Download Immunohistochemical analysis of infectious hematopoietic necrosis

AN ABSTRACT OF THE THESIS OF
Barbara S. Drolet for the degree of Doctor of Philosophy in Genetics presented on
September 15, 1994. Title: Immunohistochemical Analysis of Infectious
Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss).
Redacted for Privacy
Abstract approved:
Jo-Ann C. Leong
Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes
devastating epizootics in trout and salmon hatcheries of the Pacific Northwest, Europe
and Asia. The complete infection cycle of IHNV in fish has not been determined. It is
known that IHNV primarily infects young fish causing an acute, systemic disease
which results in mortality rates of up to 100%. The studies described herein were
designed to examine the complete pathogenesis of IHNV. The route of viral entry,
progression of the virus through the fish tissues, and the possibility of persistence of the
virus within the epizootic survivor is examined and discussed.
Two immunochemical assays were used to detect the nucleocapsid protein of
IHNV in infected cells. To relate the positive staining intensity to the degree of viral
infection, an alkaline phosphatase immunocytochemical (APIC) assay was developed to
detect IHNV in fixed tissue culture cells. The assay was published as an alternative to
current methods used to confirm IHNV in tissue culture.
To follow the IHNV infection in the fish, an alkaline phosphatase
immunohistochemical (APIH) assay was developed to detect the nucleocapsid protein
of the virus in whole, fixed, paraffin-embedded fish. Infected rainbow trout were tested
by APIH before and during the epizootic to examine the route of viral entry and the
progression of the virus, respectively. Virus entered through the gill epithelium during
normal oxygen uptake, and through the gastrointestinal epithelium during digestion.
Virus spread to virtually every organ via the circulatory system. Progression of IHNV
within and between organs is discussed.
Approximately fifty days after the epizootic, survivors are negative for
infectious virus by standard diagnostic methods and are released into the wild. The
virus source for IHNV-positive returning adults is unknown and has resulted in a
twenty year debate of secondary reservoir versus viral persistence. The last part of this
study, describes IHNV-related lesions in the kidney of rainbow trout one year after
virus exposure and persistence of the virus as shown by immunohistochemistry, PCR
amplification, and immunogold electron microscopy.
Copyright by Barbara S. Drolet
September 15, 1994
IMMUNOHISTOCHEMICAL ANALYSIS OF
INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS
IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)
by
Barbara S. Drolet
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed September 15, 1994
Commencement June 1995
Doctor of Philosophy thesis of Barbara S. Drolet presented on September 15, 1994
APPROVED:
Redacted for Privacy
Professor representing Gene
Redacted for Privacy
Director of Genetics Program
Redacted for Privacy
Dean of Gradu
School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Redacted for Privacy
Barbara S. Drolet, Author
i
ACKNOWLEDGMENTS
I thank Dr. Jo-Ann Leong, my mentor, for always having faith in my ability, and
pushing me to find the best in myself. I thank her for the kind, but firm words of
encouragement and instruction of what it takes for women to survive and grow in this
field. I thank Dr. John Rohovec for his advice, guidance, encouragement, humor and
friendship. I thank Dr. George Pearson for his guidance my first year, and for always
taking the time to listen and advise these past 5 years.
I thank my family for their constant support, and for never being further than a
phone call away. I thank Dr. Carla Mason Endres, my confidant and touchstone. I
thank her for our many hours of verbal PCR, the reality checks, and the unconditional
friendship she has given me. I thank Dan Mourich and Jon Piganelli for their friendship
and humor, which helped keep my sanity intact throughout the years. Finally, I thank
Bill Keleher for making my last year here the best.
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CONTRIBUTION OF AUTHORS
Dr. Jo-Ann C. Leong was involved in experimental design, data analysis and
interpretation, writing criticism and financial support of the work contained within each
manuscript. Dr. John S. Rohovec was involved in experimental design, writing
criticism, and financial support of the work contained within Chapters 3 and 4. Dr.
Jerry Heidel provided histological expertise, writing criticism, and financial support of
the work contained within the manuscript in Chapter 5. Pinwen (Peter) Chiou provided
PCR amplification expertise, crucial to the work contained within the manuscript in
Chapter 5.
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TABLE OF CONTENTS
Page
Chapter
Thesis Introduction
2. Literature Review
1.
Infectious Hematopoietic Necrosis Virus
Infectious Hematopoietic Necrosis
1
6
6
9
3. Serological Identification of Infectious Hematopoietic Necrosis
Virus in Fixed Tissue Culture Cells by Alkaline Phosphatase
Immunocytochemistry
Abstract
Introduction
Materials and Methods
Results and Discussion
Acknowledgments
References
4. The Route of Entry and Progression of Infectious Hematopoietic
Necrosis Virus in Oncorhynchus mykiss: A Sequential
Immunohistochemical Study
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgments
References
5. Persistence of an RNA Virus Infection by Defective Interfering Particles
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgments
References
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6. Thesis Summary
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BIBLIOGRAPHY
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APPENDIX
104
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LIST OF FIGURES
Page
Figure
2.1 Clinical signs of IHN in rainbow trout (0. mykiss)
16
3.1 Nucleocapsid protein of infectious hematopoietic necrosis virus
(IHNV) in infected cells by alkaline phosphatase immunocytochemical
staining using monoclonal antibody, 1NDW14D
31
4.1 Mortality curve of 1 g steelhead trout (0. mykiss) after immersion
infection with infectious hematopoietic necrosis virus, type 1
4.2 Photomicrographs of tissues from infectious hematopoietic necrosis
virus infected steelhead fry during the pre-epizootic period (0-5 days post
exposure) and the epizootic period (6-14 days post exposure)
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5.1 Kidney tissue from a 1 ype IHN epizootic survivor stained with Masson's
Trichrome stain
5.2 Kidney tissue from a 1 ypi IHNV survivor as tested by alkaline
phosphatase immunohistochemistry
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5.3 Cytoplasmic rhabdoviral inclusions in kidney tissue from a 1 ype IHN
epizootic survivor
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5.4 a Immunogold labeling of IHNV particles using the anti-nucleocapsid
monoclonal antibody, 1NDW14D
71
5.4 b Immunogold labeling of IHNV particles using anti-glycoprotein
monoclonal antibody, 3GH136J
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5.5 Immunogold labeled IHNV DI particles from 1 ype IHN epizootic
survivor
71
5.6 a Retrotherm PCR amplification of a 252 by fragment of IHNV N
gene from kidney tissue of a 1 year post exposure survivor
71
5.6 b Southern blot hybridization showing 252 by fragment is of IHNV N
gene origin
71
V
LIST OF TABLES
Page
Table
3.1 Virus isolates tested by alkaline phosphatase immunocytochemistry
with an anti-nucleocapsid monoclonal antibody, 1NDW14D
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4.1 Chronological appearance and staining intensity of infectious
hematopoietic necrosis virus by alkaline phosphatase
immunohistochemistry in steelhead (0. mykiss) fry
4.2 Virus-positive tissues of fish organs as tested by alkaline
phosphatase immunohistochemistry
48
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vi
DEDICATION
I dedicate this thesis to my family.
Their love and support have given me
a firm base to stand and wings to fly.
IMMUNOHISTOCHEMICAL ANALYSIS OF
INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS IN
RAINBOW TROUT (ONCORHYNCHUS MYKISS)
CHAPTER 1
THESIS INTRODUCTION
Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes
an economically important disease affecting hatcheries around the world. The lifecycle
of this virus is not completely known. The studies described in this thesis address the
pathogenesis and persistence of IHNV in rainbow trout. The complete pathogenic
pathways are known for very few organisms. The most comprehensive studies have
been done with reoviruses (Sharpe and Fei lds 1983; Nibert et al. 1991), influenza
(Brown et al. 1992) and LCM virus (Cole et al. 1971). The physical size of the host
usually prevents whole-body sequential studies often needed for extensive examination
of pathogenic pathways in vivo. Because IHNV infects primarily young fish, a
complete, whole-body examination of viral pathogenesis was accomplished.
Understanding the complete pathogenic pathway of any infectious agent is imperative
for designing effective disease treatments and vaccines.
Objectives
The objectives of these studies were to 1) determine the route of viral entry, 2)
examine the progression of virus through the tissues, and 3) determine if a latent or
subclinical form of IHNV persists in epizootic survivors.
2
Approach
To study the pathway of IHNV entry and progression, virus had to be detected
undisturbed in vivo. At the time of this study, in situ hybridization and polymerase
chain reaction (PCR) amplification technologies were not available for IHNV in fish
tissues. In addition, the instability of RNA viruses and the ubiquitous nature of
RNAses would have made in situ hybridization and PCR too labor and time intensive
for routine examination of the large number of samples needed for this type of study.
The plaque assay (Amos 1985) and immunofluorescence (LaPatra 1989b) are well
established and are used routinely to detect infectious IHNV in tissue culture and
infected tissues. However, both of these assays would require the separation and
processing of individual organs and tissues from the host to examine progression of
virus within the fish, which destroys the necessary in vivo nature of the study. In
addition, the processing of tissues for tissue culture assays results in dilution of the
samples which decreases the assay's sensitivity. Direct, in vivo immunofluorescent
staining of tissues was attempted for this study, but not pursued because of the shortlived, unstable reaction product, the necessity of examining all sections under a
fluorescent microscope, and the results, similar to those reported by Yamamoto et al.
(1989), of autofluorescing materials in fish tissues. Because no existing IHNV
detection assay was suitable for this study, an immunohistochemical assay was
developed which detected stable viral proteins, in vivo, resulting in a permanent color
reaction. There was no minimal sample size, and the reaction was highly specific and
sensitive.
Fixation of tissue
Formaldehyde, a cross-linking fixative, was used to immediately immobilize
and stabilize tissue and viral components in their native conformation. Formaldehyde
3
stabilizes proteins by forming cross-links between polypeptides both within individual
proteins and between neighboring proteins. Amino acids involved in formaldehyde
cross-linking are those possessing free amino groups, thiol groups, or aromatic ring
systems. Formaldehyde fixation is pH, temperature and time dependent.
Alkaline phosphatase immunohistochemistry (APIH)
An alkaline phosphatase immunohistochemical (APIH) assay was used to detect
viral protein in fixed, paraffin-embedded fish tissues. This assay utilized a universal
monoclonal antibody which recognizes the most highly conserved, abundant protein of
all IHNV types (Ristow and Arnzen 1989), and an alkaline phosphatase (AP) enzyme
detection system from Vector Laboratories (Burlingame, CA). The AP system was
chosen over immunoperoxidase (IP) because the staining intensity of AP is not as dense
as IP, making morphological examination of the stained cells possible. The
immunohistochemical method used was derived from the immunoenzymatic studies of
Hsu et al. (1981a; 1981b) using avidin-biotin complexes for amplification of antigens in
fixed tissue. The chromogen substrate chosen in this study was Vector Red (Vector
Labs) with levamisole (Ponder and Wilkinson 1981) added to eliminate endogenous
phosphatase activity in the fish tissues.
Immunogold electron microscopy
Although infectious virus can not be detected by tissue culture in fish past 50
days post exposure (dpe), virus is detected in spawning adults. Virus in adults is
thought to be the result of reinfection during spawning migration. The unexpected
detection of viral protein in one year post exposure (ype) survivors by APIH, led to an
electron microscopy (EM) study to examine survivor fish for persisting virus. An
4
immunogold EM procedure was adapted from previous immunochemical EM studies
(Faulk and Taylor 1971; Horisberger and Rosset 1977) and used to examine fresh and
archived tissues.
Introduction to thesis chapters
Chapter 3 is a published manuscript which describes a tissue culture adaptation
of the histochemical assay used in the pathogenic study. An alkaline phosphatase
immunocytochemical (APIC) assay was used to characterize the stain precipitate of the
AP immunochemical reaction. Degrees of staining intensity were rated 1 through 5,
weakest to strongest, and were related to the degree of productive infection. This assay
is currently the only method available to confirm IHNV in fixed, stained plaque assay
plates.
Chapter 4 is a published manuscript reporting the sequential,
immunohistochemical analysis of the complete pathogenic pathway of IHNV in
rainbow trout. The objective of the study was to characterize the pathogenesis of IHNV
infection of fish by identifying the route of viral entry and the tissues in which the virus
replicates. Sequential virus distribution following immersion exposure had not been
previously defined. Virus entered both by ingestion and penetration of the gill
epithelium and was disseminated by the circulatory system.
As indicated by it's name, it is in the hematopoietic tissue that IHNV is most
productive. In fish, that organ is the kidney. Fish kidneys are functionally divided into
the anterior, or head kidney and the posterior kidney. The anterior kidney is primarily
composed of hematopoietic cells and is thought to be the major site of blood cell
formation. The relative proportions of red and white cells in the kidney varies from
month to month, but overall there are roughly equal numbers of each cell type present
(Tatner and Manning 1983a). The posterior kidney is primarily composed of nephrons
5
embedded in hematopoietic tissue and functions to conserve salt and eliminate excess
water. The spleen is considered a secondary hematopoietic organ (Grace and Manning
1980; Tatner and Manning 1983a). Infection of the hematopoietic tissue results in slow
recovery of infected or destroyed blood cells and may result in damage to the kidney if
the fish survives the epizootic.
Chapter 5 is a manuscript reporting the pathology and viral persistence observed
in IHN epizootic survivors at one ype. The health of survivors has not been closely
monitored in the wild and it is uncertain how many survive to sexual maturation.
Epizootic survivors are released into the wild and are assumed to be virus-free because
they show no clinical signs of IHN, and testing fish by tissue culture methods
consistently shows no evidence of virus. However, when adults return to spawn, they
are again positive for IHNV. For the past 20 years, fisheries' scientists have been
studying the life cycle of IHNV trying to prove or disprove its persistence in survivor
populations. In this paper, lesions in the kidney of rainbow trout one year after
exposure are examined and evidence for subclinical persistence of IHNV is
demonstrated in the detection of viral protein by immunohistochemistry, viral RNA by
PCR amplification, and IHNV DI particles by immunogold electron microscopy.
6
CHAPTER 2
LITERATURE REVIEW
Infectious Hematopoietic Necrosis Virus
Virion characteristics
Infectious hematopoietic necrosis virus (IHNV) is a member of the
Rhabdoviridae family, in the genus Lyssavirus. Rhabdoviruses are widely distributed,
causing diseases in plants, insects, fish, amphibians, reptiles, wild and domestic
mammals, and humans. The prototypic Lyssavirus is rabies virus.
Morphology. Standard particles of IFINV are bullet-shaped, with an average
length of 160 nm and a core diameter of 60-70 nm (Amend and Chambers 1970).
Virions have an enveloped, helical nucleocapsid with glycoprotein peplomers
projecting 10 nm from the surface giving a final diameter of about 80-90 nm.
Nucleic acid. The nucleic acid of IHNV was shown to be RNA by Wingfield et
al. (1969). Subsequent work showed the RNA to be single-stranded negative sense and
non-segmented (McCain et al. 1974; McAllister et al. 1974; Hill et al. 1975; McAllister
and Wagner 1977). IHNV is a unique member of the Rhabdoviridae family in that its
genome contains six genes instead of the characteristic five. The six genes of IHNV
have been shown by electrophoresis to encode five structural proteins; a nucleocapsid
(N) protein, two matrix (M1, M2) proteins, a glycoprotein (G), and an RNA dependent
RNA polymerase (L) (McAllister and Wagner 1975; Hill et al. 1975; Lenoir and de
Kinkelin 1975; Leong et al. 1981; Kurath et al. 1985). A sixth protein, which can only
be detected in virally infected cells, is designated as the nonvirion (NV) protein (Kurath
and Leong 1985). The gene order is 3'-N-M1-M2-G-NV-L-5' as determined by R-loop
7
mapping (Kurath et al. 1985). Except for the unique NV gene, the gene order of IHNV
is identical to the other members of the Lyssavirus genus and members of the
Vesiculovirus Rhabdoviridae genus.
Proteins. The nucleocapsid protein is the most abundant and highly conserved
protein of IHNV (Gilmore and Leong 1988). The N protein binds to the ssRNA
genome and provides stability and structure. It is necessary for encapsidation and
infectivity. Because of the highly conserved, abundant nature of the N protein, it is the
target antigen for many diagnostic assays and pathogenic studies. The glycoprotein has
been shown to be the sole viral antigen responsible for inducing protective immunity in
fish (Engelking and Leong 1989a; Xu et al. 1991). Antibody produced to the G protein
induces protective immunity against all five IHNV types (Engelking and Leong 1989b).
