AN ABSTRACT OF THE THESIS OF
Jiraporn Kasornchandra
for the degree of Doctor of Philosophy
in
Microbiology
Title
Characterization of a Rhabdovirus Isolated from the Snakehead
presented on
December 2. 1991
Fish (Ophicephalus striatus)
Abstract approved:
Redacted for Privacy
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)
by
Jiraporn Kasornchandra
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed December 2, 1991
Commencement June, 1992
APPROVED:
7-.
Redacted for Privacy
Profer of MicrobioCI4y in charge of Major
Redacted for Privacy
ChairmaW, DepartmentgMicrobiology
Redacted for Privacy
Dean of Grad
School (I
Date of thesis presentation
December 2, 1991
Typed by Jiraporn Kasornchandra for Jiraporn Kasornchandra
ACKNOWLEDGEMENTS
I would like to dedicate this thesis 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 warm
hospitality.
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 their
excellent 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 viral
purification and structural protein analysis. P. Caswell-Reno provided
assistance in the monoclonal antibody production in Chapter V.
TABLE OF CONTENTS
Chapter
I.
II.
Page
Introduction
1
Review of Literature
3
Literature Cited
Characterization of a Rhabdovirus Isolated from
the Snakehead Fish (Ophicephalus striatus)
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
Literature Cited
IV.
V.
Characteristics of Three Rhabdoviruses Isolated
from Snakehead Fish (Ophicephalus striatus)
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
Literature Cited
Development and Characterization of Monoclonal
Antibodies against Snakehead Rhabdovirus
Abstract
Introduction
21
32
33
34
36
43
59
61
62
66
67
69
70
76
87
90
91
95
96
97
Chapter
V.
Page
(Continued)
Materials and Methods
Results
Discussion
Acknowledgements
Literature Cited
98
104
111
113
114
SUMMARY AND CONCLUSIONS
118
BIBLIOGRAPHY
120
LIST OF FIGURES
CHAPTER III.
111.2
111.3.
111.4.
111.5.
111.6
Page
Replication of snakehead rhabdovirus at selected
temperatures, MOI = 0.1.
Replication of snakehead rhabdovirus in selected
cell lines at 27°C, MOI = 1.0.
One-step growth curve of snakehead rhabdovirus
in EPC cells incubated at 27°C, MOI = 1.0.
Thin sections of EPC cells infected with snakehead
rhabdovirus.
Electrophoretic profile of three fish
rhabdoviruses.
Enzyme immunoassay of the snakehead
rhabdovirus glycoprotein.
47
49
51
53
55
57
CHAPTER IV.
IV.1 a.
IV.1b.
Electron micrograph of an ultrathin section of
EPC cells infected with snakehead rhabdovirus.
Electron micrograph of an ultrathin section of
SHF cells infected with ulcerative disease
rhabdovirus-BP (UDRV-BP).
IV.1 c.
77
Electron micrograph of an ultrathin section of
SHF cells infected with ulcerative disease
rhabdovirus-1 9 (UDRV-1 9).
IV.2.
77
77
A comparison of the viral polypeptides of snakehead
rhabdovirus (SHRV) and the other eight fish
79
rhabdoviruses in a 10% acrylamide gel.
CHAPTER V.
V.1.
V.2.
V.3.
V.4.
Page
Immunoprecipitation of snakehead rhabdovirus
proteins with monoclonal antibodies recognized the
G protein.
107
Immunoprecipitation of snakehead rhabdovirus
recognized the N proteins.
107
Immunoprecipitation of snakehead rhabdovirus
recognized the M1 proteins.
107
Mono layer cultures of EPC cells infected with
snakehead rhabdovirus fixed at 10 h post infection
and examined by indirect fluorescent antibody test.
109
LIST OF TABLES
CHAPTER II.
II.1.
Comparison of the characteristics among the
known fish rhabdoviruses.
Page
17
CHAPTER IV.
IV.1.
IV.2.
IV.3.
The estimated molecular weights (x103 daltons) of
virion proteins of the snakehead rhabdovirus (SHRV),
infectious hematopoietic necrosis virus (IHNV), viral
hemorrhagic septicemia (VHSV), and hirame
rhabdovirus (HRV).
84
The estimated values of molecular weights
(x103 dalton) of virion proteins of the spring viremia
of carp virus (SVCV),ulcerative disease rhabdovirus
(UDRV-BP), UDRV-19, and pike fry rhabdovirus
(PFR).
85
Cross-neutralization tests for eight fish
rhabdoviruses incubated with homologous and
heterologous antibodies.
86
Characterization of a Rhabdovirus Isolated from the Snakehead Fish
(Ophicephalus striatus)
Chapter I
Introduction
A rhabdovirus, serologically different from other fish
rhabdoviruses, was isolated from snakehead fish (Ophicephalus
striatus) in Thailand during an epizootic disease characterized by a
severe ulcerative necrosis. The ulcerative disease was reported in
Australia in 1972 and then spread throughout the Indo-Pacific region
(Tonguthai, 1985). The disease caused extensive losses among wild and
pond-cultured fish in Malaysia between 1979 and 1983 and also in
central Java, Indonesia in 1983 (Tonguthai, 1985). In 1985, the disease
spread to the Lao People's Democratic Republic (PDR) and to Burma
(Boonyaratpalin 1989). A major epizootic began in southern Thailand in
late 1980 and spread throughout the country. Although the disease was
observed in several fish species, the cultured snakehead, the walking
catfish (Clarias bratachus), and the sand goby (Oxyeleotris
marmoratus) were the most noticeably affected (Hedrick et al , 1986).
