an additional 14 d incubation, flasks were frozen at -70°C and the virus titers determined.

Growth curve A growth curve for SHRV was established using a modification of the method of McAllister et al. (1974). The EPC cells were inoculated with SHRV at an MOI of 1.0. Following a 1 h adsorption, the cell sheet was rinsed and 5 ml of MEM-5 were added, then incubated at 27°C. At 2 h intervals for 24 h, four flasks were removed and the titer of free and total virus determined. To assay cell-free virus, culture medium from two flasks was pooled, centrifuged at 2200 x gravity for 15 min, and supernate frozen at - 39 70°C.Following the final harvest, viral suspensions were rapidly thawed, then titered. To determine total virus, combined cells and culture medium from two flasks were harvested, pooled and frozen at -70°C.Before titration, these preparations were rapidly thawed, homogenized, and centrifuged. The supernate was then titered.

Chloroform sensitivity Sensitivity of SHRV to lipid solvents was determined by the method of Feldman and Wang (1961).One mililiter of the clarified supernate was combined with either 0.5 ml of chloroform or 0.5 ml of MEM-0. The tubes were shaken for 10 min at ambient temperature, then centrifuged at 1000 x gravity for 10 min. Virus was titered from the aqueous phase of the treated preparation and from the MEM control.

Resistance to 5-iododeoxyuridine (IUdR) The effect of IUdR on viral replication was determined by the method of Rovozzo and Burke (1973). Mono layers of EPC cells in 96-well plates were treated with 10-4 M IUdR in BSS or with BSS alone. The cells were inoculated with ten-fold serial dilutions of virus. After 3 h incubation, the cultures were washed, and 0.1 ml of MEM-5 was added to each well. The plates were incubated at 27°C for 4 d and the titer determined. 40 Viral nucleic acid The method of Rovozzo and Burke (1973)was used to classify the viral nucleic acid. The EPC cells were infected with SHRV atan MOI of 0.1.After 12 h incubation at 27°C, the cellswere fixed with Carnoy's fixative and stained with 0.01% acridineorange in Mcllvaine's buffer. The infected cells were then mounted with buffer and viewed witha fluorescence microscope.

Stability to low pH Virus was inoculated into MEM-0 adjusted to pH 3.0or to pH 7.0. After 30 and 60 min incubation at 27°C,an aliquot was removed from each treatment. The aliquot from pH 3.0was neutralized with 0.1 N NaOH, and the viral titer of each preparation determined.

Heat inactivation The method of Rovozzo and Burke (1973)was used to determine the effect of temperature on SHRV. Tubes containing 0.5 ml aliquots of viral suspension were held in a water bath at 56°C. After 0.5, 1, 2, 4, 6, and 8 h incubation, a tube was removed, cooled rapidly, and the virus titered.

Stability to freeze-thaw A stock suspension of SHRV was titered then frozen at -70°C. Three times at weekly intervals, the virus was rapidly thawed inwarm water and an aliquot of the suspension was removed for titration. The tube was then refrozen and the process repeated. 41 Electron microscopy A confluent monolayer of EPC cellswas inoculated with SHRV at an MOI of .001.After 14 h incubation at 27°C, the monolayerwas washed three times with phosphate buffered saline (PBS) and fixedwith 2.5% glutaraldehyde (Sigma) in 0.1 M sodium cacodylate buffer,pH 7.4. Following a 2-h fixation period, the cell sheetwas washed, harvested, and centrifuged at 1000 x gravity for 10 min. The cell pelletwas post fixed with osmium, dehydrated, and embedded in Spurr'smedium (Spurr, 1969). The cells were then sectioned, stained in Reynolds'lead citrate (Reynolds, 1963), and viewed witha Philips CM12/STEM at 60 kV in transmission mode.

Virus purification One liter of spent medium was harvested from infectedEPC cultures when CPE reached completion.The culture fluid was centrifuged at 4,000 x gravity for 10 min, and the virus concentratedby centrifugation at 80,000 x gravity for 1 h.The viral pellet was resuspended in 0.01M tris-HC1 buffer pH 7.5 and purifiedon discontinuous and continuous sucrose gradientsas described by Engelking and Leong (1989).Following the final centrifugation, the purified virus was resuspended in 0.35 ml of the tris buffer and storedat -70°C.

Analysis of viral structural proteins The mini-PROTEAN II system (Bio-Rad Laboratories, Richmond, CA) was used for separation of SDS-dissociated proteins by 42 polyacrylamide gel electrophoresis (SDS-PAGE).Purified virus was mixed in sample buffer (0.5 M tris buffer pH 6.8, 10% v/v glycerol, 10% w/v SDS 0.2%, w/v 2-mercaptoethanol, 0.05% w/v bromphenol blue), and heated for 2 min at 100°C.Polypeptides were separated by electrophoresis for 45 min at constant voltage of 200 volts, using a 4.75% stacking gel and a 10% separating gel (Laemmli, 1970).After electrophoresis, the polypeptides were stained with silver stain (Bio- Rad).Purified IHNV, VHSV, and pre-stained standard protein molecular weight markers (Bio-Rad) were used in the electrophoretic comparison.

Glycosylation of the G protein The glycoprotein of SHRV was identified by enzyme-linked immunosorbant assay (ELISA).Reagents and assay materials were obtained from a commercial source as a glycan detection kit (Boehringer Mannheim Biochemica, Indianapolis, IN).The procedures were as follows: the hydroxyl groups in sugars of viral glycoconjugates were oxidized by periodate and labeled with digoxigenin (DIG). The labeled glycoconjugate was then separated by SDS-PAGE and transferred onto nitrocellulose. An ELISA assay using an alkaline phosphatase conjugated antibody was used to detect the DIG-labeled glycoconjugate. 43

Results

Optimum temperature The optimum temperature range for viral replication was 24-30°C (Fig. 1). The extent of CPE in the culture correlated with the viral titer produced over the range of 18 to 35°C. At 21 and 35°C, viral replication was slower and CPE less extensive than at 27 and 30°C.Little viral replication took place at 14°C.

Cell line susceptibility and viral replication A single stock suspension of SHRV grown in EPC cells was titered on each of nine cell lines and resulted in titers that ranged from 107.8 to 102.1 TCID50/ml. The SHF and EPC cells were most sensitive, producing titers of 107.8 and 107.5 TCID50 /ml, respectively.Titers obtained on FHM and CCO cells were 106.6 and 105.6 TOD50hnl. Titers on the BB, BF-2, and three grass carp cell lines were low, ranging between 102.8 and 102.1 TCID501m1 (Fig. 2) Extensive CPE was induced in the SHF as well as in the EPC cell lines inoculated with SHRV at an MOI of 1.0. They yielded maximum titers of 108.25 TCID 50/ml. The CPE produced in both cell lines began with the infected cells becoming rounded, followed by detachment and then lysis.The FHM cells produced a moderate titer of 106.65 TCID50 hnl. The CCO, BB, BF-2, and the three grass carp cell lines showed no CPE and yielded low titers of new virus. 44 Growth curve At 2 h post-infection, the concentration of free and total virus was low but increased in an exponential manner after 4 h. The first progeny virus was synthesized 2 to 4 h after infection. The total virus reacheda maximum titer of 108 TCID50 /ml at 12 h post-infection, and the free virus peak was 107.67 TCID50 /m1 at 14 h post-infection.The concentration of free virus was always below that of total virus during the first 12 h but reached a similar plateau between 14 and 24 h (Fig 3).

Biochemical and biophysical characterization No infectious SHRV was detected after chloroform treatment, indicating the presence of essential lipids in the viral envelope. The MEM-0 treated control had a viral titer of 107.3 TCID50/ml. Production of SHRV was not reduced in cells treated with the DNA inhibitor, IUdR. The viral genome was thereby demonstrated to be composed of RNA. When EPC cells infected with SHRV was stained with acridine orange, they were orange-red in color, indicating the presence of a single- stranded RNA genome. Incubation of SHRV for 30 min in MEM adjusted to pH 3.0 reduced the infectious virus titer to 103.67 TCID50/ml, and after a 60 min incubation period, the virus was completely inactivated. At pH 7.0, the viral titer was 107.3 TCID50/ml. A loss of infectivity of 1 log10 or greater in one hour at 56°C is indicative of heat lability (Rovozzo and Burke, 1973). The initial SHRV titer was 107.3 TCID50 /ml, but no infectious virus was detected after 30 min incubation at 56°C, indicating complete inactivation of the virus. 45 The titer of SHRV was decreased 10-fold by three freeze-thaw cycles. The titer of the original suspension was 107.3 TCID50/ml. After three repeated cycles, the viral titer was reduced to 106.4 TCID50/ml.

Electron microscopy Thin sections revealed numerous viral particles in the cytoplasm of infected EPC cells. The new virions were surrounded by an envelope and were released by budding through the EPC membranes (Fig. 4 a). Free virus particles showed a characteristic bacilliform morphology (Fig. 4 b). Twenty-five virus particles were measured with a range from 180 to 200 nm in length and 60 to 70 nm in diameter.