The glycoprotein continues to be the focus of vaccine studies for IHNV (de Kinkelin et
al. 1984; Leong et al. 1988; Leong and Munn 1991; Oberg et al. 1991; Xu et al. 1991;
Leong 1993; Leong and Fryer 1993).
Types
The IHN virus has been classified into five electropherotypes based on gel
migration patterns of the nucleocapsid protein and glycoprotein (Leong et al. 1981; Hsu
et al. 1986). Type differences in average plaque diameter (Chen et al. 1990), virulence
(LaPatra et al. 1990), antigenicity (Winton et al. 1988; Basurco et al 1993; Ristow and
de Avila 1991), and single-step growth curves (Mulcahy et al. 1984b) have been shown.
Hsu et al. (1986) reported a geographical predominance for specific isolates in certain
areas. The electropherotypes of interest to the studies described herein are Round
Butte-83 (Type 1) and Rangen (Type 2) which cause a majority of outbreaks in Oregon
and Idaho.
8
Cell culture
Permissive cell lines. Rhabdoviruses have been shown to infect a diverse group
of cell types including plants, insects, reptiles, mammals, birds, amphibians and fish
(Clark and Soriano 1974; Wagner 1975; Scott et al. 1980). IHNV was first isolated in
primary fish cell cultures from infected salmon tissues in 1980 (Pilcher and Fryer
1980). Permissive fish cell lines used routinely today include carp epithelioma
papulosum cyprini EPC cells (Fijan et al. 1983), rainbow trout gonad RTG-2 cells
(Wolf and Quimby 1962), chinook salmon embryo CHSE-214 cells (Lannan et al.
1984), steelhead trout embryo STE-137 cells (Fryer et al. 1965; Fendrick et al. 1982),
sockeye salmon embryo SSE-5 cells (Nims et al. 1970), Atlantic salmon AS cells
(Nicholson and Byrne 1973) and fathead minnow FHM cells (Gravell and Malsberger
1965). Chum salmon embryo (SE) and Yamame 0. masou kidney (YNK) cells
(Asakawa et al. 1986) have also shown susceptibility to IHNV.
Growth requirements. The survival of IHNV has been evaluated under several
environmental conditions such as dessication, pH, temperature, water hardness, and
salinity (Pietsch et al. 1977; Kamei et al. 1987; 1988). Virus grows best at temperatures
between 13-18°C (Wingfeild et al. 1969; Yasutake and Amend 1972). The low
temperature requirement may be due to the heat liability of the virion polymerase
(McAllister and Wagner 1975) or the synthesis of interferon at higher temperatures (de
Kinkelin et al. 1982).
Infection. In cell culture, the cytopathic effect (CPE) appears initially as
thickened nuclear membranes. Cells then become granular and rounded, and eventually
detach from the surface of the culture vessel. A virus titer of lx108 pfu/ml can be
harvested from the supernatant of CHSE-214 cells at peak CPE. The appearance of
9
CPE depends on the isolate being grown and the sensitivity of the cell line being used
(Kelly et al. 1978; Fendrick 1982; Mulcahy et al. 1984a; Chen et al. 1990). In one-step
growth curves with CHSE-214 cells at 18°C, viral antigens can be detected in the
cytoplasm by fluorescent antibody at two hours post infection (hpi), and infectious
virions appear by four hpi (McAllister et al. 1974).
Stability. The infectivity of II-INV is stable if stored at -70°C or -20°C, but
decreases at temperatures above 4°C (Pietsch et al. 1977). Virus is not stable in salt
water or fresh water containing high numbers of bacteria. Virus has been shown to be
sensitive to iodine. Virally contaminated eggs and water can be effectively disinfected
with an iodophor treatment (Amend and Pietsch 1972a; Bans et al. 1991). Although
IHNV is sensitive to temperatures above 18°C, raising the water temperature is only
considered to be a practical solution in controlling the temperature sensitive isolate of
IHNV at Coleman Hatchery where there is a ready source of geothermal water.
Elsewhere, rainbow trout infection experiments have shown that the higher the
temperature the shorter the survival time of the fish (Hetrick et al. 1979).
Infectious Hematopoietic Necrosis
Infectious hematopoietic necrosis is an acute, systemic, economically
devastating disease in trout and salmon populations. Although originally isolated from
feral populations, IHN is primarily a problem in hatcheries where large numbers of fish
are grown at high densities. While density does not affect survival from egg to fry, it
does effect the level of virus titer and thus results in increased mortality during viral
epizootics among fry. Lack of genetic selection for resistance was also suggested as a
cause of IHN associated mortality (Mulcahy and Bauersfeld 1983).
10
Distribution
World-wide. IHNV was first reported in 1953 as the cause of a disease among
sockeye salmon in Washington (Rucker et al. 1953) and again in 1967 in sockeye in
western Canada (Amend et al. 1969). It is endemic to the Pacific Northwest United
States and Canada (Parisot et al. 1965; Grischkowsky and Amend 1976; Traxler 1983).
Natural outbreaks have been reported in California, Oregon, Washington, Alaska
(Leong et al. 1981; Burke and Grischkowsky 1984; Grischkowsky and Amend 1976)
and British Columbia (Williams and Amend 1976). The disease was introduced into
Japan at Hokkaido in 1968 with eggs from North America (Kimura and Awakura 1977)
and rapidly spread throughout the Japanese mainland (Sano et al. 1977; Yoshimizu et
al. 1988; Yoshimizu et al. 1993). IHNV isolates have recently been reported in Korea
(Park et al. 1993), Taiwan, Belgium (Hill 1992), France (Laurencin 1987), and Italy
(Bovo et al. 1987).
Oregon. The IHN virus was the first fish virus to be isolated and identified in
the state of Oregon (Wingfield et al. 1969). It is thought that IHNV was introduced into
Oregon from Puget Sound, Washington, in the form of unpasteurized sockeye salmon
viscera which had been incorporated into the diet of the fish. In Oregon the incidence
of IHN continues to increase from 6 hatcheries and holding ponds in 1981 to 43 sites in
1994 (Engelking, personal communication).
Host range
IHNV commonly infects sockeye (Oncorhynchus nerka), kokanee (0. nerka)
chum (0. keta), and chinook (0. tshawytscha) salmon and rainbow (0. mykiss) and
steelhead (0. mykiss) trout (Pilcher and Fryer 1980; Wolf 1988). Young coho salmon
(0. kisutch) are considered resistant to IHNV infection, yet the virus has been detected
11
in adult coho in Oregon ( LaPatra et al. 1989a), Washington (Eaton et al. 1991) and
California (Hedrick et al. 1987). Cutthroat (0. clarkii), brook (Salvelinus fontinalis)
(Goldes and Mead 1992; LaPatra et al. 1992; Bootland et al. 1994), and brown (Salmo
trutta) (LaPatra and Fryer 1990) trout have shown susceptibility to experimental IHNV
infections. Experimental infection studies with non-salmonid species have shown
whitefish to be susceptible, and squaw fish, suckers and lamprey to be resistant to
IHNV infection (Bootland and Lorz, unpublished results). In addition, two marine fish
species, seabream (Archosargus rhomboidalis) and turbot (Scophthalmus maximus),
have shown susceptibility to IHNV infection by intraperitoneal injection (Castric and
Jeffroy 1991).
IHNV primarily kills young fish: alevins, fry and early juveniles weighing less
than 5 g. It has been suggested that susceptibility decreases with increased size and age
of the fish (Kasai et al. 1993). However, kokanee and rainbow trout have been shown
to be susceptible to two types of IHNV at weights up to 13 g (LaPatra et al. 1990).
Transmission
Horizontal. Horizontal transmission of IHNV is well documented (for review,
see Wolf 1988). Virus is transmitted through feces, urine, and ovarian or seminal fluid
(Pilcher and Fryer 1980; Mulcahy et a. 1983a; 1983b; Mulcahy and Batts 1987;
Nishimura et al. 1988). Virus can be detected in effluent hatchery water during large
epizootics (Leong and Turner 1979; Yoshimizu et al. 1991), and can be transmitted to
fish held down stream (Mulcahy et a. 1983b).
Vertical. Evidence for vertical transmission is circumstantial. The strongest
evidence comes from the association made between the appearance of the disease and
the shipment of eggs from infected adults into geographical areas where the virus was
12
not known to occur (Amend 1975). In nature, the greatest danger of an epizootic is in
the spring when newly hatched fry become infected with virus excreted in sex products
during spawning. Infection of hatching fry is thought to be due to virus contaminating
the surface of the egg and not virus within the egg itself. However, neutralizing
antibodies have been isolated from eggs of spawning steelhead trout (Shors and
Winston 1989a) indicating either an internal active infection or passively acquired
maternal antibody. Virus has been shown to pass in sockeye salmon from infected
adult to egg through ovarian or seminal fluid (Mulcahy and Pascho 1985; Meyers
1990), and virus has been shown to adsorb to fish sperm (Mulcahy and Pascho 1984).
A prevalence study in 1983 reported feral, introduced and hatchery-reared salmonid
species in the Pacific Northwest had ovarian fluid IHNV titers of 105 pfu/ml at 25%,
43% and 32% respectively (Mulcahy et al. 1983c). The authors suggested that the
success of the feral population was due to IHNV titers high enough to maintain the
virus in the population, but low enough to cause little transmission of lethal doses
through the eggs.
The possibility of vertical transmission was studied by Yoshimizu et al. (1989)
using the eggs of masu and chum salmon. Eggs externally contaminated with IHNV
showed no virus after one day. Virus injected into just-fertilized eggs could not be
detected by one month post injection. However, virus injected into eyed eggs caused
infection in 90% of the embryos. Several components of the yolk showed anti-viral
activity. It was suggested that vertical transmission does not occur, in that even if virus
was internalized it would not survive to the eyed stage. In addition, an extensive
fertilization study by LaPatra et al. (1991) showed no vertical transmission in steelhead
populations. Thus, vertical transmission of IHNV remains controversial.
Covert. A third transmission route, covert transmission, is currently being
studied by the Oregon Department of Fish and Wildlife pathology staff (LaPatra
13
1989b). By differentially marking progeny from carrier (virus-positive) and virus-free
parents, they hope to determine whether progeny from carrier adults return to hatcheries
as carriers of the virus.
Environmental reservoirs
Persistence of IHNV in the environment has resulted in several studies
examining possible secondary viral reservoirs. Thirty-six environmental sampling
trips, consisting of 215 benthos and 36 concentrated water samples (Leong and Turner
1979; Batts and Winton 1989a) have been taken over a three year period from 4 sites in
Oregon. Results showed no evidence of virus with the exception of 1 water sample
taken from the Metolius River (Lorz and Stevens, unpublished data). Virus has been
isolated from a freshwater leech (Piscicola salmositica) and a copepod (Salmincola sp.)
taken from the gills of infected, spawning sockeye salmon (Mulcahy et al. 1990). Virus
has also been isolated from Mayflies found near DIN epizootic sites (Shors and
Winston 1989b). It has been suggested that these organisms may act as a vector in
which the virus could overwinter and serve as reservoirs of infection, but IHNV has yet
to be shown to replicate within these hosts.
Pathogenesis
The main target of IHNV is the hematopoietic organ of the fish, which is the
kidney. Initial sites of replication have been examined by plaque assay (Yamamoto et
al. 1992), immunohistochemistry (Yamamoto et al. 1990; Drolet et al. 1994) and
electron microscopy (Yamamoto and Clermont 1990). Several infection studies have
implicated the gills as the initial site of IHNV entry and infection. Virus has been
isolated by tissue culture in gill tissue early in the infection of sockeye fry (Mulcahy et
al. 1983a) and smolts (Burke and Grischkowsky 1984). Virus was also isolated by
14
tissue culture and G protein detected by immunoperoxidase histochemistry from gill
tissue of adult kokanee and sockeye at 3 days post exposure (dpe) (Yamamoto et al.
1989). Virus was detected in gill tissue of rainbow trout at 16-20 hours post exposure
(hpe) by electron microscopy and at 24 hpe by tissue culture methods (Yamamoto and
Clermont 1990), but not until 5 dpe by immunohistochemistry (Yamamoto et al. 1990).
Yamamoto et al. (1990) also reported that the skin was the major site of IHNV
replication and that the gill played a lesser role in the initial stages of infection. Gills
have been shown to be the initial site of replication for two other fish rhabdoviruses,
spring viremia of carp virus (Ahne 1978) and viral hemorrhagic septicemia virus
(Neukirch 1984).
Entry at the gill followed by dissemination of the virus by the blood has been
suggested from tissue culture studies (Yasutake and Amend 1972; Mulcahy et al.
1983a). The circulatory system of fish is anatomically simple, compared with that of
higher vertebrates. Blood circulates from the heart to the gill arches, where it becomes
oxygenated primarily by diffusion through the epithelium of the gill lamellae. It is
distributed by way of the dorsal aorta to the arteries and peripheral capillaries and
returned to the heart through the venous system (Yasutake and Wales 1983). Viral
entry through the gills and progression via the circulatory system was recently
confirmed by immunohistochemistry (Drolet et al. 1994).
Mulcahy et al. (1983a) also reported that infectious virus was detected in the
viscera of naturally infected spawning sockeye in the absence of virus in the gills.
Moreover, virus has been detected by immunohistochemistry (Yamamoto et al. 1990)
and by electron microscopy (Yamamoto and Clermont 1990) in the visceral organs of
rainbow trout before the gills. This suggested an alternate or additional origin of virus
infection which was determined to be ingestion by Drolet et al. (1994).
15
Clinical signs
Typical IHN external signs include abdominal swelling, petechial hemorrhages
on the ventral surface and at base of pectoral and pelvic fins, exophthalmia, pale ventral
surface, dark dorsal surface and occasionally fecal casts (Amend et al. 1969) (Figure
2.1). Dark reddish areas just behind the head are thought to be due to subcutaneous
pooling of extravascated blood from the anterior kidney. Internally, the kidney and
liver are anemic, ascitic fluid may fill the peritoneal cavity, hemorrhages may be found
in the muscle, fat and liver, and the digestive tract is often devoid of food. Behavioral
signs include anorexia, lethargy, with brief episodes of frenzied swimming, avoidance
of currents, rolling, swimming vertically or circling and flashing. Microscopically,
necrosis of the hematopoietic tissues in the anterior kidney and spleen as well as
necrosis of the liver and pancreas is common. Granular cells in the lamina propria,
stratum compactum and stratum granulosum of the alimentary tract are also commonly
necrotic (Yasutake et al. 1965; Yasutake and Amend 1972). Packed cell volume,
hemoglobin, red blood cell count, and plasma bicarbonate are significantly depressed
by the forth day after exposure (Amend and Smith 1974). Spinal deformities may
occur in epizootic survivors.
16
Figure 2.1
Clinical signs of IHN in rainbow trout (0. mykiss). The three fish on top
show classic signs of IHN infection including abdominal swelling, pale
ventral surface, dark dorsal surface, petechial hemorrhages on ventral
surface and at the base of pectoral and pelvic fins (up arrow), and
exophthalmia (down arrow). On the bottom is a negative control fish.
Figure 2.1
18
Diagnostics
In the field, diagnoses are based on clinical signs, histopathological changes and
the hatchery's IHN epizootic history. In the laboratory, presumptive diagnosis requires
the isolation of the virus in cell culture with the development of typical CPE.
Plaque assay. For plaque assays, EPC cell monolayers are inoculated with
infected fish tissue homogenate (Burke and Mulcahy 1980) or mucus supernatant
(LaPatra et al. 1989c), incubated at 17°C, fixed and stained as suggested in the Fish
Health Blue Book (Amos 1985). The recommended incubation time for tissue culture
plaque assay is 10
14 days, although this can be shortened if cell monolayers are
pretreated with polyethylene glycol Watts and Winton 1989b) or the polycation
polybrene (Leong et al. 1982). Infectious virus is quantitated by counting the number
of plaques resulting from a focal viral infection. Incubation temperatures (Burke and
Mulcahy 1983) and media (Amend and Pietsch 1972b; Nishimura et al. 1985) are well
established. Plaque neutralization is commonly used to detect IHNV or antibody to
IHNV (Jorgensen et al. 1991), and has recently been used to characterized the humoral
immune response of rainbow trout to IHNV infection (LaPatra et al. 1993).