Although potentially pathogenic organisms were isolated and
identified from the diseased fish, the etiologic agent has yet to be
established. During the viral examination, two birnaviruses (Hedrick et
al., 1986, Wattanavijarn et al., 1988) and two rhabdoviruses (Frerichs et
al., 1986, Wattanavijarn et al., 1986) were isolated. The rhabdovirus
2
isolated by Wattanavijarn et al. (1986) was not completely characterized
or compared to the other fish rhabdoviruses.
The purpose of this study was as follows:
(1)
To study the biological and biochemical characteristics of the
snakehead rhabdovirus, including analysis of the viral structural
proteins.
It was first necessary to determine the appropriate methods for
propagation and assay of the virus. The studies involved the evaluation
of cell lines, incubation temperatures, and viral purification techniques.
A detailed evaluation of the biochemical characteristics of the 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 viral
identification 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 severe
ulcerative dermal necrosis has been observed in a wide variety of
freshwater and estuarine fishes, both wild and pond-cultured,
throughout the Indo-Pacific region. The origin of the disease is
unknown, but it has been suggested by several 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 completely
destroyed 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 crossneutralization 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 a
particular viral isolate was more specific to geographic area than to
host.
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 had
characteristics unique among fish rhabdoviruses. The Rio Grande
perch rhabdovirus exhibits a bacilliform morphology rather than the
bullet-shape typical of other animal rhabdoviruses. This virus later
proved to be an acute pathogen for the original host species and
Cichlasoma nigrofasciatum.
14
7.
Perch Rhabdovirus
In France, a second perch rhabdovirus, causing disease of the
central nervous system in Perca fluviatilis, was reported by Dorson et al.
(1984). The virus showed a typical bullet-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 tissue
culture 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 viral
antibodies.
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 the
surface proteins of viruses, preventing attachment, or entry into,
susceptible cells (Van Regenmortel, 1981). Using cross serumneutralization 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)
Or
temperature of
incubation °C
propagated
cell lines
heat
Diseases
IHNV
bullet
160-180x70-90
13-18
CHSE-214, RTG-2, EPC, FHM
Reaction to
low pH lipid
halogenated
solvent pyrimidine
Selected references
+
+
+
Wingfield et al. 1969
STE-137
VHSV
bullet
1 8 5x6 5
10-15
BF-2, CHSE-214, EPC, FHM,
PG, RTG-2
+
+
+
de Kinkelin and Scherrer 1970;
Zwillenberg e al. 1965
HRV
bullet
180-220x80
15-20
FHM, EPC, RTG-2, BF-2, YNK,
+
+
+
Kimura et al. 1986
SVCV
bullet
90-180x60-90
20-22
BB, BF-2, EPC, FHM, RTG-2
+
+
+
de Kinkelin et al. 1973
de Kinkelin and Le Berre 1974
PFRV
bullet
115-135x80
21-28
RTG-2, BB, FHM, EPC
+
+
+
de Kinkelin et al. 1974
EVA
bullet
100-160x75-80
15
RTG-2, EPC, FHM, BF-2, EK-1
+
+
+
Hill et al. 1980; Sano 1976;
Sano et al. 1977
EO-2
cod ulcus
syndrome
bullet
115-195x50-80
15
PS
nd
nd
nd
bacilliform
180x50
23
FHM, BF-2, RTG-2
nd
nd
+
rhabdovirus
bullet
20 Oxl 0 0
14
RTG-2
+
nd
nd
nd
Dorson et al. 1980
UDRV
bullet
110-130x75-85
25-30
BF-2, SSN-1,AS, RSN, CP
+
nd
nd
nd
Frerichs et al. 1986;
Frerichs et al. 1989
bullet
1 7 Ox6 0
15-28
CLC, SH
+
+
+
nd
Jensen et al. 1979
Rio Grande
perch
rhabdovirus
Malsberger and Lautenslager
1980
Perch
Snakehead
isolation
nd = no data
+ = sensitive
= resistant
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-linked
immunosorbent assay (ELISA) for rapid identification of IHNV and the
two eel rhabdoviruses, EVA and EVX, in cell cultures or directly from
tissues of infected fish. Although the ELISA was successfully developed
for rapid identification of IHNV and the eel rhabdoviruses, this
techniques was ineffective in detection of VHSV and SVCV. They
thought that the antisera against these two viruses, VHSV and SVCV,
had low titers. Jorgensen et al. (1991) used three different techniques,
plaque neutralization tests, immunofluorescence, and ELISA, to detect
anti-IHNV and anti-VHSV antibodies in cultured rainbow trout
surviving disease outbreaks. They reported that immunofluorescence
was the most sensitive method for detecting anti-IHNV antibodies, but
the ELISA was best in detecting anti-VHSV antibodies. They suggested
that these three serological methods are useful for IHNV and VHSV
epidemiology, even though cross-reactivity between the two viruses was
observed in sera with high titers of viral antibodies.