Analysis of structural proteins The separation of the structural proteins of SHRV by SDS-PAGE showed five virion proteins similar to those of the fish rhabdoviruses, IHNV and VHSV (Fig. 5).The five structural protein components of IHNV and VHSV are the L, G, N, M1, and M2 as described by McAllister and Wagner (1975). The G protein of VHSV was larger than that of IHNV and SHRV. The N protein of SHRV was the largest when compared to the other viruses. The M1 protein of SHRV and VHSV had the same molecular weight both of which were smaller than that of IHNV. The M2 protein of IHNV was larger than those of SHRV and VHSV. The estimated values of the molecular weights of the five structural polypeptides (L, G, N, Ml, and M2) of SHRV were >150, 69, 48.5, 26.5, and 20 Kilodaltons (Kd), respectively. The protein with the 46 molecular weight of 69 Kd proved to be the glycoprotein as demonstrated by the detection of sugars in labeled glycoconjugates using the ELISA technique (Fig. 6). 47

FigureIII.1. Replication of snakehead rhabdovirus at selected temperatures, MOI = 0.1 48

108

10 14° C PI 18° C 10 21° C a __,...._24 °C 10 Z 27 °C Ch.... 1:1 10 30°C

E 35 °C 10

10 0 2 4 6

Time (d)

Figure III.1. 49

Figure111.2. Replication of snakehead rhabdovirus in selected cell lines at 27°C, MO1 = 1 .0 50

EPC SHF MINI COO BB BF-2

GCS-2 GCF GCSB

0 2 4 6 8 Time (d)

Figure 111.2. 51

Figure111.3. One-step growth curve of snakehead rhabdovirus in EPC cells incubated at 27°C, MO1 =1.0 52

1091

1081

107-

1061 s-- Total virus 105- Free virus

104 0 10 20 30

Time (h)

Figure 111.3. 53

Figure111.4. Thin sections of EPC cells infected with the snakehead rhabdovirus. (a) Replication of the new viruses in the intracellular space and budding from membranes. Bar equal 200 nm. (b) Free viruses with bacilliform morphology. Bar equal 230 nm. 54

Figure 111.4 55

Figure111.5. Electrophoretic profile of three fish rhabdoviruses. All purified viruses were disrupted and electrophoresed in SDS-polyacrylamide gel as described in the Materials and Methods. Lane 1. Molecular Weight Marker proteins (Bio-rad) shown by arrows in descending order: phosphorylase B (110,000); bovine serum albumin (84,000); ovalbumin (47,000); carbonic anhydrase (33,000), soy bean trypsin inhibitor (24,000), and lysozyme (16,000). Lane 2. Purified snakehead rhabdovirus (SHRV). Lane 3. Purified viral hemorrhagic septicemia virus (VHSV). Lane 4. Purified infectious hematopoietic necrosis virus (IHNV). The gel was stained with Bio-rad silver stain. 56

1 2 3 4 ler-Imp" lomor- -L 110 K 84 K -G 4 7 K

33 K- -M1 24 K-

16 K

Figure 111.5 57

Figure111.6. Enzyme immunoassay of the snakehead rhabdovirus glycoprotein. The glycoprotein was detected on a nitrocellulose membrane. The viral proteins were separated on an SDS-polyacrylamide gel and transferred as described in Materials and Methods. Lane 1. Low molecular weight marker proteins. Lane 2. Purified snakehead rhabdovirus. Lane 3. Tranferrin. Lane 4. Pre-stained Molecular Weight Marker proteins shown by arrows in descending order: phosphorylase B (110,000); bovine serum albumin (84,000); ovalbumin (47,000); carbonic anhydrase (33,000); soybean trypsin inhibitor (24,000); and lysozyme (16,000). 58

1 3 4

11111111 -110K

tit 84 K G- -47K

-33 K

-24 K

16K

Figure 111.6 59 Discussion

The virus isolated from snakeheadwas determined to be a member of the Rhabdoviridae basedon its morphological and biochemical characteristics.Unlike the other fish rhabdoviruses, the SHRV particles had two rounded ends rather thana typical flat base. The Rio Grande perch rhabdovirus is the only fish rhabdovirus described as having a bacilliform morphology (Malsberger and Lautenslager, 1980). The particle size was 180-200nm in length and 60- 70 nm in diameter, and was surrounded by a lipid envelope. Like the other rhabdoviruses, the SHRV was found budding through the cell membranes of the infected cells. Resistance to halogenated pyrimidines indicated the presence of an RNA genome. The viral genomic RNAwas single stranded as demonstrated by acridineorange fluorochrome staining. The virus was labile to pH 3.0, chloroform and heat. Similar characteristics have been reported for this virus by Ahne et al.(1988). The one-step growth curve on EPC cells at 27°C showed that exponential growth began after a 4 h latent period. Both free and total viruses reached a similar plateau between 14 and 24 h, indicating that all of the cell-associated virus had been released. This replicative cycle is similar to that of the spring viremia of carp virus, SVCV, and the pike fry rhabdovirus, PFRV (de Kinkelin and Le Berre, 1974, de Kinkelin et al.,1974), and the optimum temperatures reported for both rhabdoviruses are in the same range as that of SHRV (de Kinkelin and Le Berre, 1974, de Kinkelin et al., 1974). The snakehead fish is reared at temperatures which range between 22-30°C; therefore, it is expected that 60 the SHRV should require similar incubation temperatures and fish cell lines derived from warm water fish for its replication. Results indicate that SHRV replicated in SHF, EPC, FHM and CCO cells, with SHF and EPC yielding the highest titers > 108.25 TC1D50/ml. Replication occurred optimally at 24 to 30°C and no viral replication was detected below 14°C. The SHRV contained five structural protein components similar to IHNV and VHSV, which resemble those of the Lyssavirus, the prototype virus of the family Rhabdoviridae. The estimated molecular weights of five structural polypeptides of SHRV were> 150, 69, 48.5, 26.5, and 20 Kd.The 69 Kd molecular weight protein of SHRVwas glycosylated. Although SHRV contained structural proteins similar to IHNV and VHSV, the SHRV did not grow at temperatures suitable for replication of these salmonid viruses (Wolf, 1988). A second rhabdovirus, ulcerative disease rhabdovirus (UDRV), was isolated from diseased snakehead in southeast Asia (Frerichs et al. 1986). The morphology of this virus differed from the SHRV. The SHRV morphology was bacilliform, but the UDRV had a typical bullet-shape. Moreover, the UDRV isolates produced the highest titer in BF-2 cells and were unable to grow in EPC cells (Frerichs et al., 1989); whereas, SHRV produced a moderate titer on BF-2 cells but grew very well and yielded the maximum titer in the EPC cells. Because of the differences in the morphology and cell lines supporting growth of these two viruses, we proposed the name snakehead rhabdovirus, SHRV, in order to differentiate this virus from the UDRV isolated from the same host. 61

Acknowledgements

We thank Dr. W. Wattanavijarn, Faculty of Veterinary Science, Chulalongkorn University, Thailand for providing the virus used in this study and Dr. H. M. Engelking, Oregon Department of Fish and Wildlife, Department of Microbiology, Corvallis Oregon for his technical assistance in the viral purification and structural proteins analysis. This publication is the result, in part, of research sponsored by Oregon Sea Grant with funds from the National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of Commerce, under grant No. NA 89AA-D-SG 108 (project No. R/FSD-14) and USAID under project identification No. 7-276.This is Oregon Agricultural Experimental Station Technical Paper No. 9598. 62 Literature Cited

Ahne, W., P. E. V. Jorgensen, N. J. Olesen, and W. Wattanavijarn. 1988. Serological examination of a rhabdovirus isolated from snakehead (Ophicephalus striatus) in Thailand with ulcerative syndrome. J. Appl. Icthyol. 4:194-196.

Boonyaratpalin, S. 1989. Bacterial pathogens involved in the epizootic ulcerative syndrome of fish in southeast Asia. J. Aquat. Anim. Health 1: 272-276.

Bowser, P. R., and J. A. Plumb. 1980. Fish cell lines: establishment of a line from ovaries of channel catfish. In Vitro 16: 365-368. de Kinkelin, P., and M. Le Berre. 1974. Rhabdovirus des poissons. II. Proprietes in vitro du virus de la viremie printaniere de la carpe. Ann. Microbiol. (Paris) 125A: 113-124. de Kinkelin, P., M. Le Berre, and G. Lenoir. 1974. Rhabdovirus des poissons. I. Proprietes in vitro de virus de la maladie rouge de l'alevin de brochet. Ann. Microbiol. (Paris) 125A:93-111.

Engelking, H. M., and J. C. Leong. 1989. Glycoprotein from infectious hematopoietic necrosis virus (IHNV) induces protective immunity against five IHNV types. J. Aquat. Anim. Health 1: 291-300.

Feldman, H., and S. Wang. 1961. Sensitivity of various viruses to chloroform. Proc. Soc. Exp. Biol. Med. 106: 736-738. 63 Fijan, N., D. Sulimanovic, M. Bearzotti, D. Muzinic, L. 0. Zwillenberg, S. Chilmonczyk, J. F. Vautherot, and P. de Kinkelin. 1983. Some properties of the Epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio. Ann. Virol. (Inst. Pasteur) 134E: 207-220.

Frerichs, G. N., B. J. Hill, and K.Way. 1989. Ulcerative rhabdovirus: cell-line susceptibility and serological comparison with other fish rhabdoviruses. J. Fish Dis. 12: 51-156.

