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 PrivacyAbstract 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 Snakehead Fish(Ophicephalus striatus)

byJiraporn Kasornchandra

A THESIS

submitted to

Oregon State University

in partial fulfillment ofthe 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 PrivacyDean 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 to my family- my father Pol,

my mother, Chaleourat; my sister, Chintana; my brothers, Vichai and

Thongchai for their love, guidance, and constant support, without

whom I would not have had a chance to accomplish this longtime

survival in the USA or finishing this goal. Also to Drs. Sitdhi and Mali

Boonyaratpalin for their encouragement, guidance, and support over

the years of my study.

I would like to express my deep appreciation to my major

professor, Dr. J. L. Fryer, for the opportunity to work in the Fish Disease

Laboratory and for his support, encouragement, guidance, and patience

during my three and a half years as 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 all my 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. Bob Olson, Dr. Paul

Reno, and Prudy-Caswell Reno, for their friendship and warmhospitality.

I would like to thank Micheal for proofreading this manuscript.

I do appreciate it.

To Dr. Wattanavijarn for providing the SHRV; Dr. Frerichs for

providing the UDRV-BP and UDRV-19; Dr. Kimura for providing the

HRV and PFR, and to Dr. Winton for providing the anti-VHSV

antiserum.

To Sheri Shenk and special thanks to Julie Duimstra for theirexcellent work in the electron microscopy.

This work is a result of research sponsored by grants 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 USAID project 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 viralpurification and structural protein analysis. P. Caswell-Reno provided

assistance in the monoclonal antibody production in Chapter V.

TABLE OF CONTENTS

Chapter PageI. Introduction 1

II. Review of Literature 3

Literature Cited 21

IV.

V.

Characterization of a Rhabdovirus Isolated fromthe Snakehead Fish (Ophicephalus striatus) 32

Abstract 33Introduction 34Materials and Methods 36Results 43Discussion 59Acknowledgements 61

Literature Cited 62

Characteristics of Three Rhabdoviruses Isolatedfrom Snakehead Fish (Ophicephalus striatus) 66Abstract 67Introduction 69Materials and Methods 70Results 76Discussion 87Acknowledgements 90Literature Cited 91

Development and Characterization of MonoclonalAntibodies against Snakehead RhabdovirusAbstractIntroduction

95

96

97

Chapter Page

V . (Continued)Materials and Methods 98Results 104Discussion 111

Acknowledgements 113Literature Cited 114

SUMMARY AND CONCLUSIONS 118

BIBLIOGRAPHY 120

LIST OF FIGURES

CHAPTER III.

Replication of snakehead rhabdovirus at selectedtemperatures, MOI = 0.1.

111.2 Replication of snakehead rhabdovirus in selectedcell lines at 27°C, MOI = 1.0.

111.3. One-step growth curve of snakehead rhabdovirusin EPC cells incubated at 27°C, MOI = 1.0.

111.4. Thin sections of EPC cells infected with snakeheadrhabdovirus.

111.5. Electrophoretic profile of three fishrhabdoviruses.

111.6 Enzyme immunoassay of the snakeheadrhabdovirus glycoprotein.

CHAPTER IV.

Page

47

49

51

53

55

57

IV.1 a. Electron micrograph of an ultrathin section ofEPC cells infected with snakehead rhabdovirus. 77

IV.1b. Electron micrograph of an ultrathin section ofSHF cells infected with ulcerative diseaserhabdovirus-BP (UDRV-BP). 77

IV.1 c. Electron micrograph of an ultrathin section ofSHF cells infected with ulcerative diseaserhabdovirus-1 9 (UDRV-1 9). 77

IV.2. A comparison of the viral polypeptides of snakeheadrhabdovirus (SHRV) and the other eight fishrhabdoviruses in a 10% acrylamide gel. 79

CHAPTER V. Page

V.1. Immunoprecipitation of snakehead rhabdovirusproteins with monoclonal antibodies recognized theG protein. 107

V.2. Immunoprecipitation of snakehead rhabdovirusrecognized the N proteins. 107

V.3. Immunoprecipitation of snakehead rhabdovirusrecognized the M1 proteins. 107

V.4. Mono layer cultures of EPC cells infected withsnakehead rhabdovirus fixed at 10 h post infectionand examined by indirect fluorescent antibody test. 109

LIST OF TABLES

CHAPTER II. Page

II.1. Comparison of the characteristics among theknown fish rhabdoviruses. 17

CHAPTER IV.

IV.1.

IV.2.

IV.3.

The estimated molecular weights (x103 daltons) ofvirion proteins of the snakehead rhabdovirus (SHRV),infectious hematopoietic necrosis virus (IHNV), viralhemorrhagic septicemia (VHSV), and hiramerhabdovirus (HRV). 84

The estimated values of molecular weights(x103 dalton) of virion proteins of the spring viremiaof carp virus (SVCV),ulcerative disease rhabdovirus(UDRV-BP), UDRV-19, and pike fry rhabdovirus(PFR). 85

Cross-neutralization tests for eight fishrhabdoviruses incubated with homologous andheterologous antibodies. 86

Characterization of a Rhabdovirus Isolated from the Snakehead Fish

(Ophicephalus striatus)

Chapter I

Introduction

A rhabdovirus, serologically different from other fishrhabdoviruses, 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 (Clarias bratachus), and the sand goby (Oxyeleotrismarmoratus) 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 structuralproteins.

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 virus was

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 rhabdoviruses were compared and

their antigenic relatedness determined.

(3). To develop improved methods for diagnosis and viralidentification using monoclonal antibody technology.

Previously developed polyclonal antisera were unsatisfactory for

diagnosis because of low titers and the toxic effect of these sera on

cultured fish cells. Moreover the rabbit polyclonal antisera produced

extensive non-specific binding in fluorescent antibody tests, providing a

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 by a severeulcerative dermal necrosis has been observed in a wide variety offreshwater and estuarine fishes, both wild and pond-cultured,throughout the Indo-Pacific region. The origin of the disease isunknown, but it has been suggested by several investigators that it was

first encountered in Australia in 1972 (Boonyaratpalin, 1989a; Frerichs

et al., 1989, Tonguthai, 1985) when an outbreak of a disease known as

"red spot" occurred in sea mullet and other estuarine and freshwater

fish (McKenzie and Hall, 1976). The ulcerative disease condition also

occurred in 1981 in both freshwater and estuarine species in the 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 freshwater rice 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 submerged cages 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 fish at

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 disease was 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 the appearance 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, in some cases, especially with severely

ulcerated snakehead, the pectoral or ventral fins are completelydestroyed and the internal organs eventually show pathology.

Boonyaratpalin (1989) reported that, in the advanced stage of ulceration,

the internal organs showed a low incidence of septicemia, but an

exudate was often found in the peritoneal and pericardial cavities. Little

evidence of petechial hemorrhage was noted on the surface of the liver

and intestine. Although mortality was 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 fish are

reported to be susceptible to the disease. No ulcerative disease has been

observed in true marine fish (Frerichs et al., 1989). Tonguthai (1985)

reported at least 80 species of fish were 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), Paratransversotrema sp., 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 not appear 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 Saprolegnia sp.

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 of a 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 85 nm in

width and 180 to 230 nm in length, and the plant rhabdoviruses are

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).

Rhabdoviruses consist of two structural units, 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 and a

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 fish cell 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, cell line

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 and group them into

five types (four specific and one less defined). They suggested that aparticular viral isolate was more specific to geographic area than tohost.

2. Viral Hemorrhagic Septicemia Virus

The Egtved virus, designated strain F1, was isolated from the

North American rainbow trout which were introduced into Europe

beginning in 1879 (Jensen, 1965). By electron microscopy, Zwillenberg et

al. (1965) demonstrated that Egtved virus was 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 VHSV was

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 that a 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 hadcharacteristics 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-shape as 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 from an

American eel (A. rostrata), the virus was designated EVA (Sano, 1976).

The second eel rhabdovirus was obtained from an eel (A. anguilla)

imported from Europe and was named EVEX (Sano, 1976). Both viruses

had bullet-shaped morphology; although, the EVA particles were

shorter than those of EVEX. Physicochemical characteristics, serology,

and polypeptides of the two eel rhabdoviruses were 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, Frerichs et al., 1989, Ahne et

al., 1988). No cross-neutralization was observed between UDRV and the

other fish rhabdovirus (Frerichs et al., 1988). Ahne et al. (1988) reported

that the rhabdovirus isolated by Wattanavijarn showed no serological

relatedness to any other fish rhabdovirus. Although these two 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, both in tissueculture and in infected host species. In general, rabbits are used for

producing antisera against viral agents, although goats and sheep are

sometimes employed. Sartorelli et al. (1966) suggested that ascitic fluid

from immunized mice might be another possible source for viralantibodies.

