AN ABSTRACT OF THE THESIS OF
Hamdi Ogut for the degree of Master of Science in Fisheries Sicence presented on
December 20. 1995. Title: IN VITRO HOST RANGE OF INFECTIOUS
PANCREATIC NECROSIS VIRUS (IPNV) AND ITS RELATIONSHIP TO
VIRULENCE.
Redacted for Privacy
Abstract approved:
Of 109 aquatic birnaviruses (AB) belonging to 9 serogroup A, all replicated and
produced rapid and extensive cytopathology in CHSE-214 and RTG-2 cells, whereas
only half produced significant levels of cytopathological effect (CPE) in two non-
salmonid cell lines tested (EPC and FHM). In many instances it was found that although
CPE was not visible microscopically in EPC and FHM cells, virus replication occurred.
Isolates belonging to Buhl, Canada 1, Canada 2, Ab and Tellina subtypes did not produce
CPE on EPC and FHM cells. On the other hand, WB, VR-299, Jasper, Sp, and He
subtypes replicated efficiently in both EPC and FHM cells.
Serum sensitivity test results indicated that in 49 of 109 isolates in the presence of
normal trout serum (NTS),
100 fold reduction in virus titers occured
.
Avirulent
isolates belonging to various subtypes were more effectively inhibited in vitro by 1%
normal trout serum (NTS), whereas, virulent isolates, especially Buhl subtype, were not
affected by NTS at all and some replicated more efficiently in the presence of NTS.
In an in vivo test of the relationship between virulence and serum sensitivity
brook trout fry were exposed by immersion to four highly virulent Buhl subtype IPNV
isolates which had been passed 11 times in the presence or absence of NTS. Significant
mortalities occurred in the groups that were exposed to viruses that were passed fewer
than three times after isolation from brook trout (45-70%). All four Buhl subtype virus
isolates passed 11 times in RTG-2 cells in the absence of 1% NTS lost their virulence. In
two cases (2/4), the isolates passed in the presence of 1% NTS retained their virulence.
In Vitro Host Range Of Aquatic Birnaviruses
And Their Relationship To Virulence
By
Hamdi Ogiit
A THESIS
Submitted to
Oregon State University
In partial fulfillment of
the requirements for the
degree of
Master of Science
Completed: December 20, 1995
Commencement: June 1996
Master of Science thesis of Hamdi Ogut presented on. December, 20.1995
APPROVED:
Redacted for Privacy
Fisheries Science.
Major Professor, represe 'n Fisheries
Redacted for Privacy
Head of Department of Fisheries and Wildlife.
Redacted for Privacy
Dean of
uate School
I understand that my thesis will become part of the permenant Collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Redacted for Privacy
Hamdi ()gilt, Author
ACKNOWLEDGMENTS:
I would like to extend my deepest appreciation to Prof. Dr. Paul Reno for his
advise, assistance, and encouragement during my research. Particularly, I would like to
thank Dr. Paul Reno and Dr. Bob Olson for critically reading the manuscript as the
preparation of the thesis would not have been possible without their comments and
criticisms. I also thank Dr. Rich Holt for his support at the stage of preparation of this
study.
I would like also thank our laboratory assistants for their help, advice and
especially for their patience with me. And, of course, Janet Webster , our librarian, was
also very helpful. I also thank those not mentioned here but who shared their views in a
constructive way. This study was in part funded through the Coastal Oregon Marine
Experiment Station.
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
3
History of IPN Virus
Geographic and Host Range
Epizootology
3
Virus Virulence
6
Host and Environmental Factors
In vitro models
Cell Susceptibility
Cell Culture Adapted Virus (CCA)
Persistent Infections
DI (Defective interfering) Particles
Interferon
Serum Inhibition and Virulence
MATERIALS AND METHODS
In Vitro Experiment
IPN Virus Isolates
Cell Lines
Determination of In Vitro Host Range
5
5
9
10
10
11
13
14
15
15
19
19
19
19
24
Effects of Normal Trout Serum
25
Single Passage
Multiple Passage
25
26
In Vivo Experiment
Fish
Experimental Design
RESULTS
27
27
28
29
Ability of aquatic birnaviruses to produce cytopathology (CPE)
in teleost cell lines
29
Serotypes and in vitro host range
32
Effects of normal trout serum on aquatic birnaviruses
replication
53
TABLE OF CONTENTS (Continued)
Page
Effect of in vitro passage of aquatic birnaviruses in the presence
of NTS on virulence in brook trout.
DISCUSSION
58
65
Ability of aquatic birnaviruses to produce CPE in four
teleost cell lines
65
Effects of Normal Trout Serum on Aquatic Birnaviruses
replication
70
Effect of in vitro passage of aquatic birnaviruses in the presence
of NTS on virulence in Brook trout
73
CONCLUSIONS
76
BIBLIOGRAPHY
78
APPENDICES
91
Appendix A
92
Appendix B
111
LIST OF FIGURES
Figure
1.
a)
Pages
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by 109 aquatic birnavirus isolates on four different
cell lines
b)
Geometric mean titer (GMT) (TCID50/ml.) yields of 109
aquatic birnaviruses in all four cell lines tested.
2.
a)
31
31
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by 109 aquatic birnavirus isolates in
CHSE-214 cells
b)
Geometric mean titer (GMT) (TCID50/ml.) yields of
109 aquatic birnaviruses in CHSE-214 cells
3.
a)
a)
34
Geometric mean titer (GMT) (TCID50/ml.) yields of 109 aquatic
birnaviruses in RTG-2 cells
4.
33
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by 109 aquatic birnavirus isolates in RTG-2 cells
b)
33
34
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by Buhl (n = 29) aquatic birnavirus isolates in all four
cells
b)
Geometric mean titer (GMT) (TCID50/ml.) yields of Buhl aquatic
birnaviruses in all four cells
5.
a)
36
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by VR-299 aquatic birnavirus isolates (n = 15 ) in all
four cells
b)
36
38
Geometric mean titer (GMT) (TCID50/m1.) yields of VR-299
aquatic birnaviruses in all four cells
38
LIST OF FIGURES (Continued)
6.
a)
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by WB aquaticbirnavirus isolates (n = 14) in all four
b)
7.
a)
cells
40
Geometric mean titer (GMT) (TCID50/ml.) yields of WB aquatic
birnaviruses in all four cells
40
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by Jasper aquatic birnavirus isolates (n = 4) in all four
cells
b)
Geometric mean titer (GMT) (TCID50/ml.) yields of Jasper aquatic
birnaviruses in all four cells
8.
a)
42
42
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by Sp aquatic birnavirus isolates (n = 8) in all four
cells
b)
Geometric mean titer (GMT) (TCID50/ml.) yields of Sp aquatic
birnaviruses in all four cells
9.
a)
a)
46
Geometric mean titer (GMT) (TCID50/m1.) yields of Ab aquatic
birnaviruses in all four cells
10.
44
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by Ab aquatic birnavirus isolates (n = 5) in all four
cells
b)
44
46
Box and whisker plot of mean cytopathic effects (CPE)
(MC) produced by CAN-1 aquatic birnavirus isolates (n = 5) in
four cell lines
b)
Geometric mean titer (GMT) (TCID50/ml.) yields of CAN-1
aquatic birnaviruses in four cell lines
11.
a)
48
48
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by He aquatic birnavirus isolates (n = 2)
in CHSE-214 cells
50
LIST OF FIGURES (Continued)
b)
Geometric mean titer (GMT) (TCID50/m1.) yields of He aquatic
birnaviruses in four cells
12.
a)
Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by BC aquatic birnavirus isolates (n = 4) in four
cell lines
b)
a)
a)
a)
a)
57
The mean login titer differences in the presence and
absence of NTS
16.
55
Box and whisker plot of the mean CPE differences of between
virus replication in the presence and absence of NTS.
b)
55
Geometric mean titer (GMT) (TCID50/m1.) yields of 109 aquatic
birnaviruses in FHM cells
15.
54
Box and whisker plot of mean cytopathic effects (CPE)
(MC) produced by 109aquatic birnavirus isolates in FHM cells
b)
54
Geometric mean titer (GMT) (TCID50/m1.) yields of 109 aquatic
birnaviruses in EPC cells
14.
52
Box and whisker plot of mean cytopathic effects (CPE)
(MC) produced by 109 aquatic birnavirus isolates in EPC cells
b)
52
Geometric mean titer (GMT) (TCID50/m1.) yields of BC aquatic
birnaviruses in four cell lines
13.
50
57
Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TOD50/ml. IPNV 90-11 Buhl subtype isolate
which three treatments applied to (90-11: virus passed only once
in the absence of NTS, 90-11(-) virus passed 11 times in the
absence of NTS, 90-11(+) virus passed 11 times in the
presence of NTS.)
b)
60
Virus titer (TCID50/m1.) of dead or moribund brook trout fry
after exposure to IPNV 90-11 propagated in the presence or
absence of 1% NTS.
60
LIST OF FIGURES (Continued)
17.
a)
Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TCID50/ml. IPNV H-VAT 9/86 isolate which
three (H-VAT: virus passed only once in the absence of NTS,
H-VAT(-) 11 times in the absence virus passed of NTS, H-VAT(+)
b)
18.
a)
virus passedll times in the presence of NTS.)
Virus titer (TCID50/m1.) of dead or moribund brook trout fry
after exposure to IPNV H-VAT 9/86 propagated in the presence or
absence of 1% NTS
61
61
Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TCID50/ml. IPNV csf-035-85 (35-85 = virus
passed only once in the absence of NTS, 35-85(-) = virus
passed 11 times in the absence of NTS, 35-85(+) = virus
passed 11 times in the presence of NTS.)
b)
Virus titer (TCID50/m1.) of dead or moribund brook trout fry
after exposure to IPNV csf-035-85 propagated in the presence or
absence of 1% NTS
19.
a)
b)
20.
a)
62
62
Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TCID50/ml. IPNV csf Crayfish (Crayfish =
virus passed only once in the absence of NTS, Crayfish(-) =
virus passed 11 times in the absence of NTS, Crayfish(+) =
virus passed 11 times in the presence of NTS)
Virus titer (TCID50/m1.) of dead or moribund brook trout fry
after exposure to IPNV csf Crayfish propagated in the presence or
absence of 1% NTS
63
63
The relationship between CPE differences in RTG-2 cells
infected with aquatic birnaviruses in the presence and absence
of NTS and mortalityt levels in brook trout fry
b)
72
The relationship between Logic) titer differences in RTG-2 cells
infected with aquatic birnaviruses in the presence and absence
of NTS and mortalityt levels in brook trout fry
72
LIST OF TABLES
Table
Pages
1.
Aquatic Birnavirus isolates used in the experiments
2.
Isolates used in the multiple passage of IPNV in the presence and
absence of NRTS and its effect on virulence
3.
20
26
Cytopathic effects (CPE) of aquatic birnaviruses on four
teleost cell lines
30
LIST OF APPENDIX TABLES:
Table
A.1.
A. 2.
A. 3.
Pages
Virus yields and destruction of cell monolayers by Buhl subtype
IPNV isolates
93
Virus yields and destruction of cell monolayers by VR-299
subtype IPNV isolates
94
Virus yields and destruction of cell monolayers by WB
subtype IPNV isolates
A. 4.
Virus yields and destruction of cell monolayers by Jasper
subtype IPNV isolates
A. 5.
A. 8.
A. 9.
97
Virus yields and destruction of cell monolayers by Ab and EVE
subtype IPNV isolates
A. 7.
96
Virus yields and destruction of cell monolayers by Sp
subtype IPNV isolates
A. 6.
95
98
Virus yields and destruction of cell monolayers by CAN -1, CAN-2
and CAN-3 subtype IPNV isolates
99
Virus yields and destruction of cell monolayers by He and Te
subtype IPNV isolates
100
Shows CPE and virus yields of isolates whose serological identity
have not yet investigated
101
A. 10. Probabilities of differences among 13 subtypes of virus in their
ability to produce CPE in EPC cells
102
A. 11. Probabilities of differences among 13 subtypes of virus in their
ability to produce CPE in FHM cells
A. 12. ANOVA table for geometric mean titers obtained in EPC cells
infected with 13 different subtypes of IPNV subtypes of IPNV
103
104
LIST OF APPENDIX TABLES (Contunied)
A. 13. ANOVA table for geometric mean titers obtained in FHM cells
infected with 13 different subtypes of IPNV
105
A. 14. ANOVA table of CPE and virus yield responses of aquatic
birnaviruses, and significance levels in different cell ines
B. 1.
Replication of IPNV Buhl subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence
B. 2.
B. 4.
Replication of IPNV WB subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence
B. 7.
B. 8.
B. 9.
B.9
114
115
Replication of IPNV Sp subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence
B. 6.
113
Replication of IPNV CAN subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence
B. 5.
112
Replication of IPNV VR 299 subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence
B. 3.
106
116
Replication of IPNV Jasper and BC subtype isolates in the
presence and absence of 1% NTS and their relationship to virulence
117
Replication of IPNV Ab and EVE subtype isolates in the presence
and absence of 1% NTS and their relationship to virulence
118
Replication of IPNV He and Te subtype isolates in the presence
and absence of 1% NTS and their relationship to virulence
119
Replication of IPNV (subtypes unknown) isolates in the presence
and absence of 1% NTS and their relationship to virulence
120
Species from which IPNV isolated
121
In Vitro Host Range Of Aquatic Birnaviruses
And Their Relationship To Virulence
INTRODUCTION
Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae,
can cause mortality as high as 90-100% in 1-4 month-old rainbow trout fry (Frantsi and
Sayan, 1971; McAllister, 1983). There have been hundreds of studies on IPNV
characteristics since it was first isolated in 1957, yet there is still no effective vaccine or
any other control method, except prevention of exposure. There are more than 3,000
IPNV isolates obtained from different aquatic hosts worldwide. Most were obtained from
non-diseased fish.
Knowledge on the virulence characteristics of IPNV is very limited. After
isolating IPNV from a host, it is necessary to carry out a number of time consuming in
vivo tests to determine whether it is virulent to trout fry or not. One of the main
objectives of this study was to examine the relationship, if any, between in vivo and in
vitro characteristics of the serotype of virus related to the virulence. The second main
objective was to examine the effects of normal trout serum (NTS) on virus growth. Some
studies suggest that serum obtained from normal rainbow trout (NRT) interacts with
IPNV virus inactivating it and changing its virulence characteristics (Dorson et al., 1978;
Hill, 1981 ). How the serum interacts with the virus and inactivates is unknown. Dorson
et. al. (1978) suggested that although acquired sensitivity to NTS is not the only way that
virus loses its virulence, it must be taken into account in the production of virus for
infection trials. It has been suggested that passage of the virus in the presence of NTS
prevents loss of virulence compared to virus passed in the absence of NTS (Hill, 1982).
2
In the first part of present study, the correlation between virulence of IPNV and its
ability to replicate in certain fish cell lines was determined and described. Also, a study of
the relationship between cytopathic effects (CPE) and virus titers in various cell lines was
made. The second objective was studied by determining if virulence was correlated with
the ability of the virus to replicate in the presence of NTS.
3
LITERATURE REVIEW
History of IPN Virus:
M'Gonigle (1941) first reported the signs of infectious pancreatic necrosis (IPN)
disease in Canadian brook trout naming the disease " acute catarrhal enteritis". Later on,
IPN disease was discovered and described in the US. by Wood et al., (1955) and named
by Snieszko (Snieszko et al., 1957). Isolation of IPNV using finite teleost cell cultures
occurred in 1957 (Wolf and Dunbar, 1957). Then, Wolf et al. (1959 and 1960) established
its etiology as the first known viral disease of fish.
Research on various aspects of IPN disease continued in 1960s. The development
of the following continuous teleost cell lines during 1960s simplified studies of the virus:
Rainbow trout gonad (RTG-2) (Wolf and Quimby, 1962), grunt fin (GF) (Beasly et al.,
1965), fathead minnow (FHM) (Malsberger, 1965). The susceptibility of RTG-2 cells, as
well as many other teleost cells to IPNV was also reported (Wolf and Mann, 1980).
Early studies on the morphology of IPNV were controversial. Although originally
described as 25 nm in diameter (Cerini and Malsberger, 1965), the correct size of the
reovirus-like virion, 69 nm, was reported by Lightner and Post (1969), and confirmed by
other researchers (Moss and Gravel, 1971). Another unresolved issue was whether the
RNA genome of the virus is single or double stranded. Kelly and Loh (1972) reported that
IPNV was a single stranded RNA virus. When the double stranded nature of the genome
was described by Moss and Gravel (1971), other researchers agreed with and then verified
the findings of Moss & Gravel (Argot and Malsberger, 1972; Cohen, 1973). Further
biophysical studies proved that, IPNV has a double stranded RNA genome (Dobos, 1977;
Dobos et al., 1977) consisting of two segments. These segments are RNA segment A and
RNA segment B (Brown, 1986). RNA segment A encodes a major capsid protein (Vp2 =
54kd), a second structural protein (Vp3 = 31 kd) and a nonstructural protein (Vp4 = 29kd).
4
The RNA segment B encodes a putative viral RNA polymerase (Vpl = 105kd),
(McDonald & Dobos, 1981; Persson and MacDonald, 1982).
Susceptibility of different species of trout to IPN disease has also been investigated
(Silim et al., 1982). Seven different trout groups which have been chosen from three
different species from different areas were tested. Brook trout had highest susceptibility to
IPNV (30.0 to 46.25% mortalities). Lake trout were the most resistant (1.0 to 1.3%
mortalities) and rainbow trout were less resistant than lake trout (6.0 to 10% mortalities).
Almost all techniques for viral detection in terms of their reliability and rapidness
have been compared and the superiority of one to another reviewed. The techniques for
rapid detection and identification of IPNV used so far include fluorescent antibody
(Swanson & Gillspie, 1981; Hattori, 1983), ELISA (Dixon & Hill, 1983; Caswell and
Nicholson, 1986), coagglutination (Kimura et al., 1981), and immunodot blot (CaswellReno et al., 1989; Lipipun et al, 1989; Ya-li Hsu, 1989).
During the 1980s and 1990s, serotyping of IPNV using monoclonal antibodies
aided the classification of IPNV isolates. A polyclonal antibody classification system of
IPNV strains based on virus neutralization was developed by Hill and Way (1988).
According to this classification system, IPNV strains are divided into two serogroups;
Serogroup A and Serogroup B. Serogroup A consists of 9 serotypes, Sp, Ab, West
Buxton (WB), Jasper (Ja), Hecht (He), Tellina (Te), Canada 1 (C1), Canada 2 (C2) and
Canada 3 (C3). It includes more than 2,000 isolates. Serogroup B comprises only one
serotype and 10 isolates. Epitopes of these serotypes were defined using monoclonal
antibodies (Caswell-Reno et al., (1986, 1989); Dobos (1976); Hetrick (1983)). The Ni
strain previously was defined as a different serotype (Cristie et al., 1988) but later
concluded to be a member of Sp serotype (Melby and Cristie, 1994).
5
Geographic and Host Range:
IPNV has a wide geographic distribution. The first reports of IPNV disease came
from Canada (M'Gonigle, 1941), and then the US (Wood et al., 1955). It was not reported
in Europe until 1965 (Besse and DeKinkelin, 1965). It has since been reported from many
other places: Japan (Sano, 1976), South Africa (Bragg and Combrink, 1989), Chile
(MacAllister, 1983), Korea (Hah et al., 1984), Taiwan (Hedrick et al., 1983), China (Jaing
et al., 1989), United Kingdom (Ball, et al., 1971) and Scandinavia and British Isles (Besse
and de Kinkelin, 1965)
In the countries given above not all waters are contaminated with IPNV. Many
waters still are IPNV free. It is generally thought that IPNV has been transferred from
country to country by shipping of IPNV infected trout eggs.
Early on, it was thought that IPNV caused acute disease only in salmonid fishes.
However, many studies have proven that IPNV could be isolated from non-salmonids.
(Adair and Ferguson, 1981). IPNV was isolated from such molluscans as Asian clam
(Corbicula fluminou), scallop (Patella vulgate), periwinkle (Littorina litorea) , mussel
(Mytilus edulis), American oyster (Crassostrea gigas), (Hill, 1982). IPNV was isolated
from crustacea such as shore crab (Carcinus maenas) and Japanese shrimp (Peanaeus
japonica) (Bovo et al., 1984). It was also isolated from lamprey (Lampetra fluviatilis)
(Munro et al., 1976) and other teleosts (See Appendix B. Table 10, Paul W.Reno, personal
communication). The vast mojority of aquatic birnavirus isolates were obtained from
salmonid fishes.
Epizootiology:
IPN disease causes high mortality in young salmonids (Wolf et al., 1960;
MacAllister 1983, 1995). Survivors are life time carriers of IPNV (Dorson, 1982). These
asymptomatic, persistently infected fishes carry the virus in their visceral organs and shed
6
periodically (Hill, 1982), although the fish mounts a humoral immune response
(Yamamoto, 1971; Reno 1976).
IPNV can be transmitted both horizontally and vertically (Wolf et al., 1963). Billi
and Wolf (1969) demonstrated that IPNV could be shed from feces and reproductive
organs. Horizontal transmission has been then confirmed by many other researchers (Hill,
1982 ; Hedrick and Fryer, 1982; Boot land, 1986). Vertical transmission by carrier fish to
progeny has also been shown by many researchers (Ahne & Negele, 1985; Dorson &
Torchy, 1985; Boot land, 1991).
Smail et al., (1992) reported that there has been an increased association of IPNV
Sp strain with clinical disease in sea cage of Atlantic salmon post-smolts in Scotland. Peak
mortalities occurred five weeks after transfer smolts to the sea sites. Mortality at the cage
site rose from 0.3% to 2.4% and then again dropped to 0.2% over a period of 3 weeks.
The worst mortalities (10-13%) were in the adjacent cages. Twelve out of 12 pools were
positive for IPNV. Results of plaque neutralization tests from Atlantic salmon farms
indicated that the strain was Sp. It was also suggested that IPNV Sp isolate is associated
with clinical disease and there might have a synergistic effect with the infectious agent of
exocrine pancreatic disease (PD).
Virus Virulence:
Virulence, a relative term describing the disease causing ability of a virus, is
affected by different factors, including the physiological state of the animal, environmental
factors and the nature of virus itself. To designate a virus as virulent, that virus must have
ability to enter the host cell, replicate, overcome host cell defense mechanisms and destroy
the cells which are important in the physiology of animal. In the case of an avirulent state,
one or more stage described above may be blocked.
The physical location responsible for virulence on the virion surface is variable
among different kinds of viruses. Leavy et al., (1994) indicated that virulence of a virus is
7
often due to a single surface protein causing inhibition of synthesis of host cell
macromolecules.
Studies with rabies virus showed that antigenic alterations may influence viral
pathogenesis (Detzschold et al., 1983). Highly pathogenic strains of rabies virus were
selected by the presence of neutralizing monoclonal antibody. Avirulent strains of rabies
virus were exposed to mice to confirm their avirulence. Sequencing of glycoprotein of the
avirulent and virulent variants showed that substitution of a single amino acid altered
virulence. In the case of arenaviruses, virulence is associated with the presence of the large
[L] RNA segment. L segment encodes a large protein [L], presumably representing the
viral polymerase (Riviere et al., 1985). In the case of bunyavirus, an RNA virus, surface
glycoprotein is responsible for virulence (Bishop and Shop, 1980). Reassortment between
virulent and avirulent strains were used in the determination of bunyavirus virulence
(Janssen et al., 1986). Both the viral L genomic segment which encodes polymerase and S
segment which encodes nucleocapsid protein (N) and small non-structural protein (NSs)
can modulate the effect of the M segment on virulence.