Hsu and Leong (1985) described methods for characterizing
different strains of IHNV by using solid phase direct binding assays
with 125lodinated Protein A or with immunoperoxidase staining. They
suggested that both immunological methods were highly specific and
sensitive to viral protein.
Schultz et al. (1985) successfully produced a monoclonal antibody
(MAb) against IHNV. Although the MAb showed little neutralizing
activity (a titer of 1:50 against 50 TCID50 of IHNV), it reacted with IHNV
20
by ELISA but did not recognize 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
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southeast Asia fishes. Bull. Eur. Ass. Fish Pathol. 8:106.
Wingfield, W. H., and L. D. Chan. 1970. Studies on the Sacramento
River chinook disease and its causative agent. In: A Symposium
of 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 the
sockeye salmon virus (Oregon strain). Proc. Soc. Exp. Biol. Med.
130:1055-11059.
Winton, J. R., W. N. Batts, and T. Nishizawa. 1989. Characterization of
the first north American isolates of viral hemorrhagic septicemia
virus. FHS/AFS Newsletter. 17:2-3.
31
Winton, J. R., C. K. Arakawa, C. N. Lannan, and J. L. Fryer. 1985
Neutralizing monoclonal antibodies recognize antigenic variants
among isolates of infectious hematopoietic necrosis virus. Dis.
Aquat. Org. 4:199-204.
Wunner, W. H., B. Dietzschold, and T. J. Wiktor. 1985. Antigenic
structure of rhabdoviruses. In: Immunochemistry of Viruses.
The Basis for Serodiagnosis and Vaccines (eds. by M. H. V. van
Regenmortel and A. R. Neurath), pp 367-388. Elsevier Science
Publ. 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 haemorrhagic
septicemia 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. Rohovecl
and J. L. Fryers
1Department of Microbiology, Oregon State University,
Nash Hall 220, Corvallis, Oregon 97331-3804 USA
2Laboratory for Fish Disease Research, Department of Microbiology,
Oregon State University, Mark 0. Hatfield Marine Science Center,
Newport, Oregon 97365-5296 USA.
Submitted for publication in the Proceeding of the Second International
Symposium on Viruses of Lower Vertebrates
33
Abstract
During a severe epizootic among the snakehead fish,
Ophicephalus striatus, in Thailand, a virus belonging to the
Rhabdoviridae 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 initially
characterized by raised areas of induration and erythema, followed by
erosion of the skin. Large, deep, ulcerative lesions are formed on the
body surface, especially on the head and jaws of diseased fish.
Moribund fish collected during the outbreak in Thailand were
examined for bacteria, fungi, viruses, and higher parasites.
Aeromonas hydrophila, a gram-negative opportunistic bacterial
pathogen, was most commonly found in association with the ulcerative
diseased fish (Boonyaratpalin, 1989). Although various types of parasitic
organisms were observed, no species was specifically identified as the
vector or pathogen (Tonguthai, 1985). During the viral examination, two
35
birnaviruses (Hedrick et al., 1986; Wattanavijarn et al., 1988) and two
rhabdoviruses (Frerichs et al., 1986, Wattanavijarn et al., 1986) were
isolated. In this report, the virus isolated by Wattanavijarn et al. (1986)
is characterized.
36
Materials and Methods
Viruses
The snakehead rhabdovirus (SHRV) was isolated from diseased
snakehead in Nakorn Pathom Province, Thailand (Wattanavijarn et al.,
1986). Two rhabdoviruses from fish, infectious hematopoietic necrosis
virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) were
used as reference for comparison of viral structural proteins. In
addition to IHNV, the birnavirus, infectious pancreatic necrosis virus
(IPNV), and the herpesvirus, channel catfish virus (CCV), were 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 was
centrifuged 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 by
Engelking 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 (BioRad).
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 singlestranded 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
48
108
10
14° C
18° C
PI
10
21° C
a
__,...._ 24 °C
10
Z
1:1
E
Ch....
10
27 °C
30°C
35 °C
10
10
0
2
4
Time (d)
Figure III.1 .
6
49
Figure
111.2.
Replication of snakehead rhabdovirus in selected cell
lines at 27°C, MO1 = 1 .0
50
EPC
SHF
MINI
COO
BB
BF-2
GCS-2
GCF
GCSB
0
2
4
Time (d)
Figure 111.2.
6
8
51
Figure
111.3.
One-step growth curve of snakehead rhabdovirus in
EPC cells incubated at 27°C, MO1 =1.0
52
109 1
108
1
107 -
1061
s-- Total virus
Free virus
105104
0
10
20
Time (h)
Figure 111.3.
30
53
Figure
111.4.
Thin sections of EPC cells infected with the snakehead
rhabdovirus. (a) Replication of the new viruses in the
intracellular space and budding from membranes.
Bar equal 200 nm. (b) Free viruses with bacilliform
morphology. Bar equal 230 nm.
54
Figure 111.4
55
Figure
111.5.
Electrophoretic profile of three fish rhabdoviruses.
All purified viruses were disrupted and
electrophoresed in SDS-polyacrylamide gel as
described in the Materials and Methods. Lane 1.