Serum neutralization tests are commonly used in identification of

viral agents (Rovozzo and Bruke, 1973). The tests measure the loss of

viral infectivity resulting from binding of antibody molecules to thesurface 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 toOr incubation °C cell lines heat low pH lipid halogenatedDiseases solvent pyrimidine

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

VHSV bullet 1 8 5x6 5 10-15 BF-2, CHSE-214, EPC, FHM, + + +PG, RTG-2

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

SVCV bullet 90-180x60-90 20-22 BB, BF-2, EPC, FHM, RTG-2 + + +

PFRV bullet 115-135x80 21-28 RTG-2, BB, FHM, EPC + + +

EVA bullet 100-160x75-80 15 RTG-2, EPC, FHM, BF-2, EK-1 + + +EO-2

cod ulcussyndrome bullet 115-195x50-80 15 PS nd nd nd nd

Rio Grandeperchrhabdovirus bacilliform 180x50 23 FHM, BF-2, RTG-2 nd nd +

Perchrhabdovirus bullet 20 Oxl 0 0 14 RTG-2 + nd nd nd

UDRV bullet 110-130x75-85 25-30 BF-2, SSN-1,AS, RSN, CP + nd nd nd

Snakeheadisolation bullet 1 7 Ox6 0 15-28 CLC, SH + + +

nd = no data+ = sensitive

= resistant

Selected references

Wingfield et al. 1969

de Kinkelin and Scherrer 1970;Zwillenberg e al. 1965

Kimura et al. 1986

de Kinkelin et al. 1973de Kinkelin and Le Berre 1974

de Kinkelin et al. 1974

Hill et al. 1980; Sano 1976;Sano et al. 1977

Jensen et al. 1979

Malsberger and Lautenslager1980

Dorson et al. 1980

Frerichs et al. 1986;Frerichs et al. 1989

Ahne et al. 1988

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 slight cross 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 to any

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 and Hill (1984) developed an enzyme-linkedimmunosorbent 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 IPNV or 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 geographic areas rather

than with host species. LaPatra et al. (1990) succesfully developed an

FAT for detection of IHNV in infected tissues using either MAb or

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. (1988)

developed MAbs against four VHSV strain F1 structural proteins (G, N,

M 1 and M2). None of the MAbs neutralized VHSV in plaque

neutralization tests, but showed good binding activities. In addition,

Lorenzen et al. (1988) successfully developed an ELISA for VHSV

detection using the MAbs.

21

Literature Cited

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

Ahne, W., H. Mahnel, and P. Steinhagen. 1982. Isolation of pike fryrhabdovirus from tench, Tinca tinca L., and white bream, Bliccabjoerkna (L). J. Fish Dis. 5:535-537.

Amend, D. F., and V. C. Chambers. 1970. Morphology of certainviruses of salmonid fishes. I. in vitro studies of some virusescausing hematopoietic necrosis. J. Fish. Res. Board Can. 27:1285-1293.

Amend, D. F., W. T. Yasutake, and R. W. Mead. 1969. A hematopoieticvirus disease of rainbow trout and sockeye salmon. Trans. Am.Fish. Soc. 98:796-804.

Bachman, P. A., and W. Ahne. 1974. Biological properties andidentification of the agent causing swim bladder inflammation incarp. Arch. Gesamte Virusforsch. 44:261-269.

Banerjee, A. K. 1987. Transcription and replication of rhabdoviruses.Microbiol. Rev. 51:66-87.

Bishop D. H. L., and M. S. Smith 1977. Rhabdoviruses. In: TheMolecular Biology of Animal Viruses ( ed. by D. P. Nayak),pp 383-434. Marcel Dekker, Inc. New York.

Boonyaratpalin, S. 1987. Bacterial pathogens involved in the epizooticulcerative syndrome of fish in Asia. Department of Fisheries,Ministry of Agriculture and Cooperatives, Bangkok, Thailand. 10p.

22

Boonyaratpalin, S. 1988. A survey of an ulcerative disease condition offish in Sri Lanka. Department of Fisheries, Ministry ofAgriculture and Cooperatives, Bangkok, Thailand. 13p.

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

Boonyaratpalin, S. 1989b. A report on epizootic ulcerative syndromeand recommendations for setting up a fish disease study programin Nepal. Department of Fisheries, Ministry of Agriculture andCooperatives, Bangkok, Thailand. 4p.

Boonyaratpalin, S. 1989c. A report on epizootic ulcerative syndrome offish in India. Department of Fisheries, Ministry of Agricultureand Cooperatives, Bangkok, Thailand. 4p.

Boonyaratpalin, S. (National Institute of Coastal Aquaculture,Songkhla, Thailand). 1990. Personal Communication.

Bootsma, R., and C. J. A. H. van Vorstenbosch. 1973. Detection of abullet-shaped virus in kidney sections of pike fry (Esox lucius L.)with red disease. Neth. J. Vet. Sci. 98:86-90.

Brunson, R., K. True, and J. Yancey. 1989. VHS virus isolated atMakah Nation Fish Hatchery. AFS/FHS Newsletter 17:3-4.

Castric, J., and C. Chastel. 1980. Isolation and characterizationattempts of three viruses from European eel, Anguilla anguilla:preliminary results. Ann. Virol. (Paris) 13E:435-448.

Castric, J., D. Rasschaert, and J. Bernard. 1984. Evidence ofLyssaviruses among rhabdovirus isolates from the European eelAnguilla anguilla. Ann. Virol. (Paris) 135E:35-55.

23

Clerx, J. P. M., B. A. M. van der Zeijst, and M.C. Horzinek. 1975. Somephysicochemical properties of pike fry rhabdovirus RNA. J. Gen.Virol. 29:133-136.

Clerx, J. P., M. C. Horzinek, and A. D. M. E. Osterhaus. 1978.Neutralization and enhancement of infectivity of non-salmonidfish rhabdoviruses by rabbit and pike immune sera. J. Gen.Virol. 40:297-308.

Coates, D. 1984. An ulcer-disease outbreak amongst the freshwater fishpopulation of the Sepik River system, with notes on somefreshwater fish parasites. Department of Primary Industry,Fisheries Research and Surveys Branch, Port Moresby, PapuaNew Guinea, Report No. 84-02.

Cos lett, G. D., B. P. Holloway, and J. F. Obijeski. 1980. The structuralproteins of rabies virus and evidence for their synthesis fromseparate monocistronic RNA species. J. Gen. Virol. 49:161-180.

de Kinkelin, P., and Le M. 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., and R. Scherrer. 1970. Le virus d'Egtved I. Stabilite,developpement et structure du virus de la souche danoise Fl.Ann. Rech. Vet. 1:17-30.

de Kinkelin, P., G. Galimard, and R. Bootsma. 1973. Isolation andidentification of the causative agent of "red disease" of pike (Esoxlucius L. 1766). Nature 241:465-467.

de Kinkelin, P., M. Le Berre, and G. Lenoir. 1974. Rhabdovirus despoissons. I. Proprietes in vitro de virus de la maladie rouge del'alevin de brochet. Ann. Microbiol. (Paris) 125A:93-111.

24Dixon, P. F., and B. J. Hill. 1984. Rapid detection of fish rhabdoviruses

by the enzyme-linked immunosorbent assay (ELISA).Aquaculture 42:1-12.

Dorson, M., C. Torchy, S. Chilmonczyk, P. de Kinkelin, and C. Michel.1984. A rhabdovirus pathogenic for perch, Perca fluviatilis L.:isolation and preliminary study. J. Fish Dis. 7:241-245

Emerson, S. U. 1985. Rhabdoviruses. In: Virology (eds. by B. N.Fields,D. M. Knipe, R. M. Chanock, J. L. Melnick, B. Roizman, and R.E. Shope), pp 1119-1132. Raven Press, New York.

Faisal, M., and W. Ahne. 1984. Spring viraemia of carp (SVCV):comparison of immunoperoxidase, fluorescent antibody and cellculture isolation techniques for detection of antigen. J. Fish Dis.7:57-64.

Fijan, N., Z. Petrinec, D. Sulimanovic, and L. 0. Zwillenberg. 1971.Isolation of the viral causative agent from the acute form ofinfectious dropsy of carp. Vet. Arch. 41:125-138.

Frerichs, G. N., B. J. Hill, and K. Way. 1989. Ulcerative diseaserhabdovirus: cell line susceptibility and serological comparisonwith other fish rhabdoviruses. J. Fish Dis. 12:51-56.

Frerichs, G. N., S. D. Millar, and M. Alexander. 1989. Rhabdovirusinfection of ulcerated fish in South-East Asia. In: Viruses ofLower Vertebrates (eds. by W. Ahne and E. Kurstak), pp 369-410.Springer-Verlag, Berlin.

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

25

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

Hill, B. J., B. 0. Underwood, C. J. Smale, and F. Brown. 1975. Physico-chemical and serological characterizations of five rhabdovirusesinfecting 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 tworhabdoviruses isolated from eels. Interovirology 14:208-212.