Immunoassays. Recent advances in detecting IHNV (for review see Winton
1991) have taken advantage of the numerous IHNV-specific monoclonal antibodies
now available (Ristow and de Avila 1991). Immunodot (McAllister and Schill 1986;
Eaton et al. 1991; Ristow et a. 1991) and immunoblot (Schultz et al. 1989) assays have
been developed to detect IHNV in cell culture fluid. Viral antigen can also be rapidly
detected from cell culture or infected fry tissue homogenate supernatants with enzymelinked immunosorbent assays (Dixon and Hill 1984; Way and Dixon 1988; Medina et
al. 1992).
19
A staphylococcal coagglutination test has been developed by Boot land and
Leong (1992) primarily for use in the field. This assay detects all five types of IHNV in
cell cultures and infected fish in 15 min, but a titer of 106 pfu/ml is required to obtain a
positive reaction.
Immunofluorescence is used to rapidly detect IHNV using both monoclonal and
polyclonal antisera (LaPatra et al. 1989b; Arnzen et al. 1991; JOrgensen et al. 1991).
Immunofluorescence or plaque neutralizations are routinely done to confirm the
infectious agent as IHNV. Recently, an alternative to these subculturing confirmatory
methods has been developed. An alkaline phosphatase immunocytochemical assay can
now be used to confirm IHNV in fixed, stained tissue culture cells, alleviating the
necessity of subculturing (Drolet et al. 1993).
Immunohistochemical techniques have been developed to detect IHNV in vivo.
Yamamoto et al. (1990) used a horse-radish peroxidase technique to detect
multiplication sites of IHNV in rainbow trout. Most recently, the route of viral entry
and progression of IHNV in rainbow trout has been examined using an alkaline
phosphatase immunocytochemical assay (Drolet et al. 1994).
Techniques developed to detect the nucleic acid of IHNV include in situ
hybridization and PCR amplification. A biotinylated DNA probe has been used to
detect IHNV mRNA in infected cell cultures (Deering et al. 1991) and rainbow trout
brain tissue (Anderson et al. 1991). The N gene has been amplified by PCR from
purified genomic RNA of all five IHNV isolate types (Arakawa et al. 1990). Most
recently, PCR amplification detected viral RNA in archived tissues fixed in formalin
for 2 years or more (Chiou et al. 1994).
Although not practical as a diagnostic tool, electron microscopy (EM) has been
used to characterize virion structure (Amend and Chambers 1970) and infectivity
patterns (Yamamoto and Clermont 1990). Most recently, scanning EM has been used
to examine IHNV receptor binding and protein expression (Helmick et al. 1991).
20
Persistence of IHNV
Tissue culture. Persistence of IHNV has been shown to occur in infected tissue
culture cells. Engelking and Leong (1981) developed a cell culture model of a viruscarrier state in chinook cells. High levels of virus were released from these cells into
the medium although the cells continued to grow well in culture. Virus production
fluctuated with passage level and it was demonstrated that nearly 100% of the cells
were producing viral antigen. Furthermore, these cells were resistant to superinfection
by homologous virus but sensitive to infection by heterologous viruses. Persistence of
IHNV apparently was mediated by several factors including the development of
temperature sensitive mutants, small plaque mutants and defective interfering (DI)
particles. Interferon did not play a significant role in persistence of the virus in these
cells, although interferon was suggested as a factor in persistently infected RTG-2 cells,
(Okamoto et al. 1983). Persistent IHNV infections have also been reported in YNK
cells (Watanabe 1983).
Fish. Persistence of IHNV in fish remains controversial. Infectious virus can
only be detected in young fry and adult spawning populations. Fish are negative for
infectious virus by tissue culture methods between these two life stages. The source of
virus for adult populations returning to spawn is unknown. One theory is that returning
adults are reinfected with IHNV from currently infected fish or some secondary
reservoir during their migration upstream. The other theory is that the virus persists in
the survivor in a latent or subclinical state and is reactivated during stressful spawning
conditions. This controversy is at the core of understanding the life cycle of IHNV.
Neither theory has been proven, nor have they been shown to be mutually exclusive.
Mulcahy et al. (1982) reported that 100% of returning adult sockeye that were
examined at the Cedar Creek spawning cannel were infected and virus was present in
21
nearly all organs. In addition, there was an overall tendency for the mean concentration
to increase in many of the organs over time as the fish progressed to ripeness. Because
of the correlation of IHNV appearance in returning adult sockeye and the spawning act,
sex hormones have been suggested to have a role in susceptibility to reinfection, or
reactivation of a latent form of the virus. However Grischkowsky and Mulcahy (1982)
reported that hormones injected into naturally infected spawning adults did not
significantly alter the viral infection.
Evidence for the latency theory. The mechanism by which the carrier state may
be established and maintained in adult fish is unknown. In 1975, Amend reported that
rainbow trout that had survived an IHN epizootic and were held in pathogen-free water,
became positive for virus upon sexual maturation (Amend 1975). It was suggested that
the virus entered a latent state in the survivor and was reactivated during sexual
maturity (Amend 1975; Mulcahy et al. 1983a; Mulcahy et al. 1984a). Adult fish appear
normal and no virus can be detected in their tissues. However, high concentrations of
virus do appear in ovarian and seminal fluids during spawning (Amend 1975; Mulcahy
et al 1983c; Mulcahy and Batts 1987; Mulcahy et al. 1987).
Low positive antibody titers against IHNV have been reported in returning
spawning adults (de Avila et al. 1992), suggesting an immune memory occurring in the
immunocompetent fry (Tatner and Manning 1983b) at the time of initial virus exposure.
This may decrease the reinfection rate of adults during spawning, which would suggest
that virus reactivation was responsible for the reappearance of IHNV in the population.
The inability to define a secondary reservoir or overwintering vector has also
strengthened the latency theory. It is possible that the 100% infection rate of many
spawning populations results from virus activation in lifelong virus carriers and the
subsequent horizontal infection of the noninfected members of the returning population
(Mulcahy et al. 1982).
22
Evidence for the reinfection theory. Since 1975, fish disease laboratories have
tried to reisolate IHNV from captive survivor populations to no avail. The inability to
defmitively show the reactivation of a latent virus in numerous laboratory infection
studies has provided the majority of evidence against latency. Amos et al. (1989)
reported that no virus was isolated from adult sockeye salmon, captured as they exited
saltwater and held in pathogen-free water until sexual maturation. Simultaneously,
sockeye allowed to migrate upstream were 90-100% positive for virus by the time they
reached the hatchery. It was suggested that the high prevalence of IHNV in spawning
populations was the result of horizontal transmission from some source in the stream
and not the reappearance of virus in fish harboring a life-long latent infection.
Persistence of IHNV by defective interfering particles
Defective interfering (DI) particles may also be important in viral persistence in
IHN epizootic survivors (Drolet, unpublished results). Rhabdoviral DI particles, and
their roles in persistent tissue culture infections, are well established (for reviews, see
Reichmann and Schnitzlein 1980; Sekellick and Marcus 1980). For IHNV, defective
particles have been described in persistently infected fish cell cultures (Engelking and
Leong 1981), and autointerference has been demonstrated by serial passage at high
multiplicities of infection (McAllister and Pilcher 1974). Although DI particles and
their role in tissue culture infection have been demonstrated for almost every virus,
defective particles have not been demonstrated in animals with natural infections.
Generation of DI particles. DI particles are generated by aberrant replication
(for review, see Perrault 1981) which result in a shortened, defective genome. The
mechanism of generation can be affected by cell type (Huang and Baltimore 1970;
Holland et al. 1976), route of infection (Huang and Baltimore 1970) or multiplicity of
23
infection (Holland et al. 1976). The length of rhabdovirus particles is dependent upon
the length of genome encapsidated. Thus, IHNV DI particles, like those of the well
documented vesicular stomatitis virus, are round or short bullet-shaped.
Role in disease. The production of DI and standard particles is cyclic in nature
(Huang and Baltimore 1970). The synthesis of standard and DI particles may be in
such balance that only a very few virions are synthesized, thus, the infection would
remain subacute or latent until the balance is upset by stress or infections. The stress of
spawning and the exposure of fish to IHNV and other pathogens in the stream may
result in the resurgence of standard particles and thus, recurrence of infection. Inability
to isolate infectious virus from non-spawning survivors may be due to population
heterogeneity where excess DI particles inhibit standard virus growth and prevent the
immediate cytopathic effects needed to detect virus.
Treatment
Virally contaminated eggs and water can be disinfected with iodine derivatives
(Batts et al. 1991), but there are no antivirals or vaccines available for hatcheries to
combat this economically devastating disease. It is hoped that understanding the
infection process of IHNV will result in efficacious methods of treatment.
24
CHAPTER 3
SEROLOGICAL IDENTIFICATION OF INFECTIOUS HEMATOPOIETIC
NECROSIS VIRUS IN FIXED TISSUE CULTURE CELLS BY
ALKALINE PHOSPHATASE IMMUNOCYTOCHEMISTRY
Barbara S. Drolet
Genetics Program, Department of Microbiology
Oregon State University, Corvallis Oregon
John S. Rohovec and Jo-Ann C. Leong
Center for Salmon Disease Research
Department of Microbiology
Oregon State University, Corvallis, Oregon
Published: December 19, 1993
Journal of Aquatic Animal Health
5(4):265-269
Publication of this manuscript authorized by American Fisheries Society
5410 Grosvenor Lane, Suite 110, Bethesda, Maryland 20814-2199.
25
Abstract
An alkaline phosphatase immunocytochemical (APIC) assay was adapted for
direct detection of infectious hematopoietic necrosis virus (IHNV) in infected tissue
culture cells. The APIC assay provided a means of confirming the diagnosis of IHNV
after the tissue culture plates had been fixed with formalin and stained with crystal violet.
In the event that the original fish tissue samples had been discarded, the APIC assay was
very useful and was able to detect IHNV on plates that were more than a year old. The
assay used a broadly reactive monoclonal antibody (1NDW14D) to the IHNV
nucleoprotein to detect viral antigen in cells infected by virus isolates representing the five
known IHNV types. Likewise, the Type II-specific monoclonal antibody (2NH105B)
was able to distinguish Type 11 IHNV in plaques that had been previously fixed. No
cross-reactivity was seen with six other fish rhabdoviruses nor with a fish birnavirus,
infectious pancreatic necrosis virus (IPNV). Immunocytochemical staining was able to
distinguish between the cytopathology produced by virus infection and that induced by
the toxicity of tissue samples. In the latter case, no staining was observed. The staining
was not affected by the age of the fixed and dried plates nor by pre-treatment of cells with
polyethylene glycol. The ease, specificity, and sensitivity of the procedure made the
APIC assay an attractive alternative to the standard serum neutralization or
immunofluorescent identification of IHNV for diagnostic fish disease laboratories.
Introduction
Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that can cause
epizootics in wild and hatchery-reared salmon and trout (for review, see Wolf 1988).
Young salmonid fish are most susceptible and an IHNV outbreak can result in the
complete loss of affected fish. Epizootics of IHNV are presumptively diagnosed in the
field on the basis of several criteria, e.g. external disease signs, water temperature, fish
26
stock, geographic location, and the disease history of the stock and hatchery. Ultimately,
the final diagnosis resides on laboratory confirmation which requires virus isolation and
growth in tissue culture followed by serological identification of the virus (Amos 1985).
The recommended procedure for the laboratory diagnosis of IHNV is the
inoculation of the tissue samples onto susceptible cells followed by incubation at 15-18°C
for no less than 14 days. When the plaque assay method of Burke and Mulcahy (1980)
is used, an incubation period of 10 days is required. If virus-induced cell lysis is
observed as a plaque or as cytopathic effect (CPE), the accepted procedure for identifying
the viral agent is serum neutralization (Amos 1985). However, a number of different
procedures for identifying IHNV in tissue culture samples have been reported since these
recommendations were drafted. Several immunofluorescence tests incorporating a direct
fluorescein-conjugated anti-nucleoprotein monoclonal antibody (Ristow and Arnzen
1989) and an indirect test utilizing a monoclonal antibody to the glycoprotein of IHNV
have been developed (LaPatra et al. 1989). More recently, Ristow and colleagues
developed a quick direct fluorescent antibody test for IHNV that required only 30 min at
37°C for reacting with the viral antigen (Arnzen et al. 1991). The fluorescent antibody
tests provide quick, accurate confirmation of IHNV in tissue culture samples.
However, for many diagnostic laboratories where virus assays number in the
hundreds per week and there is often no time to screen all wells and plates for viral CPE,
a procedure was needed to confirm IHNV diagnosis after the plates had been fixed with
formalin and stained with crystal violet. An alkaline phosphatase immunocytochemical
assay (APIC) was adapted to detect IHNV antigen in fixed, stained plates. The
procedure has been useful when tissue samples had been discarded or a retrospective
analysis of a virus assay plate has revealed virus-like plaques and further analysis of a
"plaque" was required.
27
Materials and Methods
Viruses, Cell Lines, and Antibodies
The IHNV isolates used in this study were previously described and
electropherotyped by Hsu et al. (1986). Subsequently, the same isolates were
characterized by monoclonal antibody reactivity (Ristow et al. 1991). The IHNV isolates
used in this study were: Round Butte, 1983 (Type I); Rangen, 1983 (Type II); Elk
River, 1979 (Type III); Nan Scott Lake, 1971 (Type III); Coleman Hatchery, 1980 (Type
IV); Cedar River, 1981 (Type V); Karluk River, 1979 (Type V); Sacramento River
chinook virus, 1969 (untyped), and Oregon sockeye virus, 1968 (untyped). The other
fish rhabdoviruses that were included were viral hemorrhagic septicemia virus (VHSV),
pike fry rhabdovirus (PFRV), snakehead rhabdovirus (SHRV); ulcerative disease
rhabdovirus (UDRV); hirame rhabdovirus (HRV), and Rhabdovirus carpio (RC). These
viruses were obtained from C. Lannan and J. L. Fryer (Mark 0. Hatfield Marine Science
Center, Fish Disease Laboratory, Newport, Oregon). Infectious pancreatic necrosis
virus (IPNV) of the Sp serotype obtained from P. de Kinkelin (France) was also
included.
The cell line, epithelioma papillosum cyprini (EPC), obtained from F. Hetrick
(University of Maryland) was originally established by N. Fijan (Fijan et al. 1983).
These cells were used for all rhabdovirus assays at the appropriate temperature for each
virus. For the IPNV assay, chinook salmon embryo cells (CHSE-214) were used at
15°C. The original cell line was obtained from J. L. Fryer (Mark 0. Hatfield Marine
Science Center, Fish Disease Laboratory, Newport, Oregon) (Fryer et al. 1965).
The monoclonal antibodies were obtained from Sandra Ristow (Washington State
University, Pullman, Washington). The antibody preparations consisted of the tissue
culture supernatant fluid taken from hybridoma cell lines.
28
Virus Assays
The IHNV samples from fish were part of a laboratory experiment to detect
IHNV in fish during a viral epizootic. Steelhead trout (Oncorhynchus mykiss) fry
weighing 1.2 g were exposed to 103 plaque forming units/ml of IHNV (Round Butte
1983, Type I) by static immersion (13°C) for 12 h at the Salmon Disease Laboratory in
Corvallis, Oregon. The fish and tissue samples were processed as described (Amos
1985).
Virus assays were carried out by the tissue culture infectious dose assay on 96­
well tissue culture plates and by the plaque assay on 24-well tissue culture plates. Plaque
assays were performed by a slightly modified procedure of Burke and Mulcahy (1980).
Virus from homogenate samples was adsorbed to cells that either had or had not been
pre-treated with polyethylene glycol (PEG) as recommended by Batts and Winton (1989)
for 1 h at 15°C with gentle rocking. Infected cell monolayers were overlaid with 0.75%
methyl cellulose in complete MEM (Eagle's minimum essential medium, Gibco BRL,
Grand Island, NY). Plates were incubated for up to 14 days at 15°C. Cell monolayers
were then fixed and stained with crystal violet in formalin (25% formalin, 10% ethanol,
5% acetic acid, 1% w/v crystal violet), rinsed with tap water, and allowed to dry.