Molecular Weight Marker proteins (Bio-rad) shown by
arrows in descending order: phosphorylase B
(110,000); bovine serum albumin (84,000); ovalbumin
(47,000); carbonic anhydrase (33,000), soy bean trypsin
inhibitor (24,000), and lysozyme (16,000). Lane 2.
Purified snakehead rhabdovirus (SHRV). Lane 3.
Purified viral hemorrhagic septicemia virus (VHSV).
Lane 4. Purified infectious hematopoietic necrosis
virus (IHNV). The gel was stained with Bio-rad silver
stain.
56
1
2
3
4
ler -Imp" lomor-L
110 K
84 K
-G
47K
N
33 K-M1
24 K16 K
Figure 111.5
57
Figure
111.6.
Enzyme immunoassay of the snakehead rhabdovirus
glycoprotein. The glycoprotein was detected on a
nitrocellulose membrane. The viral proteins were
separated on an SDS-polyacrylamide gel and
transferred as described in Materials and Methods.
Lane 1. Low molecular weight marker proteins.
Lane 2. Purified snakehead rhabdovirus. Lane 3.
Tranferrin. Lane 4. Pre-stained Molecular Weight
Marker proteins shown by arrows in descending
order: phosphorylase B (110,000); bovine serum
albumin (84,000); ovalbumin (47,000); carbonic
anhydrase (33,000); soybean trypsin inhibitor (24,000);
and lysozyme (16,000).
58
1
3
4
11111111
-110K
tit
G-
84 K
-47K
-33 K
-24 K
16K
Figure 111.6
59
Discussion
The virus isolated from snakehead was determined to be a
member of the Rhabdoviridae based on its morphological and
biochemical 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 both
rhabdoviruses are in the same range as that of SHRV (de Kinkelin and
Le Berre, 1974, de Kinkelin et al., 1974). The snakehead fish is reared at
temperatures which range between 22-30°C; therefore, it is expected that
60
the SHRV should require similar incubation temperatures and fish cell
lines derived from warm water fish for its replication. Results indicate
that SHRV replicated in SHF, EPC, FHM and CCO cells, with SHF and
EPC yielding the highest titers > 108.25 TC1D50/ml. Replication occurred
optimally at 24 to 30°C and no viral replication was detected below 14°C.
The SHRV contained five structural protein components similar
to IHNV and VHSV, which resemble those of the Lyssavirus, the
prototype virus of the family Rhabdoviridae. The estimated molecular
weights of five structural polypeptides of SHRV were > 150, 69, 48.5, 26.5,
and 20 Kd.
The 69 Kd molecular weight protein of 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
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Frerichs, G. N., B. J. Hill, and K.Way. 1989. Ulcerative rhabdovirus:
cell-line susceptibility and serological comparison with other fish
rhabdoviruses. J. Fish Dis. 12: 51-156.
Frerichs, G. N., S. D. Millar, and R. J. Roberts. 1986. Ulcerative
rhabdovirus in fish in south-east Asia. Nature 322: 216.
Gravell, M., and R. G. Malsburger. (1965). A permanent cell line from
the fathead minnow (Pimephales promelas). Ann. N. Y. Acad.
Sci. 126:555-565.
Hedrick, R. P., W. D. Eaton, J. L. Fryer, W. G. Groberg Jr. , and S.
Boonyaratpalin. 1986. Characteristics of a birnavirus isolated
from cultured sand goby Oxyeleotris marmoratus. Dis. Aquat.
Org. 1:219-225.
Kasornchandra, J., S. Boonyaratpalin, and U. Saduadee. 1988. Fish
cell line initiated from snakehead fish (Ophicephalus striatus,
Bloch) and its characters. National Inland Fisheries Institute,
Department of Fisheries. Technical Paper No. 90. 8p.
Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (London) 227:
680-685.
Lu, Y., C. N. Lannan, J. S. Rohovec, and J. L. Fryer. 1990. Fish cell
lines: establishment and characterization of three new cell lines
from grass carp (Ctenopharyngodon idella). In Vitro 26:275-279.
64
Malsberger, R. G., and G. Lautenslager. 1980. Fish viruses:
rhabdovirus isolated from a species of the Family Cichlidae. Fish
Health News 9: i-ii.
McAllister, P. E., and R. R. Wagner. 1975. Structural proteins of two
salmonid rhabdoviruses. J. Virol. 15:733-738.
McAllister, P. E., J. L. Fryer, and K. S. Pilcher. 1974. Further
characterization of infectious hematopoietic necrosis virus of
salmonid fish (Oregon strain). Arch. Gesamte. Virusforsch.
44:270-279.
Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty
percent end points. Amer. J. Hygiene 27: 493-497.
Reynolds, E. S. 1963. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17:208-212.
Rovozzo, G. C., and C. N. Burke. 1973. A Manual of Basic Virological
Techniques. Prentice-Hall, Englewood Cliffs, New Jersy.
Spurr, A. R. 1969. A low viscosity epoxy resin embedding medium for
electron microscopy. J. Ultrastructure Res. 26: 31-43.