Hopper, K. 1989. The isolation of VHSV from chinook salmon atGlenwood Springs, Orcas Island, Washington. AFSIFHSNewsletter 17:1.

Hsu, Y. L., and J. C. Leong. 1985. A comparison of detection methodsfor infectious hematopoietic necrosis virus. J. Fish Dis. 8:1-12.

Hsu, Y., H. M. Engelking, and J. C. Leong. 1986. Occurrence ofdifferent types of infectious hematopoietic necrosis virus in fish.Appl. Environ. Microbiol. 52:1353-1361.

Jensen, M. H. 1965. Research on the virus of Egtved disease. Ann. N.Y. Acad. Sci. 126:422-426.

Jensen, N. J., and J. L. Larsen. 1982. The ulcus-syndrome in cod(Gadus morhua). IV. Tramission experiments with two virusesisolated from cod and Vibrio anguillarum. Nord. Vet. Med.34:136-142.

26Jensen, N. J., B. Bloch, and J. L. Larsen. 1979. The ulcus-syndrome in

cod (Gadus morhua). III. A preliminary virological report.Nord. Vet. Med. 31:436-442.

Jorgensen, P. E. V., N. J. Olesen, N. Lorenzen, J. R. Winton, and S. S.Ristow. 1991 Infectious hematopoietic necrosis (IHN) and viralhemorrhagic septicemia (VHS): detection of trout antibodies to thecausative viruses by means of plaque neutralization,immunoflourescence, and enzyme-liked immunosorbent assay.J. Aquat. Anim. Health. 3:100-108.

Kelley, R. K., B. W. Souter, and H. R. Miller. 1978. Fish cell lines:comparisons of CHSE-214, FHM, and RTG-2 in assaying IHN andIPN viruses. J. Fish. Res. Board Can. 35:1009-1011.

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

Kimura, T., M. Yoshimizu, N. Oseko, and T. Nishizawa. 1989.Rhabdovirus olivaceus (Hirame Rhabdovirus) In: Viruses ofLower Vertebrates (eds. by W. Ahne and E. Kurstak), pp 388-395.Springer-Verlag, Berlin.

Kurath, G., and J. C. Leong. 1985. Characterization of infectioushematopoietic necrosis virus mRNA species reveals a nonvirionrhabdovirus protein. J. Virol. 53:462-468.

LaPatra, S. E., K. A. Roberti, J. S. Rohovec, and J. L. Fryer. 1989.Fluorescent antibody test for the rapid diagnosis of infectioushematopoietic necrosis. J. Aquat. Anim. Health. 1:29-36.

27

Larsen, J. L., and N. J. Jensen. 1977. An Aeromonas speciesimplicated in ulcer-disease of the cod (Gadus morhua). Nord.Vet. Med. 29:199-211.

Lenoir, G., and P. de Kinkelin. 1975. Fish rhabdoviruses: comparativestudy 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) virus may beidentified by structural protein differences. Dev. Biol. Stand. 49:43-55.

Lorenzen, N., N. J. Olesen, and P. E. V. Jorgensen. 1988. Productionand characterization of monoclonal antibodies to four Egtvedvirus structural proteins. Dis. Aquat. Org. 4:35-42.

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

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

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

McCain, B. B., J. L. Fryer, and K. S. filcher. 1974. Antigenicrelationships in a group of three viruses of salmonid fish by crossneutralization. Proc. Soc. Exp. Biol. Med. 137:1042-1046.

McKenzie, R. A., and W. T. K. Hall. 1976. Dermal ulceration of mullet(Mugil cephalus). Aus. Vet. J. 52:230-231.

28

Nishizawa T., M. Yosliimizu, J. Winton, W. Ahne, and T. Kimura.Characterization of structural proteins of hirame rhabdovirus,HRV. Dis. Aquat. Org. 10:167-172.

Pittayangkula, S., and V. Bodhalamik. 1983. The study of Achlya spp.of fish disease in Ophicephalus striatus. In: The Symposium onFreshwater Fish Epidemic: 1982-83, p 23, ChulalongkornUniversity, June 23-24, 1983.

Ristow, S. S., and J. A. de Avila. 1991. Monoclonal antibodies to theglycoprotein and nucleoprotein of infectious hematopoieticnecrosis virus (II-INV) reveal differences among isolates of thevirus by fluorescence, neutralization and electrophoresis. Dis.Aquat. Org. 11:105-115.

Roberts, R. J. 1986. Pathological findings. In: Field and LaboratoryInvestigations into Ulcerative Fish Diseases in the Asia-PacificRegion (ed. by R. J. Roberts), pp 29-35. Technical Report of FAOProject TCP/RAS/4508. Bangkok, Thailand.

Roy, P., H. F. Clark, H. P. Madore, and D. H. L. Bishop. 1975. RNApolymerase associated with virions of pike fry rhabdovirus.J.Virol. 15:338-347.

Rucker, R. R., W. J. Whipple, J. R. Parvin, and C. A. Evans. 1953. Acontagious disease of salmon possibly of virus origin. U.S. FishWildl. Ser. Fish. Bull. 76:35-46.

Sano, T. 1976. Viral diseases of cultured fishes in Japan. Fish Pathol.10:221-226.

Sano, T., and H. Fukuda. 1987. Principle microbial diseases ofmariculture in Japan. Aquaculture 67:59-70.

29

Sano, T., T. Nishimura, N. Okamoto, and H. Fukuda. 1976. Isolation ofrhabdovirus from European eels (Anguilla anguilla) at Japaneseport of entry. Fish Health News. 5:5-6.

Sano, T., T. Nishimura, N. Okamoto, and H. Fukuda. 1977. Studies onviral diseases of Japanese fishes. VII. A rhabdovirus isolatedfrom European eel, Anguilla anguilla. Bull. Jpn. Soc. Sci. Fish.43:491-495.

Sartorelli, A. C., D. S. Fischer, and W. G. Downs. 1966. Use of sarcoma180/TG to prepare hyperimmune ascitic fluid in the mouse. J.Immunol. 96:676-682.

Schultz, C. L., B. C. Lidgerding, P. E. McAllister, and F. M. Hetrick.1985. Production and characterization of monoclonal antibodyagainst infectious hematopoietic necrosis virus. Fish Pathol.20:339-341.

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

Tonguthai, K. 1986. Parasitological findings. In: Field and LaboratoryInvestigations into Ulcerative Fish Diseases in the Asia-PacificRegion (ed. by R. J. Roberts), pp 35-42. Technical Report ofFAO Project TCP/RAS/4508. Bangkok, Thailand.

Van Regenmortel M. H. 1981. Serological methods in the identificationand characterization of viruses. In: Comprehensive Virology(eds. by H. Fraenkel-Conrat and R. R. Wagner), Vol. 17. pp 183-244. Plenum Plublishing Corp., New York.

30Wagner, R. R. 1975. Reproduction of rhabdoviruses. In:

Comprehensive Virology (eds. by H. Fraenkel-Conrat and R. R.Wagner), Vol. 4. pp 1-93. Plenum Plublishing Corp., New York.

Wagner, R. R., L. Prevec, F. Brown, D. F. Summers, F. Sokol, and R.MacLeod. 1972. Classification of rhabdovirus proteins: aproposal. J. Virol. 10:1228-1230.

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 ViralDiseases on the Development of Asian Countries, December 7-13,1986. p 121. Ambassador Hotel, Bangkok, Thailand.

Wattanavijarn, W., C. Torchy, J. Tangtronpiros, and P. de Kinkelin.1988. Isolation of a birnavirus belonging to Sp serotype, fromsoutheast Asia fishes. Bull. Eur. Ass. Fish Pathol. 8:106.

Wingfield, W. H., and L. D. Chan. 1970. Studies on the SacramentoRiver chinook disease and its causative agent. In: A Symposiumof Disease of Fishes and Shellfishes (ed. by S. F. Snieszko),Special Publication No. 5. p 307-318. Washington D.C.

Wingfield, W. H., J. L. Fryer, and K. S. Filcher. 1969. Properties of thesockeye salmon virus (Oregon strain). Proc. Soc. Exp. Biol. Med.130:1055-11059.

Winton, J. R., W. N. Batts, and T. Nishizawa. 1989. Characterization ofthe first north American isolates of viral hemorrhagic septicemiavirus. FHS/AFS Newsletter. 17:2-3.

31

Winton, J. R., C. K. Arakawa, C. N. Lannan, and J. L. Fryer. 1985Neutralizing monoclonal antibodies recognize antigenic variantsamong isolates of infectious hematopoietic necrosis virus. Dis.Aquat. Org. 4:199-204.

Wunner, W. H., B. Dietzschold, and T. J. Wiktor. 1985. Antigenicstructure of rhabdoviruses. In: Immunochemistry of Viruses.The Basis for Serodiagnosis and Vaccines (eds. by M. H. V. vanRegenmortel and A. R. Neurath), pp 367-388. Elsevier SciencePubl. B. V. (Biochemical Division), England.