Alkaline phosphatase immunocytochemistry (APIC)
Plaque assay plates were destained with several changes of 70% ethanol and
allowed to dry. All procedures were carried out at room temperature. Non-specific
binding sites were first blocked with 5% nonfat powdered milk (Saco Foods, Madison,
WI) in phosphate buffered saline (PBS: 137 mM NaC1, 2.7 mM KC1, 4.3 mM
NaHP03.7 H2O, 1.4 mM KHPO4), pH 7.4, with gentle rocking. The blocking solution
was removed after 1 h when the murine anti-nucleocapsid protein monoclonal antibody
(Mab), 1NDW14D, was used after 12-14 h when the Mab 2NH105B was used (Ristow
29
and Arnzen 1989; Ristow et al. 1991). The antibody was adsorbed to the antigen by
gently rocking the plate for 1 h. Unbound antibody was removed by two 5 min washes
with PBS. The plates were stained according to instructions for alkaline phosphatase
immunohistochemistry in the Vectastain-ABC mouse IgG kit of Vector Laboratory
(Burlingame, CA). In brief, the bound anti-N monoclonal antibody (N-Mab) was
detected by incubation with a biotinylated goat anti-mouse polyclonal antibody for 30 min
with rocking. Unbound goat antibody was removed by washing with PBS as previously
described and the plates were treated with phosphatase-conjugated avidin-biotin
complexes for 1 h with rocking. The cells were then rinsed with PBS as previously
described and incubated with 100 mM Tris-HC1, pH 8.2, for 2 min. Cells were then
incubated in the dark without rocking for 10-20 min with Vector-Red phosphate substrate
(Vector, Burlingame, CA, kit III) containing Levamisole (Vector, Burlingame, CA). The
substrate reaction was stopped with a distilled water rinse, crystal mount (Biomeda,
Foster City, CA) was added, and the cells were allowed to dry. The cells were examined
for red (positive) staining with an inverted microscope.
The APIC assay was also tested with other IHNV isolates and rhabdoviruses.
These virus isolates had been previously grown in tissue culture and the virus containing
supernatant fluid had been stored at -70°C. When these assays were performed, the
plaques were allowed to develop at the appropriate temperature for each virus (Kimura et
al. 1986; Kasornchandra et al. 1991, 1992; Fijan et al. 1983). When IPNV was tested,
the plaque assay was performed on CHSE-214 cells. These cells were overlaid with agar
and incubated for 7 days at 15°C.
Results and Discussion
The APIC staining of IHNV plaques resulted in bright red plaques that were
visible by eye. Microscopically, the APIC stained plaque appeared as an area which had
30
rounded positive cells surrounded by a darkly stained ring of positive cells. The adjacent
negative monolayer was clear (no stain) (Figure 3.1). The darkly staining red cells
enabled the examiner to detect microscopic virus plaques as early as 4 to 6 days post-
infection. Individual or groups of IHNV-infected cells were detected as early as 24 h
post-infection by microscopic examination.
The APIC assay was specific. It distinguished plaques caused by IHNV from
those caused by other fish rhabdoviruses or the fish birnavirus, IPNV. No positive
staining was seen for these viruses (Table 3.1). These observations were expected since
Ristow and colleagues had shown that the N-Mab reagent, 1NDW14D, recognized a
group-specific epitope on the IHNV nucleoprotein. The APIC assay also distinguished
IHNV Type II isolates from the other IHNV virus types with the N-Mab, 2NH105B.
This N-Mab reagent recognized a type-specific epitope on the virus nucleoprotein
(Ristow and Arnzen 1989). These findings indicate that the epitopes recognized by
1NDW14D and 2NH105B were not drastically altered by fixation in 25% formalin, 10%
ethanol and 5% acetic acid or by washing with 70% ethanol. Ristow and Arnzen (1989)
and Ristow et al. (1991) have previously demonstrated the specificity of the 1NDW14D
N-Mab using fluorescence indicators and immunodot assays. The APIC procedure
described here confirms their results.
31
Figure 3.1 Nucleocapsid protein of infectious hematopoietic necrosis virus (IFINV)
in infected cells by alkaline phosphatase immunocytochemical staining
using monoclonal antibody, 1NDW14D. IHNV-positive cells (arrows)
in viral plaque (P) stained red.
32
Figure 3.1
33
Table 3.1
Virus isolates tested by alkaline phosphatase immunocytochemistry with
an anti-nucleocapsid monoclonal antibody, 1NDW14D.
IHNV Isolate
Typea APICb
Round Butte
Rangen
Elk River
Nan Scott
Coleman 2
Cedar River
Karluk River
+
+
+
III
+
III
+
IV
+
V
+
V
+
Untyped
Untyped +
SRCRVC
OSV
I
II
Non-Rhabdovirus
Non-IHNV
Rhabdovirusesc APIC Pathogens APIC
VHSV
PFRV
SHRV
UDRV
HRV
SVCRV
IPNV
aAs determined by electropherotyping (Hsu et al. 1986)
b(+) indicates viral antigen detected, () indicates no viral antigen detected
cSee Methods for complete viral names.
34
No other non-specific positive reactions were seen. The toxicity of some fish
tissue homogenates can cause virus-like CPE in cell monolayers and this cytopathology
may be falsely interpreted as diagnostic of virus infection. This is especially true for
tissue homogenates prepared from digestive organs taken from larger fish. The APIC
assay did not stain cells that had been killed by toxicity (data not shown). In addition,
plaques on plates up to 2 years old showed no decrease in intensity of staining. Pre­
treatment of cells with PEG had no apparent affect on N-Mab staining by APIC; whereas,
Bootland and Leong (1992) did report that cells resuspended in PEG gave non-specific
positive reactions in an IHNV coagglutination assay.
Although the APIC procedure was not tested with different Mabs, the procedure
could be adapted easily for detecting the IHNV glycoprotein as well. LaPatra et al.
(1989) used an anti-G Mab to detect IHNV by fluorescence; the G-Mab used in that study
should be able to detect IHNV by APIC if the reactive G epitope is not altered by fixation
and staining. The N-Mabs were used because the IHNV nucleoprotein is the earliest and
most abundantly expressed protein in the infected cell (Leong et al. 1983). Also, several
reports had shown that the IHNV nucleoprotein contains conserved antigenic
determinants (Hill et al. 1975; Lenoir and de Kinkelin 1975; McAllister and Wagner
1975; Leong et al. 1981; Hsu et al. 1986).
Confirmatory identification of viruses by specific reaction with an antibody
reagent is a standard procedure in the virus diagnostic laboratory. The immunological
assay can be a virus neutralization assay (McCain et al. 1971; Hsu and Leong 1985;
Winton et al. 1988) or an assay that measures the specific reactivity of an antibody with a
viral antigen (for review of IHNV assays, see Winton 1991). For IHNV, this specific
reactivity may be detected by direct and indirect immunofluorescence as described by
Ristow et al. (1991), LaPatra et al. (1989), and JOrgensen et al. (1991). Staphylococcal
coagglutination (Boot land and Leong 1992), an immunodot method (McAllister and
Schill 1986; Eaton et al. 1991), and an enzyme-linked immunosorbent assay (ELISA)
35
(Dixon and Hill 1984) have been described using polyclonal rabbit antisera to IHNV. All
of these assays have several advantages and disadvantages. The assay of choice for any
laboratory will vary with the convictions and experiences of the investigators as well as
their available equipment.
In our experience, the APIC procedure was easy to perform, did not require
special equipment, and a positive result was easy to detect. Complete plaque formation
was not necessary because areas of infected cell monolayers were visible under the
microscope as early as 24 h post infection. Thus, the APIC assay might be useful for
early detection and identification of IHNV infection. It will probably have its greatest use
with virus samples that have been lost as a result of laboratory error or a severe reduction
in sample viability. It may also be used when a laboratory has to screen hundreds of
samples and the examination of individual plates for plaques before fixation and staining
is not possible. The assay is simple and may be adapted for early detection or
confirmation of many human and veterinary viral pathogens.
Acknowledgments
The authors thank Sandra Ristow for providing the monoclonal antibodies
1NDW14D and 2NH105B, and IHNV Type V, and Cathy Lannan for providing several
of the virus isolates used in this study. This work was supported by Bonneville Power
Administration Contract DE-FG79-88BP92431, Project No. 88-152; the United States
Department of Agriculture to the Western Regional Aquaculture Consortium under grant
no.92-38500-7195, Project No. 92080441; United States Department of Agriculture
Grants 91-34123-6124 and 92-37204-7997; and Oregon Sea Grant with funds from the
National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of
Commerce, under grant no. NA89AA-D-SG108, Project No. R/FSD-16. Oregon
Agricultural Experiment Station Technical Paper No. 10,056.
36
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Burke, J.A. and D. Mulcahy. 1980. Plaguing procedure for IHNV. Appl. Environ.
Microbiol. 39:872-876.
Dixon, P.F. and B.J. Hill. 1984. Rapid detection of fish rhabdoviruses by the enzymelinked immunosorbent assay (ELISA). Aquaculture 42:1-12.
Eaton, W.D., J. Hulett, R. Brunson and K. True. 1991. The first isolation in North
America of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic
septicemia virus (VHSV) in coho salmon from the same watershed. J. Aquat.
Anim. Health 3:114-117.
Fijan, N., D. Sulimanovic, M. Bearsotti, D. Musinie, L.D. Zwillengerg, S.
Chilmonczyk, J.F. Vantherot and P. de Kinkelin. 1983. Some properties of the
Epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio. Ann.
Virol. 134:207-220.
Fryer, J.L., A. Yusha and K.S. Pilcher. 1965. The in vitro cultivation of tissue and cells
of Pacific salmon and steelhead trout. Ann. N.Y. Acad. Science 126:566-586.
Hill, B.J., B.O. Underwood, C.J. Smale and F. Brown. 1975. Physicochemical and
serological characterization of five rhabdoviruses infecting fish. J. Gen. Virol.
27:369-378.
Hsu, Y.L., H.M. Engelking and J.C. Leong. 1986. Occurrence of different types of
infectious hematopoietic necrosis virus in fish. Appl. Environ. Microbiol.
52:1353-1361.
Hsu, Y.L., and J.C. Leong. 1985. A comparison of detection methods for infectious
hematopoietic necrosis virus. J. Fish Dis. 9:287-290.
37
JOrgensen, P.E.V., N.J. Olesen, N. Lorenzen, J.R. Winton and S.S. Ristow. 1991.
Infectious hematopoietic necrosis (IHN) and viral hemorrhagic septicemia (VHS):
detection of trout antibodies to the causative viruses by means of plaque
neutralization, immunofluorescence, and enzyme-linked immunosorbent assay. J.
Aquat. Anim. Health 3:100-108.
Kasornchandra, J., H.M. Engelking, C.N. Lannan, J.S. Rohovec and J.L. Fryer.
1992. Characteristics of three rhabdoviruses from snakehead fish Ophicephalus
striatus. Dis. Aquat. Org. 13:89-94.
Kasornchandra, J., C.N. Lannan, J.S. Rohovec and J.L. Fryer. 1991. Characterization
of a rhabdovirus isolated from the snakehead fish (Ophicephalus striatus). Proc.
Sec. Int. Symp. Vir. Low. Verteb. 2:175-182.
Kimura, T., M. Yoshimizu and S. Gorie. 1986. A new rhabdovirus isolated in Japan
from cultured hirame (Japanese flounder) Paralichthys olivaceus and ayu
Plecoglossus altivelis. Dis. Aquat. Org. 1:209-217.
LaPatra, S.E., K.A. Roberti, J.S. Rohovec and J L. Fryer. 1989. Fluorescent antibody
test for the rapid diagnosis of infectious hematopoietic necrosis. J. Aquat. Anim.
Health 1:29-36.
Lenoir, G. and P. de Kinkelin. 1975. Fish rhabdoviruses: comparative study of protein
structure. J. Virol. 16:259-262.
Leong, J.C., Y.L. Hsu and H.M. Engelking 1983. Synthesis of the structural
proteins of infectious hematopoietic necrosis virus, pp. 61-70. In: J. Crosa (ed.),
Bacterial and viral diseases of fish. Molecular studies. University of Washington
Press, Seattle, Washington.
Leong, J.C., Y.L. Hsu, H.M. Engelking and D. Mulcahy. 1981. Strains of infectious
hematopoietic necrosis (IHN) virus may be identified by structural protein
differences, pp. 43-55. In: D.P. Anderson and W. Hennessen (eds.),
International Symposium on Fish Biologics: Serodiagnostics and Vaccines,
Development of Biological Standards. S. Karger, Basel, Switzerland.
McAllister, P.E. and W.B. Schill. 1986. Immunoblot assay: a rapid and sensitive method
for identification of salmonid fish viruses. J. Wildl. Dis. 22(4):468-474.
McAllister, P.E. and R.R. Wagner. 1975. Structural proteins of two salmonid
rhabdoviruses. J. Viro1.15:733-738.
McCain, B. B., J.L. Fryer and K.S. Pilcher. 1971. Antigenic relationships in a group
of three viruses of salmonid fish by cross-neutralization. Proceedings of the
Society of Experimental Biology and Medicine 146:630-634.
Ristow, S.S., and J.M. Arnzen. 1989. Development of monoclonal antibodies that
recognize a Type-2 specific and a common epitope on the nucleoprotein of
infectious hematopoietic necrosis virus. J. Aquat. Anim. Health 1:119-125.
38
Ristow, S.S., N. Lorenzen and P.E.V. JOrgensen. 1991. Monoclonal antibodybased immunodot assay distinguishes between viral hemorrhagic septicemia virus
(VHSV) and infectious hematopoietic necrosis virus (IHNV). J. Aquat. Anim.
Health 3:176-180.
Winton, J.R. 1991. Recent advances in detection and control of infectious hematopoietic
necrosis virus in aquaculture. Ann. Rev. Fish Dis.1:83-93.
Winton, J.R., C.K. Arakawa, C.N. Lannan and J.L. Fryer. 1988. Neutralizing
monoclonal antibodies recognize antigenic variants among isolates of
infectious hematopoietic necrosis virus. Dis. Aquat. Org.4:199-204.
Wolf, K., editor. 1988. Infectious hematopoietic necrosis virus, pp. 83-114. In : Fish
viruses and fish viral diseases. Cornell University Press, Ithaca, New York.
39
CHAPTER 4
THE ROUTE OF ENTRY AND PROGRESSION OF INFECTIOUS
HEMATOPOIETIC NECROSIS VIRUS IN
ONCORHYNCHUS MYKISS (WALBAUM):
A SEQUENTIAL IMMUNOHISTOCHEMICAL STUDY
Barbara S. Drolet
Genetics Program, Department of Microbiology
Oregon State University, Corvallis Oregon
John S. Rohovec and Jo-Ann C. Leong
Center for Salmon Disease Research
Department of Microbiology
Oregon State University, Corvallis, Oregon
Published: July, 1994
Journal of Fish Diseases
17(4):337-347
Publication of this manuscript authorized by Blackwell Scientific
Publications Ltd., Osney Mead, Oxford, OX2 OEL.
40
Abstract
Steelhead trout (Oncorhynchus mykiss) fry were experimentally infected with
infectious hematopoietic necrosis virus (IHNV) Round Butte 1983 (Type 1). Fry were
sampled daily, before and during the epizootic. Fish tissues were tested for infectious
virus by tissue culture assay and for IHNV nucleocapsid protein by alkaline
phosphatase immunohistochemistry (APIH). The progression of virus through the
tissues was followed by APIH until the 14th day. Viral infection progressed from two
major sites, from the gills into the circulatory system, and from the oral region into the
gastrointestinal tract and then into the circulatory system. Once in the blood, virus was
disseminated to virtually every organ. Progression of IHNV within and between organs
is discussed.
Introduction
Infectious hematopoietic necrosis virus (IHNV) causes an acute, systemic
disease in trout and salmon. The complete infection cycle of IHNV in fish has not been
determined. It is known that young fish in the wild, or more commonly in aquaculture,
may be lethally infected with IHNV. Virus can be isolated from both viscera and
mucus using established tissue culture methods (Burke and Mulcahy 1980; Hsu and
Leong 1985; Winton 1988; LaPatra et al. 1989a; LaPatra et al. 1989b).