Tonguthai, K. 1985. A preliminary account of ulcerative fish diseases
in the Indo-Pacific region. Department of Fisheries, Ministry of
Agriculture and Cooperatives, Bangkok. 39p.
Wattanavijarn, W., J. Tangtronpiros, and K. Wattanodorn. 1986.
Viruses of ulcerative diseased fish in Thailand, Burma and Laos.
In: First International Conference on the Impact of Viral
Diseases on the Development of Asian Countries, Ambassador
Hotel, p 121. December 7-13, 1986. Bangkok, Thailand.
65
Wattanavijarn, W., C. Torchy, J. Tangtronpiros, and P. de Kinkelin.
1988. Isolation of a birnavirus belonging to Sp serotype, from
south east Asia fishes. Bull. Eur. Assoc. Fish Pathol. 8:106.
Wolf, K. 1988. Fish Viruses and Fish Viral Diseases. Cornell Press,
Ithaca, New York. 476p.
Wolf, K., and M. C. Quimby. 1966. Lymphocystis virus: isolation and
propagation in centrarchid fish cell lines. Science 151: 1004-1005.
66
CHAPTER IV
Characteristics of Three Rhabdoviruses Isolated from
Snakehead Fish (Ophicephalus striatus)
J. Kasornchandral , H. M. Engelking2, C. N. Lannan3, J. S. Rohovecl ,
and J. L. Fryerl
1Department of Microbiology, Oregon 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 fish
rhabdoviruses. The SHRV exhibited a bacilliform morphology while
UDRV-BP and UDRV-19 showed typical rhabdoviral bullet-shaped
particles.
Using sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), the virion protein profile patterns for
UDRV-BP and UDRV-19 were indistinguishable but differed from that of
SHRV. The protein profile of UDRV-BP and -19 resembled that of
vesicular stomatitis virus.
However, SHRV had a viral protein
composition similar to that of rabies virus, the prototype rhabdovirus of
the Lyssavirus genus. The estimated molecular weights of the five
SHRV polypeptides were >150, 68, 42, 26.5 and 20 Kilodaltons (Kd). The
estimated molecular weights of the five proteins of both UDRV-BP and
UDRV-19 were >150, 71, 53, 48, and 22 Kd. The glycosylated proteins
were those with molecular weights of 68 Kd (SHRV) and 71 Kd (UDRV-
BP and UDRV-19). The UDRV-BP and UDRV-19 were serologically
identical but distinct from the six known fish rhabdoviruses, including
SHRV. The SHRV was serologically unrelated to any of the other
rhabdoviruses as determined by cross neutralization tests. The SHRV
68
was different morphologically, serologically, and in protein composition
from UDRV-BP and UDRV-19 even though 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 for
comparison.
All rhabdoviruses, except UDRV-BP and UDRV-19, were
propagated and titered in the Epithelioma papulosum cyprini (EPC) cell
line (Fijan et al., 1983), cultured in Eagle's minimum essential medium
with Earle's salts (MEM) (Flow Laboratories Inc., McLean, VA),
supplemented with 5% fetal bovine serum (FBS) (Hyclone Laboratories,
Logan, UT), and 1% L-glutamine (Flow). The UDRV-BP and UDRV-19
were grown in snakehead (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 by
polyacrylamide 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 SDSdenaturing 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 1
month 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 at
20°C; and SHRV, UDRV-BP and UDRV-19 at 27°C. Incubation was
continued until obvious CPE had developed in the control wells. The
neutralizing titers of the eight fish 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.
Electron micrograph of an ultrathin section of SHF
cells infected with ulcerative disease rhabdovirus-BP
(UDRV-BP). Complete virions have typical bullet-
shaped morphology (Bar=143 nm).
Figure IV.1c.
Electron micrograph of an ultrathin section of SHF
cells infected with ulcerative disease rhabdovirus
(UDRV-19). Complete virions exhibit typical bullet-
shaped particles (Bar=97 nm).
78
Figure IV.1
79
Figure W.2.
A comparison of the viral polypeptides of snakehead
rhabdovirus (SHRV) and the other eight fish
rhabdoviruses in a 10% acrylamide gel. The viral
proteins were denatured and 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
7
111111111
8
111111
oar
z
Figure IV.2
9
10
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 the
L, G, N, Mi., and M2 structural proteins of IHNV, VHSV, and HRV
(Hsu et al., 1985, Kimura et al., 1989, McAllister & Wagner, 1975). In
contrast to SHRV, the UDRV-BP and UDRV-19 consisted of four major
structural proteins with estimated molecular weights of >150, 71, 53,
and 22 Kd. These may correspond to the L, G, N and 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.
84
Table W.1.
The estimated molecular weights (x103 daltons) of
virion proteins of the snakehead rhabdovirus (SHRV),
infectious hematopoietic necrosis virus (IHNV), viral
hemorrhagic septicemia (VHSV), and hirame
rhabdovirus (HRV)
Virus
Polymerase
Glycoprotein Nucleoprotein
Matrix protein
L
G
N
M1
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
M2
85
Table 1V.2.