Yoshimizu, M., T. Nishizawa, N. Oseko, and T. Kimura. 1987.Rhabdovirus disease of hirame (Japanese flounder). Fish Pathol.22:54-55.

Zwillenberg, L. 0., M. H. Jensen, and H. H. L. Zwillenberg. 1965.Electron microscopy of the virus of viral haemorrhagicsepticemia of rainbow trout (Egtved virus). Arch. Virusforsch.17:1-19.

32

CHAPTER III

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

J. Kasornchandral, C. N. Lannan2, J. S. Rohovecland 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 InternationalSymposium on Viruses of Lower Vertebrates

33

Abstract

During a severe epizootic among the snakehead fish,Ophicephalus striatus, in Thailand, a virus belonging to theRhabdoviridae was isolated in cell culture. The virus was bacilliform

60-70 nm in width and 180-200 nm in lenght and surrounded by a 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 polypeptides were > 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 initiallycharacterized 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 used as

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 at an MOI of

0.1. After 12 h incubation at 27°C, the cells were fixed with Carnoy's

fixative and stained with 0.01% acridine orange in Mcllvaine's buffer.

The infected cells were then mounted with buffer and viewed with a

fluorescence microscope.

Stability to low pH

Virus was inoculated into MEM-0 adjusted to pH 3.0 or to pH 7.0.

After 30 and 60 min incubation at 27°C, an aliquot was removed from

each treatment. The aliquot from pH 3.0 was 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 in warm

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 cells was inoculated with SHRV at

an MOI of .001. After 14 h incubation at 27°C, the monolayer was

washed three times with phosphate buffered saline (PBS) and fixed with

2.5% glutaraldehyde (Sigma) in 0.1 M sodium cacodylate buffer, pH 7.4.

Following a 2-h fixation period, the cell sheet was washed, harvested,

and centrifuged at 1000 x gravity for 10 min. The cell pellet was post

fixed with osmium, dehydrated, and embedded in Spurr's medium

(Spurr, 1969). The cells were then sectioned, stained in Reynolds' lead

citrate (Reynolds, 1963), and viewed with a Philips CM12/STEM at 60 kV

in transmission mode.

Virus purification

One liter of spent medium was harvested from infected EPC

cultures when CPE reached completion. The culture fluid wascentrifuged at 4,000 x gravity for 10 min, and the virus concentrated by

centrifugation at 80,000 x gravity for 1 h. The viral pellet was

resuspended in 0.01M tris-HC1 buffer pH 7.5 and purified on

discontinuous and continuous sucrose gradients as described byEngelking and Leong (1989). Following the final centrifugation, the

purified virus was resuspended in 0.35 ml of the tris 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-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 reached a

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

Figure III.1 . Replication of snakehead rhabdovirus at selected

temperatures, MOI = 0.1

108

10

PI10

a10

Z1:1 10

E10

100

__,...._

Ch....

2 4

Time (d)

Figure III.1 .

6

48

14° C

18° C

21° C

24 °C

27 °C

30°C

35 °C

49

Figure 111.2. Replication of snakehead rhabdovirus in selected cell

lines at 27°C, MO1 = 1 .0

0 2 4

Time (d)

Figure 111.2.

6 8

50

EPC

SHF

MINI

COO

BB

BF-2

GCS-2

GCF

GCSB

51

Figure 111.3. One-step growth curve of snakehead rhabdovirus in

EPC cells incubated at 27°C, MO1 =1.0

109 1

108 1

107 -

1061

105-

104

s-- Total virusFree virus

0 10 20

Time (h)

Figure 111.3.

30

52

Figure 111.4.

53

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

Figure 111.5.

55

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

110 K

84 K

4 7 K

33 K-

24 K-

16 K

1 2 3 4ler-Imp" lomor-

-L

-G

N

-M1

Figure 111.5

Figure 111.6.

57

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

G-

3 4

11111111

-110K

tit 84 K

-47K

-33 K

-24 K

16K

Figure 111.6

59

Discussion

The virus isolated from snakehead was determined to be amember of the Rhabdoviridae based on its morphological andbiochemical characteristics. Unlike the other fish rhabdoviruses, the

SHRV particles had two rounded ends rather than a 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 180-200 nm 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 RNA was

single stranded as demonstrated by acridine orange 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 bothrhabdoviruses 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 SHRV was

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 fromsnakehead (Ophicephalus striatus) in Thailand with ulcerativesyndrome. J. Appl. Icthyol. 4:194-196.

Boonyaratpalin, S. 1989. Bacterial pathogens involved in the epizooticulcerative 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 aline 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 despoissons. I. Proprietes in vitro de virus de la maladie rouge del'alevin de brochet. Ann. Microbiol. (Paris) 125A:93-111.

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

Feldman, H., and S. Wang. 1961. Sensitivity of various viruses tochloroform. 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) cellline 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 fishrhabdoviruses. J. Fish Dis. 12: 51-156.

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

Gravell, M., and R. G. Malsburger. (1965). A permanent cell line fromthe 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 isolatedfrom cultured sand goby Oxyeleotris marmoratus. Dis. Aquat.Org. 1:219-225.

Kasornchandra, J., S. Boonyaratpalin, and U. Saduadee. 1988. Fishcell 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 theassembly 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 celllines: establishment and characterization of three new cell linesfrom 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. FishHealth News 9: i-ii.

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

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

Reed, L. J., and H. Muench. 1938. A simple method of estimating fiftypercent 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 VirologicalTechniques. Prentice-Hall, Englewood Cliffs, New Jersy.

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

Tonguthai, K. 1985. A preliminary account of ulcerative fish diseasesin the Indo-Pacific region. Department of Fisheries, Ministry ofAgriculture 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 ViralDiseases on the Development of Asian Countries, AmbassadorHotel, 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, fromsouth 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 andpropagation 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 State University, 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 of Microbiology,

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 during a 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 to five known fishrhabdoviruses. 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 they were isolated from the

same host.

69

Introduction

Since 1980, a severe epizootic disease has occurred among both

wild and cultured snakehead fish (Ophicephalus striatus) in southeast

Asia. A necrotizing ulcerative condition was 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 snakehead were used in this

study. The SHRV was obtained from diseased snakehead in Thailand as

described by Wattanavijarn et al. (1986); UDRV-BP and UDRV-19 were

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 of carp

virus (SVCV); and pike fry rhabdovirus (PFR) were used forcomparison.

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 (Ophicephalus striatus) 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 dilution assay using

96-well plates with six wells per dilution. The cells were 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 Reed and

Muench (1938).

Electron microscopy

A confluent monolayer of EPC cells was inoculated with SHRV at

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. Following a 2-h fixation

period, the cell sheet was washed three times with 0.2 M cacodylate

buffer pH 7.3, harvested with a cell scraper, 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 cells were then sectioned,

stained with Methylene Blue-Azure II-Basic Fuchsin, and viewed with a

Zeiss EM10/A at 60 kV. The UDRV-BP and UDRV-19 were 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 bypolyacrylamide gel electrophoresis (SDS-PAGE). The 0.5 mm 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) prepared as

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 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 digoxygenin (DIG). The

labeled viral glycoconjugate was then electrophoresed by SDS-PAGE and

transferred onto a nitrocellulose membrane. An ELISA assay using an

alkaline phosphatase conjugated antibody was used to detect the DIG-

labeled glycoconjugate on the membrane. Transferrin was 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 30 gg of viral protein

mixed with Freund's incomplete adjuvant (Sigma) were given at 1month intervals. Four days after the second booster, the mice were

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 SHF cells 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 PFR at20°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 rhabdoviruses were calculated and

compared by logic, neutralization indices (LNI) which were 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.

Figure IV.1c.

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).

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).

Figure IV.1

78

Figure W.2.

79

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 electrophoresed as

described in Materials and Methods. The gel was

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

z

7 8 9 10

111111111 111111

oar

Figure IV.2

81

The relative mobility of the marker proteins was inversely related

to the logarithm of the respective molecular weights in the SDS-PAGE

system. The estimated molecular weight of the five virion protein

components of SHRV were > 150, 68, 42, 26.5 and 20 Kilodaltons (Kd).

These proteins likely correspond to those previously characterized as theL, 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 M proteins of the

SVCV and PFR (de Kinkelin et al., 1974, Lenoir, 1973). A minor protein

of both UDRV-BP and UDRV-19 with a 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, differences in the

individual proteins were observed. The G protein of SHRV was

intermediate in size to VHSV, the largest, and the smaller G proteins of

IHNV and HRV. The N protein of SHRV was larger than those of the

other three viruses, but the SHRV M2 protein was the smallest of the

four. The M1 proteins of SHRV and VHSV were 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 isolates were

intermediate in size between those of SVCV and PFR, and the UDRV G

protein was smaller than either of the other two.

The glycosylation of the G protein

The major structural protein of SHRV with a 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 with a

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 differences among 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 CPE was 1:16.