The 51 gene encoding the outer capsid protein is responsible for virulence of
reoviruses (Fields and Greene, 1982). In most reported cases, attenuation of a reovirus
occurred because the viral hemagglutinin lost its ability to react with host cell receptors
(Fields and Greene, 1982). Two factors determine virulence of reovirus: The
haemagglutinin is responsible for the host cell tropism and destruction. It is the major
antigen for cellular and humoral immune responses. Second is the outer capsid protein
(µ1C) providing a second site on the surface of virion that can be altered by mutation. g.1C
protein alterations significantly reduces effectiveness of virus replication (Fields and
Greene, 1982). Gene sequence analysis of Si gene taken from several virulent and
avirulent variants indicated that the attenuated and wild types differ only in the single amino
acid substitution at positions of sigma-1 protein (Bassel-Duby et al., 1985). The
8
importance of this single amino acid substitution on the cell tropism and attenuated
neurovirulence was also confirmed using reassortants (Kaye et al., 1986).
In the case of IPNV, the portion of the virion responsible for virulence is unknown.
Sano et al., (1992) and Dorson et al., (1978) reported that virulence is associated with
RNA segment A which codes for VP2 and VP3, not RNA segment B which codes for
VP1. But which part of the genome is responsible for virulence is not known.
Darragh and McDonald (1982) studied IPNV-Jasper and IPNV-Oyster virus 3
noting the difference in the ability to infect FHM and CHSE-214 cells is related to RNA
segment A. Dorson et al. (1978) reported that plaque size was not related to the virulence
of IPN virus although this is the case in some other viruses such as poliovirus,
coxsackievirus A9 and Venezuelan equine encaphalitis (VEE) virus (Holland, 1964).
Intertypic reassortants of Buhl and EVE isolates were compared in terms of their plaque
size (Sano and Okamoto, 1994). The genotype of P/E (P: RNA segment A from Buhl, and
E: RNA segment B from EVE) produced large plaques (1.74 - 1.41 mm) and IPNV-Buhl
also produced large plaques (1.48±0.35 mm). However, E/P and EVE (E/E) formed only
small plaques (0.98 0.28 mm) indicating that plaque size is related to RNA segment A. In
virulence experiments, there was no difference in virulence between parental and reassorted
groups. Large plaque clones caused 44% average mortality (five large plaque clone
groups) in rainbow trout and small plaque clone led to 48% mortality (average of five small
plaque clones) showing no difference between two treatments (Sano and Okamoto, 1994).
Virulent and avirulent isolates of IPNV are found within the same serotype (Sano et
al., 1992). MacAllister and Owens, (1995) tested 15 different IPNV isolates for their
virulence. The virulence was not associated with the species from which they were
originally associated: salmonid, non-salmonid or molluscan hosts. Ab serotype isolates
were found to be avirulent for brook trout, whereas Sp and VR-299 serotypes had high
virulence (Vestegard-Jorgensen, 1971). Some of non-salmonid isolates were highly
virulent for trout fry, while others were avirulent. For example, sand goby virus isolate
9
(Oxyeleotris mannoratus) was avirulent and striped bass isolate (Morone saxatilis ) showed
high virulence. Out of three eel isolates tested, two were avirulent (0-3%) and one was
virulent (87%). There was high variability in molluscan isolates in terms of their virulence
on trout fry as well. Five molluscan isolates were tested, only one of them was virulent for
brook trout (MacAllister and Owens, 1995).
In short, virulence and plaque formation of IPNV are related to RNA segment A,
but it is not know which part of genome is responsible for virulence.
Host and Environmental Factors:
Host factors. Host factors are important for the outcome of infectious disease and
its severity (Fenner, 1968). Factors such as host cell background, immune status, genetic
background, age of animal and nutritional status were studied intensively in vesicular
stomatitis virus (Sabin, and Olitsky 1937). Hormones (Lodmell, 1983), nutritional status,
which exerts a marked influence on the outcome of a disease (Chandra, 1979), the role of
host immunological responses such as inflammation (Roberts, 1979), host cell enzymes
(Scheid and Choppin, 1984) have also proven to be important in viral infection outcomes.
The virulence of IPNV usually depends on the particular host species infected (Hill,
1982). Even in salmonids which are known to be susceptible to IPNV, there are marked
differences between species in susceptibility to disease. Silim et al., (1982) reported that
brook trout (Salvelinus fontinalis ) was the most susceptible species and lake trout
(Salvelinus naymakush) the most resistant, and rainbow trout (Oncorhynchus mykiss ) of
moderate susceptibility to IPNV.
Fish age is also an important factor in IPNV disease outbreaks. There is a strong
negative correlation between fish age and prevalence of IPNV (Dorson and Torchy, 1981;
Bootland et al., 1990). IPNV is known to cause disease in 1-5 month-old fry (Wolf et al.,
1960; MacAllister, 1983). Larger fish are often carriers of the virus (Dorson and Torchy,
1985; Ahne and Negele, 1985). Mortality caused by the PEM-PI-IPNV strain was 83% in
10
1-month-old brook trout, 75% in 2 month-old brook trout, in 4-month-old trout only 45%
mortality (Frantsi and Savan, 1971). The mortality in 6 month-old trout was negligible. In
another report, Lapierre et al., (1988) reported that IPNV caused mortality was highest in
brook trout 6 to 11 weeks of age, and after 15 weeks of age, the fish did not seem to be
sensitive to virus. MacAllister (1995) reported that the mortality caused by IPNV was
highest (.70%) in 27-56 day old brook trout fry with mortality peaking in 44 day-old fry.
Temperature: Intense IPNV disease outbreaks occur at a temperature range optimum for
efficient IPNV replication in the host cell. Dorson and Torchy (1981) and Hill (1982)
reported that the temperature range of 8-12 °C at fish hatcheries is an optimum range for
IPNV replication as well as for fry growth.
However, according to Frantsi and Savan (1971), the PEM-PI strain of IPNV
caused 74% mortality of 2 month-old brook trout at 10 °C and 46% at 15 °C, while the VR-
299 strain caused only 31% mortality in 2 month-old brook trout at 15.5 °C and caused no
mortality at 10 °C and 4 °C. IPN disease mortality in rainbow trout fry was lower at 6 °C
and 10 °C than at 14 °C (Sano 1972). In a in vitro study reported by Dorson et al. (1978),
the amount of virus released decreased with increasing temperature. It was highest at 14 °C
and 9 °C and lowest at 20 °C.
These studies above also indicates that other factors beside temperature affect the
disease outcome. Other factors include; age, immune status of host fish, nutrition, and
characteristics of virus that affect the disease outcome.
In vitro Models:
Cell susceptibility:
IPN virus is routinely replicated in such cell lines as AS, BF-2, CHSE-214 (Nims
et al., 1970; Wolf and Mann, 1980), EPC (Novao et al., 1993), and FHM and RTG-2
11
(Wolf and Quimby, 1962; Kelly, 1968; Novao et al., 1993). These cell lines have been
compared in their susceptibility to IPNV and to virus yield.
All IPNV isolates grow on CHSE-214 cell lines (Kelly, 1978). In terms of time
required for destruction of cell monolayers, Kelly (1985) also demonstrated that assays
using CHSE-214 cells were terminated after 3 days, whereas titrations in FHM cells
enumerated after 5 days. The most susceptible cell lines to the turbot isolate were found to
be CHSE-214, FHM and RTG-2 (Novao et al., 1993, Sherrer and Cohen, 1987). EPC
and BB cell lines were less susceptible to this particular turbot IPNV isolate and the AS cell
line was refractory. The titer of lysate from FHM at 15 °C was 5x106 which was not as
high as the value obtained from CHSE-214 (1.8x107) and RTG-2 (1.0x107). Further, the
time required for monolayer destruction was longer in FHM cells than CHSE-214 cells
(Novao et al., 1993; Lannan, 1984; Yamamoto, 1974).
In a study comparing of RTG-2 to FHM, twelve out of twenty-four diagnostic
samples (VR-299 and Buhl) inoculated onto RTG-2 cells yielded detectable IPNV, whereas
only three out of twenty -four samples assayed in FHM were positive to IPNV after
transfer (Kelly, 1978). In the same study it was concluded that the CHSE-214 cell line
was more sensitive than FHM cell line and at least as sensitive as RTG-2 cell line.
Rodriguez et al., (1993) reported that all IPNV isolates tested replicated (VR-299, Ab and
Sp) on CHSE-214 and RTG-2 cell lines (n=194). 81.9% of them were positive on FHM
cell line, and in EPC cells, 86.17% replicated. The IPN virus for this was isolated from
carrier stocks of rainbow trout, however subtype of the virus was not determined.
Cell Culture Adapted Virus (CCA):
The cell line that is used for virus propagation is important for any studies with the
virus. There are number of observations indicating that the virus loses its ability to destroy
host cells after passage in different cell lines. It was reported that IPNV grown in RTG-2
cells adsorbs less efficiently to FHM cells than to RTG-2 cells, although there was no
12
difference in size, morphology and density of virions (Sheffer and Cohen, 1975).
Adsorption of RTG-2-IPNV to RTG-2 cells was 29%, whereas the rate of adsorption to
FHM cells was only 7%. It was stated that adsorption to FHM cells was significant, but
virus did not replicate. Nicholson (1979) also reported that after passing VR-299 IPNV
once in RTG-2 cell line, the plaque titer of RTG-2 IPNV in FHM cells was reduced by a
factor of approximately 10-fold in comparison to the plaque titer in RTG-2 cell lines. In a
further step, Nicholson et al., (1979) tried to isolate a FHM variant to compare wild type
virus to a variant which passed only in FHM cells. Buhl, Idaho and Bonami, France
isolates did not produce any plaques in FHM cells. Although undiluted virus was used, no
FHM variant could be obtained. It was concluded that different isolates differ considerably
in their ability to generate the FHM variant.
Another important factor in virus virulence is the passage number of virus. Fewer
than five passages of virus on cell monolayer did not alter the virulence of IPNV according
to Hill and Dixon (1977). Kohlmeyer et al. (1986) studied Sp and Ab European strains in
terms of their virulence, plaque formation and immunogenicity. Sp passed 11 times was
characterized by small plaques and had low virulence (9%). The He and Ab isolates
produced both small (0.52 mm) and large (>0.52 mm) plaques. In vivo tests indicated
that Sp passed 5 times produced 50% mortality, whereas Ab caused 8%, and He was
avirulent. Initial virulence of these isolates were not reported. Vestegard-Jorgensen (1971)
reported that Sp caused 90% mortality in rainbow trout and 10% mortality was observed
from Ab isolate.
CCA (cell culture adapted) virus does not always mean that the virus has entirely
lost its original structure. Wild type virus can be detected from cell culture adapted virus
infected cells (Hill and Dixon, 1977) and from CCA virus infected trout fry (Dorson, 1978)
(data were not provided).
13
Persistent Infection:
Under certain circumstances, while virus infected cell cultures are multiplying
normally and appear normal, they release significant amounts of virus. This phenomenon
which occurs both in vivo and in vitro is called persistent infection (Joklik, 1985).
Persistent infections are divided into two groups on the basis of the mechanism of
persistance (Porter, 1985). One group is defined by the presence of the infectious virus
which can be recovered in cell cultures. In the second group, the viral genome is present
but infectious virus is not generally produced except during intermittent episodes of
reactivation which are called latent infections. In order to be persistent, first, a virus under
certain circumstances must not be cytolytic and must regulate its lytic potential (Fields and
Knipe, 1990). Second, in vivo it must avoid detection and elimination by host immune
system.
There are many reports of viral persistence in cell monolayers. Parama virus in The
Syrian hamster fibroblast cell line (BHK 21), (Staneck et al., 1972), vesicular stomatitis
virus (VSV) in Aedes aegypti and A. albopictus cells (Artshop and Spence, 1974) and
measles in Buffalo green monkey (BGM) cells (Menna et al., 1975 a, b) produced
persistent infections. After a number of subcultures, the yield of infectious virus was
decreased. It was suggested that passage of persistently infected cells might be the cause of
decrease in the appearance of CPE (Rima 1976).
There are at least three mechanisms that are involved with development of persistent
infections in certain cell lines (Joklik, 1977). One is interferon production. Production of
interferon controls cytopathic effects which accompany virus production (Sekellic &
Marcus, 1978; Hedrick & Fryer, 1981). Second is selection of non-cytolytic mutant
viruses (Preble and Younger, 1975) and the third one is integration of provirus into host
cell by certain RNA viruses (Zhadnow, 1975; Simpson and linuma, 1975). In addition,
defective interfering (DI) particles are another known factor on development of persistent
infections (Hedrick and Fryer, 1981; Chu-Fang Lo et al., 1995)
14
Defective Interfering (DI) particles:
Hedrick and Fryer (1981) mentioned that at least two mechanisms are involved with
the absence of monolayer destruction in IPNV infected cells; interferon and DI virus
production. They may function alone or together to spare PI (persistently infected) cell
lines from cytocidal effects of infectious virus.
Homologous viral interference is believed to affect the viral propagation of IPNV
on cell monolayers ( Nicholson and Dunn, 1974; MacDonald and Yamamoto, 1977;
Hedrick and Fryer, 1981, 1982). Lo et al., (1995) investigated the sequential changes of
viral polypeptides of the virus preparation at high multiplicities of infection. The viral
polypeptide VP2 was smaller in undiluted virus preparations than diluted preparation which
caused CPE. The proportion of smaller VP2 and VP1 to normal molecules increased as the
number of passage increased.
Dorson et. al. (1978) reported an interesting observation on IPNV replication in
RTG-2 cells. Cell culture adapted (CCA) virus, which was obtained by passing 13 times,
prevented or reduced wild type virus replication on the cell monolayer. It was suggested
that it might be because of having higher affinity receptors on the cell surfaces and or
higher numbers of non-infecting interfering particles in CCA virus suspensions.
Passage of IPNV in RTG-2 and CHSE-214 at high multiplicity of infection resulted
in homologous viral interference (Nicholson and Dunn, 1974). The interference was
described by reductions in CPE and infectious virus production. It was concluded that
there is an evidence for production of defective interfering virus production (Hedrick and
Fryer, 1982). There is, according to Nicholson and Dexter, (1975), evidence that the in
vitro characteristics of DI particles are also true for in vivo conditions. Low dilutions of
virus taken from carrier fish, did not cause any CPE which suggested that DI particles are
responsible for absence of CPE (Nicholson and Dexter, 1975).
Hedrick and Fryer, (1981) reported that they could not prove the presence of DI
particles in CHSE-214 cells. However, STE-137 and RTG-2 cell lines which have similar
15
characteristics with CHSE-214 presented DI particles in autointerference. It was suggested
that if DI mediates persistent infections in CHSE-214 cells, it must be unique mechanism
for this cell line.
Interferon:
There are many reports on interferon or interferon-like substances. Some cell lines
release interferon which exerts its activity intracellularly, some do not. It was found that
the RTG-2 cell line releases interferon when infected with either IPNV or IHNV (DeSena
and Rio, 1975; DeKinkelin and Dorson, 1973). An interferon-like substance has been
found to be produced by FHM cells infected with IPNV (Gravel and Malsberger, 1965).
MacDonald and Kennedy (1979) demonstrated that CHSE-214 were defective in interferon
production in response to lytic infections with IPNV. It was also stated that persistent
infection is mediated by DI (defective interfering) particles. This study also indicated that
interferon may not be the only mechanism in prevention of destruction of cell monolayer.
Both CHSE-214 and RTG-2 cell lines have approximately the same susceptibility, while
one produce interferon, the other does not.
Serum Inhibition and Virulence
There are conflicting reports about the effects of normal trout serum (NTS) on in
vitro virus propagation of IPNV. Vestegard-Jorgensen (1973) and Dorson and De
Kinkelin (1974) reported that the serum taken from normal rainbow trout caused
inactivation of cell culture adapted virus (CCA). Inhibition was due to an antibody-like
non-virus-induced protein having 6S sedimentation coefficient. It was different from IgM
which has 16S sedimentation coefficient. Sp strain IPNV was neutralized by serum taken
from IPNV-free rainbow trout stock. Dorson (1975) also demonstrated that virulent Sp
strain of IPNV when passaged in the absence of trout serum became avirulent. This
16
avirulent virus, which was originally resistant to inactivation by serum, became sensitive to
inactivation by NTS. Hill and Dixon (1977) further analyzed the 6S factor. One virulent
and three avirulent strains of IPNV were passed in RTG-2 cells in the presence (passed 10
times) or absence (passed 10 times) of NTS. The originally avirulent isolates passed in the
absence of NTS remained avirulent, but passage of these avirulent strains in the presence of
NTS increased virulence. However, specific data were not provided in this study. Hill et
al., (1982) performed further work on 6S serum inhibition of IPNV. Three high passages
of virus strains which were 6S sensitive and avirulent for fry, were passed in the presence
of NTS. Two 6S sensitive viruses developed 6S resistance and virulence, the third
avirulent strain remained avirulent, but developed 6S resistance indicating that 6S resistance
does not always correlate with virulence.
Cloning of four 6S resistant viruses in the absence of NTS resulted in 6S sensitive virus
(No data were provided). Hill (1982) mentioned that normally virus in its 6S sensitive
stage does not induce neutralizing antibody in rainbow trout, while 6S resistant virus does.
However, Hill and Dixon, (1977) reported that a naturally avirulent strain of IPNV in its
6S (after serial passing in the presence of NTS) resistant form did not induce antibody
production It was suggested that the virus may need to have virulence factor itself in order
to induce immunity.
CCA virus and wild type virus were compared for both plaque size and sensitivity
to NTS (Dorson et al., 1978). It was found that CCA virus could be neutralized 50% by
NTS at dilutions from 1/ 1,000 to 1/ 10,000 (Dorson, 1978). However, the wild type
virus was not affected by 1/10 NTS. Cell culture adapted virus produced both small and
large plaques sensitive to NTS indicating that plaque size is not related to the serum
sensitivity. Kelly and Nielsen (1985) also demonstrated that adsorption of IPNV Sp
subtype to FHM cells can be prevented by 1-2% rainbow trout serum. After incubation of
FHM cells with virus in the presence of 1.3% NTS for 120 minutes, about 97% of virus
17
remained unadsorbed to the FHM cells, whereas in the absence of NTS 45% virus was
adsorbed to FHM cells.
Serum inhibition does not depend on the certain serotype or groups. Different
subtypes of IPNV were tested for serum activity against IPNV on CHSE-214 and FHM
cells (Kelly and Nielsen, 1985). Fish serum obtained from normal trout (Qu'Appelle)
showed the greatest activity against Tellina virus (TV) (512). Anti-IPNV serum titers for
VR-299 and Jasper were 128 and 256. Buhl isolate had the lowest titer (<8). Moreover,
the 6S factor does not depend on geographic areas. Fish stocks from five different areas of
the USA presented significant neutralizing activity against Sp and VR-299 isolates.
Studies with radioisotope-labeled virus showed that inactivation of IPNV by serum
is due to inhibiting of binding to host cell surface (Kelly and Nielsen, 1985) but it occurs
prior to attachment. They also stated that this inhibition is not due to induction of interferon
or binding to cellular receptors. Moreover, it does not depend on some cell variants, the
viruses obtained by passing in different cell lines, like FHM-IPNV or RTG-2-IPNV.
Overall serum titers in CHSE-214 and FHM cells were low but identical showing that
serum activity was directed against FHM-IPNV or RTG-2 IPNV variants.
There are some suggestions indicating that growth of an IPNV isolate in the
presence of NTS is an important aspect of virus pathogenesis and must be considered in the
infection trials (Dorson, 1978; Hill, 1981).
On the other hand, MacAllister and Owens, (1986) passed IPNV VR-299 15 times
in CHSE-214 cells in the presence (5% NTS in MEM) and absence (5% FBS in MEM) of
NTS. Replicate groups of 50 fish (brook trout) were exposed to viruses passed 1, 5, 10
and 15 times in the presence and absence of NTS. It was found that virulence of virus was
not conserved by passing in the presence of NTS. Seventy percent mortality was observed
after passing once in both treatments. Virulence was significantly decreased by passing 5
times in the present and absence of NTS. At passage number 5, mortalities from NTS
treatment were 50% and with no NTS were 20%. At tenth passage, mortalities
18
respectively, were 20% and 5%. At 15th passage mortalities caused by two different
treatments were 5% in NTS treatment and 2% in no NTS treatment indicating that the
virulence, which was originally 70%, was not conserved by passing 15 times with or
without NTS. However, the study also indicated that there was a significant difference in
with and without NTS treatments at passage number 5 and even at passage number 10.
19
MATERIAL AND METHODS
In vitro Experiment:
IPNV isolates:
IPNV isolates were available at Laboratory for Fish Disease Research at Hatfield
Marine Science Center, Newport, OR. Isolates were stored at -80 °C, or in the liquid
nitrogen. Viruses were isolated from various species, geographic areas and belonged to
several serotypes (Table 1). Their characteristics are listed in Table 1.
Cell lines:
The following cell lines were used in this study:
- CHSE-214 (Chinook salmon embryo cells, Fryer, 1965.)
EPC (Epithelioma papullosum cyprini, Fijan et al., 1979).
- FHM (Fathead minnow, Gravel & Malsberger, et al., 1965).
-RTG-2 (Rainbow trout gonad, Wolf and Quimby, 1962)
Cells were grown in Eagle's Minimum Essential Medium (MEM) supplemented with 10%
fetal bovine serum (HyClone Laboratories, inc.). The procedures for the passage of these
cell lines were carried out as described in Caswell-Reno (1989) and Lannan (1994).
Medium:
Cultures were grown in MEM supplemented with 10% fetal bovine serum (FBS).
FBS was stored at -20 °C, thawed at room temperature and kept at 4 °C prior to use. PH of
the medium which was buffered with HEPES (2.4%) was 7.2-7.4 and was also kept at 4
°C incubator. No antibiotics were used at the any stage of subculturing .
20
Table 1. Aquatic Birnavirus isolates used in the experiments. The data includes the
name of isolate, serotype and subtype (by monoclonal antibody analysis), the fish species
from which virus was originally isolated, country /state from which the virus was isolated
and whether it was isolated from diseased or non-diseased fish.
Coun./state d/nd(*)
Isolate
Subtype Serotype
Host species
94-445
Unknown Unknown
Unknown
Idaho
Unk.
Domsea
Unknown
Al
Oncorhynchus mykiss
Idaho
nd
VR-299
VR-299
Al
0. mykiss
W.Virginia
d
Crayfish
Buhl
Al
Astarus astarus
Idaho
nd
CSF 35-85
Buhl
Al
0. mykiss
Idaho
d
H-VAT
Buhl
Al
Salvelinus malma
Idaho
Unk.
OLD-CSF
Buhl
Al
0. mykiss
Idaho
Unk.
009-94-3
Buhl
Al
0. mykiss
Idaho
d
183-82
Buhl
AI
0. mykiss
Idaho
d
191-77
Buhl
Al
0. mykiss
Idaho
Unk.
266-89
Buhl
Al
0. mykiss
Idaho
nd
89-200
Buhl
Al
0. mykiss
Idaho
nd
89-237
Buhl
Al
0. mykiss
Idaho
nd
89-238
Buhl
Al
0. mykiss
Idaho
nd
90-236
Buhl
0. clarki
Idaho
Unk.
90-76
Buhl
Al
Al
0. mykiss
Idaho
nd
91-114
Buhl
Al
0. mykiss
Idaho
nd
91-137
Buhl
Al
Idaho
nd
93-5
Buhl
Al
0. mykiss
0. nerka
Idaho
Unk.
93-507
Buhl
0. mykiss
Idaho
nd
93-7
Buhl
0. nerka
Idaho
nd
94-167
Buhl
0. mykiss
Idaho
nd
94-187
Buhl
Al
Al
Al
Al
Idaho
d
94-273
Buhl
Al
0. mykiss
0. clarki
Idaho
nd
93-3
Buhl
Al
0. nerka
Idaho
nd
Buhl
Buhl
Al
0. mykiss
Idaho
d
CAL.Mojave
Buhl
Al
0. mykiss
Ca
d
Cal.stoddard
Buhl
Al
d
Ca
0. mykiss
nd:non-diseased; d:diseased; Unk.: Unknown
21
Table 1 (continued)
Isolate
Subtype Serotype
Host species
Coun./state
d/nd
CSF 64-93
Buhl
Al
0. mykiss
Idaho
d
90-11
Buhl
Al
0. mykiss
Idaho
nd
ID-PASS-0
Buhl
Al
0. mykiss
Idaho
Unk.