The estimated values of molecular weights (x103
daltons) of virion proteins of the spring viremia of carp
virus (SVCV), ulcerative disease rhabdovirus (UDRVBP), UDRV-19, and pike fry rhabdovirus (PFR)
Virus
Polymerase
Glycoprotein
L
G
N
M
Nucleoprotein
Matrix protein
SVCV
>150
85
45
23
UDRV-BP
>150
71
53
22
UDRV-1 9
>150
71
53
22
PFR
>150
80
43
24
Table IV.3.
Cross-neutralization tests for eight fish rhabdoviruses: SHRV, IHNV, VHSV, HRV,
SVCV, UDRV-BP, UDRV-19 and PFRV incubated with homologous and heterogous
antibodies.
polyclonal antibodies
Viruses
neutralizating titer*
Snakehead rhabdovirus
(SHRV)
Infectious hematopoietic necrosis
virus
(IHNV)
Viral hemorrhagic septicemia virus
(VHSV)
Hirame rhabdovirus
(HRV)
Spring viremia of carp virus
(SVCV)
Ulcerative disease rhabdovirus
(UDRV-BP)
Ulcerative disease rhabdovirus
(UDRV-19)
Pike fry rhabdovirus
(PFRV)
SHRV
IHNV
1:112
1:100
2.0**
0
0
VHSV
HRV
1:256
1:64
1:126
0
0
0
1.8
0
0
0
0
1.8
0
0
0
SVCV
UDRV-BP
UDRV-19
PFRV
1:64
1:96
0
0
0
0
0
0
0
0
0
0
0
0
0
1.8
0
0
0
0
0
0
0
2.0
0
0
0
0
0
0
0
0
2.0
1.9
0
0
0
0
0
0
1.85
2.0
0
0
0
0
0
0
0
0
1.8
1:64
* The neutralizing titer against homologous virus
**Cross-neutralization tests for eight of fish rhabdoviruses are expressed as
log of neutralization index = log TClD50 of control titer TCED50 of neutralization titer ]; Rovozzo & Burke (1973)
87
Discussion
Six rhabdovirus isolations were made from the diseased
snakehead fish in Thailand and Burma 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 study
demonstrated that the structural protein patterns and the molecular
weights of the two UDRV isolates were identical, indicating that the
UDRV-BP and UDRV-19 are, in fact, isolations of 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
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Boonyaratpalin, S. 1989. Bacterial pathogens involved in the epizootic
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Health. 1: 272-276.
Cartwright, B., C. J. Smale, F. Brown, and R. Hull. 1972. A model for
vesicular stomatitis virus. J. Virol. 10:256-260.
Coslette, G. D., B. P. Holloway, and J. F. Obijeski. 1980. The structural
proteins of rabies virus and evidence for their synthesis from
separate monocistronic RNA species. J. Gen. Virol. 49: 161-180.
Engelking, H. M., and J. C. Leong. 1989. Glycoprotein from infectious
hematopoietic necrosis virus (IHNV) induces protective
immunity against five IHNV types. J. Aquat. Anim. Health 1:
291-300.
de Kinkelin, P., M. Le Berre, and G. Lenoir. 1974. Rhabdovirus des
poissons. I. proprietes in tro du virus de la maldie ronge de 1'
alevin de brochet. Ann. Microbiol. (Inst. Pasteur). 125A:93-111.
Fijan, N., D. Sulimanovic, M. Bearzotti, D. Muzinic, L. 0. Zwillenberg,
S. Chilmonczyk, J. F. Vautherot, and P. de Kinkelin. 1983. Some
properties of the Epithelioma papulosum cyprini (EPC) cell line
from carp Cyprinus carpio. Ann. Virol. (Inst. Pasteur) 134E:
207-220.
Frerichs, G. N., B. J. Hill, and K. Way. 1989. Ulcerative rhabdovirus:
cell-line susceptibility and serological comparison with other fish
rhabdoviruses. J. Fish Dis. 12:151-156.
Frerichs, G. N., S. D. Miller, and R. J. Roberts. 1986. Ulcerative
rhabdovirus in fish in south-east Asia. Nature 322: 216.
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Hedrick, R. P., W. D. Eaton, J. L. Fryer, W. G. Groberg Jr., and S.
Boonyaratpalin. 1986. Characteristics of a birnavirus isolated
from cultured sand goby Oxyeleotris marmoratus. Dis. Aquat.
Org. 1:219-225.
Hetrick, F. M. 1989. Fish viruses. In: Methods for the microbiological
examination of fish and shellfish, (eds. by B. Austin and D. A.
Austin), pp.216-239. Ellis Horwood Limited, West Sussex,
England.
Hill, B. J., B. 0. Underwood, C. J. Smale, and F. Brown. 1975. Physicochemical and serological characterizations of five rhabdoviruses
infecting fish. J. Gen. Virol. 27:369-378.
Hill, B. J., R. F. Williams, C. J. Smale, B. 0. Underwood, and F. Brown.
1980. Physico-chemical and serological characterization of two
rhabdoviruses isolated from eels. Interovirology 14:208-212.
Hsu, Y., H. M. Engelking, and J. C. Leong. 1985. Analysis of the
quantity and synthesis of the virion proteins of the infectious
hematopoietic necrosis virus. Fish Pathol. 20:331-338.