The anti-IHNV and anti-VHSV rabbit antisera had neutralizing titers of

1:100 and 1:256, respectively. The eight fish rhabdoviruses were

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 antibodies were also highly

specific to their homologous viruses and all gave LNI greater than 1.7

(Table 5). No cross neutralizations were observed among these fish

rhabdoviruses. However, between the UDRV-BP and UDRV-19, strong

cross neutralization occurred, giving a LNI greater than 1.8.

Table W.1.

84

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 Glycoprotein Nucleoprotein Matrix 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

Table 1V.2.

85

The estimated values of molecular weights (x103

daltons) of virion proteins of the spring viremia of carp

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 heterogousantibodies.

polyclonal antibodies

Viruses

neutralizating titer*

SHRV

1:112

IHNV

1:100

VHSV

1:256

HRV

1:64

SVCV

1:126

UDRV-BP

1:64

UDRV-19

1:64

PFRV

1:96

Snakehead rhabdovirus(SHRV) 2.0** 0 0 0 0 0 0 0Infectious hematopoietic necrosisvirus(IHNV) 0 1.8 0 0 0 0 0 0Viral hemorrhagic septicemia virus(VHSV) 0 0 1.8 0 0 0 0 0Hirame rhabdovirus(HRV) 0 0 0 1.8 0 0 0 0Spring viremia of carp virus(SVCV) 0 0 0 0 2.0 0 0 0Ulcerative disease rhabdovirus(UDRV-BP) 0 0 0 0 0 2.0 1.9 0Ulcerative disease rhabdovirus(UDRV-19) 0 0 0 0 0 1.85 2.0 0Pike 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 diseasedsnakehead fish in Thailand and Burma during a 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 viruses was the same with a length

ranging from 110 to 130 nm and width from 75 to 85 nm. In contrast,

SHRV exhibited bacilliform morphology with a range of 180 to 200 nm 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 of a

rhabdovirus (Rio Grande perch rhabdovirus) with a 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 two groups

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 showed a 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 has a

single M protein and an additional NS protein (Wagner, 1975).

The glycoprotein of rhabdoviruses has been identified as 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 (data not

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 SHRV was unique. The

UDRV-BP and UDRV-19 were serologically identical and distinct from

the other fish rhabdoviruses examined. In addition, our studydemonstrated 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 the same rhabdovirus.

Although the SHRV and UDRV were isolated from the same 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 epizooticulcerative 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 forvesicular stomatitis virus. J. Virol. 10:256-260.

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

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

de Kinkelin, P., M. Le Berre, and G. Lenoir. 1974. Rhabdovirus despoissons. I. proprietes in tro du virus de la maldie ronge de 1'alevin de brochet. Ann. Microbiol. (Inst. Pasteur). 125A:93-111.

Fijan, N., D. Sulimanovic, M. Bearzotti, D. Muzinic, L. 0. Zwillenberg,S. Chilmonczyk, J. F. Vautherot, and P. de Kinkelin. 1983. Someproperties of the Epithelioma papulosum cyprini (EPC) cell linefrom 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 fishrhabdoviruses. J. Fish Dis. 12:151-156.

Frerichs, G. N., S. D. Miller, and R. J. Roberts. 1986. Ulcerativerhabdovirus 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 isolatedfrom cultured sand goby Oxyeleotris marmoratus. Dis. Aquat.Org. 1:219-225.

Hetrick, F. M. 1989. Fish viruses. In: Methods for the microbiologicalexamination 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 rhabdovirusesinfecting 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 tworhabdoviruses isolated from eels. Interovirology 14:208-212.

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

Kasornchandra, J, S. Boonyaratpalin, and U. Saduadee. 1988. Fish cellline 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 rhabdovirusisolated 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 theassembly of the head of bacteriophage T4. Nature 227:680-685.

93

Lenoir, G. 1973. Structural proteins of spring viremia virus of carp.Biochem. Biophys. Res. Commun. 51:895-898.

Lenoir, G., and P. de Kinkelin. 1975. Fish rhabdoviruses: comparativestudy 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) virus may beidentified 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.

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

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

Rovozzo, G. C., and C. N. Burke. 1973. A Manual of Basic VirologicalTechniques. Prentice-Hall, New Jersey. 287p.

Tonguthai, K. 1986. Parasitological findings. In: Field and laboratoryinvestigations into ulcerative fish diseases in the Asia-Pacificregion, p35-42. Technical Report of FAO Project TCP/RAS/4508.

Wagner, R. R. 1975. Reproduction of rhabdoviruses. In:Comprehensive Virology (eds. by H. Fraenkel-Conrat and R. R.Wagner), Vol. 4. pp 1-93. Plenum Plublishing Corp., New York.

94

Wagner, R. R., L. Prevec, F. Brown, D. F. Summers, F. Sokol, and R.MacLeod. 1972. Classification of rhabdovirus proteins: aproposal. J. Virol. 10:1228-1230.

Wattanavijarn, W., J. Tangtronpiros, and K. Wattanadorn. 1986.Viruses of ulcerative diseased fish in Thailand, Burma, andLaos. In: First International Conference on the Impact of ViralDiseases 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 and characterized byimmunofluorescence, neutralization tests, and their ability to

immunoprecipitate viral proteins. Of the fifteen MAbs that were

successfully developed, nine were isotyped as IgG1 and six were IgG2a.

Eight of the MAbs recognized the viral glycoprotein in an

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, serologically different from other fishrhabdoviruses (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. The cultures were fed every 7 d

with GM-HAT until screened for antibodies specific to SHRV. After two

weeks, culture fluids were screened using a dot-immunobinding assay

(Hawkes et al., 1982). Hybridomas eliciting a positive reaction were

cloned at least twice by limiting dilution. The culture 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 hybridomas were collected and frozen in

aliquots at -70°C until used. The immunoglobulin class and subclass of

the monoclonal antibody produced by each hybridoma were determined

by an enzyme-linked immunosorbent assay (Mouse monoclonalsubisotyping kit; Hyclone Laboratories).

Infection and radioactive labeling of SHRV

The method for viral infection and labeling was described by

Engelking (1988) and Ristow and de Avila (1991). Briefly, a 75 cm2 flask

of EPC cells was infected with SHRV at an MOI of 10. After 2 hadsorption, the cultures were rinsed twice with methionine-freeDulbecco's modified Eagle's medium (Calbiochem, San Diego, CA)

(DMEM-Met free medium). Fifteen milliliters of the same 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 mCi of 35S methionine

(Calbiochem) was added. The culture was incubated for 8 h at 27°C,

then rinsed once with DMEM to remove the excess radiolabel.Uninoculated cells were labeled in the same manner and included as

controls. The monolayer was harvested and pelleted at 200 x gravity for

101

10 min. The pellet was suspended in lysis buffer (0.867 g 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 centrifugation at134,000 x gravity for 30 min. The lysate was aliquoted and stored at -

20 °C. The purified SHRV was labeled and prepared as described by

Engelking (1988).

Immunoprecipitation and gel electrophoresis

Immunoprecipitation of viral proteins was carried out by the solid

phase immunoisolation technique (SPIT) described by Tamura et 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 incubate at 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% bovine serum 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 supernatant was added to the wells (3

wells each), and incubated at 4°C for 5 h. The plate was then washed

three times with PBS-T. Then, 100 11.1 of labeled cell lysate was added to

each well and allowed to incubate at 4°C overnight. The lysate was 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 of sample

buffer containing 1% SDS, 1% 2-mercaptoethanol, 50 mM Tris, pH 6.8,

10% glyceral, and 0.01% bromphenol blue was added to each well and

allowed to incubate for 15 min. The three wells for each MAb were

combined, then heated for 3 min. The preparations (1,000 counts per

lane) were electrophoresed for 45 min at constant voltage of 200 volts on

discontinuous denaturing SDS-polyacrylamide gels as described by

Laemmli (1970). A 4.75% acrylamide stacking gel and 10% acrylamide

separating gel were used. The gel was fixed in 30% methanol and 10%

acetic acid in double distilled water for 30 min, dried, and then applied to

Hyperfilm-MP (Amersham Corporation, Chicago, IL). The film was

developed after 5 to 7 d.

Neutralization tests

Neutralizing antibody titers were determined using a

modification of the Alpha procedure of Rovozzo and Burke (1973).

Briefly, hybridoma supernate was diluted 1:5 in MEM-0 and mixed in an

equal volume with ten-fold serial dilutions of SHRV. After incubation

for 1 h at 22°C, the viral mixture was inoculated into a 96-well plate

containing a monolayer of EPC cells. The log10 neutralization index was

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 with seven fishrhabdoviruses, 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. Autoradiograms of the viral protein

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 logs 0 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 nucleus were

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 lines was

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.

Figure V.1.

Figure V.2.

Figure V.3.

107

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.

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.

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.