Sawtooth
Buhl
Al
0. clarki
Idaho
Unk.
86-Q
VR-299
Al
Unknown
Unknown
Unk.
Berlin
VR-299
Al
Salvelinus fontinalis
N.Hampshire
d
Obanion
VR-299
Al
0. mykiss
Nevada
Unk.
CL-214
VR-299
Al
0. kisutch
Oregon
nd
Coho
VR-299
Al
0. kisutch
Oregon
nd
lava lake
VR-299
Al
S. fontinalis
Oregon
nd
LH(PA)
VR-299
Al
S. fontinalis
Pa
d
Menhaden
VR-299
Al
Brevoortia tyrranus
Maryland
d
Oswayo
VR-299
Al
S. fontinalis
Pennsylvania
d
Pelton D.
VR-299
Al
0. tsawytscha
Oregon
nd
Reno
VR-299
Al
0. clarki
Nevada
d
SBV
VR-299
Al
Morone saxitilis
Maryland
d
R.River
VR-299
Al
0. mykiss
Oregon
nd
92-326
WB
Al
S. fontinalis
Idaho
nd
94-158
WB
nd
WB
0. clarki
0. mykiss
Idaho
94-434
Al
Al
Idaho
nd
94-435
WB
Al
0. clarki
Idaho
d
92-429
WB
Al
Idaho
nd
CTI'
WB
Oregon
nd
Tai
WB
Al
Al
0. mykiss
0. clarkii
0. mykiss
Taiwan
d
WB
WB
Al
S. fontinalis
Maine
nd
93-321
WB
Al
0. mykiss
Idaho
nd
94-446
WB
Al
S. fontinalis
Idaho
nd
FR-21
Sp
A2
0. mykiss
France
d
OV-7
Sp
A2
Ostrea edulis
Uk
nd
22
Table 1. (continued)
Isolate
Subtype Serotype
Host species
Coun./state
d/nd
Sp
Sp
A2
0. mykiss
Denmark
d
C-18
Sp
A2
Salmo salar
Norway
nd
C-36
Sp
A2
Salmo salar
Norway
nd
C-39
Sp
A2
Salmo salar
Norway
nd
NC-33
Sp
A2
Salmo salar
Norway
nd
NH-44
Sp
A2
Salmo salar
Norway
nd
SGV
Ab
A3
Oxyeleotris
Thailand
nd
marmoratus
Ab
Ab
A3
0. mykiss
Denmark
d
EVE
EVE
A3
Anguilla anguilla
Japan
d
he(c/wb)
He
A4
Salmo salar
Scotland
Hecht
He
A4
Esox lucius
Germany
nd
AS
C-1
A6
Salmo salar
Canada
nd
C-1
C-1
A6
Salmo salar
Canada
nd
OPI-3-88
C-1
A6
Salmo salar
Me
nd
Doug Ramsey
C-1
A6
Salmo salar
Me
nd
C-2
C-2
A7
0. mykiss
Canada
nd
C-3
C-3
A8
S. alpinus
Canada
nd
93-511
BC
A9
Wa
nd
BC 89-362
BC
A9
0. kisutsch
Salmo salar
Canada
nd
HR
BC
A9
pleuronectidae
Korea
Unk.
W17
Jasper
A9
0. shawytscha
Idaho
Unk.
CTH-9/86
Jasper
A9
0. clarki
Idaho
Unk.
Jasper
Jasper
A9
S. fontinalis
Canada
d
91-63
Unknown Unknown
0. mykiss
Idaho
nd
14' boot
Unknown Unknown
Unknown
Unknown
Unk.
31-75
Unknown Unknown
Unknown
Unknown
Unk.
0. clarki
Idaho
d
0. mykiss
Idaho
nd
93-330
94-021
WB
Al
Unknown Unknown
23
Table 1. (continued)
Isolate
Subtype Serotype
Host species
Coun./state
d/nd
ID-93-15
Unknown Unknown
Unknown
Idaho
Unk.
92-326
Unknown Unknown
Unknown
Unknown
Unk.
99-E8
Unknown Unknown
Unknown
Unknown
Unk.
C-17
Sp
A2
Salmo salar
Norway
d
Chilean (tl)
VR-299
Al
0. mykiss
Chile
nd
Dry Mills
WB
Al
S. fontinalis
N.Hampshire
d
DPL
Sp
A2
Ophicephalus striatus
Thailand
nd
0. mykiss
Korea
d
DRT
Unknown Unknown
FV-
VR-299
Al
S. fontinalis
Maine
nd
G.Wilder
Jasper
A9
S. fontinalis
N. Hampshire
nd
HAH
Unknown Unknown
Unknown
Unknown
Unk.
ID-3-31-93
Unknown Unknown
0. mykiss
Idaho
Unk.
18'
Unknown Unknown
0.keta
Korea
Unk.
LKE
Ab
A3
A. japonica
Taiwan
Unk.
LKT
Ab
A3
Tilapia mosambica
Taiwan
Unk.
N-1265
Sp
A2
Salmo salar
Norway
Unk.
N-73AH
Sp
A2
Salmo salar
Norway
Unk.
Nordic
C-1
A6
Salmo salar
Maine
nd
Unknown
Unk.
nd
SF-1284
Te(c/wb)
Te
AS
Tellina tenuis
Unknown
Scotland
Thai
Sp
A2
Ophicephalus striatus
Thailand
nd
VR-299(carp) Unknown Unknown
Carassius carassius
Korea
Unk.
VR-299-(gold) Unknown Unknown
Carasssius aurata
Korea
Unk.
Japanese flounder
Korea
Unk.
Ys
BC
A9
24
Determination of In Vitro Host Range:
Passage of cell lines:
Cell growth was carried out as described by Caswell-Reno, (1989) and Lannan,
(1994). Each cell line was grown in 75 cm2 tissue culture flasks (Corning) which received
15 ml of MEM supplemented with 10% FBS for regular maintenance. Subcultures of each
cell line were incubated at 21 °C. A combination of trypsin and versene was used for
routine passage of all four cell lines.
For viral infectivity assays, monolayer cultures of each cell line were prepared in
24-well plates. For each assay ten 24 well plates (Corning) for 10 different IPNV isolates
and one @24 well plate to use as a control were prepared. Half ml of cell suspension was
added into each well. Four replicates were used from each cell line. They were incubated
at room temperature for at least 2 days to allow formation of a complete cell monolayer.
Virus inoculation:
All IPNV isolates were titered prior to use in this experiment and adjusted to
a concentration of 105 TCID50/ml.. One tenth ml of virus was added into each well of a 24
well plate. Only one IPNV isolate was used on each 24 well plate to avoid cross
contamination. After inoculation with virus, the 24 well plates were incubated for 1-2
hours to allow for virus adsorption. They were washed three times with 1-2 ml. MEM
without fetal bovine serum, and then 1.0 ml of MEM-10 was added into each well. On the
second day to the 7th day, CPE was checked and recorded daily. After 7 days incubation at
18 °C, culture medium from four replicates was pooled, aliquots were taken and held at 4
°C to assay for virus within 48 hrs. Moreover, at the last day of incubation, the cells on all
24-well plates were preserved using 10% formalin and used later for microphotography.
Titrations were carried out as described in Caswell-Reno (1986) on CHSE-214 cell
line using 50% tissue culture infectious dose (TClD50/m1) method. This method
25
(Sperman-Karber method) uses CPE formation as a indicator. Limits of detection are 101.5
to 101°.5 TCID50 virus/ml.
Effects of Normal Trout Serum(NTS):
Single Passage:
Serum:
Serum was pooled from the IPNV free steelhead trout weighing approximately 50
g, obtained from Alsea hatchery. Each pool included blood from at least 12 fish. (Fish in
this hatchery had been tested annually and no IPNV was detected within the last 15 years).
After allowing to clot for 24 hours at 4°C, the blood was centrifuged (1000 x g 20 min) to
separate blood cells from serum. Aliquots were taken and stored at -80°C (Kelly, 1985).
The serum pools were checked for toxicity on RTG-2 and FHM cell lines. Then, 1%
NTS-MEM was prepared by mixing 1 volume of serum and 99 volume of MEM and then
sterilized by passing through a 0.2 gm filter.
IPNV isolates
All of the isolates as described in Table 1. above were also used in this experiment.
Cell lines:
The RTG-2 cell line was used in this experiment. Subculturing and virus
inoculation were carried out using the same protocols used in the previous experiment.
Methods:
The RTG-2 cell line was used for virus propagation in the presence and absence of
NTS. For the culture of this cell line, protocols described by Caswell-Reno (1989) and
Lannan (1994) were used. Eight wells of RTG-2 cell lines were prepared as two rows on
26
each 24 well plate. One row was used for virus propagation in the presence of NTS and
the other one in the absence of NTS. The virus that was used for serum sensitivity test was
diluted in MEM-0 with 1% NTS. One tenth ml of 105 TCID50/m1 was added to each
replicate. After incubating for 1-2 hours, the RTG-2 cell monolayers were washed three
times with MEM-0 and 1 ml of MEM-0 with 1% NTS was added into four virus inoculated
wells. These 8 rows were also checked everyday for CPE along with cell susceptibility
experiment. Uninoculated controls were also prepared using 1% NTS in MEM-10. The
same treatments, such as washing three times and using media from the same stock, were
applied onto the cell monolayers on control plate. Cells were incubated at 18 °C for seven
days. On the last day, aliquots were taken for titration and wells were preserved with 10%
formalin.
Multiple passage:
Four highly virulent Buhl-IPNV isolates (Maret, unpublished results) were used in
this part of experiment. They were passed 11 times in the presence or absence of NTS on
RTG-2 cell monolayers in 25 cm2 tissue culture flasks.
Isolates:
The isolates used and their characteristics are listed in Table 2.
Table 2: Isolates used in the multiple passage of IPNV in the presence and absence of
NRTS and its effect on virulence.
Isolates
Serotype
Subtype
S pp.
state/coun.
d/nd
CSF Crayfish
Al
Buhl
Astarus astacus
Idaho
nd
H-Vat 9/10/86
Al
Al
Al
Buhl
Salvelinus. malma
Idaho
unknown
Buhl
0. mykiss
0. mykiss
Idaho
nd
Idaho
d
90-11
CFS 035-85
Buhl
d: diseased; nd: non-diseased
27
Methods
RTG-2 cell monolayers were prepared in 25 cm2 tissue culture flasks. Three to
four day-old RTG-2 cell monolayers were exposed to virus. Dilutions of each isolate were
prepared in MEM-0, with and without NTS. One ml of each virus dilution was added into
corresponding 25 cm2. After incubating for 1-2 hrs., virus suspension was decanted and
cell monolayers were washed three times with MEM-0. Then, 5 ml of MEM-10 with and
without NTS were added into each corresponding flask. Flasks were incubated at
18 °C until at least 75% cell monolayer destruction occurred. After that, each was
harvested, diluted to 10-1 and transferred onto new RTG-2 cell monolayers. Finally, after
passing 11 times in the presence and absence of NTS aliquots were taken, and put at
-80 °C to use in vivo experiment.
In Vivo Experiment:
Treatment groups:
These treatment groups of brook trout were used in this experiment;
Treatment I:
Virus isolates passed 11 times in the presence of NTS.
Treatment II: Virus isolates passed 11 times in the absence of NTS
Treatment III: Virus isolates which had not been passed more than twice after isolation
from fish.
Controls: No virus
Fish:
Brook trout (Salvelinus fontinalis) for these experiments were obtained from Fall
River Hatchery, Oregon. Fish were held in 1 liter tanks at the Laboratory for Fish Disease
Research at the Mark 0. Hatfield Marine Science Center, Newport, Oregon. This
laboratory provides dechlorinated and filtered Newport city water. Effluents used in
28
experiments are kept in a closed system and prechlorinated with 5.tg/L chlorine. Prior to
release to the Yaquina Bay, it was again chlorinated with 3-41.1.g/L chlorine for 2h.
Experimental Design:
Approximately 50 fish weighing approximately 0.9 grams, were randomly placed
into each 1 liter tank. Two tank replicates were used for each isolate chosen after an
acclimation period of 5 days in these 1 lt. tanks. Enough virus was put into each tank to
yield 104 TCID50/ml. Aeration of each individual tank was provided continuously during
the five hour exposure with virus as in MacAllister and Owens (1993).
Feeding of fish and cleaning of tanks were carried out daily. Dead fish were also
removed daily to be titered for virus concentration. If there were more than five fish dead,
five fish were pooled to be titered. For the first five days, mortalities were recorded and
titered daily. Subsequently five dead fish pooled and titered every three days, until the end
of the experiment.
Visceral organs were taken for viral sampling. After diluting 10-1 with MEM,
samples were homogenized using a "stomacher" (Colworth, Cincinnati) and assayed in 96
well microtiter plates seeded with barely confluent monolayers of CHSE-14.
29
RESULTS:
Ability of Aquatic Birnaviruses to produce cytopatholoey (CPE) in teleost
cell lines:
Of the 109 aquatic birnavirus isolates tested in four continuous teleost cell lines, all
produced CPE in two salmonid cell lines, CHSE-214 and RTG-2. However, only half
produced CPE in two non-salmonid cell lines, EPC and FHM (Table 3).
As can be seen in Figure 1a, all IPNV isolates tested efficiently replicated on
CHSE-214 and RTG-2 cells, and damaged 100% of the cell monolayers after 7 days. The
levels of CPE produced both in FHM (mean CPE (MC) = 1.95) and in EPC cells
(MC = 1.72) were not statistically different from each other (Wald-Wotvitz Runs Test (W-
WRT); z = 0.7502, p = 0.45). However, the viral CPE produced in EPC (MC = 1.72)
was significantly lower than the MC produced by CHSE-214 cells (W-WRT; z = 7.06,
p 5 0.0001) and RTG-2 cells (W-WRT; z = 5.91, p 5_ 0.0001) (MC = 3.95). Similarly,
the mean level of CPE produced by Buhl subtype aquatic birnaviruses on FHM cells were
also significantly lower than the MC values obtained in CHSE-214 (MC = 4) (W-WRT; z =
5.09, p 5 0.0001) and RTG-2 (W-WRT; z = 3.91, p = 0.001) cells (Appendix A.
Table 14.).
The virus titers produced by various isolates in the cell lines tested were also
compared. Virus yields were the highest in CHSE-214 cells (geometric mean titer (GMT)
= 108.43 TCID50/m1.), and RTG-2 cells with a GMT of 107.56 TOID50/ml. The GMTs of
EPC (104.87 TCID50/m1.) and FHM (105.14 TO:Dm/mi.) were not significantly different
from each other (W-WRT; z = 0.068, p = 0.95), but were significantly lower than the
GMTs of both CHSE-214 and RTG-2 cells (D. T.; F= 90.82, p 5_ 0.0001)
30
Table 3. Cytopathic effects (CPE) of aquatic birnaviruses on four teleost cell lines.
Serotype refers to the classification schema of Hill and Way (1988). (Parenthesis indicates
the archetypes of serotype, and n: the number of isolates tested).
Number producing CPE:
Serotype
n
EPC
CHSE-214
FHM
RTG-2
Al (WB)
A2 (Sp)
58
29
58
29
58
12
10
12
9
12
A3 (Ab)
A4 (He)
A5 (Te)
A6 (CAN-1)
A7 (CAN-2)
A8 (CAN-3)
A9 (Jasper)
N.S. (*)
6
0
6
0
5
2
2
2
2
2
1
0
1
0
1
5
0
5
0
5
2
0
2
1
2
1
0
1
0
1
8
8
8
8
8
14
6
14
6
14
Total
109
55
109
54
109
(*) N.S.: Not serotyped
31
a)
4
+1.96*SE
+SE
Mean
3
-SE
-1.96"SE
2
1
0
ERC
CHSE-214
FHM
RTG-2
Cell lines
b)
12
+1.96*SE
+SE
10
Mean
5
0
bjJ
-SE
-1.96*SE
L..
4.)
EPC
CHSE-214
FHM
RTG-2
Cell lines
Figure 1. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by 109 aquatic bimavirus isolates on four different teleost cell
lines (0= no CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%)
(Values indicate mean (dot), standard error of the mean (box) and 1.96
standard errors of mean (whisker))
b) Geometric mean titer (GMT) (TCID50/m1.) yields of 109 aquatic
birnaviruses in all four cell lines tested.
32
Serotypes and In Vitro Host Range:
It was of interest to determine whether the in vitro host range of aquatic
birnaviruses was correlated with the serotype of virus. As indicated in Table 3, the
following results were obtained; all viruses produced complete CPE (mean = 4) in both
salmonid cell lines tested (CHSE-214 and RTG-2 cells), whereas only half were capable of
destroying the two nonsalmonid cell lines tested (EPC and FHM). Aquatic birnaviruses
belonging to the Al (WB), A2 (Sp), A4 (He) and A9 (Ja) serotypes were generally capable
of causing CPE in the non-teleost cells, whereas the others were not.
The ability of each subtype of virus to produce CPE and replicate in the teleost cell
lines will be discussed separately below. All of the subtypes, except Te subtype, caused
significant amounts of cell destruction and all of the subtypes tested on both CHSE-214
and RTG-2 cell lines replicated efficiently (see Figures 2 ab. and 3 ab.). Therefore, these
two cell lines will not be discussed further. Tables relating to each subtype are in the
appendix listed as Appendix A. Table 1., Appendix A. Table 2., Appendix B. Table 1.,
Appendix B. Table 2., etc.
Buhl:
Aquatic birnaviruses belonging to Buhl subtype were the main focus of our study,
since they were isolated primarily from salmonids and have been frequently isolated in fish
farms of Idaho and the western US.
Both the ability to produce CPE and the ability to replicate in each of the cell lines
was assessed. Twenty-nine-out of 29 Buhl subtype isolates produced rapid and extensive
CPE in CHSE-214 and RTG-2 cells.
33
a)
VR-299
BUHL
CAN-1
JASPER
WB
CAN-2
BC
CAN-1
FE
EVE
AB
SP
TE
Subtypes
b)
10
L
VR-299
BUHL
WB
CAN-1
JASPER
BC
CAN-3
CAN-2
AB
FE
EVE
SP
Subtypes
it
Figure 2. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by 109 aquatic birnavirus isolates in CHSE-214 cells (0= no
CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%) (Values
indicate mean (dot), standard error of the mean (box) and 1.96 standard
errors of mean (whisker))
b) Geometric mean titer (GMT) (TC1D50/m1.) yields of 109 aquatic
birnaviruses in CHSE-214 cells.
34
a)
VR-299
CAN-1
JASPER
WB
BUHL
BC
CAN-3
CAN-2
AB
FE
EVE
SP
TE
Subtypes
b)
12
10
8
6
4
2
0
i
VR-299
BUHL
CAN-1
JASPER
WB
BC
CAN-3
CAN-2
AB
FE
EVE
SP
TE
Subtypes
Figure 3. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by 109 aquatic birnavirus isolates in RTG-2 cell s (0= no CPE;
1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%) (Values indicate
mean (dot), standard error of the mean (box) and 1.96 standard errors of
mean (whisker))
b) Geometric mean titer (GMT) (TCID50/m1.) yields of 109 aquatic
bimaviruses in RTG-2 cells.
35
Generally, Buhl subtype isolates did not efficiently replicate in EPC and FHM cells
(Appendix A. Table 1.). A single isolate (94-273) produced CPE in both EPC and FHM
cells. In the remaining 28 isolates, there was no CPE present in either EPC or FHM cells
infected with Buhl subtype isolates. However, virus replication was detected in 10 out of
29 isolates inoculated onto EPC cells, despite the absence of CPE. Similarly, the same
pattern was observed on FHM cells: in fourteen out of 29 cases, FHM cells presented
detectable amounts of virus (_ 101.5 TC11350/m1.) in the absence of CPE. With other Buhl
isolates such as 183-82, 89-200 and 64-93, the absence of detectable virus coincided with
the absence of CPE.
The amount of CPE produced in EPC (MC = 0.071) and FHM (MC = 0.14) cells
was significantly lower than the CPE produced in CHSE-214 (MC = 4) and
RTG-2 cells (MC = 3.96) (Duncan test (DT); F(109,3) = 690.43, p _..0.0001) (Figure 4a.).
The MC of RTG-2 and CHSE-21 were not statistically different from each other (p = 0.76)
as was also the case between EPC and FHM cells (p = 0.23).
In comparison to other subtypes of IPNV, Buhl subtype isolates produced lower
amounts of CPE in both EPC and FHM cells (Appendix A. Table 10, Appendix A. Table
11). In EPC cells, Duncan's test indicated that the level of cytopathology produced by
Buhl subtype isolates was significantly lower than the amount of CPE produced by VR-
299, WB, Sp, Jasper, He and BC (DT; F(82,12) = 21.85, p
__
0.0001) (Figure 13b.). In
FHM cells, a low level of CPE (MC = 0.214) was produced by Buhl subtype isolates and
was significantly lower than the MC of VR-299, WB, Jasper, CAN-2, Sp, He, and BC
subtypes of aquatic birnaviruses on FHM cells (Appendix A. Table 10, Figure 14a.).
Similar to the production of CPE in the cell lines tested, Buhl subtype isolates
produced higher titers in CHSE-214 (GMT = 108.6TO:1350/m1.) and RTG-2 (GMT =
108.17 TCID50/m1.) cells than in EPC (GMT = 102.09) and FHM (GMT = 103.15
TCID50/ml.) cells (DT; F(180,3) = 168.03, p = 0.0001) (Figure 4b. ).
36
a)
+1.96*SE
+SE
Mean
3
-SE
-1.96*SE
2
i.
EPC
CHSE
FHM
RIG -2
Cell lines
b)
10
+1.96*SE
+SE
Mean
-SE
-1.96*SE
6
EPC
CHSE-214
FHM
RTG-2
Cell lines
Figure 4. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by Buhl aquatic birnavirus isolates (n = 29) in all four cells
(0= no CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%)
b) Geometric mean titer (GMT) (TOD50/m1.) yields of Buhl aquatic
birnaviruses in all four cells. (Values indicate mean (dot), standard error of
the mean (box) and 1.96 standard errors of mean (whisker))
37
The amount of virus produced by Buhl subtype isolates in both EPC and FHM was
also lower than certain other subtypes of IPNV. In EPC cells, VR-299, WB, Jasper, Sp,
He, and BC subtypes produced significantly more virus than Buhl subtype isolates
(Appendix A. Table 13, Figure 13a, 14a). Similarly, FHM cells infected with Buhl
subtype isolates produced (GMT = 103.152 TCID50/ml.) significantly less virus than those
infected with VR-299 Jasper, CAN-2 and He(DT; F(81,12) = 6.64, p
0.000) (Appendix
A. Table 13 and Figures 13b, 14b).
In CHSE-214 and RTG-2 cells, all of the isolates replicated efficiently and no
statistical differences were detected in MC or GMTs of different subtypes tested in these
cell lines (See Figures 2 and 3).
VR-299:
Sixteen VR-299 subtype isolates were tested (Appendix A. Table 2.). All of the
VR-299 subtype aquatic birnavirus as destroyed 100% cell monolayers of all four cell lines
(CHSE-214, RTG-2, EPC and FHM), except the AAHL-FV-987 isolate which did not
replicate in either EPC or FHM cells.
As can be seen in Figure 5a, the MCs produced on the four cell lines were not
statistically different (DT; F(56,3) = 2.15, p = 0.10). All of the VR-299 isolates tested
caused CPE on the monolayers of CHSE-214 and RTG-2 cells (Appendix A. Table 2.).
With the exception of FV isolate, all of the other VR-299 isolates also completely destroyed
the monolayers of FHM cells MC = 3.73). In EPC cells, only the VR-299 and Chilean
isolates produced extensive CPE, the other isolates caused various degrees of CPE
formation which were above 50% destruction of monolayers.
When comparing to the other subtypes of IPNV tested, VR-299 subtype isolates
inoculated onto EPC cells caused higher levels of CPE (MC = 3.5) than the IPNV subtypes
of Buhl, CAN-1, Ab, CAN-2, CAN-3, EVE and Te subtypes of IPNV (Appendix A.