Frerichs, G. N., S. D. Millar, and R. J. Roberts. 1986. Ulcerative rhabdovirus in fish in south-east Asia. Nature 322: 216.

Gravell, M., and R. G. Malsburger. (1965). A permanent cell line from the fathead minnow (Pimephales promelas). Ann. N. Y. Acad. Sci. 126:555-565.

Hedrick, R. P., W. D. Eaton, J. L. Fryer, W. G. Groberg Jr.,and S. Boonyaratpalin. 1986. Characteristics of a birnavirus isolated from cultured sand goby Oxyeleotris marmoratus. Dis. Aquat. Org. 1:219-225.

Kasornchandra, J., S. Boonyaratpalin, and U. Saduadee. 1988. Fish cell line initiated from snakehead fish (Ophicephalus striatus, Bloch) and its characters. National Inland Fisheries Institute, Department of Fisheries. Technical Paper No. 90. 8p.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685.

Lu, Y., C. N. Lannan, J. S. Rohovec, and J. L. Fryer. 1990. Fish cell lines: establishment and characterization of three new cell lines from grass carp (Ctenopharyngodon idella). In Vitro 26:275-279. 64 Malsberger, R. G., and G. Lautenslager. 1980. Fish viruses: rhabdovirus isolated from a species of the Family Cichlidae. Fish Health News 9: i-ii.

McAllister, P. E., and R. R. Wagner. 1975. Structural proteins of two salmonid rhabdoviruses. J. Virol. 15:733-738.

McAllister, P. E., J. L. Fryer, and K. S. Pilcher. 1974. Further characterization of infectious hematopoietic necrosis virus of salmonid fish (Oregon strain). Arch. Gesamte. Virusforsch. 44:270-279.

Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent end points. Amer. J. Hygiene 27: 493-497.

Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron- opaque stain in electron microscopy. J. Cell Biol. 17:208-212.

Rovozzo, G. C., and C. N. Burke. 1973. A Manual of Basic Virological Techniques. Prentice-Hall, Englewood Cliffs, New Jersy.

Spurr, A. R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastructure Res. 26: 31-43.

Tonguthai, K. 1985. A preliminary account of ulcerative fish diseases in the Indo-Pacific region. Department of Fisheries, Ministry of Agriculture and Cooperatives, Bangkok. 39p.

Wattanavijarn, W., J. Tangtronpiros, and K. Wattanodorn. 1986. Viruses of ulcerative diseased fish in Thailand, Burma and Laos. In: First International Conference on the Impact of Viral Diseases on the Development of Asian Countries, Ambassador Hotel, p 121. December 7-13, 1986. Bangkok, Thailand. 65 Wattanavijarn, W., C. Torchy, J. Tangtronpiros, and P. de Kinkelin. 1988. Isolation of a birnavirus belonging to Sp serotype, from south east Asia fishes. Bull. Eur. Assoc. Fish Pathol. 8:106.

Wolf, K. 1988. Fish Viruses and Fish Viral Diseases. Cornell Press, Ithaca, New York. 476p.

Wolf, K., and M. C. Quimby. 1966. Lymphocystis virus: isolation and propagation in centrarchid fish cell lines. Science 151: 1004-1005. 66

CHAPTER IV

Characteristics of Three Rhabdoviruses Isolated from Snakehead Fish (Ophicephalus striatus)

J. Kasornchandral,H. M. Engelking2, C. N. Lannan3, J. S. Rohovecl, and J. L. Fryerl

1Department of Microbiology, Oregon StateUniversity, Nash Hall 220 Corvallis, Oregon 97331-3084 USA

2Oregon Department of Fish and Wildlife,Department of Microbiology, Oregon State University, Nash Hall 220, Corvallis, Oregon 97331-3804 USA

3Laboratory for Fish Disease Research, Department ofMicrobiology, Oregon State University, Mark 0. Hatfield Marine Science Center, Newport, Oregon 97365-5296 USA

Submitted for publication in the Diseases of Aquatic Organisms 67 Abstract

Three rhabdoviruses were isolated duringa severe epizootic among populations of snakehead fish, Ophicephalus striatus, in southeast Asia. Two were isolated from fish in Thailand, the snakehead rhabdovirus (SHRV) and the ulcerative disease rhabdovirus (UDRV-BP). A third isolate, UDRV-19 was obtained from fish in Burma. The protein profiles and serological characteristics of these three viruses were determined and compared tofive known fish rhabdoviruses.The SHRV exhibited a bacilliform morphology while UDRV-BP and UDRV-19 showed typical rhabdoviral bullet-shaped particles. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the virion protein profile patterns for UDRV-BP and UDRV-19 were indistinguishable but differed from that of SHRV. The protein profile of UDRV-BP and -19 resembled that of vesicular stomatitis virus.However, SHRV had a viral protein composition similar to that of rabies virus, the prototype rhabdovirus of the Lyssavirus genus.The estimated molecular weights of the five SHRV polypeptides were >150, 68, 42, 26.5 and 20 Kilodaltons (Kd). The estimated molecular weights of the five proteins of both UDRV-BP and UDRV-19 were >150, 71, 53, 48, and 22 Kd. The glycosylated proteins were those with molecular weights of 68 Kd (SHRV) and 71 Kd (UDRV- BP and UDRV-19). The UDRV-BP and UDRV-19 were serologically identical but distinct from the six known fish rhabdoviruses, including SHRV. The SHRV was serologically unrelated to any of the other rhabdoviruses as determined by cross neutralization tests. The SHRV 68 was different morphologically, serologically, and in protein composition from UDRV-BP and UDRV-19 even though theywere isolated from the same host. 69 Introduction

Since 1980, a severe epizootic disease has occurredamong both wild and cultured snakehead fish (Ophicephalus striatus) in southeast Asia. A necrotizing ulcerative conditionwas observed in freshwater fish species in Malaysia in late 1980. A major epizootic began in southern Thailand in 1981 and spread throughout the country. By 1985, it had extended to the Lao People's Democratic Republic (PDR) and Burma (Boonyaratpalin, 1989). Various kinds of bacteria, fungi and parasitic organisms, including rhabdoviruses and birnaviruses, were found to be associated with the disease (Boonyaratpalin, 1989, Frerichs et al., 1989, Hedrick et al., 1986, Tonguthai, 1985, Wattanavijarn et al., 1986). At least six rhabdovirus isolations were reported from diseased snakehead fish. The snakehead rhabdovirus (SHRV) was isolated in Thailand by Wattanavijarn et al. (1986), and five isolations of ulcerative disease rhabdovirus (UDRV) were made, three in Thailand and two in Burma by Frerichs et al. (1989) who demonstrated that the five UDRV isolates were serologically homologous.Although the viruses have been individually described in some detail, no comparative studies have been conducted.In this report, the morphology, structural proteins and serological characteristics of the representative isolates (SHRV and UDRV-BP, isolated in Thailand and UDRV-19 from Burma), are described and compared. 70 Materials and Methods

Viruses and cell lines Three rhabdoviruses isolated from snakeheadwere used in this study. The SHRV was obtained from diseased snakehead in Thailandas described by Wattanavijarn et al. (1986); UDRV-BP and UDRV-19were isolated from pooled organs of diseased fish in Thailand and Burma, respectively (Frerichs et al., 1986).The other fish rhabdoviruses, infectious hematopoietic necrosis virus (IHNV); viral hemorrhagic septicemia (VHSV); hirame rhabdovirus (HRV); spring viremia ofcarp virus (SVCV); and pike fry rhabdovirus (PFR)were used for comparison. All rhabdoviruses, except UDRV-BP and UDRV-19,were propagated and titered in the Epithelioma papulosum cyprini (EPC) cell line (Fijan et al., 1983), cultured in Eagle's minimum essential medium with Earle's salts (MEM) (Flow Laboratories Inc., McLean, VA), supplemented with 5% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), and 1% L-glutamine (Flow). The UDRV-BP and UDRV-19 were grown in snakehead (Ophicephalusstriatus) fin (SHF) (Kasornchandra et al., 1987) in Leibovitz's L-15 medium (Gibco Laboratories, Grand Island, NY), supplemented with 5% FBS.In addition, all culture media contained 100 U penicillin and 100 Rg streptomycin/ml (Sigma Chemical Co., St. Louis, MO). 71 Virus infectivity assays The viral titer was determined by end-point dilutionassay using 96-well plates with six wells per dilution. The cellswere incubated for 7 d at 27°C. The cytopathic effect (CPE)was examined and the 50% tissue culture infectious dose (TCID50) calculated by the method of Reedand Muench (1938).

Electron microscopy A confluent monolayer of EPC cells was inoculated with SHRVat a multiplicity of infection (MOI) of 0.1, and incubated at 27°C for 10 h. The culture medium was then decanted, the cell sheet washed three times with Hanks' balanced salt solution (HBSS, Flow), pH 7.4 and fixed with 2.5% glutaraldehyde (Sigma) in HBSS. Followinga 2-h fixation period, the cell sheet was washed three times with 0.2 M cacodylate buffer pH 7.3, harvested with a cellscraper, and centrifuged at 1000 x gravity for 10 min. The cell pellet was post fixed with 1% osmium tetroxide in 0.2 M sodium cacodylate buffer for 1 h, washed, dehydrated, and embedded in Medcast-Aradite 502. The cellswere then sectioned, stained with Methylene Blue-Azure II-Basic Fuchsin, and viewed witha Zeiss EM10/A at 60 kV. The UDRV-BP and UDRV-19were prepared in the same manner, except these viruses were propagated in SHF cells.