L

G

M1

M2

1 2 3 4 5 6 7 8 9

Figure V.1

L

G

N

M2

1 2 3 4 5 6

Figure V.2

108

L

G

N

M1-1

M2

1 2 3

Figure V.3

i

Figure V.4.

109

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 themammalian 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 inimmunoprecipitation 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

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

Bergmann, J. E., K. T. Tokoyasu, and S. J. Singer. 1982. Passage of anintegral membrane protein, the vesicular stomatitis virusglycoprotein, through the Golgi apparatus en route to plasmamembrane. Proc. Natl. Acad. Sci. USA. 78:1746-1750.

Caswell-Reno, P., P. W. Reno, and B. L. Nicholson. 1986. Monoclonalantibodies to infectious pancreatic necrosis virus: analysisof viral epitopes and comparison of different isolates. J. Gen.Virol. 67:2193-2205.

Cox, J. H., B. Dietzschold, and L. G. Schneider. 1977. Rabies virusglycoprotein II. biological and serological characterization.Infect. Imm. 16:754-759.

Dietzschold, W. H., T. J. Wikor, A. D. Lopes, M. Lafon, C. Smith, and H.Koprowski. 1983. Characterization of an antigenic determinantof the glycoprotein that correlates with pathogenicity of rabiesvirus. Proc. Natl. Acad. Sci. USA 80:70-74.

Engelking, H. M. 1988. Properties of the glycoprotein of infectioushematopoietic necrosis virus. Ph.D. thesis, Oregon StateUniversity, Corvallis, Oregon. 153p.

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

1 1 5

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) cellline from carp Cyprinus carpio. Ann. Virol. (Inst. Pasteur)134E: 207-220.

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

Galfre, G., S. C. Howe, C. Milstein, G. W. Butcher, and J. C. Howard.1977. Antibodies to major histocompatibility antigens produced byhybrid cell lines. Nature 266:550-552.

Hawkes, R., E. Niday, and J. Godon. 1982. A dot-immunoblotting assayfor monoclonal and other antibodies. Anal. Biochem. 119:142-147.

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

Kasornchandra, J., C. N. Lannan, J. S. Rohovec, and J. L. Fryer. 1991.Characterization of a rhabdovirus isolated from the snakeheadfish (Ophicephalus striatus) In: Proceeding of the SecondInternational Symposium on Viruses of Lower Vertebrates, July29-31, 1991, Corvallis, Oregon.

Kelley, J. M., S. U. Emerson, and R. R. Wagner. 1972. The glycoproteinof vesicular stomatitis virus is the antigen that gives rise to andreacts with neutralizing antibody. J. Virol. 10:1231-1235.

1 1 6

LaPatra, S. E., K. A. Roberti, J. S. Rohovec, and J. L. Fryer. 1989.Fluorescent antibody test for the rapid diagnosis of infectioushematopoietic necrosis. J. Aquat. Anim. Health. 1:29-36.

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

Lorenzen, N., N. J. Olesen, and P. E. V. Jorgensen. 1988. Productionand characterization of monoclonal antibodies to four Egtvedvirus structural proteins. Dis. Aquat. Org. 4:35-42.

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

Ristow, S. S., and J. A. de Avila. 1991. Monoclonal antibodies to theglycoprotein and nucleoprotein of infectious hematopoieticnecrosis virus (IHNV) reveal differences among isolates of thevirus by fluorescence, neutralization and electrophoresis. Dis.Aquat. Org. 11:105-115.

Rovozzo, G. C., and C. N. Burke. 1973. A Manual of Basic VirologicalTechniques. Prentice-Hall, New Jersey. 287p.

Seif, I., P. Conlon, P. E. Rollin, and A, Flamand. 1985. Rabiesvirulence: effect of pathogenicity and sequence characterization ofrabies virus mutations affecting antigenic site III of theglycoprotein. J. Virol. 53:926-934.

Tamura, G. S., M. 0. Dailey, W. M. Gallatin, M. S. McGrath, I. L.Weissman, and E. A. Pillemer. 1984. Isolation of moleculesrecognized by monoclonal antibodies and antisera: the solid phaseimmunoisolation technique. Anal. Biochem. 136:458-464.

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Vandepol, S. B., L. Lefrancois, and J. J. Holland. 1986. Sequences of themajor antibody binding epitopes of the Indiana serotypes ofvesicular stomatitis virus. Virology 148:312-325.

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 ViralDiseases on the Development of Asian Countries, AmbassadorHotel, p 121. December 7-13, 1986. Bangkok, Thailand.

Wehland, J., M. C. Willingham, M. G. Gallo, and I. Pastan. 1981. Themorphologic pathway of exocytosis of the vesicular stomatitisvirus G protein in cultured fibroblast. Cell 28:831-841.

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SUMMARY AND CONCLUSIONS

1. A new virus was isolated from snakehead fish in Thailandduring the ulcerative disease outbreak. Based on morphology andbiochemical characteristics, this virus is a member of thefamily Rhabdoviridae. The virus was named snakeheadrhabdovirus (SHRV).

2. The virus replicated optimally at 24-30°C in snakehead fin (SHF)and epithelioma papulosum cyprini (EPC) cell lines. Thecytopathic effect produced by this virus began with infected cellsbecoming rounded, followed by detachment and then lysis.Exponential growth of SHRV on EPC cells at 27°C began after a 4h latent period and reached a maximum plateau at 12 h postinfection.

3. SHRV has a bacilliform morphology. The complete virionwas 60-70 nm in width and 180-200 nm in lenght and surroundedby a lipid envelope.

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

5. SHRV consisted of five structural proteins with molecular weightof >150, 69, 48.5, 26.6, and 20 Kilodaltons. The viral proteinpattern was similar to rabies virus, suggesting SHRV is amember of genus Lyssavirus.

6. The SHRV was serologically unrelated to any of the other fishrhabdoviruses (IHNV, VHSV, HRV, SVCV, UDRV-BP, andUDRV-19) as determined by cross neutralization tests.

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7. Fifteen monoclonal antibodies (MAbs) were produced againstSHRV. Eight of the MAbs recognized the viral glycoprotein in animmunoprecipitation assay. Of these, three had neutralizingactivity. Twelve MAbs showed good binding activity in SHRV-infected EPC cells by immunofluorescence. Five of these MAbsrecognized the G protein, five recognized the N protein, and tworecognized the M1 protein of SHRV.

8. In an indirect fluorescence assay, the MAbs gave varied stainingpatterns depending upon the viral structural proteins recognized.MAb against the G protein provided uniform staining throughoutthe cell cytoplasm including the cisternae-like structure close tothe nucleus. MAbs against N and M1 proteins provided a coarsegranular staining in the cytoplasm of the infected cells. Inaddition, anti-M1 also stained the cell membranes.

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BIBLIOGRAPHY

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

Ahne, W., H. Mahnel, and P. Steinhagen. 1982. Isolation of pike fryrhabdovirus from tench, Tinca tinca L., and white bream, Bliccabjoerkna (L). J. Fish Dis. 5:535-537.

Amend, D. F., and V. C. Chambers. 1970. Morphology of certainviruses of salmonid fishes. I. in vitro studies of some virusescausing hematopoietic necrosis. J. Fish. Res. Board Can. 27:1285-1293.

Amend, D. F., W. T. Yasutake, and R. W. Mead. 1969. A hematopoieticvirus disease of rainbow trout and sockeye salmon. Trans. Am.Fish. Soc. 98:796-804.

Bachman, P. A., and W. Ahne. 1974. Biological properties andidentification of the agent causing swim bladder inflammation incarp. Arch. Gesamte Virusforsch. 44:261-269.

Banerjee, A. K. 1987. Transcription and replication of rhabdoviruses.Microbiol. Rev. 51:66-87.

Bergmann, J. E., K. T. Tokoyasu, and S. J. Singer. 1982. Passage of anintegral membrane protein, the vesicular stomatitis virusglycoprotein, through the Golgi apparatus en route to plasmamembrane. Proc. Natl. Acad. Sci. USA. 78:1746-1750.

1 2 1

Bishop D. H. L., and M. S. Smith 1977. Rhabdoviruses. In: TheMolecular Biology of Animal Viruses ( ed. by D. P. Nayak),pp 383-434. Marcel Dekker, Inc. New York.

Boonyaratpalin, S. 1987. Bacterial pathogens involved in the epizooticulcerative syndrome of fish in Asia. Department of Fisheries,Ministry of Agriculture and Cooperatives, Bangkok, Thailand. 10p.

Boonyaratpalin, S. 1988. A survey of an ulcerative disease condition offish in Sri Lanka. Department of Fisheries, Ministry ofAgriculture and Cooperatives, Bangkok, Thailand. 13p.

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

Boonyaratpalin, S. 1989b. A report on epizootic ulcerative syndromeand recommendations for setting up a fish disease study programin Nepal. Department of Fisheries, Ministry of Agriculture andCooperatives, Bangkok, Thailand. 4p.