Many questions remain concerning the route of viral entry. Vertical
transmission of IHNV is controversial; however, waterborne or horizontal transmission
has been reported for several species of salmon and trout, and is used routinely in
artificial infection experiments. Several infection studies have implicated the gills as
the initial site of IHNV entry and infection. Infectious hematopoietic necrosis virus has
been isolated from gill tissue of adult kokanee and sockeye at 3 days post exposure
(dpe). Direct staining of viral glycoprotein by immunoperoxidase histochemistry was
41
also made at this time (Yamamoto et al. 1989). Virus has also been isolated by tissue
culture in gill tissue early in the infection of sockeye fry (Mulcahy et al. 1983) and
smolts (Burke and Grischkowsky 1984). Virus has been detected in gill tissue of
rainbow trout at 16-20 hours post exposure (hpe) by electron microscopy and at 24 hpe
by tissue culture methods (Yamamoto and Clermont 1990), but not until 5 dpe by
immunohistochemistry (Yamamoto et al. 1990). Yamamoto et al. (1990) also reported
that the skin was the major site of IHNV replication and that the gill played a lesser role
in the initial stages of infection. Gills have been shown to be the initial site of
replication for two other fish rhabdoviruses, spring viremia of carp virus (Ahne 1978)
and viral hemorrhagic septicemia virus (Neukirch 1984).
Progression of IHNV infection through the fish is unknown. It has been
suggested that IIINV spreads from the gills to other organs via the blood. Initial
infection of the gill followed by viremia was reported by Yasutake and Amend (1972).
Mulcahy et al. (1983) reported that a titer 1 x 105 plaque forming units per gram (pfu/g)
was required in the gills of artificially infected sockeye before virus was seen in other
organs. In the same report, however; infectious virus was detected in the viscera of
naturally infected spawning sockeye in the absence of virus in the gills. Moreover,
virus has been detected by immunohistochemistry (Yamamoto et al. 1990) and by
electron microscopy (Yamamoto and Clermont 1990) in the visceral organs of rainbow
trout before the gills. This suggests an alternate or additional origin of virus infection.
We report the results of an experiment designed to demonstrate the route(s) of initial
viral entry and the progression of LHNV through fish tissues.
An alkaline phosphatase immunohistochemical assay (APIH) was used to detect
the nucleocapsid protein of IHNV in formalin- fixed, paraffin-embedded lg steelhead
trout, Oncorhynchus mykiss, which were sampled before and during an artificially
induced IHN epizootic. The nucleocapsid protein was the antigen of choice because it
is the most abundant, conserved viral antigen among the five known IHNV types (Hill
42
et al. 1975; Lenoir and de Kinkelin 1975; McAllister and Wagner 1975; Leong et al.
1981; Hsu et al. 1986). With this assay, we were able to examine viral pathogenesis in
very small tissue samples, in samples with low concentrations of virus, and in specific
cell types of each tissue.
Materials and Methods
Infection
One thousand steelhead fry, Oncorhynchus mykiss, 1 g were exposed to 103
pfu/ml IHNV (Round Butte, Type I) by static immersion (13°C) for 12 h at the Salmon
Disease Laboratory, Corvallis, OR. Asymptomatic fry were taken during the pre­
epizootic period (0-5 dpe), and moribund and dead fry were taken during the epizootic
period (6-14 dpe). Three fish were homogenized for tissue culture and three were fixed
for APIH daily.
Cell culture
Epithelioma papillosum cyprini (EPC) cells (Fijan et al. 1983) were grown to
confluency in 24-well plates with MEM containing 10% fetal bovine serum, 2%
penicillin-streptomycin (lunit/ug), 2% 200 mM L-glutamine, 2% fungizone, 1%
gentamycin (50 mg/ml), buffered with 1M Tris buffer to pH 7.0. For viral assays, cell
monolayers were inoculated with 0.1 ml of serially diluted samples.
Tissue, mucus samples
Whole steelhead fry were diluted 1:10 (w/v) in Hanks' balanced salt solution
(Gibco BRL, Gaithersberg, MD) and homogenized in a Stomacher 80 homogenizer
43
(Tekmar, Cincinnati, OH). The homogenate was centrifuged at low speed for 10 min to
sediment tissue debris. The supernatant was diluted 1:1 (v/v) in Eagle's minimum
essential media (MEM; Gibco BRL) containing antibiotic-antimycotic (10 µg/ml
penicillin G sodium, 10 µg/m1 streptomycin sulfate and 25 µg/ml amphotericin B as
fungizone in 0.85% saline, Gibco BRL) and stored at 4°C overnight (Amend and
Pietsch 1972). Mucus samples from infected fish were taken with cotton swabs and put
directly in the antibiotic media and stored at 4°C overnight (LaPatra 1989).
Plaque assay
Virus from tissue and mucus samples was adsorbed to cells for 1 h at 15°C with
gentle rocking. Mono layers were overlaid with 0.75% methyl cellulose (in complete
MEM) and incubated for up to 14 days at 15°C. Cells were fixed and stained (25%
formalin, 10% ethanol, 5% acetic acid, 1% crystal violet), rinsed with tap water and
allowed to dry. Plaques were counted and virus titers were calculated as pfu per mucus
swab or pfu/g of tissue.
Fixation and embedding of fish for APIH
Fish were fixed in buffered formalin (10% formalin, 33 mM sodium phosphate
monohydrate, 46 mM sodium phosphate dibasic), dehydrated in an alcohol-xylene
series and embedded in Paraplast paraffin (60°C) (Oxford Labware, St. Louis, MO).
Blocks were sectioned sagittally (6 gm) and fixed (40° C overnight) to coated slides
(0.1% gelatin, 0.1% chromic potassium sulfate).
44
Alkaline phosphatase immunohistochemistry (APIH)
All reagents for the APIH assay were diluted in 5.8 mM phosphate buffered
saline, pH 7.4 (PBS) and excess reagent was removed by rinsing with PBS between
incubations. All incubations were done at room temperature unless otherwise
indicated. Tissue sections were deparaffinized by standard methods and rehydrated for
10 min in distilled water and 20 min in PBS. Nonspecific binding sites were blocked
for 1 h with 5% nonfat powdered milk (Saco Foods, Madison, WI) in PBS. Blocking
solution was blotted off and sections were incubated for 1 h with a universal anti­
nucleocapsid monoclonal antibody (1NDW14D) which is specific for a highly
conserved region of the nucleocapsid protein and has been shown by direct
immunofluorescence and immunoblotting to recognize all five IHNV types (Ristow and
Arnzen 1989; Ristow et al. 1991). Slides were then incubated for 30 min with
biotinylated goat anti-mouse polyclonal antibody, followed by a 1 h incubation with
phosphatase-conjugated avidin-biotin complex (Vectastain-ABC mouse IgG kit, Vector
Laboratories, Burlingame, CA). Sections were equilibrated with 100 mM Tris-HC1, pH
8.2 (substrate buffer), and incubated with Vector-Red phosphate substrate (Vector) for
10-20 min in the dark. Levamisole (Vector) was added to the substrate solution to
inhibit endogenous phosphatase activity. The substrate reaction was stopped with
distilled water and sections were counterstained in hematoxylin for 1-2 min and 0.2%
ammonium hydroxide in 70% ethanol solution for 30 s. Sections were covered with
Crystal Mount (Biomeda, Foster City, CA), Cytoseal mounting media (Stephens
Scientific, Riverdale, NJ) and a coverslip and examined by light microscopy.
45
Alkaline phosphatase immunocytochemistry (APIC)
Infectious hematopoietic necrosis virus from tissue homogenates was confirmed
in viral plaques by APIC (Drolet, et al. 1993). Briefly, formalin-fixed, crystal violetstained cell monolayers were destained with 70% ethanol and cells were incubated with
the same reagents and antibodies as described for APIH.
Results
Infection
Deaths in the infected population started 5 dpe, peaked at 6-14 dpe, then
decreased with time (Figure 4.1). Cumulative percent mortality at 40 dpe was 42%.
Clinical signs of the IHNV infection included distended abdomens, petechial
hemorrhages on ventral surface and near the base of fins, darkened body coloration and
exophthalmia. Behavioral signs included lethargy interspersed with frenzied
swimming, vertical drifting, whirling and anorexia.
Tissue culture assays
Virus was first detected by plaque assay in both mucus and whole fish
homogenates at 5 dpe. During the epizootic (6-14 dpe), swab virus titers ranged from
1x104 to 4x108 pfu per mucus swab and tissue homogenate titers ranged from 4x104 to
5x109 pfu/g. Cells at the periphery of plaques stained positive (red) for IHNV by
APIC. Infectious virus was not detected in mucus or tissue homogenates after 54 dpe.
46
Detection of IHNV by APIH
The degree of IHNV infection in individual cells of tissues was rated by the
intensity of staining on a scale of 1 to 5. For those tissues that were intact and stained a
light red or pink, the rating of 1-2 was used and interpreted to indicate either early
stages of infection or transient infection. Intact tissues that were stained a bright red
indicated a productive infection and were given ratings of 3-4. The tissues that were
stained bright red and showed obvious signs of damage were given a rating of 5. The
relationship between intensity of staining and degree of viral infection was also
observed in the immunocytochemical staining of viral plaques (Drolet et al. 1993).
PreEpizootic Epizootic
Post Epizootic
40
co
- C
30
715
a)
CI
20
co
O
10"
0
I
0
20
10
30
.
I
40
Days Post Exposure
Figure 4.1
Mortality curve of 1 g steelhead trout (0. mykiss) after immersion
infection with infectious hematopoietic necrosis virus, type 1.
The pre-epizootic period is from day 0-5 and the epizootic period
is from day 6-14.
47
Pre-epizootic period
Circulatory pathway. Sequential progression of virus to the organs via the
circulatory system is shown in Table 4.1. Epithelial cells of gill lamellae were positive
by day 2 (Figure 4.2 A). In the kidney, virus was also first seen at day 2 in the anterior
portion in the lumen and epithelial cells of the renal tubules (Figure 4.2 C). The
surrounding hematopoietic tissue was negative until day 4 (Figure 4.2 D). The Hassel's
corpuscles (thymic bodies) of the thymus were positive by 3 dpe, and in the spleen, the
connective tissue stroma throughout the organ was positive at 5 dpe, but the
hematopoietic cells were negative. In the pancreas, the supporting stroma was positive,
and the acinar cells were negative. Muscle cells of the heart ventricle were positive on
day 4. In the brain, virus was detected in the meninges of the forebrain, optic lobe, and
cerebellum by day 5.
Gastrointestinal pathway. The sequential progression of virus in gastrointestinal
organs is also shown in Table 4.1. Virus-positive red staining was first observed from
2-4 dpe in the epithelial cells of the oral region, pharynx, esophagus, stomach, pyloric
caeca (Figure 4.2 E) and intestine. Beginning on day 5, the sub-mucosal and lamina
propria layers of these organs (Figure 4.2f) were positive and the epithelial cells were
negative. Virus was first detected in liver hepatocytes on day 3.
Other tissues. Epithelial cells of the ventral epidermis and head region were
weakly positive 1 dpe. Muscle cells consistently gave low background staining and
were not considered positive unless particulate dark red staining could be seen in the
connective tissue surrounding the skeletal muscle. By 3 dpe, positive cartilage was
seen in the head region and the connective tissue surrounding adipose cells of the
abdomen was positive.
48
Table 4.1
Chronological appearance and staining intensity of infectious
hematopoietic necrosis virus by alkaline phosphatase
immunohistochemistry in steelhead (0. mykiss) fry.
Pre-Epizootic
Pathway:
Days Post Exposure 0.5 1
# Sections Examined 15 6
Circulatory:
Gills
Anterior Kidney
Posterior Kidney
Thymus
Spleen
Pancreas
Heart
Brain
Gastrointestinal:
Oral region
Pharynx
Esophagus
Stomach
Pyloric Caeca
Liver
Intestine
Other:
Skin
Muscle
Cartilage
Adipose
Epizootic
9 10 11 12
2
3
4
11
6
6 23
22 30 6 17 6
6
6 3
NN 2
2
3
5
5
5
5
5 5
3
4
5
5
5
5
5
4
5 5
5
5
5 5
NN 1
5
NNN 2 3 5
NN N N33
NNNNN 3
NNNNN 4
NNNNN 4
NNNNN 4
NN 1
NN 1
NN 1
NN 1
NN 1
6
7 8
13 14
3
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5 5 5
5
5
5
5
5
5
5 5 5
5
5
5
5
5
5
5 5 5
5
5
5
5
5
5
5 5 5
5
5
5 5 5
2
3
4
5
5
5
5
5
5 5
5 5
2
2
2
2
4
4
5
5
5
5
5
5
5
5
5
5
5 5
5 5
5 5
5 5
2
3
3
4
5
5
5
5
5 5
5 5
2
4
NNN 2
5
5
5
5
5 5
5 5
5
5
5
5
2
4
3
5
5
5
5
5 5
NNNN 2
4
5
5
5
5
5
5 5
5 5
3
3
4
4
4
3 3
2
3
5
5
5
5
5
5 5
5 4
3
2
5
5
5
5
5
5
5 5
5 5
5
5
5
4 4
5 4
Ni
2
3
3 3
NNN 2 3
NNN 2 3
NNN N 2
4
2
* Relative intensity of positive staining; 1 = weak, 5 = strong, N = no vir us detected.
49
Figure 4.2
Photomicrographs of tissues from infectious hematopoietic necrosis
virus infected steelhead fry during the pre-epizootic period (0-5 days
post exposure) and the epizootic period (6-14 days post exposure). A
positive reaction is indicated by arrows and red staining cells. (A) Pre­
epizootic gill with virus-positive lamellar and filament epithelial cells
(ep) (40x). (B) Epizootic gill with virus-positive lamellar and filament
endothelium (en) (40x). (C) Pre-epizootic anterior kidney with viruspositive tubule (t) and negative hematopoietic cells (40x). (D)
Epizootic anterior kidney with negative tubule and virus-positive
hematopoietic cells (h) (40x). (E) Pre-epizootic pyloric caeca with
virus-positive columnar epithelial cells (ep) (40x). (F) Epizootic
intestine with negative epithelia and virus-positive lamina propria (1p)
(40x). (G) Epizootic brain with virus-positive glial (g) cells in the
granular layer of the optic lobe (100x). (H) Epizootic eye with viruspositive retina (r) (10x).
51
Circulatory pathway. In the gill, the lamellar vessel endothelia and the
connective tissue of the filament were strongly positive, whereas the epithelial cells
were negative (Figure 4.2 B) during the epizootic. In the kidney, epithelial cells of the
anterior renal tubules were necrotic with positive tubule basement membranes and
strongly positive, necrotic hematopoietic tissue (Figure 4.2 D). The posterior kidney
showed the same distribution of positive staining; however, the negative epithelial cells
of the tubules remained intact and the hematopoietic tissue was strongly positive, but
less necrotic. Virus-positive cells in hemorrhaged blood pools were primarily
macrophage, with little or no red blood cell involvement. The Hassel's corpuscles of
the thymus were positive throughout the epizootic and in the spleen, there was a diffuse
positive reaction which spared the majority of lymphocytes. In the pancreas, the
supporting stroma was positive and the acinar cells were positive and necrotic. In at
least 33% of the fish sampled on each day of the epizootic, virus was detected in glial
cells in localized regions of the forebrain, optic lobe (Figure 4.2 G), cerebellum,
medulla oblongata and spinal cord and in the retinal cells of the eye (Figure 4.2 H).
Gastrointestinal pathway. During the epizootic, the epithelial cells of the oral
region, pharynx, esophagus, stomach, pyloric caeca and intestine were negative, but the
submucosal and loose connective tissue (lamina propria) of these organs was strongly
positive (Table 4.1). The mucosal epithelial cells located at the tips of villi in the
pyloric caeca and intestine were often destroyed and infected cell debris could be seen
in the lumen (Figure 4.2 F). Epithelial cells at the base of the villi were negative and
intact. The basally adjacent submucosa and lamina propria were strongly positive, as
were specific areas of muscle of the cardiac and pyloric stomach. Distinct foci of viruspositive hepatocytes were seen throughout the liver.
52
Other tissues. During the epizootic, Positive epidermis was primarily seen on
the ventral surface and rarely on the head or dorsal surface. Cross section of muscle
cells showed positive staining on the inside of the cells as well as in the connective
tissue surrounding the cell. Positive regions of cartilage were seen in the head and
along the spine. Positive staining was seen in the connective tissue surrounding adipose
cells of the abdomen.