38
a)
+1.96*SE
+SE
Mean
-SE
-1.96*SE
ai
02
EPC
FHM
CHSE
RTG-2
Cell lines
b)
10
+1.96"SE
+SE
Mean
8
-SE
-1.96*SE
6
413
O
4
2
0
I33C
CHSE-214
FHM
RTG-2
Cell lines
Figure 5. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by VR-299 aquatic birnavirus isolates (n = 15 ) in all four cells
(0= no CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%)
b) Geometric mean titer (GMT) (TC1D50/m1.) yields of VR-299 aquatic
birnaviruses in all four cells. (Values indicate mean (dot), standard error of
the mean (box) and 1.96 standard errors of mean (whisker))
39
Table 10). The amount of CPE produced by VR-299 subtypes in FHM cells was also
higher than some subtypes of IPNV Buhl CAN-1, Ab , CAN-3, EVE (Figures 13a, 14a.)
(Appendix A. Table 11).
The highest virus yield was detected in CHSE-214 cells (GMT =
108.68
TCID50/ml.). Titers obtained from FHM (GMT = 107.3 TCID50/ml.), EPC (GMT =
107.42 TCID50/ml.) and RTG-2 (GMT =107.38 TCID50/ml.) cells were similar to each
others (Figure 5b). The amount of virus produced in CHSE-214 cells was statistically
higher than the amounts obtained from other three cell lines tested (DT; F(60,3) = 5.04, p =
0.003).
In terms of virus yields, VR-299 subtype isolates produced significantly more virus
in EPC cells (GMT = 107.14 TCID50/m1.) than Buhl, CAN-2, CAN-3, EVE and Te (Figure
13b). In FHM cells, the amount of virus obtained in HIM cells infected with VR-299
subtypes was only significantly higher than the amount obtained from EVE (GMT =
102.75), and Te (GMT = 101.5 TCID50/ml.) (Figure 14b).
WB:
Fourteen WB subtype isolates were tested (Appendix A. Table 3.). The vast
majority of isolates belonging to this subtype replicated in all four cell lines tested, except
for isolate Idaho 93-321which did not produce any CPE in EPC and FHM cells (Appendix
A. Table 14.). Idaho isolate 94-434 was the another exception in terms of causing CPE in
EPC, but not in HIM cells. The amount of CPE produced by the WB isolates tested in
EPC cells (MC = 3) was lower than in CHSE-214 (MC = 4), RTG-2 (MC = 4) and FHM
(MC = 3.36) cells (Figure 6a).
In comparison to the other subtypes of IPNV, WB subtype viruses in EPC cells
were able to produce more CPE (MC = 3) than Buhl, CAN-1, Ab CAN-2, CAN-3, EVE
and TE subtypes of IPNV (Appendix A. Table 10., Figure 13a.). In HIM cells, aquatic
birnaviruses belonging to WB subtype also produced more CPE than the amount of CPE
40
a)
4
+1.96*SE
+SE
Mean
3
-SE
-1.96*SE
2
1
0
MC
CHSE-214
FHM
RTG-2
Cell lines
b)
10
+1.96*SE
+SE
Mean
-SE
-1.96*SE
EPC
CHSE-214
FHM
RTG-2
Cell lines
Figure 6. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by WB aquatic birnavirus isolates (n = 14) in all four cells tested
(0= no CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%)
b) Geometric mean titer (GMT) (TOED50/m1.) yields of WB aquatic
birnaviruses in all four cells tested. (Values indicate mean (dot), standard
error of the mean (box) and 1.96 standard errors of mean (whisker))
41
produced by Buhl, CAN-1, Ab, CAN-3, EVE and TE (Figure 13a).
The greatest amount of virus was released from CHSE-214 cells (GMT = 108.39
TCID50/ml.) compared to other cell lines tested (Figure 2b.) (DT; F(52,3) = 5.73, p =
0.002). A lower amount of virus was released by EPC (GMT = 106.45 TCID50/ml.) and
FHM cells (GMT = 106.36 TCID50/ml.). The virus yields in RTG-2 cells (GMT =
107.63) did not statistically differ from virus yields of any other cell lines tested (Figure
6a.).
The amounts of virus released in EPC cells infected with WB subtype was also
higher than the amount of virus released from the same cell line infected with Buhl
(GMT = 102.09 TCID50/ml.), CAN-2 (GMT = 103.83 TCID50/m1.), and Te (GMT = 101.5
TCID50/m1.) (Appendix A. Table 12, Figure 13b). None of the IPNV subtypes tested on
FHM cells produced more virus than WB subtype (Appendix A. Table 13, Figure 14b).
Jasper:
Four Jasper subtype isolates were tested ( Appendix A. Table 4.). All of them
produced high levels of destruction of the cell monolayers of CHSE-214, RTG-2, EPC and
FHM cells, (Figure 7a.). In the presence of high degree of CPE production, high amounts
of virus were released with this subtype. Analysis by Duncan test indicated that none of
the cells tested produced CPE significantly higher or lower than the other cells tested
(Duncan Tests (DT); F(12,3) =2.45, p = 0.113) nor did they did not differ in terms of the
amount of virus released. (DT; F(12,3)= 2.82, p = 0.084).
42
a)
4
+1.96*SE
+SE
Mean
3
-SE
-1.96*SE
2
1
0
EPC
CHSE-214
FHM
RTG-2
Cell lines
b)
10
+1.96"SE
+SE
Mean
-SE
-1.96*SE
EFC
CHSE-214
FHM
RTG-2
Cell lines
Figure 7. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by Jasper aquatic birnavirus isolates (n = 4) in all four cells
tested (0= no CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%)
b) Geometric mean titer (GMT) (TCID50/m1.) yields of Jasper aquatic
birnaviruses in all four cells tested. (Values indicate mean (dot), standard
error of the mean (box) and 1.96 standard errors of mean (whisker))
43
Generally, compared to other subtypes, Jasper subtype isolates produced higher
levels of CPE in EPC cells. Geometric mean CPEs (MC) produced by Buhl, CAN-1,
CAN-3, EVE and Te were lower than MC obtained by Jasper subtype isolates (MC = 3.25)
in EPC cells (Appendix A. Table 10, Figure 13a). Similarly, MC obtained in FHM cells
infected by some subtypes (Buhl, CAN-1, Ab, and CAN-3) of IPNV was also
significantly lower than the MC obtained from Jasper isolated in FHM (Appendix A. Table
11, Figure 14a).
The GMT of Jasper subtype was the highest in CHSE-214 cells (GMT = 109.18
TO:Dm/mi.) (Figure 7b.). In the other cell lines tested, GMTs were statistically the same
(GMTs of EPC = 107.81 TCID50/ml., FHM = 107.43 TCID50/ml., and RTG-2 = 107.94
TCID50/ml.).
Buhl, CAN-1, Ab, CAN-2 and Te subtypes of aquatic birnaviruses produced far
less virus than Jasper subtype (107.81 TCID50/m1.) on EPC cells (Figure 13b) (Appendix
A. Table 12). The amount of virus produced on FHM cells by Jasper isolate (GMT =
107.43 TCID50/ml.) was also higher than the amount of virus produced by Buhl, Ab, EVE
and Te subtypes of IPNV (Appendix A. Table 13, Figure 14b).
Sp:
Twelve Sp subtype isolates were tested on four cell lines in terms of their ability to
replicate and produce CPE (Appendix A. Table 5.). As can be seen in Figure 8a, all Sp
subtype isolates produced significant amounts of CPE in both CHSE-214 and RTG-2 cells.
There was one case in EPC and two cases in FHM cells that monolayer destruction of cells
by Sp subtypes did not occur. One particular isolate , C-18, failed to produce CPE on EPC
and FHM cells and Sp isolate also failed to produce CPE on FHM cells. The amount of
CPE produced in EPC (MC = 2.62) and in FHM cells (MC = 2.46) was approximately the
same but was significantly lower than the CPE produced in CHSE-214 (MC = 4) and
RTG-2 cells (MC = 4) (DT; F(48,3), p 5. 0.0001) (Appendix A. Table 14, Figure 8b).
44
a)
4
+1.96*SE
+SE
Mean
3
-SE
-1.96*SE
2
1
0
EPC
CHSE-214
FHM
RTG-2
Cell lines
b)
10
+1.96*SE
+SE
8
Mean
gl
-SE
-1.96*SE
0
EFC
CHSE-214
FHM
RTG-2
Cell lines
Figure 8. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by Sp aquatic birnavirus isolates (n = 8) in all four cells tested
(0= no CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%)
b) Geometric mean titer (GMT) (TCID50/m1.) yields of Sp aquatic birnaviruses
in all four cells tested. (Values indicate mean (dot), standard error of the
mean (box) and 1.96 standard errors of mean (whisker))
45
In comparison to other subtypes, seven out of 13 subtypes of aquatic birnaviruses
produced lower amounts of CPE than Sp subtype (MC = 2.61) on EPC cells (Buhl, CAN1, CAN-2, EVE, and Te) (Appendix A. Table 10, Figure 13a). In FHM cells CPE
produced by Sp subtype was also higher than by isolates in the Buhl, CAN, CAN-3, EVE
and Te subtypes (Appendix A. Table 11, Figure 9b).
CHSE-214 cells (GMT = 108.11 TCID50/m1.) released the highest amount of virus
after infection with Sp subtype isolates. The GMTs of virus in EPC (GMT =
106.48
TO:Dm/mi.) and FHM (GMT = 105.42 TCID50/ml.) cells which were not statistically
different from each other, but were lower than the yields of CHSE-214 and RTG-2 cells
(GMT = 106.71 TCID50/ml.) (DT; F(48,3)= 6.22, p
0.0001).
In EPC cells, Sp subtype isolates produced more virus than the amount produced
by Buhl (GMT = 102.09 TCID50/ml.) and Te (GMT = 101.5 TCID50/ml.) (Appendix A.
Table 12, Figure 13b). Sp subtype (GMT = 102.61) replication in FHM cells was
significantly higher than Buhl (GMT = 102.089 TCID50/ml.) and Te (GMT = 101.5
TCID50/m1.) subtypes of aquatic birnaviruses (Appendix A. Table 13, Figure 13b).
Ab and EVE:
Five Ab subtype isolates were tested (Appendix A. Table 6.). Destruction of
monolayers of CHSE-214 (MC = 4) and RTG-2 cells (MC = 4) occurred at a rate of 100%
in both cell lines (Figure 9a.). There was low level of virus present in the absence of CPE
in the other Ab subtype isolates
.
The amount of virus produced in EPC (GMT = 103.75 TCID50/m1.) and FHM (GMT
= 10344 TCID50/m1.) cells was approximately the same. However, the amount of virus
produced in these two cells was significantly lower than the amount produced in CHSE-
214 (GMT = 108.56 TCID50/m1.) and RTG-2 cells (GMT = 107.68 TCID50/ml.). (DT;
F(12,3)= 20.6, p= 0.000) (Figure 7b.).
46
a)
4
+1.96*SE
+SE
Mean
3
-SE
-1.96*SE
2
1
0
ITC
CHSE-214
FHM
RTG-2
Cell lines
b)
10
+1.96*SE
+SE
Mean
-SE
-1.96*SE
6
ETC
CHSE-214
FHM
RTG-2
Cell lines
Figure 9. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by Ab aquatic birnavirus isolates (n = 5) in all four cells tested
(0= no CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%)
b) Geometric mean titer (GMT) (TCID50/ml.) yields of Ab aquatic birnaviruses
in all four cells tested. (Values indicate mean (dot), standard error of the
mean (box) and 1.96 standard errors of mean (whisker))
47
MC produced in EPC cells infected with Ab subtype isolates (MC = 0.25) was
significantly lower than the MC of VR-299, WB, Jasper Sp, He and BC subtypes of
aquatic birnaviruses (Appendix A. Table 10, Figure 13a.). Similarly, the amount of CPE
observed in FHM cells (0.25) was lower than MC of Buhl, VR-299, WB Jasper, CAN-2,
Sp He and BC subtypes in FHM cells (Appendix A. Table 11., Figure 14a.).
Except for the LKE and SGV isolates, all of the other Ab subtype aquatic
birnaviruses produced significant amounts of virus in both FHM (GMT = 103.75
TCID50/m1.) and EPC cells (GMT = 103.44TO:1)50/ml.) in the absence of CPE. The LKE
isolate caused no CPE and no virus was detected. The SGV isolate caused twenty-five
percent CPE on both EPC and FHM monolayers producing also significant amounts of
virus in these cell lines (EPC = 104.25 TCID50/m1. and FHM = 104.5 TCID50/m1.)
(Appendix A. Table 13).
The amount of virus produced by Ab subtype isolates (GMT = 103.75 TCID50/m1.)
was significantly lower than the amount of virus produced by Jasper subtype isolates
(GMT = 107.18 TCID50/m1.) in EPC cells. In FHM cells, CAN-2 (GMT = 109.5
TCID50/m1.) and He (GMT = 107.63 TCID50/m1.) produced more virus than Ab subtype
isolates (Figures 13b, 14b.).
The only EVE subtype isolate tested on four cells presented very similar pattern to
Ab subtype isolates in terms of CPE and virus production (Appendix A. Table 7). There
was no CPE in either EPC or FHM cells, but a low amount of virus was present.
Canadian (CAN-1, CAN-2 and CAN-3):
Eight Canadian isolates belonging to CAN-1 (6), CAN-2 (1) and CAN-3 (1) were
tested (Appendix A. Table 6.). CHSE-214 cells were the most susceptible to CAN-1
subtype isolates in terms of CPE and virus production. The amount of CPE produced in
CHSE-214 (MC = 4) and RTG-2 cell lines (MC = 3.83) was significantly higher than the
48
a)
+1.96*SE
+SE
Mean
-SE
-1.96*SE
EFC
CHSE-214
FHM
RTG-2
Cell lines
b)
12
+1.96*SE
+SE
Mean
-SE
-1.96*SE
EPC
CHSE-214
FHM
RTG-2
Cell lines
Figure 10. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by CAN-1 aquatic birnavirus isolates (n = 5) in four cell lines
(0= no CPE; 1= 25% or less; 2=25-50%; 3= 50-75%; 4= >75%)
(Values indicate mean (dot), standard error of the mean (box) and 1.96
standard errors of mean (whisker))
b) Geometric mean titer (GMT) (TCID50/ml.) yields of CAN-1 aquatic
birnaviruses in four cell lines.
49
CPE production in EPC (MC = 0.33) and FHM cells (MC = 0.50) (DT; F(20.3) = 42.09, p
0.0001). As shown in Figure 10a. the amount of CPE produced in EPC was identical to
the amount produced in FHM. Similarly, CHSE-214 and RTG-2 cells did not differ from
each other in terms of CPE production.
In EPC cells, the amount of CPE produced by CAN-1 subtype (MC = 0.33) was
significantly lower than IPNV subtypes of VR-299, WB, Jasper, Sp, He and BC
(Appendix A. Table 10, Figure 13a.). The same subtypes mentioned above also were
more destructive to the monolayers of HIM cells than CAN-1 subtype isolates. VR-299,
WB, Jasper, Sp, He and BC (Appendix A. Table 11, Figure 14a).
CAN-1 subtype isolates produced higher amounts of virus in CHSE-21 cells
(GMT = 108.71 TCID50/ml.) (Figure 10b.). Justifying the CPE results obtained in the cells
infected with CAN-1 subtypes, in terms of the virus yields, CHSE-214 and RTG-2 cells
(GMT = 107.91 TO: Dm/mi.) released more virus than EPC (GMT = 10604TCID50/m1.)
and FHM cells (GMT = 104.29 TCID50/ml.) infected with CAN-1 subtypes (DT; F(20.3) =
9.82, p
0.0001).
The GMT of CAN-1 subtype (103.83 TCID50/ml.) was much lower than GMTs of
Jasper (107.81 TOD50/m1.) and He (108.0 TCID50/ml.) subtypes of aquatic birnaviruses in
EPC cell lines (Appendix A. Table 12, Figure Ab). In FHM cells, it (GMT = 104.29
TCID50/ml.) was significantly lower than the GMT of CAN-2 subtype (109.5 TCID50/ml.)
(Appendix A. Table 13, Figure 14b).
I was unable to compare the other CAN-2 subtypes, since we had only one isolate
from each subtype. CAN-3 reacted in four cell lines to a degree similar to CAN-1
subtypes, whereas CAN-2 did not grow in EPC cells, but replicated efficiently in FHM
cells producing significant amount of virus (log10 titer = 9.5) (Appendix A. Table 5. )
(Appendix A. Table 13, Figures 13, 14.).
50
a)
+1.96*SE
+SE
Mean
-SE
-1.96*SE
2
EPC
CHSE-214
FHM
RTG-2
Cell lines
b)
10
E
0
O
1.4
+1.96*SE
+SE
8
Mean
-SE
6
-1.96*SE
4
4.1
2
0
EPC
CHSE-214
FHM
RTG-2
Cell lines
Figure 11. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by He aquatic birnavirus isolates (n = 2) in CHSE-214 cells
(0= no CPE; 1= 25% or less; 2=25-50%; 3= 50-75%; 4= >75%)
(Values indicate mean (dot), standard error of the mean (box) and 1.96
standard errors of mean (whisker))
b) Geometric mean titer (GMT) (TC1D50/m1.) yields of He aquatic
birnaviruses in four cells tested.
51
He:
There were only two He subtype isolates tested (Appendix A. Table 7.). The Hecth
isolate caused 100% destruction of monolayers of all four cell lines tested, whereas the He
isolate caused 75% cell destruction in both EPC and FHM cells. In terms of CPE
production there was no statistical difference between MC of all four cell lines (DT; F(4,3) =
0.61, p = 0.64) (A-14, Figure 11a).
In EPC cells, the He subtype differed from aquatic birnavirus subtypes of
Buhl, CAN-1, Ab, CAN-2, CAN-3 and EVE in producing higher amounts of CPE
(Appendix A. Table 10, Figure 13a). Similar results were deserved in FHM cells with
Buhl , CAN-1, Ab CAN-3, EVE and Te subtypes (Appendix A. Table 11, Figure 14a).
Regarding the amount of virus produced in EPC cells, He subtype viruses
produced higher amount of virus than Buhl, CAN-1, Ab, CAN-2, CAN-3 and Te subtypes
of IPNV (Appendix A. Table 12, Figure Ab). In FHM cells, the GMTs of Buhl , Ab,
EVE and Te were lower than GMT obtained in FHM cells (GMT = 107.63 TCID50/ml.)
(Appendix A. Table 13., Figure 14b).
BC:
The growth characteristics of the BC subtype in all cell lines tested were very
similar to those of Jasper subtype isolates. Interestingly, all of four BC isolates tested
replicated efficiently in CHSE-214, FHM and RTG-2 cells, while there was variation in
EPC cells (A. Table ), as was the case with Jasper subtypes. Although there was no
difference in MC of four cell lines (DT; F(12,3) = 3, p = 0.072) (Figure 12a), EPC cells
were statistically different from CHSE-214 (p = 0.031), FHM (p = 0.037) and RTG-2
(p = 0.042) (Appendix A. Table 14).
The GMTs obtained from each cell line were generally high, but statistically did not
differ (GMTs; CHSE-214 = 107.69 TCID50/m1., RTG-2 = 107.38 TCID50/ml.,
52
a)
+1.96*SE
+SE
Mean
-SE
-1.96*SE
EPC
CHSE-214
FHM
RTG-2
Cell lines
b)
10
E
bD
O
+1.96*SE
+SE
8
Mean
-SE
-1.96*SE
6
4
PI
2
0
EPC
CHSE-214
FHM
RTG-2
Cell lines
Figure 12. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by BC aquatic birnavirus isolates (n = 4) in four cell lines (0=
no CPE; 1= 25% or less; 2=25-50%; 3= 50-75%; 4= >75%) (Values
indicate mean (dot), standard error of the mean (box) and 1.96 standard
errors of mean (whisker))
b) Geometric mean titer (GMT) (TaD50/m1.) yields of BC aquatic
birnaviruses in four cell lines.
53
EPC = 106.13 TCID50/ml. and FHM = 106.15 TCID50/ml.) (DT; F(12,3) = 1.34, p = 0.31)
(Figure 12b).
The BC subtype in EPC cells differed from 7 out of 13 IPNV subtypes tested Buhl,
CAN-1, Ab, CAN-2, CAN-3 and EVE and Te, in terms of virus production (Appendix A.
Table 10, Figure 13a). In FHM cells, BC subtypes also produced higher amounts of
cytopathic destruction than Buhl, Ab, CAN-3, EVE and Te subtypes (Appendix A. Table
11, Figure 14a.).
The quantity of virus produced in EPC cells by BC subtype isolates was also
significantly higher than the EPC cells exposed to Buhl (GMT = 102.09 TCID50/ml.) and
Te (GMT = 101.5 TCID50/m1.) (Figure 13b.), similarly in FHM cells the amount of virus
produced by BC subtype isolates was higher than the amount of virus obtained from FHM
cells infected with Buhl subtype isolates (Figure 14b).
Effects of Normal Trout Serum on Aquatic Birnaviruses Replication:
The ability of 1% NTS to inhibit IPNV replication in RTG-2 and FHM cells was
examined. Preliminary experiments indicated that NTS was toxic to FHM cells but not to
RTG-2 cells. The toxic component could not be removed by pretreatment with charcoal.
Consequently, the experiments reported here were performed on RTG-2 cells only.
IPNV virus strains treated with 1% NTS on RTG-2 cells and those which had no
treatment were compared for CPE production and virus replication. Many of the isolates
tested and treated with 1% NTS had significant decreases both in amount of CPE produced
and the titer of virus produced in RTG-2 cells (Appendix B, Tables 1-9). The loss of titer
was in some instances .?.. 107 TCID50/ml. Some subtypes of IPNV differed from others in
terms of being inhibited by NTS (Figure 15b). The inhibition of CPE in many instances
that NTS-treated virus isolates used was total comparing to untreated viruses. Significant
54
a)
0 NAM
VR-299
JASPER
WB
BUHL
CAN-1
BC
CAN-3
CAN-2
AB
RE
FE
SP
II
TE
Subtypes
b)
BUHL
VR-299
WB
JASPER
BC
CAN-1
CAN-2
CAN-3
SP
AB
FE
EVE
TE
Subtypes
Figure 13. a) Box and whisker plot of mean cytopathic effects (CPEs) (MC)
produced by 109 aquatic birnavirus isolates in EPC cells (0= no CPE;
1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%) (Values indicate
mean (dot), standard error of the mean (box) and 1.96 standard errors of
mean (whisker))
b) Geometric mean titer (GMT) (TCID50/m1.) yields of 109 aquatic
birnaviruses in EPC cells tested.
55
a)
VR-299
BUHL
JASPER
BC
VvB
CAN-1
CAN-3
CAN-2
AB
I-E
SP
TE
Subtypes
b
10
L
VR-299
BUHL
VVB
JASPER
BC
L
CAN-3
CAN-2
AB
CAN-1
EVE
SP
1-E
TE
Subtypes
Figure 14. a) Box and whisker plot of mean cytopathic effects (CPE) (MC)
produced by 109 aquatic birnavirus isolates on FHM cells (0= no
CPE; 1= 25% or less; 2= 25-50%; 3= 50-75%; 4= >75%) (Values
indicate mean (dot), standard error of the mean (box) and 1.96 standard
errors of mean (whisker))
b) Geometric mean titer (GMT) (TC1D50/m1.) yields of 109 aquatic
birnaviruses in FHM cells.
56
differences among the mean CPE values of IPNV subtypes were detected (Anova; F =
4.61, p 5_ 0.0001).
In terms of differences in CPE levels produced, Buhl subtype isolates were affected
significantly by the presence of NTS less than VR-299, WB, Jasper, He, Sp and BC
subtypes. (Figure 15, Appendix A. Table 10). In terms of serum inhibition, Buhl subtype
proved to be unaffected by the presence of 1% NTS. Logio titer reductions were also
generally very low and 10/27 isolates showed an increase in titer in the presence of NTS.
(Appendix B. Table 1 ). The range of login titer reductions was between -0.75 and 2.2
.
Only one isolate (Idaho, 94-273), was significantly inhibited from replicating in the
presence of 1% NTS (Login titer difference = 7.5 ): no CPE was observed and no virus
was detected.