Boonyaratpalin, S. 1989c. A report on epizootic ulcerative syndrome offish in India. Department of Fisheries, Ministry of Agricultureand Cooperatives, Bangkok, Thailand. 4p.

Boonyaratpalin, S. (National Institute of Coastal Aquaculture,Songkhla, Thailand). 1990. Personal Communication.

Bootsma, R., and C. J. A. H. van Vorstenbosch. 1973. Detection of abullet-shaped virus in kidney sections of pike fry (Esox lucius L.)with red disease. Neth. J. Vet. Sci. 98:86-90.

Bowser, P. R., and J. A. Plumb. 1980. Fish cell lines: establishment ofaline from ovaries of channel catfish. In Vitro 16:365-368.

122

Brunson, R., K. True, and J. Yancey. 1989. VHS virus isolated atMakah Nation Fish Hatchery. AFS/FHS Newsletter 17:3-4.

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

Castric, J., and C. Chastel. 1980. Isolation and characterizationattempts of three viruses from European eel, Anguilla anguilla:preliminary results. Ann. Virol. (Paris) 13E:435-448.

Castric, J., D. Rasschaert, and J. Bernard. 1984. Evidence ofLyssaviruses among rhabdovirus isolates from the European eelAnguilla anguilla. Ann. Virol. (Paris) 135E:35-55.

Caswell-Reno, P., P. W. Reno, and B. L. Nicholson. 1986. Monoclonalantibodies to infectious pancreatic necrosis virus: analysisof viral epitopes and comparison of different isolates. J. Gen.Virol. 67:2193-2205.

Clerx, J. P. M., B. A. M. van der Zeijst, and M.C. Horzinek. 1975. Somephysicochemical properties of pike fry rhabdovirus RNA. J. GenVirol. 29:133-136.

Clerx, J. P., M. C. Horzinek, and A. D. M. E. Osterhaus. 1978.Neutralization and enhancement of infectivity of non-salmonidfish rhabdoviruses by rabbit and pike immune sera. J. Gen.Virol. 40:297-308.

Coates, D. 1984. An ulcer-disease outbreak amongst the freshwater fishpopulation of the Sepik River system, with notes on somefreshwater fish parasites. Department of Primary Industry,Fisheries Research and surveys Branch, Port Moresby, PapuaNew Guinea, Report No. 84-02.

123

Cos lett, G. D., B. P. Holloway, and J. F. Obijeski. 1980. The structuralproteins of rabies virus and evidence for their synthesis fromseparate monocistronic RNA species. J. Gen. Virol. 49:161-180.

Cox, J. H., B. Dietzschold, and L. G. Schneider. 1977. Rabies virusglycoprotein. II. Biological and serological characterization.Infect. Imm. 16:754-759.

Dietzschold, W. H., T. J. Wikor, A. D. Lopes, M. Lafon, C. Smith, and H.Koprowski. 1983. Characterization of an antigenic determinantof the glycoprotein that correlates with pathogenicity of rabiesvirus. Proc. Natl. Acad. Sci. USA 80:70-74.

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., and R. Scherrer. 1970. Le virus d'Egtved I. Stabilite,developpement et structure du virus de la souche danoise Fl.Ann. Rech. Vet. 1:17-30.

de Kinkelin, P., G. Galimard, and R. Bootsma. 1973. Isolation andidentification of the causative agent of "red disease" of pike (Esoxlucius L. 1766). Nature 241:465-467.

de Kinkelin, P., M. Le Berre, and G. Lenoir. 1974. Rhabdovirus despoissons. I. Proprietes in vitro de virus de la maladie rouge del'alevin de brochet. Ann. Microbiol. (Paris) 125A:93-111.

Dixon, P. F., and B. J. Hill. 1984. Rapid detection of fish rhabdovirusesby the enzyme-linked immunosorbent assay (ELISA).Aquaculture 42:1-12.

124

Dorson, M., C. Torchy, S. Chilmonczyk, P. de Kinkelin, and C. Michel.1984. A rhabdovirus pathogenic for perch, Perca fluviatilis L.:isolation and preliminary study. J. Fish Dis. 7:241-245

Emerson, S. U. 1985. Rhabdoviruses. In: Virology (eds. by B. N.Fields,D. M. Knipe, R. M. Chanock, J. L. Melnick, B. Roizman, and R.E. Shope), pp 1119 -1132. Raven Press, New York.

Engelking, H. M. 1988. Properties of the glycoprotein of infectioushematopoietic necrosis virus. Ph.D. thesis, Oregon StateUniversity, Corvallis, Oregon. 153p.

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

Faisal, M., and W. Ahne. 1984. Spring viraemia of carp (SVCV):comparison of immunoperoxidase, fluorescent antibody and cellculture isolation techniques for detection of antigen. J. Fish Dis.7:57-64.

Feldman, H., and S. Wang. 1961. Sensitivity of various viruses tochloroform. Proc. Soc. Exp. Biol. Med. 106: 736-738.

Fijan, N., Z. Petrinec, D. Sulimanovic, and L. 0. Zwillenberg. 1971.Isolation of the viral causative agent from the acute form ofinfectious dropsy of carp. Vet. Arch. 41:125-138.

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) cellline from carp Cyprinus carpio. Ann. Virol. (Inst. Pasteur)134E: 207-220.

125

Frerichs, G. N., B. J. Hill, and K. Way. 1989. Ulcerative diseaserhabdovirus: cell line susceptibility and serological comparisonwith other fish rhabdoviruses. J. Fish Dis. 12:51-56.

Frerichs, G. N., S. D. Millar, and M. Alexander. 1989. Rhabdovirusinfection of ulcerated fish in South-East Asia. In: Viruses ofLower Vertebrates (eds. by W. Ahne and E. Kurstak), pp 369-410.Springer-Verlag, Berlin.

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

Galfre, G., S. C. Howe, C. Milstein, G. W. Butcher, and J. C. Howard.1977. Antibodies to major histocompatibility antigens produced byhybrid cell lines. Nature. 266:550-552.

Hawkes, R., E. Niday, and J. Godon. 1982. A dot-irmriunoblotting assayfor monoclonal and other antibodies. Anal. Biochem. 119:142-147.

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

Hetrick, F. M. 1989. Fish viruses. In: Methods for the MicrobiologicalExamination 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 rhabdovirusesinfecting fish. J. Gen.Virol. 27:369-378.

126

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

Hopper, K. 1989. The isolation of VHSV from chinook salmon atGlenwood Springs, Orcas Island, Washington. AFS/FHSNewsletter 17:1.

Hsu, Y. L., and J. C. Leong. 1985. A comparison of detection methodsfor infectious hematopoietic necrosis virus. J. Fish Dis. 8:1-12.

Hsu, Y., H. M. Engelking, and J. C. Leong. 1986. Occurrence ofdifferent types of infectious hematopoietic necrosis virus in fish.Appl. Environ. Microbiol. 52:1353-1361.

Jensen, M. H. 1965. Research on the virus of Egtved disease. Ann. N.Y. Acad. Sci. 126:422-426.

Jensen, N. J., and J. L. Larsen. 1982. The ulcus-syndrome in cod(Gadus morhua). IV. Tramission experiments with two virusesisolated from cod and Vibrio anguillarum. Nord. Vet. Med.34:136-142.

Jensen, N. J., B. Bloch, and J. L. Larsen. 1979. The ulcus-syndrome incod (Gadus morhua). III. A preliminary virological report.Nord. Vet. Med. 31:436-442.

Jorgensen, P. E. V., N. J. Olesen, N. Lorenzen, J. R. Winton, and S. S.Ristow. 1991 Infectious hematopoietic necrosis (IHN) and viralhemorrhagic septicemia (VHS): detection of trout antibodies to thecausative viruses by means of plaque neutralization,immunoflourescence, and enzyme-liked immunosorbent assay.J. Aquat. Anim. Health. 3:100-108.

127

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

Kasornchandra, J., C. N. Lannan, J. S. Rohovec, and J. L. Fryer. 1991.Characterization of a rhabdovirus isolated from the snakeheadfish (Ophicephalus striatus) In: Proceeding of the SecondInternational Symposium on Viruses of Lower Vertebrates, July29- 31,1991, Corvallis, Oregon.

Kelley, J. M., S. U. Emerson, and R. R. Wagner. 1972. The glycoproteinof vesicular stomatitis virus is the antigen that gives rise to andreacts with neutralizing antibody. J. Virol. 10:1231-1235.

Kelley, R. K., B. W. Souter, and H. R. Miller. 1978. Fish cell lines:comparisons of CHSE-214, FHM, and RTG-2 in assaying IHN andIPN viruses. J. Fish. Res. Board Can. 35:1009-1011.

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

Kimura, T., M. Yoshimizu, N. Oseko, and T. Nishizawa. 1989.Rhabdovirus olivaceus (Hirame Rhabdovirus) In: Viruses ofLower Vertebrates (eds. by W. Ahne and E. Kurstak), pp 388-395.Springer-Verlag, Berlin.