Kasornchandra, J, S. Boonyaratpalin, and U. Saduadee. 1988. Fish cell
line initiated from snakehead fish (Ophicephalus striatus, Bloch)
and its characters. National Inland Fisheries Institute,
Department of Fisheries, Technical Paper No. 90. 8p.
Kimura, T., M. Yoshimizu, and S. Gorie. 1986. A new rhabdovirus
isolated in Japan from cultured hirame (Japanese flouder)
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Aquat. Org. 1:209-217.
Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-685.
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Lenoir, G. 1973. Structural proteins of spring viremia virus of carp.
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Lenoir, G., and P. de Kinkelin. 1975. Fish rhabdoviruses: comparative
study of protein structure. J. Virol. 16:259-262.
Leong, J. C., Y. L. Hsu, H. M. Engelking, and D. Mulcahy. 1981.
Strains of infectious hematopoietic necrosis (IHN) virus may be
identified by structural protein differences. Develop. Biol. Stand.
49:43-55.
Malsberger, R. G., and G. Lautenslager. 1980. Fish viruses:
Rhabdovirus isolated from a species of the Family Cichlidae.
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McAllister, P. E., and R. R. Wagner. 1975. Structural proteins of two
salmonid rhabdoviruses. J. Virol. 15:733-738.
Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty
percent end points. Amer. J. Hygiene 27:493-497.
Rovozzo, G. C., and C. N. Burke. 1973. A Manual of Basic Virological
Techniques. Prentice-Hall, New Jersey. 287p.
Tonguthai, K. 1986. Parasitological findings. In: Field and laboratory
investigations into ulcerative fish diseases in the Asia-Pacific
region, p35-42. Technical Report of FAO Project TCP/RAS/4508.
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Wagner), Vol. 4. pp 1-93. Plenum Plublishing Corp., New York.
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Wagner, R. R., L. Prevec, F. Brown, D. F. Summers, F. Sokol, and R.
MacLeod. 1972. Classification of rhabdovirus proteins: a
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Wolf, K. 1988. Fish Viruses and Fish Viral Diseases. Cornell Press,
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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 by
immunofluorescence, 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 fish
rhabdoviruses (Ahne et al., 1988, Kasornchandra et al., 1991), was
isolated from snakehead (Ophicephalus striatus) during an outbreak of
ulcerative disease in southeast Asia (Wattanavijarn et al., 1986).
Kasornchandra et al. (1991) found that the snakehead rhabdovirus
(SHRV) contained five structural proteins similar to those of infectious
hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia
virus (VHSV). Replication of this virus and production of cytopathic
effect (CPE) is rapid on susceptible cell lines. On the Epithelioma
Papulosum Cyprini (EPC) (Fijan et al., 1983) and the snakehead fin
(SHF) (Kasornchandra et al., 1988) cell lines, CPE is apparent within 14
h and complete in 24 to 48 h at 27°C (Kasornchandra et al., 1991).
Although polyclonal antibodies were developed for identification of
SHRV in a serum neutralization assay, the virus was shown to be a
particularly weak antigen. In immunofluorescence assays, the rabbit
antiserum had extensive non-specific binding with high levels of
background fluorescence both in the control and infected cell lines
tested. To improve the methods for identification of this virus,
monoclonal antibodies against SHRV have been developed.
The purpose of this study was to produce monoclonal antibodies
(MAbs) against SHRV to be used in immunodiagnosis. These MAbs
were characterized by immunoprecipitation, immunofluorescence, and
serum neutralization.
98
Materials and Methods
Virus and cell line
Snakehead rhabdovirus was propagated and titered in EPC cells
cultured in Eagle's minimum essential medium with Earle's salts
(MEM) (Flow Laboratories Inc., McLean, VA), supplemented with 5%
fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 1% Lglutamine (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) (GMHAT), 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 monoclonal
subisotyping 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 h
adsorption, the cultures were rinsed twice with methionine-free
Dulbecco'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 at
134,000 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 fish
rhabdoviruses, IHNV, VHSV, hirame rhabdovirus (HRV), spring
viremia of carp virus (SVCV), pike fry rhabdovirus (PFR), ulcerative
disease rhabdovirus (UDRV)-BP, and UDRV-19).
These tests were
103
conducted as described, except the SHF cell line was used to propagate
UDRV-BP and UDRV-19.
Fluorescence assays
The indirect fluorescent antibody test (IFAT) was modified from
that reported by LaPatra et al. (1989). Briefly, EPC cells were grown on
sterilized 18-mm-diameter cover glasses placed in separate wells of a 24well 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 SHRVinfected 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 antiimmunoglobulin 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.
107
Figure V.1.
Immunoprecipitation of snakehead rhabdoviral
(SHRV) proteins with monoclonal antibodies (MAbs).
The 35S-methionine labeled polypeptides were
recovered by immunoprecipitation from infected cell
lysates with MAbs and electrophoresed on a 10%
acrylamide gel. Lane 1 contains purified SHRV, Lane
2-9 MAbs against the G protein.
Figure V.2.
Immunoprecipitation of snakehead rhabdoviral
(SHRV)proteins with monoclonal antibodies (MAbs).
The 35S-methionine labeled polypeptides were
recovered by immunoprecipitation from infected cell
lysates with MAbs against SHRV and electrophoresed
on a 10% acrylamide gel. Lane 1 contains purified
SHRV, Lane 2-5 MAbs aganist the N protein.