Virus purification The viruses (SHRV, IHNV, VHSV, HRV, SVCV, UDRV-BP, UDRV-19, and PFR) were propagated as described. The cell monolayers were inoculated at an MOT of 0.001 for each virus and incubated at 72 suitable temperatures (Wolf, 1988) until the CPE reached completion. The culture fluid was harvested, clarified by low speed centrifigation at 4000 x gravity for 10 min, and the virus concentrated by centrifigation at 80,000 x gravity for 90 min. The viral pellet was resusupended in 0.001M tris-HC1 buffer pH 7.5 and purified on discontinuous and continuous sucrose gradients as described by Engelking and Leong (1989). Finally, the purified virus was resuspended in 0.35 ml tris-HC1 buffer and stored at -70°C.

Analysis of viral structural proteins The mini-PROTEAN II system (Bio-Rad Laboratories, Richmond, CA) was used for separation of SDS-denatured proteins by polyacrylamide gel electrophoresis (SDS-PAGE). The 0.5mm thick gels were composed of a separating gel of 10% polyacrylamide with a stacking gel of 4.75% polyacrylamide (Laemmli, 1970). Before electrophoresis, each of the purified viruses was mixed in SDS- denaturing electrophoresis buffer (0.05M tris buffer pH 6.8, 10% v/v glycerol, 10% w/v SDS, 2-mercaptoethanol, 0.05% w/v bromphenol blue), and boiled for 2 min.Polypeptides were electrophoresed under a constant 200 volts for 45 min. After electrophoretic separation, the gel was silver stained (Bio-Rad).Standard protein molecular weight markers (Bio-Rad) and vesicular stomatitis virus (VSV) were used to compare and determine the molecular weight of the viral proteins. The protein markers included phosphorylase B (94 Kd), bovine serum albumin (68 Kd), ovalbumin (43 Kd), carbonic anhydrase (30 Kd), 73 soybean trypsin inhibitor (21 Kd) and lysozyme (14 Kd) preparedas described by the manufacturer.

Glycosylation of the G protein The glycosylated proteins of SHRV, UDRV-BP, and UDRV-19 were identified by enzyme-linked immunosorbant assay (ELISA) of western blotted proteins.The viruses were purified as previously described and then stored at -70° C. All reagents andassay materials were obtained from a commercial source as a glycan detection kit (Boehringer Mannheim Biochemica, Indianapolis, IN). The procedures were as follows: the hydroxyl groups in sugars of viral glycoconjugates were oxidized by periodate and labeled with digoxygenin (DIG). The labeled viral glycoconjugate was then electrophoresed by SDS-PAGE and transferred onto a nitrocellulose membrane. An ELISAassay using an alkaline phosphatase conjugated antibody was used to detect the DIG- labeled glycoconjugate on the membrane. Transferrinwas used as a positive control and the standard molecular weight protein markers (Bio-Rad) served as a negative control.

Polyclonal antibody production The purified SHRV, HRV, SVCV, PFR, UDRV-BP, and UDRV-19 were mixed separately with an equal amount of Freund's complete adjuvant (Sigma), giving a final concentration of 301.1g/m1 viral protein. Using a Virtis 23 homogenizer equipped with a microblade, the viral mixture was homogenized until the emulsion was complete. Each viral emulsion was injected intraperitoneally into three female 8-week old 74 BALB/c mice. Two booster injections containing 30gg of viral protein mixed with Freund's incomplete adjuvant (Sigma)were given at 1 month intervals.Four days after the second booster, the micewere primed with an injection of 1.0 gg viral protein. Four days later, 3.3x106 sarcoma 180/TG cells in 0.3 ml were injected intraperitoneally using a 1 ml tuberculin syringe with a 23 gauge needle. After 10-15 d, ascitic fluid was collected using a sterile 18 gauge hypodermic needle, then clarified by centrifigation at 1,000 x gravity for 5 min to remove debris and clots. The supernatant fluid was passed through a 0.45 gm sterile membrane filter, then stored in 1.0 ml aliquots at -70°C.

Cross-neutralization test Cross-neutralization was performed by the Alpha method of Rovozzo and Burke (1973). Anti-IHNV rabbit antiserum was prepared and anti-VHSV rabbit antiserum was provided by J. R. Winton (National Fisheries Research Center, Seattle, Washington). Polyclonal antibody against SHRV, HRV, SVCV, UDRV-BP, UDRV-1 9, and PFR was prepared in mice as previously described.Cross-neutralization tests using the end-point dilution assay (TCID50) were conducted in 96-well plates with three wells per dilution. Briefly, ten-fold serial dilutions of each rhabdovirus were prepared in serum free MEM (MEM-0). The polyclonal antibodies were titered, and then stock dilutions were prepared in MEM-0 to concentrations appropriate for neutralization of 100 TCID50's of their respective homologous viruses.Each virus dilution was reacted with an equal volume of the stock polyclonal antibody at 22°C for 1 h. The virus-antibody combination was assayed for 75 infectivity by inoculating 0.2 ml of each dilution into each of three wells of a microplate containing a confluent monolayer of SHFcells for UDRV-BP and UDRV-19 or EPC cells for all other viruses. The IHNV, VHSV and HRV cultures were incubated at 14°C; SVCV and PFRat 20°C; and SHRV, UDRV-BP and UDRV-19 at 27°C.Incubation was continued until obvious CPE had developed in the control wells. The neutralizing titers of the eight fish rhabdoviruseswere calculated and compared by logic, neutralization indices (LNI) whichwere determined as the difference in exponents of the log10 TCID50 titer between the virus dilutions incubated with and without antibody (Rovozzo and Bruke, 1973). 76 Results

Electron microscopy The morphology of SHRV and UDRV was different when observed in thin section by electron microscopy. The SHRV was bacilliform (Fig. la), with a range of 180 to 200 nm in length and 60 to 70 nm in width. Numerous virus particles were found in the cytoplasm of the infected EPC cells and were being released by budding through the cell membranes (Fig. la). The morphology of UDRV-BP and UDRV-19 was identical, but distinct from SHRV, in that the particles showed a typical bullet-shape (Fig lb,c) with the size ranging from 110 to 130 nm in length and 50 to 65 nm in width. Like SHRV, both UDRV-BP and UDRV-19 particles were found in the cytoplasm of the infected cells (SHF) and were released by budding through the cell membranes.

Analysis of viral structural proteins The polypeptides of the nine rhabdoviruses (SHRV, IHNV, VHSV, HRV, SVCV, UDRV-BP, UDRV-19, PFR and VSV) separated by SDS-PAGE gave two distinct groups of patterns when stained with silver nitrate (Fig 2). The first pattern, exhibited by IHNV, VHSV, HRV and SHRV, consisted of five major polypeptides and was similar to that of rabies virus (Cos lett et al., 1980).The second pattern more closely resembled that of VSV, which had a single matrix protein and an additional non-structural protein (Wagner, 1975).The SVCV, PFR, UDRV-BP and UDRV-19 belonged to this group. 77

Figure IV. 1 a. Electron micrograph of an ultrathin section of EPC cells infected with snakehead rhabdovirus. Numerous virus particles are budding through the EPC cell membranes. Complete virions display bacilliform morphology (Bar=260 nm).

Figure IV.1 b. Electron micrograph of an ultrathin section of SHF cells infected with ulcerative disease rhabdovirus-BP (UDRV-BP). Complete virions have typical bullet- shaped morphology (Bar=143 nm).

Figure IV.1c. Electron micrograph of an ultrathin section of SHF cells infected with ulcerative disease rhabdovirus (UDRV-19). Complete virions exhibit typical bullet- shaped particles (Bar=97 nm). 78

Figure IV.1 79

Figure W.2. A comparison of the viral polypeptides of snakehead rhabdovirus (SHRV) and the other eight fish rhabdoviruses in a 10% acrylamide gel. The viral proteins were denatured and electrophoresedas described in Materials and Methods. The gelwas then stained with a silver stain. Lane 1 contains the molecular weight marker proteins. The other lanes are identified as follows: Lane 2, SHRV; Lane 3, infectious hematopoietic necrosis virus (IHNV); Lane 4, viral hemorrhagic septicemia virus (VHSV); Lane 5, hirame rhabdovirus (HRV); Lane 6, spring viremia of carp virus (SVCV); Lane 7, ulcerative disease rhabdovirus (UDRV)-BP; Lane 8, UDRV-19; Lane 9, vesicular stomatitis virus (VSV); and Lane 10, pike fry rhabdovirus (PFR). The SHRV, IHNV, VHSV, and HRV consist of five structural proteins (L, G, N, Ml, M2) similar to rabies virus. The SVCV, UDRV-BP, UDRV-19, and PFR contain polypeptides (L, G, N, and M) similar to VSV. The minor proteins of UDRV-BP and UDRV-19 with molecular weight of 48 Kilodaltons represent the NS protein. 80