Similarly, some subtypes of aquatic birnaviruses also differed from each other in
terms of being inhibited from viral replication by 1% NTS (ANOVA; F = 5.18, p
0.0001). For example, as seen in Figure 15a, Buhl and CAN-1 isolates were significantly
less inhibited by NTS than VR-299, WB, Jasper, BC, Ab, Sp, He and subtypes in terms
of inhibition of viral replication by NTS. The inhibition of replication of Buhl subtype
isolates by NTS (mean titer loss = 100.38 TCID50/ml.) was significantly lower than only
the Jasper (103.94 TCID50/m1.) and EVE (103.8 TCID50/ml.) subtypes (Figure 15).
The mean logio titer and the mean CPE difference in VR-299 subtype was not
statistically different from the mean titers of the 13 subtypes tested (Figure 15). The
highest inhibition was observed with isolates of Reno (107.75 TCID50/ml., pelton dam
(104.5 TO:Dm/mi.) and Oswayo (103.03 TO:Dm/mi.) (Appendix B Table 2). Reno was
not tested for its virulence.
Logic, titer differences in the presence and absence of NTS were generally high in
WB subtype isolates (Appendix B Table 3). In ten out of 15 isolates, the logio titer
differences were higher than 103 fold. Moreover, in five cases, virus replication was
entirely inhibited by NTS
.
57
a)
+1.96*SE
4
+SE
Mean
-SE
-1.96*SE
VR-299 JASPER
BUHL
WB
BC
CAN-1
CAN-3
EVE
AB
CAN-2
SP
FE
TE
Subtypes
b)
+1.96*SE
+SE
Mean
-SE
-1.96*SE
bA
O
a)
VR-299 JASPER
BUHL
WB
BC
CAN-1
CAN-3
EVE
AB
CAN-2
SP
Subtypes
FE
TE
Figure 15.a) Box and whisker plot of the mean CPE differences of between replicated
virus in the presence and absence of NTS. Values indicate mean (dot),
standard error of the mean (box) and 1.96 standard errors
of mean (whisker)
b) The mean login titer differences in the presence and absence of NTS.
58
Except for CAN-1 isolate which was inhibited the most by 1% NTS, the others had
high logio titer difference between the titers obtained from the virus treated with 1% NTS
and the virus that had no treatment (Appendix B Table 4). In terms of mean CPE
differences of subtypes tested, the mean CPE difference in this subtype was different from
EVE and Jasper.
There were high variations among the Jasper subtype isolates tested (Appendix B.
Table 6). The CTH-8/86 isolate was 100% inhibited by 1% NTS. In terms of CPE
produced, 3 of 4 isolates were inhibited in forming cytopathology by 1% NTS. Among 13
subtypes, the highest mean logio titer and mean CPE differences obtained from both
treatments was observed. In terms of mean logio titer differences, this subtype differed
from CAN-2 only but in terms of mean CPE differences, differed from Buhl, CAN-3 and
Te (Figure 15).
Logio titer reductions were also high (Appendix B. Table 7) in Ab subtype of
isolates. Ab subtype isolates was not statistically different from the mean logio titer
difference of any other subtype (Figure 15). The mean CPE difference also did not lead to
any significant difference from other subtypes tested in terms of serum inhibition.
Effect of in vitro passage of aquatic birnaviruses in the presence of NTS
on virulence in Brook trout:
An experiment was performed with 4 Buhl subtype IPNV isolates to determine if
passing the virus 11 times in the presence of 1% NTS had an effect on virus virulence. In
two control groups of fish unexposed to IPNV, the mortality level after 22 days was
approximately 20%. No virus was detected in any of these fish.
Significant mortality levels were detected in lg. brook trout infected with 4 different
IPNV isolates. Mortality levels in this size fish reached approximately 50-60% and were
accompanied by behavioral and gross internal signs of IPN disease.
59
It was found that the number of passage in vitro reduced virus virulence. For two
of 4 Buhl subtype isolates, the virus passed 11 times in vitro in the absence of 1% NTS
(Figures 16 and 17) lost their virulence compared to unpassaged virus, and in no case was
the mortality significantly different than that which occurred in negative controls. Crayfish
and csf-35-85 isolates appeared to lose virulence even in the presence of 1% NTS
(Figure 18, 19).
During the course of the epizootic period (5-10 days postinfection) the virus titers
from moribund fish appeared higher for unpassaged and NTS-treated viruses than for high
passage virus in the absence of NTS. (Figures 16-19).
The 90-11 virus isolate, which is a Buhl subtype isolate, was used in one trial. As
can be seen in Figure 16, the 90-11 isolate passed only once produced 45% mortality and
90-11 isolate passed 11 times in the presence of 1% NTS caused approximately the same
level of mortality. However, the 90-11 virus isolate passed 11 times in the absence of NTS
did not produced elevated mortality. The difference in mortality level between NTS treated
and untreated virus was highly significant (x2 = 15.76, p
0.0001).
Mortalities in the groups exposed to H-VAT isolate were 45% in fish infected with
the H-VAT isolate passed once and the virus passed 11 times in the presence of NTS.
These were significantly higher than the control groups (x2 = 19.71, p
0.0001) (Figure
17). The level of mortalities caused by the virus passed 11 times in the absence of NTS did
not differ from controls (z = 0.16, p = 0.43).
The mortalities caused by the csf 035-85 virus were significantly higher than the
control groups (x2 = 51.77, p
0.0001) (Figure 18). The mortalities caused by csf -035-
85 IPNV isolate passed once was significantly higher than the mortalities caused by csf035-85 isolate which had no treatment during 11 times passage in vitro (ANOVA; F(52,144 )
= 1.74, p = 0.005), whereas no difference was observed between the mortalities caused by
the virus passed 11 times in the presence and the virus passed in the absence of 1% NTS (z
= 0.16, p = 0.43) (Figure 18).
60
a)
50
40
30
20
..
p--0-0:Q.42::fr--e--- Es
A-nr
.o...c5 .04(-133- -ES- -SI
-fr-i*"
10
ES--ESE9 el Eg
-I33- -ESES
16
11
1:1 90-11
ES
days
0
90-11(+)
0
21
A
90-11(-)
Controll
Control2
b)
10
,--.
o
0
604
ai,._.
ao
-1..)
P
7.5
5
2.5
02.5
1
0
4
1
8
I
12
16
i
20
i
24
Days
Figure 16 a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TCID50/ml. IPNV 90-11 (90-11 = virus passed only
once in the absence of NTS, 90-11(-) = virus passed 11 times in the
absence of NTS, 90-11(+) = virus passed 11 times in the presence of
NTS.).
b) Virus titer (TCID5Ohnl.) of dead or moribund brook trout fry after
exposure to IPNV 90-11 propagated in the presence or absence of 1% NTS.
61
a)
50
it
40
0
a .0.O
30
.
.0. 2.estr-A--A---A-
20
o
.A
I itc--A
1.1 ,4133-133-133 -03- i33-133-1B-133
10
-ii8ZER" in
10
25
Days
0 H-VAT
03
20
15
0
0
H-VAT(+)
A
H-VAT(-)
CONTROL 1
CONTROL 2
b)
86-
4
0
.....
...43... .....
.....................
.........
............ 7......r.r8...
--.
.13-
2
0
4
1
8
12
16
20
.... 0
' . ...
24
days
Figure 17. a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TC1D50/ml. IPNV H-VAT 9/86 (H-VAT = virus
passed only once in the absence of NTS, H-VAT(-) = virus passed 11
times in the absence of NTS, H-VAT(+) = virus passed 11 times in the
presence of NTS.).
b) Virus titer (TOD50/m1.) of dead or moribund brook trout fry after
exposure to IPNV H-VAT 9/86 propagated in the presence or absence of
1% NTS.
°
62
a)
10
0 35-85
EH
0
days
35-85(+)
15
0
20
35-85(-)
25
A
control 1
control 2
b)
Figure 18. a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TCID50/ml. IPNV csf-035-85 (35-85 = virus passed
only once in the absence of NTS, 35-85(-) = virus passed 11 times in
the absence of NTS, 35-85(+) = virus passed 11 times in the presence
of NTS.)
b) Virus titer (TC1D50/m1.) of dead or moribund brook trout fry after
exposure to IPNV csf-035-85 propagated in the presence or absence
of 1% NTS.
63
a)
50
40
30
..6...k.,sal...2
20
-ES- -EB -ES- Ell -EB- 131 -EEI
10
10
0 crayfish
ES
20
15
25
days
0
crayfish(+)
0
crayfish(-)
A
control
control 2
b)
7.5
-2.5
0
1
1
4
8
1
1
12
16
20
1
24
days
Figure 19. a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by
immersion to 104 TO:DSO/mi. IPNV Csf-Crayfish (Crayfish = virus
passed only once in the absence of NTS, Crayfish(-) = virus passed 11
times in the absence of NTS, Crayfish(+) = virus passed 11 times in
the presence of NTS).
b) Virus titer (TO:DSO/mi.) of dead or moribund brook trout fry after
exposure to IPNV Csf-Crayfish propagated in the presence or
absence of 1% NTS.
1
64
As can be seen in Figure 19, no difference was detected between negative controls
and the crayfish isolate viruses passed 11 times in the presence or absence of NTS (x2 =
6.17, p = 0.46), whereas the virus passed once proved to be virulent to brook trout.
Titers:
The titers obtained in the exposure of trout to the 90-11 isolate passed in the
presence or absence of NTS were significantly different from each other (ANOVA; F(25,2)
= 3.94, p = 0.032). In all other three trials, titers obtained in different treatments did not
differ from each other as can be seen Figures 16b, 17b, 18c, 19d.
65
DISCUSSION:
Ability of aquatic birnaviruses to produce CPE in four teleost cell lines.
It has long been known that certain cells vary in their susceptibility or resistance to
certain viral agents. Even cells having identical surface receptors may differ in their
susceptibility to the same group of virus (Conrat-Fraenkel and Wagner, 1984). It is known
that certain kinds of aquatic birnaviruses also replicate better in certain cell lines than the
others. Kelly (1978) found that CHSE-214 cells were more sensitive to the Buhl isolate
than were FHM cells and at least as sensitive as RTG-2 cells.
In this study, it was found that the most susceptible cell line to 109 aquatic
birnaviruses examined was CHSE-214 (Mean CPE = 4, and GMT = 108.43 TCID50/m1.)
among four cell lines tested (RTG-2, FHM, EPC ). The number of isolates tested in this
study was the highest in the literature compared to the studies in the host range
investigations of aquatic birnaviruses. Selection of these isolates was also made from a
broad range of isolates in terms of serotypes to which they belong, and countries from
which they were isolated. All of the aquatic birnavirus isolates tested on CHSE-214 cells
replicated efficiently and produced rapid and extensive total (4) CPE. Consistently, with all
isolates tested, the virus yields were higher in this cell line than that produced by the other
salmonid cell line (RTG-2 ) and two other non-salmonid cell lines (EPC and FHM ) The
results obtained supported the results of Kelly (1978), and Novao et al. (1993).
Similarly, except for the Te subtype of IPNV, all the other aquatic birnaviruses
replicated to high levels and caused extensive cytopathology in RTG-2 cell lines as well as
in CHSE-214. The levels of CPE produced in these two cell lines were statistically the
same. This confirms that these cell lines can be used interchangeably in viral propagation
for certain viral studies and for the detection of IPN viruses in aquatic animals. As an
exception, Te subtype (one isolate) did not replicate in EPC, FHM and RTG-2 cells,
66
whereas it did in CHSE-214 cells. This result was quite unexpected, since all of the other
(108) isolates tested replicated both in CHSE-214 and RTG-2 cells.
As can be seen Figure in 14 and 15, the 109 isolates could be placed into 2 groups
on the basis of their in vitro characteristics. Group one consist of 6 subtypes (Buhl, CAN1, CAN-3, Ab, EVE, and Te), which do not efficiently replicate or do not cause any
cytopathology in EPC and FHM cells. The second group includes WB, VR-299, Jasper,
BC, He and Sp.
One interesting observation to note was that, although CAN-2 (one isolate) subtype
was not able to produce CPE and high virus yields in EPC cell lines, it did effectively
replicate in FHM cells (10" TCID50/m1.). This isolate was the only one tested from this
subtype, since no other CAN-2 isolates were available. CAN-2 and CAN-3 isolate are
identical to each other in terms of 11 of their epitopes (Caswell-Reno et. al., 1989).
Therefore, the ability of these viruses to replicate in FHM may be a way to distinguish
these two serotypes.
All of the subtypes which failed to replicate in EPC and FHM lacked the W4
epitope (Caswell-Reno et al., 1989; Appendix A. Table 20). These data indicate that the
W4 epitope may be important in the determination of host range in vitro. For example, the
Idaho 93-321 isolate, in its epitope structure, was the closest to WB subtype. However, it
was lacking W4 epitope, and in vitro tests showed that this isolate did not replicate in EPC
or FHM cells. Four of the 6 remaining subtypes posses the W4 epitope and are capable of
replicating efficiently in EPC and FHM cells. There might be a combination(s) of epitopes
that completes the viral structures of these two isolates instead of W4 epitope
This study found that 26 of 27 Buhl subtype isolates did not replicate on EPC or
FHM cells. Kelly (1978) compared the VR-299 and Buhl isolates and reported that the
Buhl isolate failed to produce virus yields and cytopathology, whereas VR-299 isolate
caused significant amounts of virus and CPE in FHM cells. In another study carried out by
Nicholson et al., (1979) no FHM variant (virus propagated in FHM cells) could be isolated
67
from Buhl subtype isolates. Darragh and McDonald (1982) also reported the failure to
isolate Buhl isolate on FHM cells. These studies strongly indicated that Buhl subtype
isolates do not replicate efficiently in FHM cells. The present study, however, used 27
isolates of the Buhl subtype rather than one as in previous studies. Since there is marked
variability in virus characteristics within a serotype (Sano, 1972), this study showed that
the host range of Buhl subtype virus isolates is consistent.
While Buhl subtype isolates did not to produce CPE in EPC and FHM cells, this
was not the case in terms of viral replication. We found that in some cases (8 of 28 in EPC
and 12 of 28 in FHM cells) significant amounts of virus were recovered from the cells even
though no CPE was observed. It was found that at levels of virus production greater than
105.5 TCID50/ml, CPE was produced in infected cells, whereas at the levels lower than
this, CPE was not evident. As can be seen in Appendix A. Table 1 , interestingly, the
amounts of virus in some cases were unusually high in FHM cells (105.75 to 106.5
TCID50/m1.). In 7/12 cases, the amount of virus recovered in the absence of CPE was
higher than the lower limit to cause CPE in EPC, CHSE-214 and RTG-2 cells. Holding
cells for a further 7 days may have resulted in the production of CPE, but this was not done
in this study. These results confirm the utility of blind passing cells to enhance the
potential for producing CPE. This should be encouraged for diagnostic work since the
absence of CPE may not mean absence of replicating virus.
The reason that some isolates of aquatic birnaviruses produce cytopathology in
certain cells and not in the others is not known. Darragh and McDonald (1982) suggested
that cellular receptors for IPNV subtypes are different on FHM and CHSE-214 cells. So
he described the failure of the adsorption of OV-7 isolate to FHM cells to a lack of
receptors. In contrast, our results indicated that in many cases IPNV entered into nonsalmonid cells and replicated to some extent but not to the level occurring in the salmonid
cells. This was not only the case in Buhl subtype isolates but also was the case with other
subtypes such as CAN-1, EVE, Ab and CAN-3. Therefore, the best cell line for the
68
propagation of isolates (Buhl CAN-1, CAN-2, CAN-3, EVE, and Ab) is CHSE-214 and
RTG-2 cell lines. As observed with Buhl subtype isolates, one CAN-1 subtype isolate
(Nordic) and the only CAN-3 subtype isolate tested produced more virus in FHM cells than
the lower limit of virus yields to form CPE in FHM cells infected with other subtypes such
as VR-299, WB, Jasper etc., but no CPE was observed.
There was no CPE observed in either EPC or FHM cells with Ab subtype isolates
with the exception of sand goby virus (SGV) isolate which caused 25% CPE in both EPC
and FHM cells. It is possible that this level of CPE was due to toxicity, unrelated to virus
production, because 1) titers in both EPC and FHM cells were below 1053 TCID50/ml
2) some low level of destruction (25%) was present after 24 hours of infection and did not
progress further during the 7 day-incubation.
Te (a mollusk) subtype isolate only replicated efficiently in CHSE-214 cells. In the
other three cell lines replication and production of cytopathology was inefficient.
Moreover, this isolate was the only one that did not replicate well in RTG-2 cells (Mean
CPE = 25% and GMT = 10").
As mentioned previously, the isolates in the second group (VR-299, WB, Jasper,
BC, and He) all replicated and produced significant amounts cytopathology in all four cell
lines. Higher virus titers, however, were obtained from CHSE-214 cells and RTG-2 cells
than from EPC and FHM cell lines. VR-299 and WB subtypes belong to the Al serotype
and reacted very similarly to one another on all four cell lines. Since its first isolation,
numerous studies on IPN disease have been carried out with VR-299 isolate. As recorded
by Wolf and Mann (1979), this isolate can be propagated in EPC and FHM cells as well as
in CHSE-214 and RTG-2 cells. The single exception among 16 isolates was the FV isolate
which did not cause any cytopathology on either EPC or FHM cells. It is possible that this
isolate might have been mislabeled or misread, so the data for this isolate were not included
in statistical tests of the VR-299 subtype.
69
Subtypes belonging to the Jasper (A9) serotype proved to replicate efficiently and
caused 100% cytopathology in CHSE-214, RTG-2 and FHM cells, while some of A9
serotype isolates did not cause extensive cytopathology in EPC cells. This isolate was also
used in the comparison of different viruses on different cell lines by Darragh and McDonald
(1982). They also demonstrated that this isolate could be propagated in FHM cells
effectively. In terms of virus production, only CHSE-214 cell lines produced more than
107 TCID50/ml, which is a desired concentration in certain test such as immunodot-blotting
(Caswell-Reno, 1989). Therefore, I suggest using CHSE-214 cells for propagation of
aquatic birnaviruses belonging to A9 serotype to obtained high amounts of virus titers.
Sp serotype viruses also have been extensively used in research on IPNV. Sp
subtype isolates are currently causing problems for Norwegian fish farms. As was reported
by Smail et al., (1992), Sp subtype isolates have been found to cause disease after transfer
of Atlantic salmon smolts to the sea. The majority of isolates tested (11 of 12) replicated in
both EPC and FHM, although the isolate, C-18, did not produce CPE in either EPC and
FHM cells. Although, isolates C-17, C-18, C-36 and C-39 were isolated from the same
cage of clinically ill Atlantic salmon, C-18 proved to different in its growth pattern. The
other two Sp subtype isolates, NC-33 and NH-44, were isolated from clinically ill (NC-33)
and healthy (NH-44) fish in adjacent cages, so they were identical to each other in terms of
CPE production and virus titers. Even they presented similarities in their reaction to NTS,
as we will be discussing in the oncoming section. As reported by Darragh and McDonald
(1982) the OV-7 isolate, an Sp subtype isolate, also did not produce extensive CPE in EPC
and FHM cells and virus yields were also low.
In conclusion, our results suggested that the best cell line for IPNV enumerating in
clinical sample testing is CHSE-214 cells. RTG-2 cells are the alternative of CHSE-214
cells. Clinical sample testing should not be carried out on either EPC or FHM cells since
they are not susceptible to some subtypes of IPNV. Since CPE formation is the target
response of clinical sample testing, false results may be obtained.
70
One of the objectives of this study was to determine the relationship between an
isolate's in vitro host range and virulence of that virus. The results indicated that there was
not any correlation between in vitro virus propagation of virus and its virulence. This
result supported the result of Sano (1972 ). For example, the vast majority of Buhl
subtype isolates which were the most virulent (Maret, thesis in progress) did not replicate
in either EPC or FHM cells. But WB and VR 299 subtype isolates grew on both of these
cell lines regardless of whether they are virulent or avirulent isolates. As we just mentioned
virulence also does not depend on certain subtypes. Buhl subtype isolates were generally
virulent, and also there were some virulent isolates belonging to different subtypes tested.
It is important to note that in our results, four isolates belonging to different
subtypes of IPNV, as we will discus further in the oncoming sections, acted totally
unexpectedly. These isolates were 94-273 (Buhl), FV (VR-299), Idaho 94-434 (WB) and
C-18 (Sp). In these situations we consider the possibility of mislabeling or misreading of
vials.
Effects of Normal Trout Serum on Aquatic Birnaviruses replication:
As can be seen in Figure 20a, there was a striking relationship observed with CPE
differences in some isolates replicated in the presence of NTS (chi-square = 21.54; df = 4;
p = 0.002) . High CPE differences coincided with low degrees of virulence of virus. The
isolates having more than 50% virulence was resistant to normal trout serum, whereas the
isolates that had low virulence or no virulence were inhibited effectively by 1% NTS.
As can be seen in Figure 20b, there was a strong relationship (F(1,65)=31.970, p
0.0001) between logio titer differences on RTG-2 cells from the virus treated with 1% NTS
and the virus which had no treatment and virulence of virus. When the inhibition by NTS
is high, the virulence of virus tends to be low, whereas especially, if virus caused greater
than 80% mortality there was no inhibition. Infact, virus yield was increased for some
Buhl subtype isolates having nearly 100% virulence. Viruses inducing moderate virulence
71
levels (50-80%) were very variable in terms of being inhibited by NTS. Most of the
isolates fit the model of that indicated a negative correlation between inhibition by NTS and
virulence. Except for a single CAN-2 isolate and Buhl subtype isolates, the other isolates
were all inhibited by NTS to some degree.
It was found that avirulent isolates were inhibited from replication on RTG-2 cells
even after a single passage in the presence of NTS. These result supported those of
Vestegard-Jorgensen (1973), Dorson and DeKinkelin (1974) and Kelly and Nielsen
(1985). This inhibition as reported by Vestegard-Jorgensen (1973), was due to an
antibody-like, nonvirus induced factor having a 6S sedimentation coefficient. This differs
from salmonid IgM which has a 16S sedimentation coefficient.
Dorson et al., (1978) reported that cell culture adapted virus could be neutralized
50% by NTS at dilutions from 1/1,000 to 1/10,000. Kelly and Nielsen (1985) also
reported that Sp subtype IPNV could be inhibited 97% by NTS on FHM cells.
It was not possible to test NTS inhibition on FHM cells because of cellular toxicity
of 1% NTS on this cell line. However, since, as reported by Kelly (1985), NTS acts prior
to attachment to cells, host cell origin differences should not affect susceptibility to NTS in
vitro. Host range might be important in observing development of 6S sensitivity relative to
virulence alterations with passage level. For example, it was reported by Hill and Dixon
(1977 ) that serum sensitivity developed faster in EPC cells than RTG-2 and BF cells. The
results that will be obtained from this kind of study may help to establish standard
procedures to produce virus for infection trials and immunization.
Our results also supported the findings of Kelly and Nielsen (1985 ) that serum
inhibition is not dependent on the virus serotype. The data presented in the present study
indicates that Buhl subtypes having highest virulence values had the lowest inhibition of
replication among the IPNV tested. WB, Jasper, Ab, Sp, Te, BC and He subtypes were
inhibited the most since most of the isolates included within these subtypes were avirulent.
For example, the 93-321 a virulent isolate belonging to WB subtype was not inhibited
72
a)
chi-square = 21.54; df= 4; p = 0.002
100
+1.96*SE
+SE
Mean
80
t'0 so
=
s
O
-SE
-1.96*SE
-4-)
e
40
20
25
0
50
75
100
% Inhibition of Cytopathology
b)
F(1,65) = 31.970, p
0
20
40
60
80
0.000
Scatterplot
100
120
Mortality( % )
Figure 20. a) The relationship between CPE differences in RTG-2 cells infected
with aquatic birnaviruses in the presence and absence
of NTS and mortality levels in brook trout fry
b) The relationship between Logio titer differences in RTG-2 cells infected
with aquatic birnaviruses in the presence and absence
of NTS and mortality levels in brook trout fry
73
(logio decrease = 0.5). On the other hand, 92-326 isolate which is also a WB subtype
isolate, but which was avirulent (21.5% ) the titer difference was also highly significant
(logio = 7.2).