Discussion
The results of this study suggest that IHNV infection progressed from two major
sites: the gills and the gastrointestinal tract. Both routes of entry eventually resulted in
systemic viremia. While IHNV readily infected epithelial cells of numerous organs, it
had a propensity for connective tissue. Included in this broad category were the
supportive stroma of the spleen, pancreas, adipose and muscle; the submucosa of the
oral cavity, pharynx, esophagus and stomach; the lamina propria of the stomach,
pyloric caeca and intestine; and the meninges of the brain. Epithelial cells in virtually
every organ were virus-positive and intact during the pre-epizootic period and virus
negative and intact during the epizootic period. This indicated a short lived or transient
infection. Results of daily samples indicate that the epithelial cells had changed from
positive to negative and not that negative epithelial cells were replacements for those
cells that had been previously lysed. Specific areas of the pyloric caeca and intestine
which had cells that were virus-positive and undergoing lysis during the pre-epizootic
period were the same areas where only virus-positive cell debris remained, suggesting
lysed cells were not replaced with new negative cells. We suggest that ingested virus
transiently infected epithelial cells lining the oral cavity, pharynx, esophagus, stomach,
pyloric caeca and intestine; and then productively infected the basally adjacent
connective tissue (submucosa and/or lamina propria) of each organ (Table 4.2). Virus
53
produced in these highly vascularized connective tissues would have been collected by
the abdominal vein and transported to the heart for systemic dissemination.
The virus-positive staining was strongest and most persistent in the kidney.
Results of the sequential sampling indicate that the virus infecting the kidney, probably
originated at the gills. Both the proposed circulatory and ingestion pathways resulted in
viremia; however, the circulatory pathway is the most direct route to the kidney. We
propose that initial infection of the kidney began with the virus infecting the gill
epithelium, then infecting the lamellar and filament endothelium, and being carried by
the blood through the dorsal aorta and reaching the kidney tissue via the renal arteries.
Positive staining of the kidney tubules prior to infection of vascularized tissues of the
gut support this hypothesis. A few strongly positive red blood cells and macrophagelike cells were observed and may indicate that the virus replicated during its
transportation. Virally contaminated blood may have been responsible for pre-epizootic
infections of the thymus (via the thymic artery), the stroma of the spleen (via the
splenic artery), and pancreas (via the parietal artery) and the meninges of the brain (via
the circle of Willis). Virus was detected in the anterior kidney one day earlier than in
the posterior kidney. This anterior to posterior sequence was also reported by Yasutake
and Amend (1972) in histopathological evaluation of IHNV infected sockeye salmon.
The APIH results indicate that the kidney was infected by virus from the capillaries
surrounding the renal tubules. Virus entered the tubules, transiently infected the
epithelial cells, budded out of the basement membrane and productively infected the
basally adjacent hematopoietic tissue. A large amount of virus was produced
throughout the hematopoietic tissue of the kidney, contributing the majority of infected
blood to be disseminated systemically.
54
Table 4.2
ORGAN
Circulatory:
Gills
Anterior kidney
Posterior kidney
Thymus
Spleen
Pancreas
Heart
Brain
Gastrointestinal:
Oral region
Pharynx
Esophagus
Stomach
Pyloric caeca
Liver
Intestine
Other
Skin
Muscle
Cartilage
Adipose
Virus-positive tissues of fish organs as tested by alkaline
phosphatase immunohistochemistry. Pre-epizootic to
epizootic progression of virus in specific tissues of each organ
examined in Table 4.1.
PRE-EPIZOOTIC
Lamellar epithelium
Tubule lumen, epithelium
Tubule lumen, epithelium
Hassel's corpuscles
Connective stroma
Connective stroma
Ventricle musculature
Meninges
Mucosal epithelium
Mucosal epithelium
Mucosal epithelium
Lumen, epithelium
Lumen, epithelium
Hepatocytes
Lumen, epithelium
Ventral
Ventral
Head
Connective stroma
EPIZOOTIC
Capillary endothelium
Hematopoietic cells
Hematopoietic cells
Hassel's corpuscles
Hematopoietic cells
Acinar cells
Ventricle musculature
Meninges and glial cells
Submucosa
Submucosa
Submucosa
Lamina propria, muscle
Lamina propria
Hepatocytes
Lamina propria
Anterior ventral, dorsal
Anterior, ventral, eye
Head, spine
Connective stroma
55
Yamamoto et al. (1990) reported that skin was the major site of IHNV
replication and that gills played a lesser role in the initial stages of infection. In our
study, the epithelial cells of the ventral and anterior epidermis were weakly positive by
APIH at 1 dpe (one fish), but the location of positive staining in the skin varied greatly
among all 18 fish sampled during the pre-epizootic period. Throughout both the pre­
epizootic and epizootic periods, the intensity of the positive signal in the skin was more
indicative of a transient infection than a productive infection. During the epizootic,
ventral muscle was often positive while the adjacent skin epithelium was negative. A
consistent progression of infection from the skin to adjacent muscle or other organs was
not seen in the sequential samples examined.
Although not the source of initial infection of the kidney, the ingestion or
gastrointestinal pathway was a highly significant route of infection. All fish sampled
from day 2 to day 4 had virus-positive staining in the lumen and regions of epithelial
cells lining the gastrointestinal tract. From 5 to 14 dpe the highly vascularized lamina
propria was strongly positive in all gastrointestinal organs. The most distinguishable
and consistent organ to organ progression of virus was from the stomach to the pyloric
caeca. The strongest positive staining and the most cell destruction was observed
nearest the stomach. Both the signal and the destruction decreased as one progressed
down to the tip of the caeca.
The most restricted infection was in the brain. During the epizootic, the distinct
positive foci were seen primarily in the granular layers of the lobes and showed no
apparent pattern of infection. Infection of the retinal cells of the eye was always near
the layer of connective tissue called the falciform process. The resulting inflammation
may be in part the cause of exophthalmia, a prominent symptom of NIN disease.
56
Results of the immunohistochemical method used in this study suggest that
IHNV exhibits a specific tissue tropism for connective tissue. While IHNV readily
infected epithelial cells, the infection appeared transient and not highly productive. The
majority of cells undergoing productive viral infection were of connective tissue origin.
These results may influence the design of vaccines, and the study of specific cell
receptors involved in IHNV infection. Post-epizootic samples are currently being
examined by APIH to determine the mechanism of viral clearing and the existence of
viral latency.
Acknowledgments
Sandra Ristow (Washington State University, Pullman, Washington) provided
the monoclonal antibody used in this study. Jerry Heidel (Oregon State University,
Veterinary Medicine, Corvallis, Oregon) gave invaluable histopathology evaluation.
Others involved in the project were John Fryer, Linda Boot land, Harriet Lorz and Pin
Wen Chiou. This study was supported by Bonneville Power Administration, project
number DE-FG79-88BP92431, Project No. 88-152; the United States Department of
Agriculture to the Western Regional Aquaculture Consortium under grant no. 92­
38500- 7195, Project No. 92080441; United States Department of Agriculture grants 91­
34123 -6124 and 92-37204-7997; and Oregon Sea Grant with funds from the National
Oceanic and Atmospheric Administration, Office of Sea Grant, Department of
Commerce, under grant no. NA89AA-D-SG108, Project No. R/FSD-16. Oregon
Agricultural Experiment Station Technical Paper No.10,303.
57
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Ahne, W. 1978. Uptake and multiplication of spring viraemia of carp virus in carp,
Cyprinus carpio L. J. Fish Dis. 1:265-268.
Amend, D.F. and J.P. Pietsch 1972. An improved method for isolating virus from
asymptomatic carrier fish. Trans. Amer. Fish. Soc. 101:267-269.
Burke, J.A. and R. Grischkowsky. 1984. An epizootic caused by infectious
hematopoietic necrosis virus in an enhanced population of sockeye salmon,
Oncorhynchus nerka (Walbaum), smolts at Hidden Creek, Alaska. J. Fish Dis.
7:421-429.
Burke, J.A. and D. Mulcahy. 1980. Plaguing procedure for infectious hematopoietic
necrosis virus. Appl. Environ. Microbiol. 39:872-876.
Drolet, B.S., J.S. Rohovec and J.C. Leong. 1993. Serological identification of
infectious hematopoietic necrosis virus in fixed tissue culture cells by alkaline
phosphatase immunocytochemistry. J. Aquat. Anim. Health 5(4):265-269.
Fijan, N., D. Sulimanovic, M. Bearsotti, D. Musinie, L.D. Zwillengerg, S.
Chilmonczyk, J.F. Vantherot, and P. de Kinkelin. 1983. Some properties of the
Epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio.
Ann. Virol. 134:207-220.
Hill, B.J., B.O. Underwood, C.J. Smale and F. Brown. 1975. Physicochemical and
serological characterization of five rhabdoviruses infecting fish. J. Gen. Virol.
27:369-378.
Hsu, Y.L., H.M. Engelking and J.C. Leong. 1986. Occurrence of different types of
infectious hematopoietic necrosis virus in fish. Appl. Environ. Microbiol.
52:1353-1361.
Hsu, Y.L. and J.C. Leong. 1985. A comparison of detection methods for infectious
hematopoietic necrosis virus. J. Fish Dis. 9:287- 290.
LaPatra, S.E. 1989. Strain differentiation and detection of infectious hematopoietic
necrosis virus, pp. 149-166. In: Ph.D. thesis, Oregon State University,
Corvallis, Oregon.
LaPatra, S.E., K.A. Roberti, J.S. Rohovec and J.L. Fryer. 1989a. Fluorescent antibody
test for the rapid diagnosis of infectious hematopoietic necrosis. J. Aquat.
Anim. Health 1:29-36.
LaPatra, S.E., J.S. Rohovec and J.L. Fryer. 1989b. Detection of infectious
hematopoietic necrosis virus in fish mucus. Fish Pathol. 24:197-202.
Lenoir, G. and P. de Kinkelin. 1975. Fish rhabdoviruses: Comparative study of protein
structure. J. Virol. 16:259-262.
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Leong, J.C., Y.L. Hsu, H.M. Engelking and D. Mulcahy. 1981. Strains of infectious
hematopoietic necrosis (IEN) virus may be identified by structural protein
differences, pp. 43-55. In: D.A. Anderson and W. Hennessen (eds.),
International Symposium on Fish Biologics: Serodiagnostics and Vaccines,
Development of Biological Standards. S. Karger, Basel, Switzerland.
McAllister, P.E. and R.R. Wagner. 1975. Structural proteins of two salmonid
rhabdoviruses. J. Virol. 15:733-738.
Mulcahy, D., R.J. Pascho and C.K. Jenes. 1983. Detection of infectious hematopoietic
necrosis virus in river water and demonstration of waterborne transmission.
J. Fish Dis. 6:321-330.
Neukirch, M. 1984. An experimental study of the entry and multiplication of viral
hemorrhagic septicaemia virus in rainbow trout, Sa lmo gairdneri Richardson,
after water-borne infection. J. Fish Dis. 7:231-234.
Ristow, S.S. and J.M. Arnzen,. 1989. Development of monoclonal antibodies that
recognize a Type-2 specific and a common epitope on the nucleoprotein of
infectious hematopoietic necrosis virus. J. Aquat. Anim. Health 1:119-125.
Ristow, S.S., N. Lorenzen and P.E.V. Jorgensen. 1991. Monoclonal
antibody-based immunodot assay distinguishes between viral hemorrhagic
septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV).
J. Aquat. Anim. Health 3:176-180.
Winton, J.R., C.K. Arakawa, C.N. Lannan and J.L. Fryer. 1988. Neutralizing
monoclonal virus. Dis. Aquat. Org. 4:199-204.
Yamamoto, T., W.N. Batts, C.K. Arakawa and J.R. Winton. 1989. Comparison of
infectious hematopoietic necrosis in natural and experimental infections of
spawning salmonids by infectivity and immunohistochemistry, pp. 411-429. In:
W. Ahne and E. Kurstak (eds.), Viruses of Lower Vertebrates. Springer-Verlag,
Berlin.
Yamamoto, T., W.N. Batts, C.K. Arakawa and J.R. Winton. 1990. Multiplication of
infectious hematopoietic necrosis virus in rainbow trout following immersion
infection: whole body assay and immunohistochemistry. J. Aquat. Anim.
Health 2:271-280.
Yamamoto, T. and T.J. Clermont. 1990. Multiplication of infectious hematopoietic
necrosis virus in rainbow trout following immersion infection: organ assay and
electron microscopy. J. Aquat. Anim. Health 2:261-270.
Yasutake, W.T. and D.F. Amend. 1972. Some aspects of pathogenesis of infectious
hematopoietic necrosis (IHN). J. Fish Biol. 4:261-264.
59
CHAPTER 5
PERSISTENCE OF AN RNA VIRUS INFECTION
BY DEFECTIVE INTERFERING PARTICLES
Barbara S. Drolet
Genetics Program, Department of Microbiology
Oregon State University, Corvallis Oregon
Pinwen P. Chiou
Department of Microbiology
Oregon State University, Corvallis Oregon
Jerry Heidel
College of Veterinary Medicine
Oregon State University, Corvallis, Oregon
Jo-Ann C. Leong
Center for Salmon Disease Research
Department of Microbiology
Oregon State University, Corvallis, Oregon
Manuscript in preparation
for submission to Journal of Virology
60
Abstract
Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes
devastating epizootics of trout and salmon fry in hatcheries around the world.
Epizootic survivors are negative for infectious virus by plaque assay at about 50 days
post exposure. Survivors are considered to be virus-free and suffering no ill effects of
the viral infection upon subsequent release into the wild. When adults return to spawn,
infectious virus can again be isolated by plaque assay. Two hypotheses have been
proposed to account for the source of virus in these adults. One hypothesis contends
that virus in the epizootic survivors is cleared and that the adults are being reinfected by
IHNV during their migration upstream. The second hypothesis contends that IHNV
persists in the survivor in a subclinical or latent form, then reactivates during the
stressful conditions of spawning. After 20 years of experiments and debate, there is no
conclusive evidence to support either of these hypotheses. In this paper, we describe
lesions in hematopoietic tissue of rainbow trout, one year post exposure, and provide
the first irrefutable evidence for subclinical persistence of virus in tissues of IHN
survivors. We demonstrate the presence of viral protein by immunohistochemistry,
viral RNA by PCR amplification, and IHNV defective interfering particles by
immunogold electron microscopy.
Introduction
The rhabdovirus infectious hematopoietic necrosis virus (IHNV) causes
devastating epizootics in hatchery-reared trout and salmon species of the Pacific
Northwest, Europe and Asia. IHNV primarily infects fry and can result in 100%
mortality. The main target of IHNV is the hematopoietic organ of the fish, the kidney.
Infectious virus can be detected in the viscera of infected fry by plaque assay until
approximately 50 days post exposure (dpe) when the virus appears to clear. It is
61
believed that epizootic survivors remain negative for infectious virus until sexual
maturity when virus can again be detected by plaque assay. It is not known whether
infectious virus isolated from spawning adults is the result of reinfection from
secondary reservoirs during their migration upstream, or the reactivation of a latent
form of IHNV. With the exception of one study (Amend 1975), the inability to detect
"reactivated" latent virus in numerous, carefully monitored, naturally and
experimentally infected fish populations has resulted in a majority of evidence against
latency. The reinfection theory has not been overwhelmingly accepted, however,
because large amounts of virus are required to infect large fish, no secondary viral
reservoirs have been found for IHNV, and adult infection studies have shown that
IHNV survivors are less susceptible than naive controls to reinfection by IHNV upon
sexual maturation (unpublished results).
The argument against persistence of IHNV has been based on the continued
negative results of plaque assays on IHN survivors held in captivity. This assay detects
only infectious virus and routinely has a limit of detection of 200 pfu/g tissue. The
possibility of viral persistence, at a level below the detection limit of plaque assays,
would help explain the resurgence of virus in adult populations where reinfection from
an unknown secondary reservoir seems unlikely as the sole source of virus.
The persistent rhabdoviral infections of vesicular stomatitis virus (VSV) and
rabies virus have been extensively studied and explained by defective interfering (DI)
particles (Holland and Villareal 1974; Kawai et al. 1975; Ramseur and Friedman 1978;
Horodyski et al. 1983). Huang and Baltimore (1970) were the first to report DI
particles, and proposed that their action was to modulate the yield of infectious virus in
a cyclical manner. Over the years, several attempts have been made to elucidate a role
for the seemingly ubiquitous DI particles of viral infections (For review, see Holland
1987; Barrett and Dimmock 1986; Lazzarini et al. 1981; Perrault 1981). VSV DI
particles have been shown to play an important role in relative virulence of different
62
serotypes and in the establishment of persistent infections in cell culture. DI particles
of many viruses have been shown to alter the pathogenesis and course of disease in cell
culture, and experimental animals given DI particles before or during an infection
(Bangham and Kirkwood 1990; Browning et al. 1991; Moscona and Peluso 1993) show
an altered disease course. Yet, defective virus has not been confirmed in natural
infections in animals and often cannot be reisolated from animals where they have been
experimentally administered to examine virus replication effects.