7 8 9 10

111111111 111111 oar z

Figure IV.2 81

The relative mobility of the marker proteinswas inversely related to the logarithm of the respective molecular weights in the SDS-PAGE system.The estimated molecular weight of the five virionprotein components of SHRV were > 150, 68, 42, 26.5 and 20 Kilodaltons (Kd). These proteins likely correspond to those previously characterizedas the L, G, N, Mi., and M2 structural proteins of IHNV, VHSV,and HRV (Hsu et al., 1985, Kimura et al., 1989, McAllister & Wagner,1975). In contrast to SHRV, the UDRV-BP and UDRV-19 consisted of four major structural proteins with estimated molecular weights of >150,71, 53, and 22 Kd. These may correspond to the L, G, N and Mproteins of the SVCV and PFR (de Kinkelin et al., 1974, Lenoir, 1973). A minorprotein of both UDRV-BP and UDRV-19 witha molecular weight of 48 Kd was also observed in this gel. The estimated molecular weights of the structural proteins of SHRV, IHNV, VHSV, and HRV were determined (Table 1).Although the banding patterns were similar for these viruses, differencesin the individual proteins were observed.The G protein of SHRVwas intermediate in size to VHSV, the largest, and the smaller G proteins of IHNV and HRV. The N protein of SHRVwas larger than those of the other three viruses, but the SHRV M2 proteinwas the smallest of the four. The M1 proteins of SHRV and VHSVwere similar in size and migrated to a position between those of IHNV and HRV. The estimated molecular weights of the viral proteins of SVCV, PFR, UDRV-BP and UDRV-19 were determined (Table 2). The proteins of the two UDRV isolates were equivalent in size. The NS proteins of SVCV and PFR could not be identified in the preparation comparing 82 these four viruses, but the N and M proteins of the UDRV isolateswere intermediate in size between those of SVCV and PFR, and the UDRVG protein was smaller than either of the other two.

The glycosylation of the G protein The major structural protein of SHRV witha molecular weight of 68 Kd proved to be the glycoprotein as demonstrated by the ELISA technique for detection of sugars in labeled glycoconjugates.Similar results were obtained with the UDRV-BP and UDRV-19 proteins witha molecular weight of 71 Kd.Transferrin, the positive control, also reacted in the test.

Cross-neutralization tests The mouse ascitic fluid contained neutralizing immunoglobulins to each of the respective fish rhabdoviruses (SHRV, HRV, SVCV, PFR, UDRV-BP, and UDRV-19) against which it had been made, and the neutralizing titers were adequate to determine differencesamong the viruses.The greatest neutralizing activity was obtained with SVCV with a titer of 1:126, and the lowest titer was from HRV at 1:64. The mouse ascitic fluid had a toxic effect on the EPC and BF-2 cells; therefore, the lowest dilution permitting detection of viral CPEwas 1:16. The anti-IHNV and anti-VHSV rabbit antisera had neutralizing titers of 1:100 and 1:256, respectively.The eight fish rhabdoviruseswere compared by the cross-neutralization assay. The LNI of anti-SHRV with its homologous virus was 2.0.The SHRV was not significantly neutralized by any neutralizing antisera to the heterologous viruses. 83 The IHNV, VHSV, HRV, SVCV and PFR antibodieswere also highly specific to their homologous viruses and allgave LNI greater than 1.7 (Table 5).No cross neutralizations were observedamong these fish rhabdoviruses. However, between the UDRV-BP and UDRV-19, strong cross neutralization occurred, giving a LNI greater than 1.8. 84

Table W.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)

Virus Polymerase GlycoproteinNucleoproteinMatrix protein

L G N M1 M2

SHRV >150 68 42 26.5 20 IHNV >150 67 40.5 28 22.5 VHSV >150 72 42 26.5 22 HRV >150 60 42.5 30 22 85

Table 1V.2. The estimated values of molecular weights (x103 daltons) of virion proteins of the spring viremia ofcarp virus (SVCV), ulcerative disease rhabdovirus (UDRV- BP), UDRV-19, and pike fry rhabdovirus (PFR)

Virus Polymerase Glycoprotein Nucleoprotein Matrix protein L G N M

SVCV >150 85 45 23

UDRV-BP >150 71 53 22

UDRV-1 9 >150 71 53 22

PFR >150 80 43 24 Table IV.3. Cross-neutralization tests for eight fish rhabdoviruses: SHRV, IHNV, VHSV, HRV, SVCV, UDRV-BP, UDRV-19 and PFRV incubated with homologous and heterogous antibodies.

polyclonal antibodies

Viruses SHRV IHNV VHSV HRVSVCV UDRV-BPUDRV-19 PFRV

neutralizating titer* 1:112 1:100 1:256 1:64 1:126 1:64 1:64 1:96

Snakehead rhabdovirus (SHRV) 2.0** 0 0 0 0 0 0 0 Infectious hematopoietic necrosis virus (IHNV) 0 1.8 0 0 0 0 0 0 Viral hemorrhagic septicemia virus (VHSV) 0 0 1.8 0 0 0 0 0 Hirame rhabdovirus (HRV) 0 0 0 1.8 0 0 0 0 Spring viremia of carp virus (SVCV) 0 0 0 0 2.0 0 0 0 Ulcerative disease rhabdovirus (UDRV-BP) 0 0 0 0 0 2.0 1.9 0 Ulcerative disease rhabdovirus (UDRV-19) 0 0 0 0 0 1.85 2.0 0 Pike fry rhabdovirus (PFRV) 0 0 0 0 0 0 0 1.8

* The neutralizing titer against homologous virus **Cross-neutralization tests for eight of fish rhabdoviruses are expressed as log of neutralization index = log TClD50 of control titer TCED50 of neutralization titer ]; Rovozzo& Burke (1973) 87 Discussion

Six rhabdovirus isolations were made from the diseased snakehead fish in Thailand and Burma duringa severe disease outbreak in southeast Asia. Characteristics of the three representative rhabdovirus isolates, snakehead rhabdovirus (SHRV) and ulcerative disease rhabdovirus (UDRV-BP) from Thailand, and UDRV-19 from Burma were described and compared. Electron microscopic observation of these viruses in thin section showed that both UDRV-BP and UDRV- 19 had a typical bullet-shape similar to that of other fish rhabdoviruses previously described (Hill et al., 1975, Hill et al., 1980, Kimura et al., 1986). The particle size of these two viruseswas the same with a length ranging from 110 to 130 nm and width from 75 to 85nm. In contrast, SHRV exhibited bacilliform morphology with a range of 180 to 200nm in length and 50 to 65 nm in width. The bacilliform-shape is commonly found in association with plant diseases (Hetrick 1989); however, Malberger and Lautenslager (1980) reported the isolation ofa rhabdovirus (Rio Grande perch rhabdovirus) witha bacilliform morphology from a fish species of the Family Cichlidae. The shape and size of the UDRV reported in this study were similar to the observations made by Frerichs et al. (1986) and were different from those of the SHRV. As with the other fish rhabdoviruses, the SHRV and UDRV virions were released by budding through host cell membranes. The eight fish rhabdoviruses (IHNV, SHRV, VHSV, HRV, SVCV, UDRV-BP, UDRV-19, and PFR) can be separated into twogroups based on their SDS-PAGE protein patterns and the molecular weight of 88 their structural proteins. The first group (IHNV, SHRV, VHSV, and HRV) was composed of virions with five structural proteins.These proteins were classified as L for the polymerase, G for the surface glycoprotein, N for the nucleocapsid, and M1 and M2 for the envelope matrix proteins (Leong et al., 1981, Hsu et al., 1985). The virion protein patterns of these viruses closely resembled that of rabies virus (the prototype virus of the Lyssavirus genus of the Family Rhabdoviridae). The second group of viruses contained SVCV, UDRV-BP, UDRV -19 and PFR which were composed of four major structural proteins.In addition, the UDRV-BP and UDRV-1 9 each showeda minor structural protein with a molecular weight of 48 Kd similar to the NS protein of VSV. By analogy with the polypeptides of VSV and the other fish rhabdoviruses, the four major bands may correspond to the L, G, N, and M proteins and the minor band may correspond to the NS protein. Because protein banding patterns of those viruses resemble that of VSV, it is possible to place this group in the Vesiculovirus genus, which hasa single M protein and an additional NS protein (Wagner, 1975). The glycoprotein of rhabdoviruses has been identifiedas the only component of the spikes present on the viral envelope (Cartwright et al., 1970) and can elicit neutralizing antibody as described by Engelking and Leong (1989). Using an ELISA technique detecting sugars in the labeled glycoconjugates, we detected the presence of a glycosylated structural protein associated with the SHRV at the molecular weight of 68 Kd and the UDRV at the molecular weight of 71 Kd. In a western blot, we found that the G protein of the IHNV, SHRV, VHSV, HRV, SVCV, PFR, and UDRV-BP reacted with a monoclonal antibody against SHRV. A similar 89 result was also observed using polyclonal antiserum to the purified G protein of IHNV which reacted with SHRV, IHNV, and VHSV (datanot shown). This suggests there are conserved epitopes in the G protein among these fish rhabdoviruses. The serological comparison of SHRV, UDRV-BP, and UDRV-19 with five other fish rhabdoviruses showed that SHRVwas unique. The UDRV-BP and UDRV-19 were serologically identical and distinct from the other fish rhabdoviruses examined.In addition, our study demonstrated that the structural protein patterns and the molecular weights of the two UDRV isolates were identical, indicating that the UDRV-BP and UDRV-19 are, in fact, isolations of thesame rhabdovirus. Although the SHRV and UDRV were isolated from thesame host, they were different and can be distinguished by their morphology, structural protein patterns, serological characteristics, and the molecular weights of virion proteins.However, both rhabdoviruses isolated from snakehead fish differ from previously described fish rhabdoviruses. Therefore, these two fish rhabdoviruses have been demonstrated to be distinct new virus isolations. 90 Acknowledgements