In short, isolates having high degree of virulence were consistently resistant to NTS
as, for instance, was the case in Buhl subtype isolates. It is suggested to propagate virus in
the presence of NTS for infection trials as suggested by Hill (1982). Before infection trials
it is important to confirm that the virus that will be used is not 6S sensitive. If it is 6S
sensitive, it will be neutralized by NTS effectively.
Effect of in Vitro Passage in the presence of NTS on IPNV virulence in
brook trout:
The number of in vitro passages is known to influence with virulence. It has been
reported by many researchers that IPNV loses its virulence to some degree through in vitro
passage. However, fewer than five passages does not significantly alter virulence of the
virus (Hill and Dixon, 1977; Dorson et al., 1978).
Kohlmeyer et al., (1986) reported that the Sp isolate passed 5 times produced 50 %
mortality, whereas at the 11th passage, it caused only 9% mortality. This isolate at the
original isolation had caused 90% virulence in trout (Vestegard-Jorgensen, 1971).
Similarly, MacAllister and Owens (1986 ) passed the VR 299 isolate, which caused 90%
mortality originally, multiple times. It was found that the VR 299 isolate passed 5 times in
the absence of NTS produced 20% mortality, and in the presence of NTS, 50% mortality
was observed. At 15th passage this isolate was avirulent regardless of the presence of
serum.
These results port here were also supportive of results mentioned above in terms of
loss of virulence by multiple passages. In four out of four cases, viruses passed 11 times
in the absence of NTS lost their virulence (See Figures 16, 17, 18 and 19). In two out of
four cases, virus isolates, 90-11 and H-VAT, passed 11 times in the presence of NTS
74
retained their virulence, whereas the other two isolates, crayfish and 35-85, lost their
virulence although they were passed in the presence of NTS. Mortalities observed from
crayfish (+) and 35-85 (+) isolates were not statistically different from mortalities observed
from negative controls although virus was isolated from these fish. This strongly
suggested that these viruses became avirulent after passing 11 times despite the presence of
1% NTS in the culture medium. In all four cases, mortalities from the isolates that passed
only once were the highest.
The effects of NTS on preservation of virulence during multiple passage remains in
question. Hill (1982) reported that virulence of aquatic birnaviruses could be preserved by
using NTS during virus propagation on cell monolayers. Moreover, he added that
avirulent isolates could be made virulent by multiple passing in the presence of NTS.
However, the avirulent isolates made virulent did not induce antibody production whereas
naturally virulent isolates induce. No specific data were provided in Hill's 1982 report or
in the later report by Hill and Dixon (1977).
In contrast, MacAllister and Owens (1986) reported IPNV virulence could not be
preserved using NTS and FBS must be used instead. According to the data provided by
MacAllister and Owens (1986), the virus passed in the presence of 5% FBS and the virus
passed in the presence of 5% NTS both lost their virulence entirely at the passage number
15. One drawback to this study was that only one isolate, VR 299, was used.
According to our data and the data provided by MacAllister and Owens (1986) we
suggest that NTS usage is important in preparation of virus stocks for in vivo experiments.
We also agree with the suggestions made by Hill (1982) that if one is investigating small
degrees of virulence between isolates, it is essential to use NTS in the culture medium to
prevent virulence lost during in vitro virus preparation.
The results we obtained do not agree with the statements made by both Hill (1982)
and MacAllister and Owens (1986). In two out of four trials, virulence was preserved
suggesting that virulence could be preserved using NTS, whereas with the other two Buhl
75
subtype isolates virulence was lost after passing 11 times in the presence of NTS. This
suggests that preservation of virulence by NTS may not always occur. It is strongly
possible that there may be some other factors related to viral mutation playing role in
resistance to NTS. Some alterations in viral components that are important in the
determination of virulence may be preserved for some passage number by NTS during
cloning.
In conclusion, according to our results, the effect of NTS on virulence is variable,
even within one subtype of IPNV (Buhl). There is evidence for some kind of effect of
NTS on preservation of virulence, but variability of effects on IPNV isolates remains
unknown.
76
CONCLUSION:
One of the objectives of the study was to determine in vitro growth characteristics
of virus and the relationship to serotype. A strong relationship between in vitro host range
of IPN virus and its serotype was detected. Buhl, CAN-1, CAN-2, CAN-3, Ab, EVE and
Te subtypes of IPNV did not produce CPE in two non-salmonid cell lines (EPC and FHM)
tested, whereas VR-299, WB, Jasper, BC, He and Sp subtypes of aquatic birnaviruses
produced significant levels of cytopathology. The formerly mentioned group of isolates
did not have W4 epitope, whereas the latter mentioned group, except Sp and He subtypes,
had this epitope. This feature migth be one of the important aspects in determination of
host range of aquatic birnaviruses.
EPC and FHM cell lines, since only half of the isolates tested produced CPE, these
cell lines should not be used for diagnostic work with IPNV.
This study is also important in terms of refinement of some standard laboratory
works with IPNV, since the host range of aquatic bimaviruses is clarified.
The results obtained indicated that there was no relationship between being 6S
sensitive of resistant and virus serotype, although it was originally thought that there was.
There was a strong negative correlation between inhibition by 1% NTS and virulence of
isolate. Virulent isolates was not affected by NTS. Moreover, the highly virulent (more
than 90%) isolates of Buhl subtype replicated better in the presence than absence of NTS.
The last objective of the study was the determination of the effects of NTS in vivo
.
Preservation of virulence using NTS in culture medium was not always possible according
to the results obtained. In two cases virulence was effectively preserved by NTS, whereas
in the other it was lost. In all four cases virus passed 11 times in the absence of 1% NTS
lost its virulence, suggesting the importance of NTS during multiple passing of virus in
vitro.
77
Recommendations For Future Research:
There are many areas to be investigated yet. The variants obtained from four
different teleost cell lines can be sequenced and compared for any significant mutational
change.
Molecular structure of 6S serum factor should be investigated. It is important in
terms of investigation of some more characteristics of virus related to its virulence.
Using the same isolates that were used in multiple pass experiment, the virulence
change in each individual pass number can be determined and the change in the virus
structure can be investigated. For further investigation, the same isolate can be pass in the
presence and absence of NTS on different teleost cell lines and change in the virulence of
isolates can be determined. In two out of four cases, NTS helped to retained the virulence
of isolates. The viral structure of virus isolates that retained and did not retained might be
determined with molecular structures.
78
BIBLIOGRAPHY:
Adair, B. M. and H. W. Ferguson. 1981. Isolation of infectious pancreatic necrosis
virus from non-salmonid fish. Journal of Fish Diseases 4:69-76
Ahne, W., and R. D. Negele. 1985. Studies on transmission of infectious pancreatic
necrosis virus via eyed eggs and sexual products of salmonid fishes. In A. E. Ellis
Ed. Fish and Shellfish Pathology 261-269. Academic Press, London.
Ahne, W. 1978. Isolation and characterization of infectious pancreatic necrosis virus
from pike (Esox lucius). Archives of Virology 58:65-69.
Argot, J., and R. G. Malsberger. 1972. Intracellular replication of infectious
pancreatic necrosis virus. Canadian Journal of Microbiology 18:865-867.
Artshop, H., and L. Spence 1974. Persistent infection of mosquito cell lines with
vesicular stomatitis virus. Acta Virology 18:331-341.
Ball, H. J., A. L. S. Munro, A. Ellis, K. G. R Elson, W. Hodgkiss, and I. S.
McFarlane, . 1971. Infectious pancreatic necrosis in rainbow trout in Scotland.
Nature 234:417-418.
Bassel-Duby R, D. R. Spriggs , K. L. Tyler , and B. N Fields. 1986. Identification of
attenuating mutants on the reovirus type 3 Si double stranded RNA segment with a
rapid sequencing technique. Journal of Virology 60:64-67.
Beasly, A. R., M. M. Siegel, and L. W. Clem. 1965. Virus carrier cell cultures from a
marine fish. Bacteriological Proceedings 14:99. Abstract.
Besse, P., and P. de Kinkelin. 1965. Surl'existence en France de la necrose pancreatique
de la truite arc-en-ciel (Salmo gairdneri) Bulletin de l'Academie Veterinaire de
France 38:185-190.
Billi, J. L. and K. Wolf. 1969. Quantitative comparison of peritoneal washes and feces
for detecting infectious pancreatic necrosis (IPN) virus in carrier brook trout.
Journal of Fisheries Research Board of Canada 26:1459-1465.
79
Bonami, J. R., F. Couserans, M. Weppe, and B. J. Hill. 1983. Mortalities in
hatchery-reared sea bass fry associated with a birnavirus. Bull. Eur. Assoc. Fish
Pathol. 3: 41
Boot land, L. M., P. Dobos, and R. M. W. Stevenson. 1986. Experimental induction of
the carrier state in yearling brook trout: A model challenge protocol for IPNV
immunization. Veterinary Immunology and Immunopathology 12:365-372.
Boot land, L. M., P. Dobos, and R. M. W. Stevenson 1991. The IPNV carrier state and
demonstration of vertical transmission in experimentally infected brook rout.
Diseases of Aquatic Organisms 10:13-21.
Bovo, G., G. Giogetti, and G. Geshia. 1985. Comparative sensitivity of five cell lines to
wild infectious pancreatic necrosis viruses in northeastern Italy. In Ellis, A. (Ed.)
Fish and Shellfish Pathology pp. 289-294.
Bragg, R. R. and M. E. Combrink. 1989. Isolation and identification of trout viruses in
South Africa. Journal of Veterinary Resources n:3 55:139-143.
Brown, F. 1986. The classification and nomenclature of viruses: summary of results of
meeting of the international community on taxonomy of viruses in Sendai,
September, 1984. Intervirology 25:114-143.
Caswell-Reno, P., P. W. Reno, and B. L. Nicholson. 1986. Monoclonal antibodies to
infectious pancreatic necrosis virus: Analysis of viral epitopes and comparison of
different isolates. Journal of General Virology 67:2193-2205
Caswell-Reno, P. Lipipun V., Reno P. W., and Nicholson B. L. 1989. Use of a groupreactive and other monoclonal antibodies in an enzyme immunodot assay for
identification and presumptive serotyping of aquatic birnaviruses, Journal of
Clinical Microbiology 27:1924-1929.
Chandra, R. 1979. Nutritional deficiency and susceptibility to infection. Bull WHO
57:167-177.
80
Cohen, J., A. Poinsard, and R. Scherrer. 1973. Physico-chemical and morphological
features of infectious pancreatic necrosis virus after isolation in cell culture,
Canadian Journal of Fisheries and Aquatic Sciences 40:2200-2203.
Cristie, K. E., L. S. Havarstein, H. 0. Djupvic , S. Ness, and C. Endersen. 1988.
Characterization of infectious pancreatic necrosis virus isolated from Atlantic
salmon (Salmo salar). Archives of Virology 103:167-177.
Darragh, E. A., and R. D. Macdonald. 1982. A host range restriction in infectious
pancreatic necrosis virus maps to the large RNA segment and involves virus
attachment to the cell surface. Virology 123:264-272.
De Kinkelin, P., and M. Dorson. 1973. Interferon production in rainbow trout (Salmo
gairdneri) experimentally infected with Egtved virus. Journal of General Virology
19:125-127.
DeSena, J., and G. Rio. 1975. Partial purification and characterization of RTG-2 fish cell
interferon. Infection and Immunity 11:815-822.
Dixon, P. F., and B. J. Hill. 1983. Inactivation of infectious pancreatic necrosis virus for
vaccine use. Journal of Fish Diseases 6:399-409.
Dobos, P. 1976. Virus-specific protein synthesis in cells infected by infectious
pancreatic necrosis virus. Journal of Virology 21:242-258.
Dobos, P., R. Hallet, T. C. Kells, 0. Sorensen, and D. Rowe. 1977. Biophysical studies
of infectious pancreatic necrosis virus: analysis of viral epitopes and comparison of
different isolates. Journal of General Virology 67: 2193-2205.
Dobos, P., and D. Rowe. 1976. Peptide map comparison of infectious pancreatic
necrosis virus-specific polypeptides. Journal of Virology 24: 805-820.
Dorson, M. 1982. Infectious pancreatic necrosis of salmonids. In: Anderson, D. P.
Dorson, M., P. H. Dubourget (Eds.). Antigens of Fish Pathogens: Development
and production for vaccines and serodiagnostics, 1-32. Collection foundation
Marcel Merieux, Lyon.
81
Dorson, M., Castric J., and C.Torchy. 1978. Infectious pancreatic necrosis virus of
salmonids. Biological and antigenic features of a pathogenic strain and a nonpathogenic variant selected in RTG-2 cells. Journal of Fish Diseases
1:309-320.
Dorson, M., and C. Torchy. 1985. Experimental transmission of infectious pancreatic
necrotic necrosis virus via the sexual products in A. E. Ellis, Ed. Fish and Shellfish
Pathology 251-260. Academic Press, London.
Fenner, F. 1968 Biology of Animal Viruses 475-613. Academic Press. Orlando.
Fields, B. N., and D. M. Knipe. 1990. Molecular and genetical determinants of virulence.
In Virology Vol. 1. pp: 213-224.
Fields, B. N., and H. W. Greene. 1982. Genetically and molecular mechanisms of viral
pathogenesis implication for prevention and treatment. Nature 300:19-23.
Frankel-Conrat, H., and R. R. Wagner. 1984. Comprehensive Virology. Viral
Cytopathology Volume 19. pp. 7.
Frantsi, C. and M. Sayan. 1971. Infectious pancreatic necrotic necrosis virus-temperature
and age factors in mortality Journal of Wildlife Diseases 7:249-255.
Fryer, J. L., A. Yusha, and K. S. Pilcher. 1965. The in vitro cultivation of tissue and
cells of pacific salmon and steelhead trout. Annals Of the New York Academy Of
Science 126 (Art. 1) 566-586.
Gravell, M. and Malsberger. 1965. A permanent cell line from fathead minnow
(Pimephales promelas). Annals Of the New York Academy Of Science
126:555-565.
Hah, V., S. Hong, M. Kim, J. L. Fryer, and J. R. Winton. 1984. Isolation of
infectious pancreatic necrosis virus from goldfish (Carassius auratus) and chum
salmon (Oncorhynchus keta) in Korea. Korean Journal of Microbiol ogy
22:85-90.
82
Hattory, M., H. Kodama, S. Ishiguro, A. Honda, and H. Izawa. 1984. In vitro and in
vivo detection of infectious pancreatic necrosis virus in fish by enzyme linked
inununosorbent assay. American Journal of Veterinary Research 45:1876-1879.
Hedrick, R. P., and J. L. Fryer. 1981. Persistent infection of three salmonid cell lines
with infectious pancreatic necrosis virus (IPNV). Fish Pathology 15:163-172.
Hedrick, R. P., and J. L. Fryer. 1982. Persistent infections of salmonid cell lines with
infectious pancreatic virus (IPNV): A model for the carrier state in trout. Fish
Pathology 16(4):163-172.
Hedrick, R. P., J. L. Fryer, S. N. Chen, and G. H. Kou. 1983. Characteristics of four
birnaviruses isolated from fish in Taiwan. Fish Pathology 18:91-97.
Hedrick, R. P., J. C. Leong, and J. L. Fryer. 1978. Persistent infections in
salmonid fish cells with infectious pancreatic necrosis virus (IPNV). Journal of
Fish Diseases 1:297-308.
Hill, B. J. 1976. Properties of virus isolated form bivalve mollusk, Tellina tenuis
(Dacosta), In L. A. Ed. Ellis.Wildlife Diseases 445-452. Plenum Press,
New York.
Hill, B. J. 1982. Infectious pancreatic necrosis virus and its virulence In R. J. Roberts ed.
Microbial Diseases of Fish 91-114. Academic Press, London.
Hill, B. J. and K. Way. 1988. Proposed standardization of the serological classification of
the aquatic birnaviruses. In: Conference handbook, International fish Heath
Conference 151-163. Vancouver, Canada.
Hill, B. J., and P. F. Dixon 1977. Studies on IPN virulence and immunization.
Bulletin de l'office International des epizooties 85:425-427.
Huang, A. S., and D. Baltimore. 1970. Defective viral particles and viral disease
Processes. Nature 226:325-327.London.
83
Holland, J. J. 1964. The symposium of the Society for General Microbiology 12:257
Hsu, Y. L., Chiang, S. Y., Lin, S. T. and Wu, J. L. 1989. The specific detection of
infectious pancreatic necrosis virus in infected cells and fish by immunodot blot
method . Journal of Fish Diseases 12: 561-571.
Janssen R., N. Nathanson, M. Endres, and F. Gonzalez-Scarano. 1986. Virulence of La
Crosse virus is under polygenic control. Journal of virology 59:1-7.
Jorgenson, P. E. V. 1973. The nature and biological activity of IPN virus neutralizing
antibodies in normal and immunized rainbow trout (Salmo gairdneri). Archiv Fuer
die Gesamte Virusforschung 42:9-20.
Joklik, W. K., 1977. Mechanisms of establishment and maintenance of persistent
infections. Schlesinger, D. Ed. Microbiology 434-438. Wash., DC.
Kaye K., D. Springgs , R. Bassel-Duby, B. N. Fields, K. Tyler. 1986. Genetic basis for
altered pathogenesis of an immune-selected antigenic variant of reovirus type 3
(Dearing). Journal of Virology 59:90-97.
Kelly, R. K., and 0. Nielsen. 1985. Inhibition of infectious pancreatic necrosis virus by
serum from normal trout (Salmo gairdneri) in Canadian hatcheries. Fish
Pathology 19:245-251.
Kelly, R. K., and P. C. Loh. 1975. Electron microscobical and biochemical
characterization of infectious pancreatic necrosis virus. Journal of Virology
10:824-834.
Kelly, R. K., B. W. Souter, and H. R. Miller 1968. Fish cell lines: comparisons of
CHSE-214, FHM And RTG-2 in assaying IHN and IPN Viruses. Journal of
Fisheries Research Board of Canada. 30:913-916.
Kimura, T., M. Yoshimizu, and H. Yasuda. 1984. Rapid, simple serological
diagnosis of infectious pancreatic necrosis virus by coagglutination test using
antibody-sensitized staphylococci. Fish Pathology 19:25-33.
84
Kohlmeyer, G., W. Ahne, and I. Thomsen, 1986. Plaque sizes, virulence and
immunogenicity of the IPNV subtypes Sp, Ab, and He: IPNV subtypes Sp, Ab and
He occurring in th Federal Republic of Germany have been studied in the view of
plaque size, virulence and immunogenicity. Tieraerztlander umschau 41:512-541.
Kou, G. H., and S. N. Chen 1984. Pathogenicity of birnavirus isolated from loach,
Misgurnus anguillicaubulatus. In international seminar on fish pathology
for the
20. Anniversary of the Japanese of fish pathologist Tokyo, Japan. September 810. Abstract.
Lannan, C. N. 1994. Fish cell culture: A protocol for quality control. Journal of
Tissue Culture Methods 16:95-98.
Lannan, C. N., J. R. Winton, and J. R. Fryer. 1984. Fish Cell Lines: Establishment and
characterization of nine cell lines from salmonids. In Vitro 20:671-676.
Lapierre, J ; Larrrivee, D., and Berthiaume, L. 1988. Influence of water temperature and
fish age on mortality in brook trout (Salvelinus fontinalis) infected with Infectious
pancreatic necrosis virus (IPNV)Aquaculture 59-81-92.
Lightner, D., and G. Post. 1969. Morphological characteristics of Infectious pancreatic
necrosis virus in trout pancreatic tissue. Journal of Fisheries Research Board of
Canada 26:2247-2250.
Lo, C. F., Chi, S., Tien, N. Y., Yu, P. J., Chou, C. M., S. N., Wang, C. H., Kou, G.
H. 1995. The sequential changes of the virion polypeptides of CV HB1 virus
(Birnaviridae) during serial passaging. Aquaculture 132:73-80.
Lodmell, D. 1983. Genetic control of resistance to street rabies virus in mice.
Experimental Medicine 175:451-460.
M'Gonigle, R. H. 1941. Acute catarrhal enteritis of salmonid fingerlings. Transactions of
the American Fisheries Society
70:297-303.
85
Macdonald, R. D., and J. C. Kennedy. 1979. Infectious pancreatic virus persistently
Infects chinook salmon embryo cells independent of interferon. Virology
95:260-264.
Macdonald, R. D., and P. Dobos. 1981. Identification of proteins encoded by each
genome segment of infectious pancreatic necrotic virus. Virology 114: 414-422.
Macdonald, R. D., and T. Yamamoto. 1977. Quantitative analysis of defective
Interfering particles in infectious pancreatic virus. Archieves of Virology
57:77-89.
Mackelvie, R. M., and H. Artsob. 1969. Infectious pancreatic necrosis virus in young
salmonids of the Canadian Maritime provinces. Journal of Fisheries Research
Board of Canada 26: 3259-3262.
Malsberger, R. G., and C. P. Cerini. 1965. Multiplication of infectious pancreatic
necrosis virus. Annalls of New York Academy and Sciences 126:320-327.
MacAllister, P. E. 1983. Infectious pancreatic necrosis virus (IPNV) of salmonids.
Fish Diseases Leaflet 65, US. Department of the Interior, Fish and Wildlife
Service, 12p.
MacAllister, P. E. and W. J. Owens. 1995. Infectious pancreatic necrosis virus: A
protocol for a standard challenge in brook trout. Transactions of the American
Fisheries Society 115:466-470.
MacAllister, P. E. and W. J. Owens. 1995. Assessment of the virulence of fish and
molluscan isolates of infectious pancreatic necrosis virus for salmonid fish by
challenge of brook trout, Salvelinus fontinalis. Journal of Fish diseases
18:97-103.
Menna J. H., Collins, A. R., and Flanagan, T. D. 1957a. Characterization of an in vitro
persistent-state measles infection. Establishment and virological characterization of
the BGM/ MW cell line. Infection and Immunity 11: 152-158.
86
Menna J. H., Collins, A. R., and Flanagan, T. D. 1957b Characterization of an in vitro
persistent state measles infection: Species characterization and interference in the
BGM/ MW cell line. Infection and Immunity 11:159-163.
Melby, H. P. and K. E. Christie. 1994. Antigenic analysis of reference strains and
Norwegian field strains of aquatic birnaviruses by the use of six monoclonal
antibodies produced against the infectious pancreatic necrosis virus strain. Journal
of Fish Diseases 17:409-415.
Moss, L. H., and M. Gravel. 1968. Morphology of Infectious pancreatic necrosis virus.
Bacteriological Proceedings 104:162.
Nicholson, B. L. 1971. Macromolecule synthesis in RTG-2 cells following
infection with infectious pancreatic necrosis virus (IPNV). Journal of General
Vrology 13:369-372.
Nicholson, B. L., and R. Dexter. 1975 Possible interference in the isolation of IPNV
virus from carrier fish. Journal of Fisheries Research Board of Canada.
32:1437-1439.
Nicholson B. L., and J. Dunn. 1974. Homologous viral interference in trout and Atlantic
salmon cell cultures infected with infectious pancreatic virus. Journal of Virology.
14:180-182.
Nicholson B. L., G. W. Thorne, Carlyle J., and C. Janike. 1979. Studies on a host range
variant from different isolates of infectious pancreatic virus (IPNV). Journal of
Fish Diseases. 2:367-379.
Nims, L., J. L. Fryer, and K. S. Pilcher. 1970. Studies of replication of four selected
viruses in two cell lines derived from salmonid fish. Proceedings of the Society
For Experimental Biology and Medicine 135:6-12.
Novoa, B., A. Figueras, C. F. Puentes, A. Ledo, A. E. 1993. Characterization of a
birnavirus isolated from diseased turbot cultured in Spain. Diseases of Aquatic
Organisms 15:163-169.
87
Olesen, N. J., P. E. Vestegard-Jorgensen, B. Bloch, and S. Mellergard. 1988.