Two studies have come close to detecting DI particles in natural infections.
Pedley et al. (1984) isolated rotaviruses with altered genomes from chronically infected
immunodeficient children, but the particles were not confirmed due to the difficulty in
culturing the rotavirus. Bean et al. (1985) found "DI-type" RNAs in an avirulent strain
of type A influenza virus after they had been passed in embryonated eggs twice.
Although it explained the virulence difference, the possibility that the DI-type RNA
was made in the eggs was never ruled out.
IHNV DI particles have been described in persistently infected fish (McAllister
and Pilcher 1974; Engelking and Leong 1981) and insect cell cultures (Scott et al.
1980). These particles caused the establishment of persistent cell culture infections and
inhibited subsequent viral infection in susceptible fish. It is possible that IHN disease is
modulated by DI particles in natural infections and this may explain the apparent
disappearance and reappearance of the virus during the life cycle of its host.
Although IHN epizootic survivors have not been closely monitored in the wild,
they are assumed to be virus-free and suffering no ill effects of the viral infection upon
release. In this paper we describe the lesions found in the kidney of rainbow trout one
year after exposure to IHNV and the evidence for persistence of virus in these survivors
as shown by immunohistochemistry, PCR amplification, and immunogold electron
microscopy.
63
Materials and Methods
Fish infection and sampling
Thirty rainbow trout (Oncorhynchus mykiss), which had been exposed to 103
pfu/ml (LD50) of IHNV (Rangen, type-2) as fry by static immersion were maintained in
pathogen-free water at the Center for Salmon Disease Research fish facility (Corvallis,
Oregon). IHN epizootic survivors were lethally sampled at 90 d, 180 d, 1 y and 2 y
post exposure (ype). At each sampling time, pieces of kidney, spleen, and liver tissues
were pooled for each fish and tested for infectious virus by plaque assay as suggested in
the Fish Health Blue Book (Amos 1985) on EPC cells (Fijan et al. 1983). In addition,
pieces of 11 different organs from each fish were fixed in 10% neutral buffered
formalin (10% formalin, 33 mM sodium phosphate monohydrate, 46 mM sodium
phosphate dibasic), dehydrated in an alcohol-xylene series and embedded in Paraplast
paraffin (60°C) (Oxford Labware) for immunohistochemistry. Mock-infected fish,
which were exposed to non-infected cell culture media, were used as negative controls.
For elucidation of EM immunogold staining parameters, fresh positive and
negative tissue was used from a second groups of fish which were similarly infected.
Fresh, epizootic kidney tissues were cut into 1-2 mm3 blocks, fixed in
paraformaldehyde/gluteraldehyde (2% paraformaldehyde, 1% gluteraldehyde in 0.1 M
sodium cacodylate buffer) (Williams 1985), dehydrated with an ethanol series and
embedded in LR White resin (Ted Pella, Inc.). Kidney tissue from 1 ype survivors
which had been fixed in formalin for two years, but never embedded in paraffin were
treated as fresh tissue. Kidney tissue from 1 ype survivors which had been previously
embedded in paraffin were cut into blocks, deparaffinized and rehydrated in a
xylene/ethanol series, and treated as fresh tissue. Ultrathin sections of these tissues
were collected on nickel or gold grids (Ted Pella, Inc.).
64
Histology
Several histological stains were used to examine 7 gm paraffin sections of
kidney from 1 ype survivors. Sections were stained with hematoxylin and eosin stain
(Lillie and Fullmer 1976; Gill et al. 1974), Masson's trichrome stain (Masson 1929),
periodic acid-Schiff (PAS) stain (McManus 1948), PAS stain with a diastase pre­
digestion and a PAS stain with phenyl hydrazine HC1 (Leppi and Spicer 1967),
mucicarmine stain (Mallory 1961), Perl's iron stain (Highman 1942), and DunnThompson hemoglobin stain (Dunn and Thompson 1945).
Immunohistochemistry
Immunohistochemistry was done as described by Drolet et al. (1994). Briefly, 7
p.m paraffin-embedded sections were deparaffinized, hydrated, blocked with 5% nonfat
milk and incubated with a monoclonal antibody (Mab; 1NDW14D) made to a
conserved region of the highly conserved nucleocapsid (N) protein of IHNV. Sections
were then incubated with a biotinylated goat anti-mouse antibody, phosphatase
conjugated avidin-biotin complexes, and a red phosphate substrate (Vector). Sections
were then counterstained with hematoxylin for 1 min and 0.2 % ammonium hydroxide
for 1 min. Mock-infected fish tissues were used as negative controls and 8-10 day post
exposure infected fish were used as positive controls.
Immunogold staining
All incubations were done in 0.5 ml microfuge tubes with volumes of 25 IA and
all rinses were done in disposable glass dilution tubes. All reagents were filtered
through a 0.2 j.tm Acrodisc membrane (Gelman Sciences). Grids were blocked with
10% normal goat serum in Tris buffer (20 mM Tris, 20 mM NaN3, 225 mM NaC1,
65
0.1% bovine serum albumin, pH 8.2) for 10 mM, blotted and incubated in 1NDW14D
Mab in Tris buffer overnight. Grids were rinsed in 5 changes of Tris buffer, then
incubated with 5 nm gold-conjugated goat anti-mouse Ig (H+L) (Ted Pella, Inc.) in Tris
buffer for 1 hour. Grids were rinsed in two changes of 10X salt Tris buffer (20 mM
Tris, 20 mM NaN3, 2.25 M NaC1, 0.1% bovine serum albumin, pH 8.2) (Giberson and
Demeree 1994), five changes of Tris buffer, and five changes of nanopure water. Grids
were air dried and stained on droplets of 1% uranyl acetate and 0.2% lead citrate for 5
min each. Grids were examined by transmission electron microscopy (Phillips CM12).
PCR sample preparation
Eight 7 gm paraffin sections were placed, four each, in 1.5 ml microfuge tubes.
Sections were deparaffinized with two changes of xylene and two changes of 100%
EtOH, centrifuged for 5 mM between each step and air dried (Wright and Manos 1990).
RNA was extracted by RNAzolTm (Tel-Test, Inc.) treatment followed by choroform
extraction and isopropanol precipitation. RNA was washed twice in 75% ethanol and
air dried. The RNA in the two tubes were combined by dissolving the pellets in the
same 10 gl volume of tris EDTA (TE; 50mM Tris, 1mM EDTA, and 0.5% Tween 20,
pH 7.5). Mock-infected fish were used as negative controls and mock-infected fish
with purified IHNV genomic RNA added were used as positive controls.
PCR and southern hybridization
Retrotherm PCR was done as described by Chiou et al. (1994) with sense
(nucleotides 319-338) and antisense (nucleotides 570-552) N gene primers to obtain a
252 by N gene product (Arakawa et al. 1990). PCR reactions were incubated in an
automatic thermal cycler (Coy Laboratory Products), electrophoresed on 1.5% GTG
agarose gel (FMC Bioproducts) and blotted to nytran plus (Schleicher & Schuell) by
66
standard methods (Sambrook et al. 1989). PCR products were probed with a DIG endlabeled (Schmitz et al. 1991) internal N gene primer (nucleotides 456-427) and detected
by chemiluminescence (Holtke et al. 1992).
Results
Tissue culture
Rainbow trout (Oncorhynchus mykiss) fry which had been infected with IHNV
were positive throughout the epizootic for infectious virus by plaque assay until 46 days
post exposure. Plaque assay results continued to be negative for the 90 d, 180 d, and
1 y samples.
Histology
Kidneys of 1 ype survivors had significant periglomerular and peritubular
fibrosis as well as fibrotic foci within the hematopoietic tissue (Figure 5.1). Survivor
kidneys contained greater than 50% fewer hematopoietic cells than those of age-
matched control fish. Glomeruli showed thickening and splitting of the Bowman's
capsule and adherence of the glomerular tuft to the capsule. Renal tubules often
contained large numbers of epithelial blebs, indicative of ongoing tissue injury. The
PAS carbohydrate stain showed polysaccharide-positive deposits in the kidney tubules
and in foci of the hematopoietic tissue. A diastase digestion done before the PAS stain
which destroys the staining reaction in glycogen did not alter the PAS positive staining
indicating that the deposits were not glycogen. Phenyl hydrazine HC1-PAS which
destroys the staining reaction in glycoproteins did clear the positive staining reaction
indicating the presence of glycoprotein in the deposits. Hemosiderin deposition and
nephrocalcinosis were prominent throughout the kidneys. Kidneys from mock-infected
67
Figure 5.1
Kidney tissue from a 1 ype IHN epizootic survivor stained with
Masson's Trichrome stain. Fibrotic connective tissue stained
blue(arrows). Note the extensive fibrosis surrounding the renal
tubule (T) and occluding the hematopoietic tissue (H).
...4
a)
vi
a
L.
4:
69
negative controls showed no fibrosis or glomerular lesions. Hematopoietic tissue was
dense, few if any epithelial blebs were seen in tubules and PAS and Perl's hemosiderin
stains were negative. There was little or no nephrocalcinosis in negative controls.
Immunohistochemistry
Dense, positive deposits were seen within renal tubules and hematopoietic tissue
of survivor kidneys at 90 d, 180 d, 1 y and 2 ype when tested with 1NDW14D (Figure
5.2). No evidence of virus was seen in any of the other tissues, with the exception of
one positive liver sample at 90 dpi. Weaker positive results were also seen with a
second N-specific Mab (N-Mab; 1NCO27G) and two G-specific Mabs (G-Mab;
3GH135L, 3GH136J). The G-Mab APIH showed positive staining in individual
hematopoietic cells, whereas the N Mabs showed positive staining in individual
hematopoietic cells as well as a highly positive reaction in deposits located in renal
tubules and hematopoietic interstitium. No positive staining was seen in mockinfected, age-matched negative control fish when tested with all 4 monoclonal
antibodies.
EM examination of 1 ype kidneys
Negative staining of survivor kidney tissue previously embedded in paraffin and
processed for EM contained intracytoplasmic rhabdoviral inclusions (Ghadially 1988)
(Figure 5.3). Negative staining of survivor kidney tissue which had been held in
formalin for two years then processed for EM contained 50-60 nm If-INV-like DI
particles in hematopoietic tissue.
70
Immunogold
Immunogold labeling parameters were defined using kidney tissue samples
from IHNV infected rainbow trout 8 dpe (Figure 5.4 a and b). IHNV -like DI particles
in 1 ype survivors were confirmed as IHNV in origin by positive labeling with an antiIHNV immunogold reaction (Figure 5.5).
PCR and southern analysis
IHNV RNA was detected in the kidney tissue of lypi survivors by PCR
amplification. A 252 by band was detected in an ethidium bromide stained gel (Figure
5.6 a). The IHNV specificity of this band was confirmed by hybridization with an N
gene specific probe to a region within the 252 by amplified fragment (Fig. 5.6 b).
Discussion
The histological staining with hematoxylin and eosin showed prominent lesions
in MN survivor kidneys at 1 ype. Glycoprotein was detected in deposits within the
renal tubules and hematopoietic interstitium by staining with PAS-phenyl hydrazine.
Epithelial blebs and hemosiderin deposits were prominent throughout the kidney
indicating ongoing renal tissue injury and destruction of red blood cells. These results
suggest that renal and possibly hematopoietic function in IHN survivors is
compromised. The Dunn-Thompson hemoglobin stain showed no indication of
formalin-hemoglobin artifacts which can occur during the fixation of blood-rich tissues.
Although no infectious virus was detected in survivors after 46 dpe, intense
IHNV positive staining was seen by immunohistochemistry in deposits in renal tubules
and hematopoietic interstitium of samples taken at 90 d, 180 d, 1 y and 2 ype. Antigen
was seen in fibrotic regions, renal tubules and in close association with hematopoietic
71
Figure 5.2
Kidney tissue from a 1 ype IHN epizootic survivor as tested by alkaline
phosphatase immunohistochemistry. Note the highly positive deposits
in the renal tubules (T) (straight arrows) and in the sparse hematopoietic
tissue (H) (curved arrows). Deposits stained red on microscopic slides.
Figure 5.3
Cytoplasmic rhabdoviral inclusions in kidney tissue from a 1 ype IHN
epizootic survivor. Tissue was negatively stained with uranyl acetate
and lead citrate.
Figure 5.4 a
Immunogold labeling of IHNV particles using the anti-nucleocapsid
monoclonal antibody, 1NDW14D.
Figure 5.4 b
Immunogold labeling of IHNV particles using the anti-glycoprotein
monoclonal, 3GH136J.
Figure 5.5
Immunogold labeled IHNV DI particles from 1 ype IHN epizootic
survivor.
Figure 5.6 a
Retrotherm PCR amplification of a 252 by fragment of IHNV N gene
from kidney tissue of a 1 year post exposure survivor. Lanes from left to
right contained 123 by ladder marker, IHNV genomic RNA, and RNA
extracted from a 1 ype epizootic survivor. The bright band at the bottom
of each lane is excess primers and nucleotides.
Figure 5.6 b
Southern blot hybridization showing 252 by fragment is of IHNV N
gene origin. Lane 1 contained 123 by ladder marker. Lane 2 contained
a 10-4 dilution of IHNV genomic RNA. Lane 3 contained a 10-3 dilution
of IHNV genomic RNA. Lane 4 contained a linearized plasmid
containing the IHNV N gene as a positive control. Lane 5 contained the
IHNV N gene digested from the positive control plasmid. Lane 6
contained RNA extracted from a negative control fish. Lane 7 contained
RNA extracted from a 1 ype survivor kidney sample. The 500 by band
in lanes 2 and 3 is the result of self priming (Arakawa et al. 1990).
73
loonm
Figure 5.3
"7 4
Figure 5.4 a
75
S.
50 nm
Figure 5.4 b
76
Cr
1 ype Survivor
IHNV RNA
Marker
78
1
2
3
4
5
6
7
500 by
252 by
Figure 5.6 b
79
cells. As to the possibility of reinfection, survivors showed no signs of IHN infection
during their lifetime and were maintained in pathogen-free water. No viral
contaminants were detected by tissue culture testing of 11 organs, every 3-6 months,
throughout their captivity. Bacterial contaminants, such as Renibacterium
salmoninarum the causative agent of bacterial kidney disease, which causes salmonid
renal lesions (Sami et al. 1992) were seen in survivor kidneys. Lesions observed were
therefore assumed to be due to IHNV.
All survivor kidney samples examined had moderate to severe precipitation of
calcium complexes in renal tubules. This condition is commonly known as
nephrocalcinosis and is associated with excessive carbon dioxide levels in the water
(Harrison and Richards 1979; Smart et al. 1979). Age-matched mock-infected negative
controls had mild or no nephrocalcinosis. Routine sampling of laboratory rainbow trout
populations at the Center for Salmon Disease Research fish facility (Corvallis, Oregon)
have shown a 30-60% incidence of nephrocalcinosis in IHN survivors versus 0-10%
incidence in age-matched, mock-infected survivors (data not shown). The increased
incidence of nephrocalcinosis in fish that survive IHN epizootics suggests that the
general impairment of kidney function from IHNV infection predisposes fish to
nephrocalcinosis. This, in turn, may result in the death of survivors before sexual
maturation.
Cytoplasmic rhabdoviral inclusions, like those reported in rabies virus infections
(Ghadially 1988), were detected in a kidney sample which had been embedded in
paraffin before processing for EM. We were not able to confirm the cytoplasmic
rhabdoviral inclusions as IHNV in origin because the tissue was unsuitable for
immunogold testing. Rhabdoviral DI particles are typically spherical and one-third the
size of standard virus particles (Pattniak and Wertz 1991). Round 50 nm particles,
approximately one-third the size of standard IHNV particles (160 x 90 nm; Amend and
Chambers 1970) were seen in kidney tissues of 1 ype survivors which had been in
80
formalin for 2 years. The DI particles were confirmed as IHNV in origin by
immunogold labeling (Figure 5.5).