We thank Dr. W. Wattanavijarn, Chulalongkorn University, Thailand for providing the SHRV; Dr. T. Kimura, Hokkaido University, Japan for providing the viruses, HRV and PFR; and Dr. G. N. Frerichs, University of Stirling, UK for providing the UDRV-BP and UDRV-19. We thank Dr. J. R. Winton, National Fisheries Research Center, Seattle,Washington for providing the anti-VHSV antiserum. We also thank Dr. J. L. Bartholomew for assistance in immunizing mice, and J. R. Duimstra, Veterinary Medicine, OSU for preparation of samples for the transmission electron microscope. This publication is the result, in part, of research sponsored by Oregon Sea Grant with funds from the National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of Commerce, under grant No. NA 89AA-D-SG 108 (project No. R/FSD-14) and USAID under project identification No. 7-276. This is Oregon Agricultural Experiment Station Technical Paper No. 9654. 91 Literature Cited

Boonyaratpalin, S. 1989. Bacterial pathogens involved in the epizootic ulcerative syndrome of fish in southeast Asia. J. Aquat. Anim. Health. 1: 272-276.

Cartwright, B., C. J. Smale, F. Brown, and R. Hull. 1972. A model for vesicular stomatitis virus. J. Virol. 10:256-260.

Coslette, G. D., B. P. Holloway, and J. F. Obijeski. 1980. The structural proteins of rabies virus and evidence for their synthesis from separate monocistronic RNA species. J. Gen. Virol. 49: 161-180.

Engelking, H. M., and J. C. Leong. 1989. Glycoprotein from infectious hematopoietic necrosis virus (IHNV) induces protective immunity against five IHNV types. J. Aquat. Anim. Health 1: 291-300. de Kinkelin, P., M. Le Berre, and G. Lenoir. 1974. Rhabdovirus des poissons.I. proprietes in tro du virus de la maldie ronge de 1' alevin de brochet. Ann. Microbiol. (Inst. Pasteur). 125A:93-111.

Frerichs, G. N., B. J. Hill, and K. Way. 1989. Ulcerative rhabdovirus: cell-line susceptibility and serological comparison with other fish rhabdoviruses. J. Fish Dis. 12:151-156.

Frerichs, G. N., S. D. Miller, and R. J. Roberts. 1986. Ulcerative rhabdovirus in fish in south-east Asia. Nature 322: 216. 92 Hedrick, R. P., W. D. Eaton, J. L. Fryer, W. G. Groberg Jr., and S. Boonyaratpalin. 1986. Characteristics of a birnavirus isolated from cultured sand goby Oxyeleotris marmoratus. Dis. Aquat. Org. 1:219-225.

Hetrick, F. M. 1989. Fish viruses. In: Methods for the microbiological examination of fish and shellfish, (eds. by B. Austin and D. A. Austin), pp.216-239. Ellis Horwood Limited, West Sussex, England.

Hill, B. J., B. 0. Underwood, C. J. Smale, and F. Brown. 1975. Physico- chemical and serological characterizations of five rhabdoviruses infecting fish. J. Gen. Virol. 27:369-378.

Hill, B. J., R. F. Williams, C. J. Smale, B. 0. Underwood, and F. Brown. 1980. Physico-chemical and serological characterization of two rhabdoviruses isolated from eels. Interovirology 14:208-212.

Hsu, Y., H. M. Engelking, and J. C. Leong. 1985. Analysis of the quantity and synthesis of the virion proteins of the infectious hematopoietic necrosis virus. Fish Pathol. 20:331-338.

Kasornchandra, J, S. Boonyaratpalin, and U. Saduadee. 1988. Fish cell line initiated from snakehead fish (Ophicephalus striatus, Bloch) and its characters. National Inland Fisheries Institute, Department of Fisheries, Technical Paper No. 90. 8p.

Kimura, T., M. Yoshimizu, and S. Gorie. 1986. A new rhabdovirus isolated in Japan from cultured hirame (Japanese flouder) Paralichthys olivaceus and ayu Plecoglossus altivelis. Dis. Aquat. Org. 1:209-217.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. 93 Lenoir, G. 1973. Structural proteins of spring viremia virus ofcarp. Biochem. Biophys. Res. Commun. 51:895-898.

Lenoir, G., and P. de Kinkelin. 1975. Fish rhabdoviruses: comparative study of protein structure. J. Virol. 16:259-262.

Leong, J. C., Y. L. Hsu, H. M. Engelking, and D. Mulcahy. 1981. Strains of infectious hematopoietic necrosis (IHN) virusmay be identified by structural protein differences. Develop. Biol. Stand. 49:43-55.

Malsberger, R. G., and G. Lautenslager. 1980. Fish viruses: Rhabdovirus isolated from a species of the Family Cichlidae. Fish Health News 9:i-ii.

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Wattanavijarn, W., J. Tangtronpiros, and K. Wattanadorn. 1986. Viruses of ulcerative diseased fish in Thailand, Burma, and Laos. In: First International Conference on the Impact of Viral Diseases on the Development Countries, Ambassador Hotel, December 7-13, 1986. p. 121. Bangkok, Thailand.

Wolf, K. 1988. Fish Viruses and Fish Viral Diseases. Cornell Press, Ithaca, New York. 476p. 95

CHAPTER V

Development and Characterization of Monoclonal Antibodies Against the Snakehead Rhabdovirus

J. Kasornchandral,P. Caswell-Reno2, C. N. Lannan2, and J. L. Fryer'

'Department of Microbiology, Oregon State University, Nash 220, Corvallis, OR 97331-3804

2Laboratory for Fish Disease Research, Department of Microbiology, Oregon State University, Mark 0. Hatfield Marine Science Center, Newport, OR 97365-5296

Submitted for publication in the Journal of Aquatic Animal Health 96 Abstract

Monoclonal antibodies (MAbs) directed against the snakehead rhabdovirus (SHRV) were produced andcharacterized by immunofluorescence, neutralization tests, and their abilityto immunoprecipitate viral proteins.Of the fifteen MAbs that were successfully developed, nine were isotypedas IgG1 and six were IgG2a. Eight of the MAbs recognized the viral glycoprotein inan immunoprecipitation assay. Three of these, designated E1-9A, PlOC, and 010F, had neutralizing activity. Twelve MAbs showed good binding activity in SHRV-infected EPC cells by immunofluorescence.In an indirect fluorescence assay, the MAbs gave varied staining patterns depending upon the viral structural proteins recognized. 97 Introduction

A rhabdovirus,serologicallydifferent from other fish rhabdoviruses (Ahne et al., 1988, Kasornchandra et al., 1991), was isolated from snakehead (Ophicephalus striatus) during an outbreak of ulcerative disease in southeast Asia (Wattanavijarn et al., 1986). Kasornchandra et al. (1991) found that the snakehead rhabdovirus (SHRV) contained five structural proteins similar to those of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV).Replication of this virus and production of cytopathic effect (CPE) is rapid on susceptible cell lines. On the Epithelioma Papulosum Cyprini (EPC) (Fijan et al., 1983) and the snakehead fin (SHF) (Kasornchandra et al., 1988) cell lines, CPE is apparent within 14 h and complete in 24 to 48 h at 27°C (Kasornchandra et al., 1991). Although polyclonal antibodies were developed for identification of SHRV in a serum neutralization assay, the virus was shown to be a particularly weak antigen.In immunofluorescence assays, the rabbit antiserum had extensive non-specific binding with high levels of background fluorescence both in the control and infected cell lines tested.To improve the methods for identification of this virus, monoclonal antibodies against SHRV have been developed. The purpose of this study was to produce monoclonal antibodies (MAbs) against SHRV to be used in immunodiagnosis. These MAbs were characterized by immunoprecipitation, immunofluorescence, and serum neutralization. 98 Materials and Methods

Virus and cell line Snakehead rhabdovirus was propagated and titered in EPC cells cultured in Eagle's minimum essential medium with Earle's salts (MEM) (Flow Laboratories Inc., McLean, VA), supplemented with 5% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 1% L- glutamine (Flow), 100 U penicillin, and 100 j.ig streptomycin/ml (Sigma Chemical Co., St. Louis, MO). The viral titer was determined by end- point dilution (Rovozzo and Burke, 1973).The 50% tissue culture infectious dose (TCID50) was calculated by the method of Reed and Muench (1938).