Isolation of a IPNV-like virus belonging to the serogroup II. of the aquatic
birnaviruses from dab, Limanda limanda. L. Journal of Fish Diseases.
11: 449-451.
Persson, R. H., and R. D. Macdonald. 1982. Evidence that infectious pancreatic
necrosis has genome-linked protein. Journal of Virology 44:437-443.
Piper, D., Nicholson B. L., and J. Dunn. 1973. Immunofluorescent study of the
replication of Infectious pancreatic necrosis virus in trout and Atlantic salmon
cell cultures. Infection And Immunity 8:249-254.
Preble, 0. T., and J. S. Youngner. 1975. Temperature sensitive viruses and the
etiology of chronic and inapparent diseases. Journal of Infectious Diseases
131: 467-472.
Rima, B. K., and J. S. Martin. 1976. Persistent infection of tissue culture cells by RNA
viruses, Medical Microbiology Immunology 162:89-118
Reno, P. W. 1976. Quantitative and qualitative aspects of the infectious pancreatic
necrosis (IPN) virus carrier state. Ph. D. Dissertation. University of Guelph,
Guelph, Ontario.
Riviere, Y., R. Ahmed, P. Southern, M. Buchmeier, and M. Oldstone. 1985.
Choriomeningitis virus pathogenicity. Virulence in guinea pigs is associated with
the L RNA segment. Journal of Virology 1985, 55:704-709.
Robert, N. 1979 Temperature and host defense. Microbiological Reviews 43:241-259.
Rodriguez, S., P. Vilas and S. Perez. 1993. A viral diagnostic survey of Spanish rainbow
trout farms : I sensitivity of four cell lines to wild IPNV isolates. Bull. Eur. Ass.
Fish Pathol., 13:119-122.
Sabin, A., and P. Olitsky. 1937. Pathological evidence of axonal and transsynaptic
progression of vesicular stomatitis and eastern equine encephalomyelitis viruses
Am. J. Pathol. 13-165.
88
Sano T. 1971. Studies on viral diseases of Japanese fishes II. Infectious pancreatic
necrosis virus of rainbow trout, pathogenicity of isolates. Bulletin of the Japanese
Society of Scientific Fisheries 37:499-503.
Sano T. 1976. Viral diseases of cultured fishes in Japan. Fish Pathology 10:221-226.
Sano, M., N. Okamoto, H. Fukuda, M. Saneyoshi, and T. Sano. 1992. Virulence of
infectious pancreatic necrosis virus associated with the large RNA segment (RNA
segment A). Journal of fish Diseases. 15: 283-293.
Sano, M., and N. Okamoto. 1994. Infectious pancreatic necrosis virus plaque size
depends on the larger RNA segment and is not linked to virulence. Journal of
Fish Diseases 17:657-659.
Sekellic, M. J., and P. I. Markus. 1978. Persistent infection. I. interferon inducing
defective interfering particles as mediators of cell sparing; possible role in the
persistent infection by vesicular stomatitis virus. Virology 85:175-186.
Sheid, A., and P. W. Choppin. 1984. Proteolytic cleavage and viral pathogenesis. In:
Notkins A. L., Oldstone M. B. A., eds. Concepts in Viral Pathogenesis New
York: Springer-Verlag, 26-31
Sheffer, R., and J. Cohen. 1975. Studies on infectious pancreatic necrosis virus
interactions with RTG-2 and FHM cells. Journal of General Virology 28:9-20.
Silim, A., and Y. Lagace 1982. Susceptible of trouts of different species and origins to
various isolates of infectious pancreatic necrosis virus. Canadian Journal of
Fisheries and Aquatic Sciences 39:1580-1584.
Simpson, R. W., and M. Iinuma. 1975. Recovery of infectious proviral DNA from
mammalian cells infected with respiratory syncytial virus. Proceedings of National
Academy of Sciences 72:3230-3234
Smail, D. A., W. D. Bruno, G. Dear, L. A. McFarlane, and K. Ross. 1992. Infectious
pancreatic virus Sp serotype in farmed Atlantic salmon, Salmo salar. L., associated
with mortality and clinical disease. Journal of Fish Diseases 15:77-83.
89
Snieszko, S. F., E. M. Wood, and W. T. Yasutake. 1957. Infectious pancreatic
necrosis virus in trout. Archives of Pathology 63:229-233.
Sonstegard, R. A., and L. A. MacDermott. 1972. Isolation of infectious pancreatic
necrosis virus from white suckers (Catostomus commersoni). Nature
236:174-175. London.
Sorimachi, M., and T. Hah. 1985. Characteristics and pathogenicity of a virus isolated
from yellowtail fingerlings showing ascites. Fish Pathology 19:231-238
Staneck, L. D., R. S. Troubridge, R. M. Welsh, E. A. Wright, and C. J. Pfau. 1972.
Arena viruses: Cellular response to long term in vitro infection with parana and
lymphocytic choriomeningitis viruses. Infection and Immunity 6:44-450
Swanson, R. N., and J. H. Gillspie. 1981. And indirect fluorescent antibody test for the
rapid detection of infectious pancreatic necrosis virus in tissues.
Journal of Fish Diseases 4:309-315.
Toranzo, A. E., and F. M. Hedrick. 1982. Comparative study of two salmonid viruses
and poliovirus in fresh estaurine and marine waters. Journal of Fish Diseases
5:223-231.
Vestegard-Jorgensen, P. E. 1972. Freund's adjuvants: their influence on the
specificity of viral antisera. Acta Pathologica et Microbiologica Scandinavica [B]
80:931-933.
Vestegard-Jorgensen, P. E., and Kehlet. 1971. Infectious pancreatic necrosis (IPN)
viruses in Danish rainbow trout. Nordsk Veterenary Medizin. 23:568-575.
Wolf, K., and C. E. Dunbar. 1957. Cultivation of adult teleost tissues in vitro.
Proceedings of the Society For Experimental Biology and Medicine 95:455-458.
Wolf, K., C. E. Dunbar, and S. F. Snieszko 1960. Virus nature of infectious
pancreatic necrosis virus in trout. Proceedings of the Society For Experimental
Biology and Medicine 104: 105-108.
90
Wolf, K., and J. A. Mann 1980. Poikiliotermic vertebrate cell lines and viruses. In Vitro
16:168-179.
Wolf, K., M. C. Quimby, and A. D. Bradford. 1963. Egg-associated transmission of
IPNV virus of trouts. Virology 21: 317-321.
Wolf, K., and M. C. Quimby. 1962. Established eurythermic cell line of fish cell in
vitro. Science 135:1060-1066
Wolf, K., S. F. Snieszko and C. E. Dunbar. 1959. Infectious pancreatic necrosis
virus-caused disease of fish. Excerpta Medica International Series 13:228
Abstract.
Wood, E. M., S. F. Snieszko , and W. T. Yasutake. 1955. Infectious pancreatic
necrosis virus in brook trout. Archieves of pathology 60:26-28.
5: 223-231.
Yamamoto, T. 1974. Infectious pancreatic necrosis virus occurrence at a hatchery in
Alberta. Journal of Fisheries Research Board of Canada. 31:397-402.
Zhadnov, V. M. 1975. Integration of viral genomes. Nature
256:471-473. London.
91
APPENDICES
92
Appendix A
93
Appendix A. Table 1. Virus yields and destruction of cell monolayers by Buhl subtype
IPNV isolates.
ISOLATE
EPC
CPE Titer
CPE and TITERS (Log10 /m1)
FHM
CHSE-214
CPE
Titer
CPE
Titer
0
0
4.75
6.50
0
NVD
0
0(a)
5.00(b)
4
009-94
0
NVD(c)
4
183-82
0
NVD
4
89-200
0
NVD
4
7.50
8.50
7.75
8.75
89-237
0
1.75
4
8.5
89-238
0
NVD
4
90-11
0
2.50
90-236
0
90-76
Buhl
RTG-2
CPE Titer
3
4
6.25
8.50
7.50
NVD
4
4
0
NVD
4
8.25
8.75
0
5.75
4
4
10.50
0
6.25
4
NVD
4
8.25
0
NVD
4
8.00
9.25
8.50
0
NVD
4
0
3.25
4
7.75
91-114
0
NVD
4
8.00
8.75
0
3.25
4
8.75
91-137
0
NVD
4
8.25
0
NVD
4
8.75
93-3
0
NVD
4
0
NVD
4
8.25
93-5
0
NVD
4
0
NVD
93-507
0
NVD
4
8.50
8.25
8.50
0
NVD
4
4
8.00
7.50
94-167
0
2.00
4
8.25
0
5.25
4
8.25
94-187
0
NVD
8.75
4
2
8.25
7.50
4
Cal.Mojave
0
NVD
4
0
4
0
1.75
94-273
4
4
NVD
4
Stoddard
csf-crayfish
csf-064-93
0
4
0
6.25
6.25
4
0
2.50
3.50
0
NVD
4
0
NVD
4
csf-35-85
0
1.75
4
0
3.50
4
csf-191-77
csf-266-89
0
NVD
4
0
NVD
4
0
NVD
4
0
NVD
HAH
0
6.00
4
7.50
8.25
8.50
0
6.00
4
4
H-VAT-9-10-86
0
NVD
4
9.75
0
NVD
4
7.75
7.50
8.25
8.25
9.05
8.25
8.75
9.00
7.75
6.00
8. 50
ID-PASS-0
0
NVD
4
8.50
0
NVD
4
8.25
Obanion
0
2.75
4
8.25
0
5.50
4
0
NVD
4
8.75
0
NVD
4
7.50
8.50
Sawtooth
4
9.25
7.75
9.00
9.75
8.25
10.0
0
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Login finl. of virus titer from pooled samples from four wells.
(c) NVD= No virus detected.
4
8.25
94
Appendix A. Table 2. Virus yields and destruction of cell monolayers by VR-299
subtype IPNV isolates..
CPE and TITERS (Login /m1)
ISOLATE
EPC
CPE Titer
CHSE-214
CPE Titer
FHM
CPE Titer
RTG-2
CPE Titer
2(a)
4.75(b)
4
7.50
4
3.50
4
5.50
AAHL-1=FV-987
0
3.00
4
8.25
0
4
6.75
AAHL-6=LH(PA)
4
8.00
4
8.50
4
4
5.75
86-Q
4
4
8.75
4
4
8.25
Berlin
4
4
4
2
CL-214
4
8.50
4
Coho
4
8.50
4
Lava lake
4
4
Menhaden
4
8.00
5.75
Vold" csf
4
4
Oswayo
Pelton Dam
4
4
7.25
9.00
7.25
4
Roaring R.
4
4
4
5.50
6.00
8.50
8.00
7.25
8.50
8.00
8.50
7.50
8.25
Reno
4
9.00
8.25
4
4
7.75
Striped Bass
4
7.75
4
7.50
6.50
9.00
9.00
8.75
8.75
9.75
9.25
8.50
9.75
8.50
8.75
4
Chilean (tl)
7.75
5.75
5.75
4.00
7.00
7.75
5.50
6.50
8.75
8.25
7.25
7.75
8.50
8.50
9.75
8.50
7.75
7.25
4
7.50
VR-299
4
4
4
4
4
4
4
4
4
4
4
4
4
4
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Login /ml. of virus titer from pooled samples from four wells.
4
4
4
4
4
4
4
4
95
Appendix A. Table 3. Virus yields and destruction of cell monolayers by WB subtype
IPNV isolates.
CPE and TITERS (Log10 /m1)
ISOLATE
EPC
CPE Titer
CHSE-214
CPE Titer
FHM
CPE Titer
4
6.25
4
7.25
4
6.75
4
6.50
7.50
6.00
7.25
4
4
7.50
4
0
NVD
4
4
4
4
7.25
7.50
7.75
6.75
7.00
4
7.50
7.50
7.00
8.00
8.25
8.00
7.50
7.50
4
4
4
9.00
9.00
7.25
4
7.75
4
4
4
4
8.50
8.50
9.75
8.00
8.00
9.25
8.25
8.00
7.50
8.75
92-326
4
93-030
4
93-7
4
94-158
2
94-434
2
94-435
3
94-446
3
CTT
4
Dry Mills
2
ID-3-31-93
4
4.00(b)
7.50
7.25
8.50
6.75
6.00
7.75
7.75
8.00
5.75
6.00
Idaho-92-429
3
7.00
4
10.00
4
7.50
Idaho-93-321
0
NVD(c)
4
8.25
0
NVD
Taiwan
4
4
6.50
3
7.75
WB
4(a)
6.50
4
4
4
4
4
4
4
4
4
4
4
4
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Login /ml. of virus titer from pooled samples from four wells.
(c) NVD= No virus detected.
RTG-2
CPE Titer
4
4
4
96
Appendix A. Table 4.Virus yields and destruction of cell monolayers by Jasper
and BC subtype IPNV isolates..
CPE and TITERS (Logio/m1)
CHSE-214
FHM
CPE Titer CPE Titer
ISOLATE
EPC
CPE Titer
Jasper
3(a)
8.00(b)
4
8.75
4
7.25
4
8.00
8.75
4
9.50
4
7.25
4
W17
4
4
4
10.0
4
4
N. H.
2
4
BC 89-362
2
2
4
8.50
5.50
7.75
4
4
93-511
HR
4
6.50
5.50
5.75
7.00
4
8.75
4
9.00
6.25
5.00
5.50
7.50
Ys KOREA
4
6.25
4
8.75
4
8.00
CF-CTH-9/86
4
4
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Loge /ml. of virus titer from pooled samples from four wells.
RTG-2
CPE Titer
4
4
4
4
4
7.25
9.00
8.00
7.25
5.75
8.00
7.25
8.50
97
Appendix A. Table 5. Virus yields and destruction of cell monolayers by Sp subtype
IPNV isolates.
CPE and TITERS (Log10 /m1)
ISOLATE
EPC
CPE Titer
CHSE-214
CPE Titer
FHM
CPE Titer
1(a)
5.75(b)
4
10.00
0
C-17
2
5.75
4
5.75
C-18
0
4.50
4
C-36
3
6.50
C-39
2
DPL
4
FR-21
RTG-2
CPE Titer
4
2
4.75
5.75
7.75
0
NVD(c)
4
4
5.75
4
5.75
4
6.75
4
3
5.25
4
4
4
7.25
4
4
7.75
7.00
4
6.50
9.25
7.75
4
6.75
4
N-73A1 1
4
7.25
4
9.25
3
4
NC-33
4
8.75
4
4
NH-44
4
7.75
4
OV-7
2
4
Thailand
4
5.00
8.75
9.00
9.50
7.75
9.50
6.00
7.75
7.25
2.50
7.00
Sp
4
4
1
3
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Login iml. of virus titer from pooled samples from four wells.
(c) NVD= No virus detected.
4
4
4
4
4
7.00
6.00
5.50
5.75
4.75
8.25
7.00
6.75
7.50
7.50
6.00
8.75
98
Appendix A. Table 6. Virus yields and destruction of cell monolayers by Ab and EVE
subtype IPNV isolates.
ISOLATE
EPC
CPE Titer
CPE and TITERS (Logio/m1)
FHM
CHSE-214
CPE Titer CPE Titer
0(a)
3.50(b)
4
LKT
0
5.75
4
SGV
1
4.25
4
LKE
0
NVD(c)
4
N-12G5
0
2.75
4
EVE
0
4.75
4
Ab
9.00
8.75
8.00
8.50
7.75
9.25
4
1
4.00
3.75
4.50
0
NVD
4
0
3.00
2.75
4
8.50
8.00
7.00
7.25
6.50
4
9.00
0
0
0
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Login /ml. of virus titer from pooled samples from four wells.
(c) NVD= No virus detected.
RTG-2
CPE Titer
4
4
99
Appendix A. Table 7. Virus yields and destruction of cell monolayers by CAN -1,
CAN-2 and CAN-3 subtype IPNV isolates.
ISOLATE
EPC
CPE Titer
CPE and TITERS (Logio/m1)
FHM
CHSE-214
CPE Titer CPE Titer
0(a)
2.50(b)
4
9.25
0
AS
0
4
0
AAHL-3=0P1-3-88
0
6.00
4
9.00
9.00
8.50
0
Doug Ramsey
2.50(c)
NVD
AAHL-9=Nordic
0
4
8.00
0
CAN-2
0
4
10.25
4
CAN-3
0
4.75
3.50
3.50
4
7.50
0
CAN-1
4
0
0
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Login /ml. of virus titer from pooled samples from four wells.
(c) NVD= No virus detected.
RTG-2
CPE Titer
2.50
2.50
3.50
4.25
3
6.50
9.50
6.50
4
4
4
4
4
4
8.50
7.00
8.25
8.50
7.25
8.00
7.50
100
Appendix A. Table 8. Virus yields and destruction of cell monolayers by He and Te
subtype IPNV isolates.
CPE and TITERS (Login /m1)
ISOLATE
He(cw/b)
Hecht
Te
EPC
CPE Titer
3(a)
4
7.00(b)
9.00
CHSE-214
CPE Titer
FHM
CPE Titer
4
8.75
3
6.75
4
8.00
4
9.00
4
8.50
4
NVD
1
8.00
4.00
4
8.75
0
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
0
RTG-2
CPE Titer
NVD(c)
(b) Logio /ml. of virus titer from pooled samples from four wells.
(c) NVD= No virus detected.
101
Appendix A. Table 9. Shows CPEs and virus yields of isolates whose serological
identity have not yet investigated.
ISOLATE
EPC
CPE Titer
94-445
4(a)
Domsea
CPE and TITERS (Login /ml)
FHM
CHSE-214
CPE Titer CPE Titer
RTG-2
CPE Titer
7.75
8.50
8.75
7.25
8.00
8.50
8.00
6.75
4
10.50
4
6.75
4
0
7.50(b)
NVD(c)
4
9.25
0
6.25
4
94-021
0
NVD
4
0
NVD
4
91-63
0
0
NVD
4
0
6.50
4
NVD
4
0
1.75
0
2.75
4
0
2
5.75
4
3
4.75
6.50
0
NVD
4
9.00
7.50
9.25
8.75
8.50
6.00
0
NVD
4
4
4
4
3
4
6.75
4
0.5
4
4
6.00
7.50
0
5.50
4
8.00
5.75
3
4
5.75
8.00
6.00
0
6.50
5.75
6.00
4
6.00
8.50
4
6.00
6.00
4
5.75
6.00
7.25
6.00
93-15
SF-1284
14' boot
18'
31-75
DRT
0
8.00
4.50
3.50
VR-299(carp)
2
5.50
4
99-E8
4
5.75
4
92-325
3
4
HAH
0
7.30
6.00
VR-299-(gold)
1
4
4
4
0
(a) 0= none CPE 1= 25% or less 2= 25-50% 3= 50-75% 4= >75%
(b) Login /ml. of virus titer from pooled samples from four wells.
(c) NVD= No virus detected.
4
4
Appendix A. Table 10.
Probabilities of differences among 13 subtypes of virus in their ability to produce
CPE in EPC cells.
'SUBTYPES Means BUHL
BUHL
0.071
VR-299
3.733 0
3
0
WB
CAN-1
0.333 0.78
JASPER
3.25 0
0.25 0.84
0 0.94
0 0.94
2.615 0.01
0 0.94
3.5 0
AB
CAN-2
CAN-3
SP
EVE
HE
BC
3
0
TE
0
0.93
VR-299
0.4587
0.0005
0.6021
0.0004
0.0002
0.0002
0.2659
0.0002
0.7877
0.4466
0.0002
SUBTYPES
CAN-1
WB
0.004
0.788
0.004
0.003
0.003
0.657
0.002
0.604
1
0.002
0.002
0.923
0.749
0.744
0.01
0.738
0.001
0.005
0.73
JASPER AB
0.0021
0.0013
0.0012
df
Effect
Effect
1
12
df
Effect Error
18.7
81
EVE
HE
0.8
Error
F
0.854
21.85
p-level
BC
1
0.009
0.008
1
1
6E-04
0.003
1
0.008
5E-04 0.371 5E-04
0.003 0.678 0.003
1
0.007
1
0.59
4E-04
EPC cells and CPEs
MS
MS
SP
0.81
0.5104 0.01
0.0011 0.8
0.7729
0
0.7729
0
0.0011 0.79
STATISTIC summary of all effects; design:
GENERAL 1-SUBTYPE
MANOVA
CAN-2 CAN-3
STATIST Duncan test; CPE
GENERA Probabilities for Post-hoc Tests
MANOV, MAIN EFFECT: SUBTYPE
0.002
TE
I
Appendix A. Table 11.
Probabilities of differences among 13 subtypes of virus in their ability to produce
CPE in FHM cells.
I SUBTYPES Means
0.214
BUHL
VR-299
WB
CAN-1
JASPER
BUHL VR-299
4
0
4
0
4
0
3.357 0
0.5 0.78
0.25 0.97
AB
CAN-2
CAN-3
0.84
2.462 0.03
0 0.84
3.5 0
0
SP
EVE
HE
BC
4
0
0 0.82
TE
0.5336
0.0011
1
0.0005
1
0.0003
0.1494
0.0003
0.6046
1
0.0003
SUBTYPES
WB
0.005
0.55
0.003
0.56
0.002
0.355
0.002
0.882
0.568
0.002
CAN-1
0.001
JASPER
0.796
0.0006
0.658
0.045
0.0004
0.1599
0.0003
0.6284
0.001
0.651
0.004
0.002
0.642
1
1
0.0003
STATISTIC summary of all effects; design:
GENERAL 1-SUBTYPE
MANOVA
Effect
1
MS
df
MS
df
Error
Effect Effect Error
81 1.061
12 21.3
AB
0
0.82
0.03
0.82
0
0
0.81
CAN-2
4E-04
0.168
4E-04
0.642
1
3E-04
CAN-3
SP
EVE
HE
BC
0.651
0.001
4E-04
0.027
0.025
0.002 0.314 0.002
4E-04 0.174 4E-04
1
0.023
1
1
FHM cells and CPEs
STATIST Duncan test; CPE
GENERA Probabilities for Post-hoc Tests
F
20.11
MANOV, MAIN EFFECT: SUBTYPE
p-level
0
TE
I
Appendix A. Table 12.
ANOVA table for geometric mean titers obtained in EPC cells
infected with 13 different
SUBTYPES
I SUBTYPES Means
2.089
7.417
6.446
3.833
7.813
3.75
3.5
3.5
6.481
4.75
BUHL
VR-299
WB
CAN-1
JASPER
AB
CAN-2
CAN-3
SP
EVE
HE
BC
8
BUHL
0
0.01
0.29
0
0.3
0.33
0.36
0.01
0.11
0
6.125 0.02
1.5 0.68
TE
V R -299
WB
0.5309
0.0299
0.7845
0.0283
0.0214
0.102
0.396
0.099
0.0201
0.5182
0.1031
0.7066
0.4222
0.0004
0.081
0.076
0.981
0.272
0.346
0.824
0.003
STATISTIC summary of all effects; design:
GENERAL 1-SUBTYPE
MANOVA
MS
df
MS
df
Error
Effect Effect Error
Effect
1
12
39
81
2.384
CAN-1 JASPER
0.017
0.954
0.837
0.83
0.106
0.527
0.013
0.138
0.163
0.0157
0.0115
0.0108
0.3893
0.0651
0.897
0.3061
0.0002
AB
0.87
0.86
0.1
0.52
0.01
0.14
0.17
CAN-2
CAN-3
SP
EVE
HE
1
0.081
0.45
0.009
0.116
0.195
0.077
0.437
0.008
0.109
0.212
0.281
0.345 0.053
0.819 0.343
0.003 0.053
0.264
1E-04
TITERS AND EPC
STATIST Duncan test; CPE
GENERA Probabilities for Post-hoc Tests
F
16.37
MANOV, MAIN EFFECT: SUBTYPE
p-level
0
BC
0.006
TE
I
Appendix A. Table 13.