Kurath, G., and J. C. Leong. 1985. Characterization of infectioushematopoietic necrosis virus mRNA species reveals a nonvirionrhabdovirus protein. J. Virol. 53:462-468.

128

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

LaPatra, S. E., K. A. Roberti, J. S. Rohovec, and J. L. Fryer. 1989.Fluorescent antibody test for the rapid diagnosis of infectioushematopoietic necrosis. J. Aquat. Anim. Health. 1:29-36.

Larsen, J. L., and N. J. Jensen. 1977. An Aeromonas speciesimplicated in ulcer-disease of the cod (Gadus morhua). Nord.Vet. Med. 29:199-211.

Lenoir, G. 1973. Structural proteins of spring viremia virus of carp.Biochem. Biophys. Res. Commun. 51:895-898.

Lenoir, G., and P. de Kinkelin. 1975. Fish rhabdoviruses: comparativestudy 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) virus may beidentified by structural protein differences. Dev. Biol. Stand. 49:43-55.

Lorenzen, N., Olesen, N. J., and P. E. V. Jorgensen. 1988. Productionand characterization of monoclonal antibodies to four Egtvedvirus structural proteins. Dis. Aquat. Org. 4:35-42.

Lu, Y., C. N. Lannan, J. S. Rohovec, and J. L. Fryer. 1990. Fish celllines: establishment and characterization of three new cell linesfrom grass carp (Ctenopharyngodon idella). In Vitro 26:275-279.

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

129

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

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

McCain, B. B., J. L. Fryer, and K. S. Pilcher. 1974. Antigenicrelationships in a group of three viruses of salmonid fish by crossneutralization. Proc. Soc. Exp. Biol. Med. 137:1042-1046.

McKenzie, R. A., and W. T. K. Hall. 1976. Dermal ulceration of mullet(Mugil cephalus). Aus. Vet. J. 52:230-231.

Nishizawa T., M. Yoshimizu, J. Winton, W. Ahne, and T. Kimura.Characterization of structural proteins of hirame rhabdovirus,HRV. Dis. Aquat. Org. 10:167-172.

Pittayangkula, S., and V. Bodhalamik. 1983. The study of Achlya spp.of fish disease in Ophicephalus striatus. In: The Symposium onFreshwater Fish Epidemic: 1982-83, p 23, ChulalongkornUniversity, June 23-24, 1983.

Reed, L. J., and H. Muench. 1938. A simple method of estimating fiftypercent 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.

130

Ristow, S. S., and J. A. de Avila. 1991. Monoclonal antibodies to theglycoprotein and nucleoprotein of infectious hematopoieticnecrosis virus (IHNV) reveal differences among isolates of thevirus by fluorescence, neutralization and electrophoresis. Dis.Aquat. Org. 11:105-115.

Roberts, R. J. 1986. Pathological findings. In: Field and LaboratoryInvestigations into Ulcerative Fish Diseases in the Asia-PacificRegion (ed. by R. J. Roberts), pp 29-35. Technical Report of FAOProject TCP/RAS/4508. Bangkok, Thailand.

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

Roy, P., H. F. Clark, H. P. Madore, and D. H. L. Bishop. 1975. RNApolymerase associated with virions of pike fry rhabdovirus.J.Virol. 15:338-347.

Rucker, R. R., W. J. Whipple, J. R. Parvin, and C. A. Evans. 1953. Acontagious disease of salmon possibly of virus origin. U.S. FishWildl. Ser. Fish. Bull. 76:35-46.

Sano, T. 1976. Viral diseases of cultured fishes in Japan. Fish Pathol.10:221-226.

Sano, T., and H. Fukuda. 1987. Principle microbial diseases ofmariculture in Japan. Aquaculture 67:59-70.

Sano, T., T. Nishimura, N. Okamoto, and H. Fukuda. 1976. Isolation ofrhabdovirus from European eels (Anguilla anguilla) at Japaneseport of entry. Fish Health News. 5:5-6.

1 3 1

Sano, T., T. Nishimura, N. Okamoto, and H. Fukuda. 1977. Studies onviral diseases of Japanese fishes. VII. A rhabdovirus isolatedfrom European eel, Anguilla anguilla. Bull. Jpn. Soc. Sci. Fish.43:491-495.

Sartorelli, A. C., D. S. Fischer, and W. G. Downs. 1966. Use of sarcoma180/TG to prepare hyperimmune ascitic fluid in the mouse. J.Immunol. 96:676-682.

Schultz, C. L., B. C. Lidgerding, P. E. McAllister, and F. M. Hetrick.1985. Production and characterization of monoclonal antibodyagainst infectious hematopoietic necrosis virus. Fish Pathol.20:339-341.

Seif, I., P. Conlon, P. E. Rollin, and A. Flamand. 1985. Rabiesvirulence: effect of pathogenicity and sequence characterization ofrabies virus mutations affecting antigenic site III of theglycoprotein. J. Virol. 53:926-934.

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

Tamura, G. S., M. 0. Dailey, W. M. Gallatin, M. S. McGrath, I. L.Weissman, and E. A. Pillemer. 1984. Isolation of moleculesrecognized by monoclonal antibodies and antisera: the solid phaseimmunoisolation technique. Anal. Biochem. 136:458-464.

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

132

Tonguthai, K. 1986. Parasitological findings. In: Field and LaboratoryInvestigations into Ulcerative Fish Diseases in the Asia-PacificRegion (ed. by R. J. Roberts), pp 35-42. Technical Report ofFAO Project TCP/RAS/4508. Bangkok, Thailand.

Vandepol, S. B., L. Lefrancois, and J. J. Holland. 1986. Sequences of themajor antibody binding epitopes of the Indiana serotypes ofvesicular stomatitis virus. Virology 148:312-325.

Van Regenmortel M. H. 1981. Serological methods in the identificationand characterization of viruses. In: Comprehensive Virology(eds. by H. Fraenkel-Conrat and R. R. Wagner), Vol. 17. pp 183-244. Plenum Plublishing Corp., New York.

Wagner, R. R. 1975. Reproduction of rhabdoviruses. In:Comprehensive Virology (eds. by H. Fraenkel-Conrat and R. R.Wagner), Vol. 4. pp 1-93. Plenum Plublishing Corp., New York.

Wagner, R. R., L. Prevec, F. Brown, D. F. Summers, F. Sokol, and R.MacLeod. 1972. Classification of rhabdovirus proteins: aproposal. J. Virol. 10:1228-1230.

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 ViralDiseases on the Development of Asian Countries, December 7-13,1986. p 121. Ambassador Hotel, Bangkok, Thailand.

Wattanavijarn, W., C. Torchy, J. Tangtronpiros, and P. de Kinkelin.1988. Isolation of a birnavirus belonging to Sp serotype, fromsouth east Asia fishes. Bull. Eur. Assoc. Fish Pathol. 8:106.

133

Wehland, J., M. C. Willingham, M. G. Gallo, and I. Pastan. 1981. Themorphologic pathway of exocytosis of the vesicular stomatitisvirus G protein in cultured fibroblast. Cell 28:831-841.

Wingfield, W. H., and L. D. Chan. 1970. Studies on the SacramentoRiver chinook disease and its causative agent. In: A Symposiumof Disease of Fishes and Shellfishes (ed. by S. F. Snieszko),Special Publication No. 5. p 307-318. Washington D.C.

Wingfield, W. H., J. L. Fryer, and K. S. Pilcher. 1969. Properties of thesockeye salmon virus (Oregon strain). Proc. Soc. Exp. Biol. Med.130:1055-11059.

Winton, J. R., W. N. Batts, and T. Nishizawa. 1989. Characterization ofthe first north American isolates of viral hemorrhagic septicemiavirus. FHS/AFS Newsletter. 17:2-3.

Winton, J. R., C. K. Arakawa, C. N. Lannan, and J. L. Fryer. 1985Neutralizing monoclonal antibodies recognize antigenic variantsamong isolates of infectious hematopoietic necrosis virus. Dis.Aquat. Org. 4:199-204.

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 andpropagation in centrarchid fish cell lines. Science 151: 1004-1005.

Wunner, W. H., B. Dietzschold, and T. J. Wiktor. 1985. Antigenicstructure of rhabdoviruses. In: Immunochemistry of Viruses.The Basis for Serodiagnosis and Vaccines (eds. by M. H. V. vanRegenmortel and A. R. Neurath), pp 367-388. Elsevier SciencePubl. B. V. (Biochemical Division), England.

134

Yoshimizu, M., T. Nishizawa, N. Oseko, and T. Kimura. 1987.Rhabdovirus disease of hirame (Japanese flounder). Fish Pathol.22:54-55.

Zwillenberg, L. 0., M. H. Jensen, and H. H. L. Zwillenberg. 1965.Electron microscopy of the virus of viral haemorrhagicsepticemia of rainbow trout (Egtved virus). Arch. Virusforsch.17:1-19.