Antigen preparation The SHRV was propagated as described.A cell monolayer, inoculated at an multiplicity of infection (MOI) of 0.001, was incubated at 27°C until CPE reached completion.Culture fluid was harvested, clarified by low speed centrifugation (10 min, 4,000 x gravity) and the virus pelleted at 80,000 x gravity for 60 min in an SW 28 rotor (Beckman Instruments, Palo Alto, CA).The viral pellet was resuspended in 0.001M tris-HC1 buffer, pH 7.5, and purified by centrifugation on discontinuous and continuous sucrose gradients as described by Engelking and Leong (1989). The final pellet was resuspended in 0.4 nil tris-HC1 buffer and stored at -70°C. 99 Immunization Three, 8-week-old BALB/c mice were injected intraperitoneally with 0.3 ml of purified SHRV mixed 1:1 with Freund's complete adjuvant (Sigma), giving a final concentration of 30 gghnl viral protein. Two booster injections of purified SHRV mixed 1:1 with Freund's incomplete adjuvant (Sigma) were given at 1 month intervals. Serum was collected from all mice and tested for antibody production using the neutralization assay (Alpha procedure) as described by Rovozzo and Burke (1973). Four days after the second booster, the mice received 0.3 ml intraperitoneally containing 5.6x107 TCID50/ml of SHRV. Five days later, the spleen of an immunized mouse was removed and then fused with myeloma cells.

Fusion and screening of hybridomas Hybridomas were produced by a modification of the methods described by Galfre et al. (1977) and Caswell-Reno et al. (1986). Briefly, spleen cells were fused with Sp2/0 mouse myeloma cells at a ratio of 10:1 with 50% polyethylene glycol 1500 (Sigma) in serum-free growth medium (GM). The GM contained Dulbecco's modified Eagle's MEM (DMEM) supplemented with 10% NCTC 109 medium (Sigma), 1% bovine pancreatic insulin (0.2 units/ml), oxalacetic acid (1mM) and pyruvic acid (0.45mM) (OPI) (Sigma), and 1% L-glutamine (Sigma). Fused cells were resuspended at a concentration of 2.5 x 105 myeloma cells/ml in selective medium (DMEM growth medium supplemented with 20% fetal bovine serum plus hypoxanthine, aminopterin, and thymidine) (GM- HAT), distributed in 96-well microtiter plates (Corning Inc., Corning, 100 NY), and incubated at 37°C in 10% CO2. Thecultures were fed every 7 d with GM-HAT until screened for antibodiesspecific to SHRV. After two weeks, culture fluids were screened usinga dot-immunobinding assay (Hawkes et al., 1982). Hybridomas elicitinga positive reaction were cloned at least twice by limiting dilution. Theculture fluids from single clones were again tested for positive antibodies.Selected hybridomas were expanded into 75 cm2 flasks and maintained in GM plus 10% FBS. Culture fluids from selected hybridomaswere collected and frozen in aliquots at -70°C until used. The immunoglobulinclass and subclass of the monoclonal antibody produced by each hybridomawere determined by an enzyme-linked immunosorbentassay (Mouse monoclonal subisotyping kit; Hyclone Laboratories).

Infection and radioactive labeling of SHRV The method for viral infection and labelingwas described by Engelking (1988) and Ristow and de Avila (1991). Briefly,a 75 cm2 flask of EPC cells was infected with SHRVat an MOI of 10.After 2 h adsorption, the cultureswere rinsed twice with methionine-free Dulbecco's modified Eagle's medium (Calbiochem,San Diego, CA) (DMEM-Met free medium). Fifteen milliliters of thesame medium was added, and the cells incubated for 30 min. Then,5 ml of DMEM-Met free medium supplemented with 5% FBS and 0.5 mCiof 35S methionine (Calbiochem) was added. The culturewas incubated for 8 h at 27°C, then rinsed once with DMEM toremove the excess radiolabel. Uninoculated cells were labeled in thesame manner and included as controls. The monolayer was harvested and pelletedat 200 x gravity for 101

10 min. The pellet was suspended in lysis buffer (0.867g NaC1, 1.17 g EDTA, 0.48 g Tris, 0.5 ml Nonldet P40 in 100 ml distilled water; pH 7.0) and sonicated for three 5 s pulses at 4°C, followed by centrifugationat 134,000 x gravity for 30 min. The lysatewas aliquoted and stored at - 20 °C. The purified SHRV was labeled and preparedas described by Engelking (1988).

Immunoprecipitation and gel electrophoresis Immunoprecipitation of viral proteins was carried out by the solid phase immunoisolation technique (SPIT) described by Tamuraet al. (1984). A 96-well ELISA flat-bottom microtiter plate (Nunc Inter-Med, Denmark) was coated with 100 Ill goat anti-mouse Ig [200 µg/ml (1:50)] in Tris coating buffer (0.1M Tris, pH 9.6), and allowed to incubateat 4°C overnight. The well was washed five times in phosphate buffered saline, pH 7.2, with 0.05% Tween 20 and 0.1% sodium azide (PBS-T), then air dried. The well was blocked by adding 100 gi 1% bovineserum albumin (BSA) in PBS, and incubated for 1 h at ambient temperature. The plate was washed three times with PBS-T and air dried.One hundred microliters of undiluted MAb supernatantwas added to the wells (3 wells each), and incubated at 4°C for 5 h. The platewas then washed three times with PBS-T. Then, 100 11.1 of labeled cell lysatewas added to each well and allowed to incubate at 4°C overnight. The lysatewas then removed, the well washed five times with NET-NO (150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM Tris, pH 8.0, 0.1% BSA, 0.2% sodium azide) and twice with NET-N (NET-NO without BSA). 102

For SDS-polyacrylamide gel electrophoresis, 50 pi ofsample buffer containing 1% SDS, 1% 2-mercaptoethanol, 50 mM Tris,pH 6.8, 10% glyceral, and 0.01% bromphenol bluewas added to each well and allowed to incubate for 15 min. The three wells for each MAbwere combined, then heated for 3 min. The preparations (1,000counts per lane) were electrophoresed for 45 min at constant voltage of 200 voltson discontinuous denaturing SDS-polyacrylamide gelsas described by Laemmli (1970). A 4.75% acrylamide stacking gel and 10% acrylamide separating gel were used. The gel was fixed in 30% methanol and10% acetic acid in double distilled water for 30 min, dried, and then appliedto Hyperfilm-MP (Amersham Corporation, Chicago, IL).The film was developed after 5 to 7 d.

Neutralization tests Neutralizing antibody titers were determined usinga modification of the Alpha procedure of Rovozzo and Burke (1973). Briefly, hybridoma supernate was diluted 1:5 in MEM-0 and mixed inan equal volume with ten-fold serial dilutions of SHRV. After incubation for 1 h at 22°C, the viral mixturewas inoculated into a 96-well plate containing a monolayer of EPC cells. The log10 neutralization indexwas determined as the difference in exponents of the logo TCID50 titer of the virus dilutions incubated with and without antibody. Three neutralizing MAbs were used in cross-neutralization tests withseven fish rhabdoviruses, IHNV, VHSV, hirame rhabdovirus (HRV), spring viremia of carp virus (SVCV), pike fry rhabdovirus (PFR), ulcerative disease rhabdovirus (UDRV)-BP, and UDRV-19).These tests were 103 conducted as described, except the SHF cell line was used to propagate UDRV-BP and UDRV-19.

Fluorescence assays The indirect fluorescent antibody test (IFAT) was modified from that reported by LaPatra et al. (1989). Briefly, EPC cells were grown on sterilized 18-mm-diameter cover glasses placed in separate wells of a 24- well plate (Corning). A stock suspension containing 2.8x103 TCID50/m1 of SHRV was inoculated into each well and allowed to adsorb at 24°C for 1 h. One milliliter of MEM-5 was added. After 10 h incubation at 27°C, the medium was removed and cover glasses rinsed with phosphate buffered saline (PBS), pH 7.2, for 5 min on a rocker platform. The cultures were then fixed with cold acetone (-70°C) for 5 min.Fifty microliters of each MAb supernate was applied and the fixed cells incubated at room temperature for 1 h in a dark, humidified chamber. The cover slips were rinsed for 5 min with PBS and fluorescein isothiocyanate-conjugated goat antimouse serum was applied. After 1 h incubation, they were rinsed with PBS, air dried, mounted in glycerol pH 9.0, and examined with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). 104

Results

Screening of hybridomas Twelve days after fusion, approximately 21% of the 672 wells selected contained hybridomas.In the initial dot-immunobinding assay, about 46% of these hybridomas produced detectable antibodies against SHRV. Subcultivation, followed by repeated screening yielded 15 positive hybridomas (23% of those positive in the first screening). Three MAbs possessed neutralizing activity against homologous virus, and 12 MAbs had binding activity detected by immunoflorescence with SHRV- infected cells.

Immunoglobulin isotype determination Two different subclasses were observed among 15 MAbs produced against SHRV. Nine MAbs were of the IgG1, and six were determined to be of the IgG2a subclass.

Specificity of MAbs to viral polypeptides To determine the specific SHRV polypeptides recognized by the MAbs, the antibodies were bound to an ELISA plate via an anti- immunoglobulin reagent.Labeled cell lysates were reacted with the specific antibody, eluted with a denaturing sample buffer, and then electrophoresed. Autoradiogramsoftheviralprotein immunoprecipitations by the MAbs are presented in Figs. 1, 2, and 3. Of the 15 MAbs tested, eight recognized the G protein, five the N protein, and two the Mi. protein. 105 Titration of antibody Hybridoma culture fluid containing MAb was diluted 1:5 and reacted against SHRV. Three MAbs (E1-9A, P1 0C, and 010F), all of which recognized the G protein, gave logs0 neutralization indices ranging from 1.8 to 1.7. Twelve MAbs were unable to neutralize SHRV even though five of them were bound to the G protein.No toxicity was observed at this dilution. In cross-neutralization tests, the neutralizing MAbs did not react with any heterologous fish rhabdoviruses tested.