ANOVA table for geometric mean titers obtained in FHM cells
infected with 13 different
'SUBTYPES Means BUHL VR-299
BUHL
3.152
7.3
6.375
4.292
7.438
3.438
9.5
VR-299
WB
CAN-1
JASPER
AB
CAN-2
CAN-3
6
5.423
2.75
7.625
6.5
SP
EVE
HE
BC
1.5
TE
0.05
0.11
0.55
0.04
0.87
0
0.15
0.25
0.82
0.03
0.1
0.38
0.6246
0.14
0.938
0.0599
0.2614
0.5084
0.3514
0.0289
0.8638
0.6507
0.0051
WB
0.288
0.589
0.142
0.125
0.832
0.615
0.078
0.536
0.944
0.018
CAN-1
SUBTYPES
JASPER AB
0.128
0.629
0.012
0.365
0.522
0.433
0.0538
0.2742
0.4763
0.3262
0.0254
0.11 0.9155
0.272 0.6199
0.163 0.0043
CAN-2 CAN-3
0
0.19
0.29
0.72
0.05
0.13
0.32
0.089
0.049
0.001
0.29
0.134
1E-04
SP
EVE
HE
0.744
0.11 0.182
0.429 0.29
0.792 0.584
0.027 0.052
0.021
0.071
0.48
0.567
0.003 0.016
STATISTIC summary of all effects; design:
TITERS AND EPC
GENERAL 1-SUBTYPE
MANOVA
df
MS
STATIS1 Duncan test; CPE
GENERA Probabilities for Post-hoc Tests
Effect
1
df
MS
Error
Effect Effect Error
12 24.9
81 3.551
F
7.001
MANOV, MAIN EFFECT: SUBTYPE
p-level
0
BC
TE
Appendix A. Table 14.
ANOVA table of CPE and virus yield responses of aquatic birnaviruses, and
significance levels in different cell ines.
BUHL
BUHL
CELL LINES
CHSE-: FHM
CELL LINES MEAN EPC
0.071
EPC
4
CHSE-214
RTG-2
CHSE-214
0.214 0.23 6E-05
0 0.765
3.964
Effect
MS
df
MS
df
F
p-level 'Effect
Effect Effec. Error Error
3
138
108 0.199 690.4
01
VR-299
CELL LINES
MEAN
3.73
EPC
4
4
4
CHSE-214
FHM
RTG-2
Effect
1
EPC
0.04
0.05
0.06
CELL LINES
CHSE-: FHM
RTG-2
1
0
1
CELL LINES MEAN
1E-04
MS
df
MS
I
p-level
Effec. Error
Error
F
01
3
317
108 1.882 168.3
EPC
7.42
8.68
0
7.3
0.8
7.38 0.94
RTG-2
MS
MS
df
df
p-level 'Effect
F
Effect Effec. Error Error
56 0.124 2.154 0.1041
3 0.27
0
0.765
TITER
CHSE-214
FHM
1
3.964
df
EPC
1
0
Effect
VR-299
CPE
4
0.214 0.23
FHM
RTG-2
1E-04
RTG-2
0.071
EPC
0
CELL LINES
CHSE-2' FHM
CELL LINES MEAN EPC
FHM
RTG-2
1
TITER
CELL LINES
CHSE-2' FHM
0.003
0.005
RTG-2
0.845
MS
MS
df
df
Error
F
p-level
Effect Effec. Error
56 1.342 4.871
0
3 6.54
I
1
Appendix A. Table 14. (Continued)
TITER
CELL UNES
MEAN
CELL UNES
CHSE-: FHM
EPC
EPC
3
CHSE-214
4
0.01
4
0.01
Effect
1
1
CHSE-214
FHM
0.09
RTG-2
df
MS
df
MS
Effect Effec Error Error
F
p-level 'Effect
3 3.45
52
0.87 3.964 0.0131
CAN-1
df
CELL UNES MEAN
EPC
0.333
EPC
CHSE-214
4
1
FHM
RTG-2
0.5 0.71 7E-05
3.833
0
0.71 2E-04
Effect
df
MS
df
MS
Effect Effec. Error Error
3 24.6
20 0.583
1
FHM
RTG -2
42.1
df
p-level Effect
01
EPC
3.833
8.708
0
4.292 0.57
7.917
0
CHSE-214
F
RTG-2
0.044
I
F
5.734
p-level
01
TITER
EPC
0
0.002
0.188
MS
df
MS
Effec Error
Error
3 13.3
52 2.321
CELL LINES MEAN
RTG-2
CELL LINES
CHSE-2' FHM
Effect
CAN-1
CELL LINES
CHSE-: FHM
EPC
6.446
8.393
0
6.375 0.9
7.625 0.05
EPC
3.357 0.32 0.074
FHM
RTG-2
CELL LINES MEAN
RTG-2
1E-04
0.333
RTG-2
3E-04
MS
df
MS
I
Effec. Error
Error
F
p-level
3
37
20 1.907 19.38
0
Effect
1
CELL LINES
CHSE-2' FHM
Appendix A. Table 14. (Continued)
JASPER
CPE
JASPER
CELL UNES
MEAN
3.25
EPC
CHSE-214
4
4
4
FHM
RTG-2
Effect
1
CELL LINES
CHSE-: FHM
EPC
0.05
0.06
0.06
TITER
CELL UNES MEAN
RTG-2
CHSE-214
FHM
1
RTG-2
1
1
df
MS
df
MS
Effect Effec. Error Error
F
p-level 'Effect
12
0.229
2.455
0.1131
3 0.56
CELL UNES
MEAN
CELL LINES
CHSE-: FHM
EPC
0.25
EPC
CHSE-214
FHM
4
0.25
4
RTG-2
Effect
1
1
0
1
1 E -04
df
MS
df
MS
Effect Effec. Error Error
3 18.8
12 0.125
F
150
df
Effect
p-level 'Effect
01
EPC
3.75
8.563
0
3.438 0.71
7.688
0
CHSE-214
FHM
RTG-2
2E-04
2.31
12
RTG-2
0.472
MS
Error
I
F
p-level
0.818 2.822 0.08
TITER
EPC
0
3
1
CELL LINES MEAN
RTG-2
0.025
0.074
df
MS
df
Effect Effec. Error
Ab
Ab
EPC
7.813
9.188 0.06
7.438 0.57
7.938 0.85
EPC
CELL LINES
CHSE-2' FHM
1
3
CELL LINES
CHSE-2' FHM
1E-04
0.314
RTG-2
4E-04
df
MS
I
Effec. Error
Error
F
p-level
28
12 1.387 20.16
01
MS
8
co
Appendix A. Table 14. (Continued)
Sp
SP
CELL UNES
MEAN
CELL LINES
CHSE-: FHM
EPC
RTG-2
CELL LINES MEAN
2.615
EPC
CHSE-214
0
4
0
df
Effect
1
1
8.115 0.02
5.423 0.1
6.712 0.72
CHSE-214
FHM
RTG-2
0.002
MS
df
Effect Effec. Error Error
48 1.298
3 9.31
MS
7.17
p-level Effect
5E-041
RTG-2
2E-04
0.03
0.057
df
MS
I
Effec Error Error
F
p-level
3
16
48 2.563 6.223
0
df
Effect
I
F
EPC
CELL LINES
CHSE-2' FHM
6.481
EPC
4
2.462 0.73 0.002
FHM
RTG-2
TITER
1
MS
TITER
CELL UNES
MEAN
3.5
EPC
CHSE-214
Effect
1
EPC
4
0.38
4
0.38
3.5
FHM
RTG-2
CELL UNES
CHSE-: FHM
1
RTG-2
CELL UNES MEAN
1
8.875 0.41
7.625 0.71
8.125 0.9
CHSE-214
0.38
FHM
RTG-2
MS
df
MS
df
F
p-level 'Effect
Effect Effec. Error Error
4
0.25 0.667 0.6151
3 0.17
RTG-2
8
EPC
0.374
EPC
CELL LINES
CHSE-2' FHM
0.263
0.473
0.63
df
MS
MS
df
Effect Effec. Error
Error
F
p-level
4 0.898 0.612 0.641
3 0.55
I
1
Appendix A. Table 14. (Continued)
TITER
CELL LINES
CELL LINES
MEAN
3
4
4
4
EPC
CHSE-214
FHM
RTG-2
Effect
1
EPC CHSE: FHM
0.03
0.04
0.04
RTG-2
CELL LINES MEAN
6.125
7.688 0.13
6.5 0.68
7.375 0.21
EPC
CHSE-214
FHM
1
1
RTG-2
1
MS
df
MS
df
Effect Effec Error Error
3
1
12 0.333
F
p-level Effect
3 0.0731
EPC
df
Effect
1
CELL LINES
CHSE-2' FHM
0.23
0.733
RTG-2
0.347
df
MS
Effec. Error
Error
F
p-level
3 2.14
12 1.598 1.339 0.31
MS
STATIST Duncan test; TITER
GENERA Probabilities for Post-hoc Tests
MANOV, MAIN EFFEG Cell line
111
Appendix B
112
Appendix B. Table 1. Replication of IPNV Buhl subtype isolates in the presence and
absence of % NTS and their relationship to virulence.
ISOLATE
CPE and Titers(logio) In
Without NTS
With NTS
CPE Titer CPE Titer
Logo
Reduction
IPN
%
Disease Mortality
0.00
0.75
Y
5.75
7.00
1.75
Y
1.25
Y
0.25
Y
4
8.00
8.75
-0.75
Y
9.25
4
9.75
Y
4
Y
0.25
N
4
8.50
8.50
8.75
8.50
8.50
-0.75
0.00
4
8.50
7.75
8.75
8.75
8.25
-0.5
0.00
-0.25
Y
93-5
4
8.00
4
8.25
-0.25
Y
93-507
4
4
7.75
-0.25
Y
94-167
4
4
7.75
4
4
7.5
94-273
4
0
ND
CAL.Mojave
4
4
8.25
Stoddard
csf Crayfish
4
8.25
9.5
4
4
8.50
7.25
8.00
9.00
7.75
8.75
6.00
4
8.25
0.50
0.25
7.50
0.00
-0.25
2.25
-0.25
Y
94-187
7.50
8.25
7.75
7.5
8.25
4
4
7.75
7.75
4
9.25
0
4
4
2.50
8.75
7.75
7.50
4
8.25
BUHL
3
6.25
3
6.25
009-94
4
8.5
4
7.75
183-82
4
4
89-200
4
7.50
8.25
89-237
4
8.25
4
89-238
4
8.00
90-11
4
90-236
4
90-76
91-114
4
4
91-137
4
93-3
CSF 64-93
csf 191-77
csf 266-89
csf-35-85
4
4
4
4
4
HAH
H-VAT
4
ID-PASS-0
4
Obanion
4
8.50
8.25
7.00
Sawtooth
4
8.50
4
4
4
4
4
Y
73.0
90.0
52.5
87.0
87.0
96.0
93.0
91.0
95.5
82.5
24.5
92.5
88.5
93.5
88.0
92.5
24.5
79.0
94.0
85.5
96.0
85.0
92.0
97.5
ND
ND
Y
ND
89.0
89.5
ND
Y
96.0
Y
Y
Y
Y
N
Y
Y
Y
Y
1.25
Y
0.00
-0.50
3.50
-0.25
0.50
-0.50
0.25
Y
Y
Y: Yes; N: no; ND:Not Done
113
Appendix B. Table 2. Replication of IPNV VR 299 subtype isolates in the presence
and absence of NTS and their relationship to virulence.
ISOLATE
CPE and Titers(logio) In
With NTS
Without NTS
Titer
CPE Titer CPE
VR-299
4
5.50
1
86-Q
4
8.25
0
Berlin
4
5.50
3
Chilean (t1)
4
0
CL-214
4
Coho
4
6.00
8.50
8.00
4
4
6.75
4
4
7.25
4
4
5.75
4
Menhaden
4
2
1-"old" csf
4
3
Oswayo
4
8.50
8.00
8.50
4.50
2.50
4.50
3.50
7.25
7.50
5.25
7.50
5.75
6.50
3
Pelton Dam
4
0
Roaring R.
4
Reno
4
Striped Bass
4
7.50
8.25
7.75
7.50
AAHL-1=FV-987
Lava lake
AAHL-6=LH(PA)
Logic,
Reduction
IP N
%
Disease Mortality
1.00
Y
ND
5.75
Y
77.5
1.00
Y
54.0
2.50
ND
ND
1.25
Y
0.50
Y
75.0
73.0
1.50
ND
ND
-0.25
Y
64
0.00
2.00
ND
ND
Y
86.5
6.5
1.5
N
Y
0
ND
ND
ND
4
8.75
3.00
4.50
0.50
7.75
-1.25
Y
3
5.50
3.00
7.75
33.0
80.5
66.5
84.5
ND
ND
3
Y
Y: Yes; N: no; ND:Not Done
114
Appendix B. Table 3. Replication of IPNV WB subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence.
ISOLATE
CPE and Titers(logio) In
With NTS
Without NTS
CPE Titer CPE Titer
WB
4
6.25
2
7.00
92-326
4
7.25
0
ND
93-030
93-7
4
1
2.75
4
6.75
7.50
0
ND
94-158
4
0
ND
94-434
4
1
ND
94-435
4
0
ND
94-446
CTT
4
4
0
2
2.50
5.00
Dry Mills
4
2
7.75
Idaho 92-429
4
1
2.50
Idaho 93-321
4
ID-3-31-93
4
Taiwan
4
8.50
8.00
2.00
7.50
7.00
8.00
8.25
8.00
7.50
9.00
9.00
7.50
7.25
4
4
1
IPN
Reduction Disease
Logio
%
Mortality
ND
ND
7.25
4.00
7.50
7.50
7.00
8.00
5.75
N
21.5
46.0
3.00
-0.25
-0.75
Y
Y
30.0
33.0
53.5
60.0
N
18.5
Y
62.0
ND
ND
6.50
N
11.0
0.50
-0.50
Y
84.5
ND
ND
ND
ND
5.25
Y
Y
Y
Y: Yes; N: no; ND:Not Done
115
Appendix B. Table 4. Replication of IPNV CAN subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence.
CPE and Titers(logio) In
With NTS
Logio
IPN
%
ISOLATE Without NTS
CPE Titer CPE
Titer Reduction Disease Mortality
Y
58.5
1.50
Y
0.00
-0.5 0
0.75
Y
69.0
65.0
ND
ND
ND
ND
8.75
-0.75
Y
50.5
5.75
1.75
Y
44.5
4
8.00
3
7.50
4
3
8.50
AS
4
4
Doug Ramsey
4
AAHL-9=Nordic
4
4
7.00
8.50
7.25
8.50
CAN-2
4
CAN-3
4
AAHL-3=0P1-3-88
5.00
3.50
5.50
8.50
7.75
7.75
CAN-1
1
4
4
Y: Yes; N: no; ND:Not Done
116
Appendix B. Table 5. Replication of IPNV Sp subtype isolates in the presence and
absence of 1% NTS and their relationship to virulence.
ISOLATE
CPE and Titers(logio) In
With NTS
Without NTS
Titer
CPE Titer CPE
Sp
4
C-17
4
C-18
4
C-36
4
C-39
NC-33
4
4
4
4
4
NH-44
4
OV-7
4
Thailand
4
DPL
FR-21
N-73A1 1
7.00
6.00
5.50
5.75
4.75
8.25
7.00
7.50
7.50
7.50
6.00
8.75
Logio
IP N
%
Reduction
Disease
Mortality
1.00
N
26.0
1
6.00
3.75
2.25
ND
ND
2
3.75
1.75
ND
ND
2
3.75
2.00
2.25
2.50
4.25
ND
ND
ND
ND
ND
ND
ND
ND
1.25
ND
ND
1.25
Y
1.25
Y
50.0
51.5
6.00
8.75
ND
ND
ND
ND
0
1
4
1
4
4
4
0
0
2.50
5.75
2.75
6.25
6.25
6.25
ND
ND
Y: Yes; N: no; ND:Not Done
117
Appendix B. Table 6. Replication of IPNV Jasper and BC subtypes isolates in the
presence and absence of 1% NTS and their relationship to virulence.
CPE and Titers(log) In
ISOLATE
Without NTS
CPE Titer
Jasper
CF-CTH-9/86
W17
4
N.H.
4
BC 89-362
93-511
HR
4
Ys KOREA
4
4
4
4
4
7.50
9.00
8.00
7.25
5.75
8.00
7.25
8.50
With NTS
CPE Titer
0
0
Reduction
IPN
Disease
Mortality
2.25
Y
28.5
Logio
%
5.25
ND
9.00
Y
56.5
3.50
2.50
N
31.0
0
4.50
4.75
ND
ND
0
ND
5.75
ND
ND
4
5.50
ND
ND
2
4.50
ND
ND
0
5.75
2.50
2.75
2.75
ND
ND
1
Y: Yes; N: no; ND:Not Done
118
Appendix B. Table 7. Replication of IPNV Ab and EVE subtypes isolates in the
presence and absence of 1% NTS and their relationship to virulence.
CPE and Titers(log) In
ISOLATE
Without RTS
CPE Titer
With RTS
CPE Titer
AB
5
8.5
0
5.75
LKT
4
8.00
4
3.75
SGV
4
0
ND
LICE
4
4
4.75
N-1265
4
4
5.75
EVE
4
7.00
7.25
6.50
9.00
0
6.00
%
IPN
Log
Reduction Disease Virulence
2.75
4.25
7.00
2.50
0.75
3.00
N
19.0
ND
ND
N
23.5
ND
ND
ND
ND
N
18.0
Y: Yes; N: no; ND:Not Done
119
Appendix B. Table 8. Replication of IPNV He and Te subtypes isolates in the
presence and absence of 1% NTS and their relationship to virulence.
ISOLATE
CPE and Titers(logio) In
Without NTS
With NTS
CPE Titer CPE Titer
He(c/wb)
4
Hecht
4
Te(c/wb)
1
8.00
8.25
4.00
4
4.75
1
1
IPN
Reduction Disease
Logio
%
Mortality
ND
ND
2.50
3.25
5.75
ND
ND
3.75
0.25
ND
ND
Y: Yes; N: no; ND:Not Done
120
Appendix B. Table 9. Replication of IPNV (subtypes unknown) isolates in the
presence and absence of 1% NTS and their relationship to virulence.
blanks
ISOLATE
CPE and Titers(log) In
Without RTS
CPE Titer
With RTS
CPE
Titer
Log
IPN
%
Reduction Disease Mortality
94-445
4
7.75
4
4.5
3.25
Y
34.5
DOMSEA
4
8.5
4
8.5
Y
94-021
4
8.75
4
8.5
N
86.5
22.5
91-63
4
4
96.0
4
7.75
7.75
Y
93-15
ND
ND
SF-1284
14' boot
4
ND
0
8.00
2.75
ND
4
7.25
8.00
8.50
8.00
ND
ND
18'
4
6.75
3
6.75
ND
ND
31-75
4
8.00
4
5.75
ND
ND
4
5.75
0
ND
ND
ND
DRT
4
5.75
0
3.5
ND
ND
VR-299(carp)
4
0
ND
ND
ND
99-E8
4
5.75
6.00
4
3.75
0.00
0.25
-0.50
0.25
0.5
5.25
.000
2.25
5.75
2.25
5.75
2.25
ND
ND
92-325
4
7.25
4
5.75
1.50
ND
ND
VR-299-(gold)
4
4
Y: Yes; N: no; ND:Not Done
121
Appendix B. Table 10.
CLASS
MONOGONTA
MOLLUSCA
CRUSTACEA
AGNATHA
TELEOSTEI
I
Species from which IPNV isolated.
GENUS & SPECIES
FAMILY 1
(Order) Ploima Branchionus plicatilis
Veneridae
Veneridae
Actamaeidae
Littorinidae
Mytilidae
Ostreidae
Ostreidae
Ostreidae
Tellinidae
Veneridae
Meretrix lusoria
Corbicula fluminou
Patella vulgate
Littorina littorea
Mytilus edulis
Crassotrea virginica
C. gigas
Ostrea edulis
Tellina tennuis
Mercenaria mercenaria
Portunidae
Portunidae
Penaeidae
Carcinus maenas
Macropipus depurator
Peanaeus japonica
Petromyzontidae
Clupeidae
Clupeidae
Anguillidae
Anguillidae
Anguillidae
Esocidae
Esocidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Salmonidae
Channidae
Cyprinidae
Lampetra fluviatilis
Brevoortia tyranus
Dorosoma cepedianum
A. japonica
A. rostrata
A. anguilla
Esox lucius
E. Niger
Hucho hucho
Oncorhychus mykiss
0. keta
0. kitsuch
0. clarki
0. nerka
0. tshawytchka
0. gorbushka
0. rhodurus
Salvelinus fontinalis
S. naymakush
S. alpinus
Salmo salar
S. trutta
Thymallus thymallus
Prosopium willimsoni
Ophicephalus striatus
Abramis brama
I
COMMON NAME I REF
rotifer
enamel venus shell
Asian clam
scallop
periwinkle
mussel
Amerikan oyster
Japanese oyster
European oyster
tellina
surf clam
shore crab
harbour crab
Japanese shrimp
lamprey
menhaden
gizzard shad
Japanese eel
American eel
European eel
northern pike
chain pickerel
grayling
rainbow trout
chum salmon
coho salmon
cutthroat trout
sockeye salmon
chinook salmon
pink salmon
amago trout
brook trout
lake trout
arctic char
Atlantic salmon
brown trout
grayling
whitefish
snakehead fish
bream
30
1
33
2
2
2
2
2
2
3
2
2
2
4
5
6
33
7
33
8
9
10
10
11
12
13
12
14
12
12
15
16
18
12
18
19
21
31
20
2
122
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cobitidae
Atherinidae
Sciaenidae
Carangidae
Cichlidae
Cichlidae
Percidae
Percichthyidae
Percichthyidae
Poecilidae
Gadidae
Pleuronectidae
Pleuronectidae
Pleuronectidae
Pleuronectidae
Pleuronectidae
1. Lo et al., 1981
2. Hill et al., 1982
3. Hill, 1976
4. Bovo et al., 1984
5. Munro et al., 1976
6. Stevens et al., 1983
7. Sano et al., 1981
8. Hudson et al., 1981
9. Silim et al., 1982
10. Ahne et al., 1989
11. Parisot and Yasutake, 1963
12. Wolf, 1988
Appendix B. Table 10.
(contunied)
Barbodes schwanefeldi
Blicca bjoernka
Carassius auratus
C. ceratisinis
C. carassius
Chondrostoma nasus
Cyprinus carpio
Gobio gobio
Oxyeleotris marmoratus
Hampala dispar
Phoxinus phoxinus
Rutilus rutilus
Scardinius erythrophtalmus
Barbus barbus
Brachydanio rerio
Misgurnus anguillicabulatus
Menidia menidia
Leiostomus xanthurus
Seriola quincieradiata
symphysodon discus
Tilapia mosambica
Perca fluviatilis
Dicentrarchus labrax
Morone saxatilis
Xiphophorus xiphidium
Gadus morhua
Limanda limanda
Paralichthys lethostigma
Pleuronectes fluviatilis
Hippoglossus
Scopthalmus maximus
barb
dace
goldfish
carp
carp
nase
common carp
goby
sand goby
eyespot barb
dace
roach
rudd
barbel
zebra danio
roach
silversides
drum
yellowtail
discus
tilapia
yellow perch
seabass
striped bass
platy
Atlantic cod
common dab
Southern flounder
sole
halibut
turbot
18. MacKelvi and Artsop, 1969
19. Wolf et al., 1960
20. Wattanavijarn et al., 1988
21. Ahne, 1980
22. Hill, 1988
23. Chen et al., 1984
24. Sorimachi and Hara, 1985
25. Adair and Ferguson, 1981
26. Welchsler et al., 1986
27. Olesen et al., 1989
28. McAllister et al., 1984
29. Castric et al., 1987
10
20
22
10
2
10
2
10
32
20
5
2
10
10
10
23
25
25
24
25
10
5
32
26
10
2
29
28
2
34
29
123
13. Wolf and Pettijohn, 1970
14. Traxler, 1986
15. Sano, 1973
16. Hedrick et al., 1986
30. Comps et al., 1991
31. Yamamoto and Kilistoff, 1979
32. Bonami et al., 1984
33. Plump et al., 1989
34. T. Halstein ersonal communication
17. Ljungberg and Vestegard-Jorgensen, 1972