Figure V.3.
Immunoprecipitation of snakehead rhabdoviral
(SHRV) proteins with monoclonal antibodies (MAbs).
The 35S-methionine labeled polypeptides were
recovered by immunoprecipitation from infected cell
lysates with MAbs against SHRV and electrophoresed
on a 10% acrylamide gel. Lane 1 contains purified
SHRV, Lane 2-3 MAbs against the M1 protein.
108
1
2
3
4
6
5
8
7
9
L
G
M1
M2
Figure V.1
1
1
2
3
4
5
6
L
L
G
G
N
N
M11
M2
M2
Figure V.2
Figure V.3
2
3
109
i
Figure V.4.
Mono layer cultures of epithelioma papulosum cyprini
(EPC) cells infected with snakehead rhabdovirus
(SHRV) fixed at 10 h post infection and examined by
indirect fluorescent antibody test. (a) specific
immunofluorescence to SHRV in infected cells with
MAb against the G protein. (b) SHRV-infected EPC
cells with MAb against the N protein. (c) SHRVinfected EPC cells with MAb against the M1 protein.
(d) Ml- specific MAb stained the SHRV-infected EPC
cell membranes.
110
Figure V.4
111
Discussion
Monoclonal antibodies were successfully developed against
SHRV. Two groups of MAbs, both neutralizing and non-neutralizing
MAbs, were characterized. Three neutralizing MAbs were directed
against the G protein as determined by radioimmunoprecipitation.
None of the MAbs that recognized the N and M1 viral protein had
neutralizing activity.
Like the G protein of IHNV and of the
mammalian rhabdoviruses, vesicular stomatitis virus (VSV) and rabies
virus (Cox et al., 1977, Engelking and Leong, 1989, Kelley et al., 1972), the
G protein of SHRV appeared to be responsible for inducing neutralizing
antibody as well.
Although the rhabdoviral G protein elicits neutralizing antibody,
some MAbs specific for the G protein do not produce neutralization. In
monoclonal antibody competition assays, Dietzschold et al. (1983)
demonstrated that the glycoprotein of rabies virus, CVS strain, had nine
distinct epitopes involved in immunogenic activity. Three of these
epitopes bind neutralizing MAbs (Seif et al., 1985). On the G protein of
VSV, five non-neutralizing and four neutralizing epitopes were defined
by MAbs studies (Vandepol et al., 1986). Among our MAbs developed
against SHRV, five which recognized the viral G protein in
immunoprecipitation were nonneutralizing.
In immunoprecipitation assays, the N protein of SHRV displayed
two bands, the lower band with a molecular weight of 38 Kilodaltons and
the upper band at 42 Kilodaltons, when reacted with the MAbs. The two
112
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.
113
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.
114
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SUMMARY AND CONCLUSIONS
1.
A new virus was isolated from snakehead fish in Thailand
during the ulcerative disease outbreak. Based on morphology and
biochemical characteristics, this virus is a member of the
family Rhabdoviridae. The virus was named snakehead
rhabdovirus (SHRV).
2.
The virus replicated optimally at 24-30°C in snakehead fin (SHF)
and epithelioma papulosum cyprini (EPC) cell lines. The
cytopathic effect produced by this virus began with infected cells
becoming rounded, followed by detachment and then lysis.
Exponential growth of SHRV on EPC cells at 27°C began after a 4
h latent period and reached a maximum plateau at 12 h post
infection.
3.
SHRV has a bacilliform morphology. The complete virion
was 60-70 nm in width and 180-200 nm in lenght and surrounded
by a lipid envelope.
4.
SHRV was not inhibited by 5- iodo- 2- deoxyuridine, indicating an
RNA genome, and acridine orange staining suggested the
presence of single-stranded nucleic acid. The virus was sensitive
to chloroform, pH 3, and heat (56°C), but resistant to freeze-thaw.
5.
SHRV consisted of five structural proteins with molecular weight
of >150, 69, 48.5, 26.6, and 20 Kilodaltons. The viral protein
pattern was similar to rabies virus, suggesting SHRV is a
member of genus Lyssavirus.
6.
The SHRV was serologically unrelated to any of the other fish
rhabdoviruses (IHNV, VHSV, HRV, SVCV, UDRV-BP, and
UDRV-19) as determined by cross neutralization tests.
119
7.
Fifteen monoclonal antibodies (MAbs) were produced against
SHRV. Eight of the MAbs recognized the viral glycoprotein in an
immunoprecipitation assay. Of these, three had neutralizing
activity. Twelve MAbs showed good binding activity in SHRVinfected EPC cells by immunofluorescence. Five of these MAbs
recognized the G protein, five recognized the N protein, and two
recognized the M1 protein of SHRV.
8.
In an indirect fluorescence assay, the MAbs gave varied staining
patterns depending upon the viral structural proteins recognized.
MAb against the G protein provided uniform staining throughout
the cell cytoplasm including the cisternae-like structure close to
the nucleus. MAbs against N and M1 proteins provided a coarse
granular staining in the cytoplasm of the infected cells. In
addition, anti-M1 also stained the cell membranes.
120
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