Indirect fluorescent antibody technique (IFAT) The IFAT reaction of the MAbs with SHRV-infected EPC cells 10 h post inoculation is illustrated in Fig. 4.Only the non-neutralizing MAbs displayed strong binding to SHRV-infected EPC cells. Antibody against the G protein stained most of the cell structures and provided uniform staining throughout the cell cytoplasm (Fig. 4a). In most of the infected cells, the cisternae-like structures close to the nucleuswere stained as well (Fig 4a).Antibody against the N and M1 proteins provided a coarse granular staining in the cytoplasm of the infected cells (Fig. 4b, c).In addition, the anti-M1 stained the cell membranes (Fig. 4d). No background fluorescence of EPC cells was observed. At 1:500 dilution of the MAbs, these antibodies still displayed strong binding to the SHRV-infected EPC cells.The cross-reactivity of selected MAbs (against the G protein) with six other fish rhabdoviruses (IHNV, VHSV, SVCV, PFR, HRV, UDRV-BP, and UDRV-19) in infected cell lineswas examined using IFAT.Cross-reactivity was detected between fish rhabdoviruses especially with the SVCV, PFR, UDRV-BP, and UDRV- 106 19, only at high concentration of antibodies tested. At 1:100 dilution of the MAbs, no cross-reactivity has been observed. 107

Figure V.1. Immunoprecipitation of snakehead rhabdoviral (SHRV) proteins with monoclonal antibodies (MAbs). The 35S-methionine labeled polypeptides were recovered by immunoprecipitation from infected cell lysates with MAbs and electrophoresed on a 10% acrylamide gel. Lane 1 contains purified SHRV, Lane 2-9 MAbs against the G protein.

Figure V.2. Immunoprecipitation of snakehead rhabdoviral (SHRV)proteins with monoclonal antibodies (MAbs). The 35S-methionine labeled polypeptides were recovered by immunoprecipitation from infected cell lysates with MAbs against SHRV and electrophoresed on a 10% acrylamide gel. Lane 1 contains purified SHRV, Lane 2-5 MAbs aganist the N protein.

Figure V.3. Immunoprecipitation of snakehead rhabdoviral (SHRV) proteins with monoclonal antibodies (MAbs). The 35S-methionine labeled polypeptides were recovered by immunoprecipitation from infected cell lysates with MAbs against SHRV and electrophoresed on a 10% acrylamide gel. Lane 1 contains purified SHRV, Lane 2-3 MAbs against the M1 protein. 108

1 2 3 4 5 6 7 8 9

Figure V.1

1 2 3 1 2 3 4 5 6

L L

G G

N N

M1- 1 M2 M2

Figure V.2 Figure V.3 109

i Figure V.4. Mono layer cultures of epithelioma papulosum cyprini (EPC) cells infected with snakehead rhabdovirus (SHRV) fixed at 10 h post infection and examined by indirect fluorescent antibody test. (a) specific immunofluorescence to SHRV in infected cells with MAb against the G protein. (b) SHRV-infected EPC cells with MAb against the N protein. (c) SHRV- infected EPC cells with MAb against the M1 protein. (d) Ml- specific MAb stained the SHRV-infected EPC cell membranes. 110

Figure V.4 1 1 1

Discussion

Monoclonal antibodies were successfully developed against SHRV. Two groups of MAbs, both neutralizing and non-neutralizing MAbs, were characterized.Three neutralizing MAbs were directed against the G protein as determined by radioimmunoprecipitation. None of the MAbs that recognized the N and M1 viral protein had neutralizing activity.Like the G protein of IHNV and of the mammalian rhabdoviruses, vesicular stomatitis virus (VSV) and rabies virus (Cox et al., 1977, Engelking and Leong, 1989, Kelley et al., 1972), the G protein of SHRV appeared to be responsible for inducing neutralizing antibody as well. Although the rhabdoviral G protein elicits neutralizing antibody, some MAbs specific for the G protein do not produce neutralization. In monoclonal antibody competition assays, Dietzschold et al. (1983) demonstrated that the glycoprotein of rabies virus, CVS strain, had nine distinct epitopes involved in immunogenic activity.Three of these epitopes bind neutralizing MAbs (Seif et al., 1985). On the G protein of VSV, five non-neutralizing and four neutralizing epitopes were defined by MAbs studies (Vandepol et al., 1986). Among our MAbs developed against SHRV, five which recognized the viral G protein in immunoprecipitation were nonneutralizing. In immunoprecipitation assays, the N protein of SHRV displayed two bands, the lower band with a molecular weight of 38 Kilodaltons and the upper band at 42 Kilodaltons, when reacted with the MAbs. The two 1 1 2 forms of the N protein may have a product-precursor relationship or may be a breakdown product of endonuclease cleavage. The MAbs recognizing different viral proteins gave distinct immunofluorescent staining patterns.Similar results have been reported for VHS virus which contains structural proteins similar to those of SHRV (Lorenzen et al., 1988). Lorenzen et al. (1988) discovered that MAbs specific for the G protein of VHSV gave two distinct staining patterns; one involved staining of reticular structures and the other stained cisternae-like structures.In our study, only the characteristic reticular pattern was observed. Lorenzen et al. (1988) suggested that the staining of the reticular structures is an unusual observation. Using electron microscopy and immunofluorescence, Bergmann et al. (1982) and Wehland et al. (1981) found the VSV glycoprotein present in the endoplasmic reticulum, Golgi apparatus, plasmalemma, and cell surface of the VSV-infected cells.Therefore, it is possible that MAbs against SHRV recognize the epitope of the G protein present in cytoplasmic reticulum as found in VSV. The MAbs developed against SHRV have potential as tools for immunodiagnosis and studies of pathogenesis. The standard method for viral identification requires the use of serum neutralization tests, but rabbit anti-SHRV sera have low titers and are highly toxic to the cells. However, the SHRV-neutralizing MAbs provide adequate neutralizing titers with no toxicity. The non-neutralizing MAbs have strong binding activity in IFAT and will be useful in diagnosis and studies of pathogenesis, as well. 1 1 3

Acknowledgements

We thank Dr. J. M. Bertolini, Department of Microbiology, Oregon State University, Mark 0. Hatfield Marine Science Center, Newport Oregon for providing the protocol in immunoprecipitation assay and Dr. H. M. Engelking, Oregon Department of Fish and Wildlife, Department of Microbiology, Corvallis Oregon for his assistance in the viral protein labeling. This publication is the result, in part, of research sponsored by Oregon Sea Grant with funds from the National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of Commerce, under grant No. NA 89AA-D-SG 108 (project No. RIFSD -14) and USAID under project identification No. 7-276. This is Oregon Agriculture Experimental Station Technical Paper No. 9776. 1 1 4 Literature Cited

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1. A new virus was isolated from snakehead fish in Thailand during the ulcerative disease outbreak. Based on morphology and biochemical characteristics, this virus is a member of the family Rhabdoviridae. The virus was named snakehead rhabdovirus (SHRV).

2. The virus replicated optimally at 24-30°C in snakehead fin (SHF) and epithelioma papulosum cyprini (EPC) cell lines. The cytopathic effect produced by this virus 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.

3. SHRV has a bacilliform morphology. The complete virion was 60-70 nm in width and 180-200 nm in lenght and surrounded by a lipid envelope.

4. SHRV was not inhibited by 5- iodo- 2- deoxyuridine, indicatingan RNA genome, and acridine orange staining suggested the presence of single-stranded nucleic acid. The virus was sensitive to chloroform, pH 3, and heat (56°C), but resistant to freeze-thaw.

5. SHRV consisted of five structural proteins with molecular weight of >150, 69, 48.5, 26.6, and 20 Kilodaltons. The viral protein pattern was similar to rabies virus, suggesting SHRV is a member of genus Lyssavirus.

6. The SHRV was serologically unrelated to any of the other fish rhabdoviruses (IHNV, VHSV, HRV, SVCV, UDRV-BP, and UDRV-19) as determined by cross neutralization tests. 1 1 9 7. Fifteen monoclonal antibodies (MAbs)were produced against SHRV. Eight of the MAbs recognized the viral glycoprotein inan immunoprecipitation assay. Of these, three had neutralizing activity. Twelve MAbs showed good binding activity in SHRV- infected EPC cells by immunofluorescence. Five of these MAbs recognized the G protein, five recognized the N protein, and two recognized the M1 protein of SHRV.

8. In an indirect fluorescence assay, the MAbs gave varied staining patterns depending upon the viral structural proteins recognized. MAb against the G protein provided uniform staining throughout the cell cytoplasm including the cisternae-like structure close to the nucleus. MAbs against N and M1 proteins provideda coarse granular staining in the cytoplasm of the infected cells. In addition, anti-M1 also stained the cell membranes. 120 BIBLIOGRAPHY

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