The presence of DI particles suggests that standard infectious virions are
present, but kept at levels below tissue culture detection limits. This has been well
documented with VSV and rabies virus in tissue culture (for review, see Sekellick and
Marcus 1980). Standard and DI particles may be in such balance that only a very few
virions are synthesized, resulting in a subacute or latent form of the infection, until the
balance is upset by the stressful conditions of spawning and/or exposure of the fish to
the same or other viruses. Standard infectious virus would subsequently increase to
levels detectable by plaque assay, resulting in an apparently negative survivor
becoming positive at sexual maturation. Experimentally or naturally infected fish
which are held in captivity do not experience the same stress levels as fish naturally
migrating upstream to their natural spawning grounds. Nor are they exposed to the
onslaught of pathogens (IHNV and other) which contaminate river waters. The
inability to detect infectious virus in experimentally or naturally infected, sexually
mature survivors held in captivity may be due to these environmental differences.
Until now, there has been no proof that IHNV persists in epizootic survivors
past 50 days when the last positive plaque assays are typically seen. We have shown,
unequivocally, that IHNV does persist in fish that survive IHN epizootics. IHNVspecific N and G proteins were detected by immunohistochemistry using 4 different
Mabs. In addition, a fragment of the N gene was amplified by PCR from survivor
kidney and confirmed with southern hybridization (Figure 5.6 a and b). The differential
positive staining by G-Mabs versus N-Mabs may be attributed to the G monoclonals
binding primarily to currently infected, N and G expressing, hematopoietic cells, and N
monoclonals binding primarily to the dense deposits which contained DI particles and
nucleoprotein conglomerates upon examination by immunogold EM (Figure 5.5).
81
Most, if not all, viruses have been shown to produce DI particles in tissue
culture cells. In addition, animals given DI particles before or during viral infections
show an altered infection course. Despite these studies, there has been no definite
report of DI particles from animals following a natural infection. In this study, animals
were infected by adding a moderate dose (103 standard virus particles/ml) of virus
directly to the water thereby simulating the horizontal exposure known to occur during
natural epizootics. Daily and cumulative mortality was just as expected for
experimental exposure of this species to this dose of IHNV, and clinical disease signs
were identical to those seen in natural infections. The DI particles seen in these
survivors suggest that the persistence of IHNV in salmonid populations is attributable
to the subclinical carrier status of survivors caused by DI particles. Standard virions are
detected during natural spawning because the DI/standard particle balance is upset and
results in a resurgence of standard virus. This finding does not preclude reinfection as a
source of virus in the adult. In fact, re-exposure to IHNV could be a factor in upsetting
the DI/standard particle balance.
Acknowledgments
Sandra Ristow (Washington State University, Pullman, Washington) provided
the monoclonal antibodies used in this study. Dr. Linda Boot land and Harriet Lorz
assisted in fish care, maintenance and sampling. Pat Allison provided technical
assistance with histological staining. Julie Duimstra provided technical assistance with
immunogold labeling. Al Soeldner provided technical assistance in electron
microscopy and photography.
82
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86
CHAPTER 6
THESIS SUMMARY
Serological identification of IHNV in fixed tissue culture cells
Confirmatory identification of viruses by specific reaction with an antibody
reagent is a standard procedure in the virus diagnostic laboratory. The immunological
assay can be a virus neutralization assay (McCain et al. 1971; Hsu and Leong 1985;
Winton et al. 1988) or an assay that measures the specific reactivity of an antibody with
a viral antigen (for review of IHNV assays, see Winton 1991). The APIC procedure
described in chapter 3 was easy to perform, did not require special equipment, and a
positive result was easy to detect. It will probably have its greatest use with virus
samples that have been lost as a result of laboratory error or a severe reduction in
sample viability. It may also be used when a laboratory has to screen hundreds of
samples and the examination of individual plates for plaques before fixation and
staining is not possible.
The route of IHNV entry and progression in rainbow trout
By carefully examining sequential fish samples taken before and during an
epizootic, the route of entry and the progression of IHNV within rainbow trout has been
determined. IHNV infection progressed from two major sites: the gills and the
gastrointestinal tract. Both routes of entry eventually resulted in systemic viremia.
While IHNV readily infected epithelial cells of numerous organs, it had a propensity for
connective tissue. While IHNV readily infected epithelial cells, the infection appeared
transient and not highly productive. The majority of cells undergoing productive viral
infection were of connective tissue origin. These results may influence the design of
vaccines, and the study of specific cell receptors involved in IHNV infection.
87
Detection of IHNV in epizootic survivors
The survivors from the epizootic study described in chapter 4 and survivor
rainbow trout from previous infection studies were examined by APIH for IHNV
nucleocapsid protein. Intense IHNV positive staining deposits were seen in renal
tubules and hematopoietic interstitium of samples taken at 90 d, 180 d, 1 y and 2 ype.
This unexpected positivity in fish where no infectious virus had been detected since 46
dpe was the impetus for the survivor study described in chapter 5. In addition to the N
protein, glycoprotein was detected in the deposits by staining with PAS-phenyl
hydrazine and by APIH. Histological staining with hematoxylin and eosin showed
prominent lesions in IHN survivor kidneys at 1 ype. Until now, there has been no proof
that IHNV persists in epizootic survivors past 50 days when the last positive plaque
assays are typically seen. The amplification of a fragment of the IHNV N gene by PCR
and the detection of two IHNV proteins with APIH is proof that IHNV does in fact
persist in the epizootic survivor for at least two years. This persistence is in the form of
a heterogeneic population of standard and DI particles, resulting in a subclinical carrier
status. This finding does not preclude reinfection as a source of virus in the adult. In
fact, re-exposure to IHNV could be a factor in upsetting the DI/standard particle
balance.
Future use of methods
The methods described in this thesis have already given new research tools to
ongoing IHNV infection studies. The immunocytochemical techniques are currently
being used to determine transfection efficiencies of various IHNV gene constructs and
to detect the expression of viral proteins in transgenic cells which can then be examined
for resistance to IHNV infection (Anderson and Leong 1991). The APIC assay is
currently being used by several diagnostic fish laboratories to detect IHNV in fixed
88
plaque assay plates as opposed to subculturing for immunofluorescence or plaque
neutralizations.
Future use of results
The detailed description of specific virus-positive cell types and degrees of
IHNV infection and progression of the virus in rainbow trout provided preliminary
work for a funded grant to characterize the receptor of IHNV. Documentation of the
complete pathogenic pathway of a wild type IHNV isolate could be used as a baseline
for examining alterations in pathogenesis from protein mutations, protective
monoclonal antibodies, or vaccines.
The most exciting results of these studies is the irrefutable evidence of
persistence of IHNV in epizootic survivors. For the first time, IHNV has been shown
to persist after the 50 day positive plaque assay limit. This result allows for the longheld hypothesis that adults returning to spawn are positive for virus because it was
never cleared and is somehow reactivated upon sexual maturation. This will inevitably
lead to more research on IHNV persistence and the role that it plays in maintaining the
endemic level of virus in nature. This may also change the way hatcheries have been
dealing with IHN survivors for the past 35 years. In the best scenario, the destruction
of IHN survivors will decrease levels of virus in nature to the point where this virus is
no longer a significant problem to hatcheries.
89
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APPENDIX
PROTOCOLS FOR THESIS ASSAYS
105
Alkaline Phosphatase Immunocytochemistry
for IHNV Detection in Plaque Assays
Procedure
Destain virus infected cells with several changes of 70% EtOH 20-60
1.
min (depends on stain intensity).
2.
Equilibrate cells with PBS pH 7.4
3.
Block with 5% milk in PBS for 1-12 hrs
4.
Aspirate excess block from wells, and do not rinse
5.
Incubate with Primary antibody, diluted in PBS for 1 hr
6.
Rinse with PBS (2 changes 5 min each, fill wells)
7.
Incubate with Biotinylated secondary antibody, diluted in PBS for
30 min
8.
Rinse as above
9.
Incubate with Avidin-Biotin Complexes (ABC solution) for 1 hr
10.
Rinse as above
11.
Equilibrate cells with 100 mM Tris, pH 8.2, for 3 min
12.
Incubate with Fast-Red Substrate, in 100 mM Tris, pH 8.2,
for 10-20 min
Notes
All steps are done directly in the wells of tissue culture plates
Cells must not be allowed to dry after step 1
The minimum amount of reagent volume per well for 24-well plate is 100 ul
Fixing cells without the crystal violet stain followed by destaining, results in
high background.
106
Counterstaining cells after substrate step can result in occlusion of the red
immunochemical reaction stain
Steps 1- 11. Incubations are done at room temperature on a rocker.
Steps 7. and 8. Vector Laboratories Vectastaintm Mouse IgG kit, store kit
at 4°C
Step 7. Incubation with Biotinylated antibody for longer than 30 mM,
results in high background
Step 12. Vector Alkaline Phosphatase Substrate Kit, store kit at 4°C
Incubate substrate in the dark with no rocking
Reaction can be preserved by applying Crystal Mounttm (BiOmeda, Foster
City, CA)
Reagents
Phosphate Buffered Saline (PBS) pH 7.4
1 liter 1X
1 liter 10X
137 mM NaC1
8.0 g
80.0 g
2.7 mM KC1
0.2 g
2.0 g
1.15 g
11.5 g
0.2 g
2.0 g
distilled H2O
990.0 ml
904.5 ml
5% milk Block
100 ml
4.3 mM NaHP03.7 H2O
1.4 mM KHPO4
5 % nonfat dry powdered milk
PBS
Make fresh each time or store at 4°C
5.0 g
95.0 ml
107
100 mM Tris-HC1, pH 8.2
100 ml
Trisma Base
12.1 g
distilled H2O
88.0 ml
pH 8.2 is crucial!
Primary Antibody (Anti-IHNV-nucleocapsid monoclonal antibody_)
Dilute 1NDW14D ascites in PBS at 1:4000
Sandra Ristow, Washington State University
Use 1NDW14D to detect all 5 types of IHNV
Dilute 2NH105B ascites in PBS at 1:500 1:1000
Sandra Ristow, Washington State University
Use 2NH105B to detect type 2 IHNV only
Secondary Antibody (goat anti-mouse biotinylated polyclonal antibody)
10 ml PBS
1 drop biotinylated anti-mouse, mix
Vectastain (Vector Laboratories, Burlingame, CA), Anti-mouse IgG (H+L)
Alkaline Phosphatase Kit
Avidin Biotin Complex (ABC) solution
10 ml PBS
2 drops of A, mix
2 drops of B, mix
Vectastain (Vector Laboratories, Burlingame, CA), Anti-mouse IgG (H+L)
Alkaline Phosphatase Kit
Let sit at room temp for 30 min before use
108
Fast-Red Phosphate Substrate
1 drop Levamisoletm (Vector, Laboratories), mix
2 drops #1, mix
2 drops #2, mix
2 drops #3, mix
5 ml 100 mM Tris-HC1
Vectastain (Vector Laboratories, Burlingame, CA), Vector Red Alkaline
Phosphate Substrate Kit
Make and use immediately
Formaldehyde Fixing and Paraffin Embedding Fish Tissues
Fixing
10% Neutral Buffered Forma lin
At least 24 hr at room temp
70% EtOH
At least 1 hr
85% EtOH
1 hr
95% EtOH
1 hr
95% EtOH
1 hr
95% EtOH
1 hr
100% EtOH
1 hr
100% EtOH
1 hr
100% EtOH
1 hr
100% Slidebrite or Xylene
1 hr
100% Slidebrite or Xylene
1 hr
100% Slidebrite or Xylene
1 hr
109
Paraffin 60°
2 hr
Paraffin 60°
2 hr
Notes
Tissue to fixative ratio should be a minimum of 1:10 (w/v).
If whole fish are being fixed, their ventral surface should be slit with a
sterile scalpel and their viscera pulled out slightly.
Forma lin must be kept at a neutral pH to avoid fixing artifacts.
Ethanol and xylene solutions should be changed if yellowing or particulate
sediment is seen.
Embedding
Positioning of tissues
Flat side down
Avoid bubbles
Avoid edges of block
Place long pieces of tissue at an angle so that the microtome blade will come
in contact with just one end first.
Paraffin
Paraffin temperature should not exceed 60°C. Wax that has been heated
above 60°C should be discarded.
110
Slides
1.
Dip clean microscopic slides in slide affixative
2.
Draw excess off with paper towel
3.
Air dry
Sectioning
1.
Slice 5-7 j.im sections
2.
Float sections on 45°C water bath
3.
Collect on gelatin coated slides
4.
Incubate on 40°C hot plate overnight
Reagents
10% Neutral Buffered Formalin
1 liter
Formalin
100.0 ml
33 mM NaH2PO4 H2O
4.6 g
46 mM Na2HPO4
6.5 g
distilled H2O
889.0 ml
pH 7.0-7.4
Slide Affixative
Gelatin
Hot distilled H2O
1 liter
1.0 g
1000.0 ml
Mix and cool, then add
CrK(SO4)2 12H2O
1.0 g
111
Alkaline Phosphatase Immunohistochemistry
for IHNV Detection in Paraffin Embedded Fish Tissues
Deparaffinizing and rehydrating tissue section slides
Xylene
5 min
Xylene
5 min
100% EtOH
10 dips
95% EtOH
5 min
95% EtOH
5 min
70% EtOH
5 min
Running distilled H2O
10 min
PBS
20 min
Immunohistochemistry
1.
Equilibrate tissue with PBS pH 7.4
2.
Block with 5% milk in PBS for 1 hr
3.
Blot excess block from sections
4.
Incubate with Primary antibody, diluted in PBS for 1 hr
5.
Rinse by flooding slide with PBS (2 changes, 5 min each)
6.
Incubate with Biotinylated secondary antibody, diluted in PBS for
30 min
7.
Rinse as above
8.
Incubate with Avidin-Biotin Complexes (ABC solution) for 1 hr
9.
Rinse as above
10.
Equilibrate sections with 100 mM Tris, pH 8.2, for 3 min
11.
Incubate with Fast-Red Substrate solution for 20 min in the dark
112
Counterstain tissues with Hematoxylin for 1-2 min followed by 2%
12.
ammonium hydroxide in 70% EtOH for 1-2 min
Notes
All steps are done directly on slides.
Once rehydrated, tissues must not be allowed to dry until staining is
complete.
The minimum amount of reagent volume per slide depends on section size.
The entire section must be covered. A hydrophobic marking pen such as a
grease pencil or PEPTM pen can be used to confine reagents on the slides.
Counterstaining cells after substrate step can result in occlusion of the red
immunochemical reaction stain
Steps 1- 10. Incubations are done at room temperature on a rocker.
Steps 6. and 7. Vector Laboratories Vectastaintm Mouse IgG kit, store kit
at 4°C
Step 6. Incubation with Biotinylated antibody for longer than 30 min,
results in high background
Step 11. Vector Alkaline Phosphatase Substrate Kit, store kit at 4°C
Incubate substrate in the dark with no rocking
After counterstaining tissues can be mounted with Crystal Mount
If it is necessary to manipulate the tissue after this point, Crystal Mount can
be removed by soaking the slide in PBS, with agitation, for 30 min to 1 hr.
Immersion oil will destroy Crystal Mount. If high magnification is required
apply 2-3 drops of cytoseal mounying media (Stephens Scientific,
Riverdale, NJ) on top of the Crystal Mount and cover with a coverslip.
Examine with light microscopy, RED = IHNV NUCLEOCAPSID
113
Reagents
Phosphate Buffered Saline (PBS) pH 7.4
1 liter 1X
1 liter 10X
137 mM NaC1
8.0 g
80.0 g
2.7 mM KC1
0.2 g
2.0 g
1.15 g
11.5 g
0.2 g
2.0 g
distilled H2O
990.0 ml
904.5 ml
5% Milk Block
100 ml
4.3 mM NaHP03.7 H2O
1.4 mM KHPO4
5g
Nonfat dry powdered milk
PBS
95 ml
Make fresh or store at 4°C
100 mM Tris-HC1, pH 8.2
100 ml
Trisma Base
12.1 g
distilled H2O
88.0 ml
pH 8.2 is crucial!
Primary Antibodies (Anti-IHNV-nucleocapsid monoclonal antibody)
Dilute 1NDW14D ascites in PBS at 1:4000
Sandra Ristow, Washington State University
Use 1NDW14D to detect all 5 types of IHNV
Dilute 2NH105B ascites in PBS at 1:500-1:1000
Sandra Ristow, Washington State University
Use 2NH105B to detect type 2 IHNV only
114
Secondary Antibody (goat anti-mouse biotinylated polyclonal antibody)
10 ml PBS
1 drop biotinylated anti-mouse, mix
Vectastain (Vector Laboratories, Burlingame, CA), Anti-mouse IgG (H+L)
Alkaline Phosphatase Kit # 5001
Avidin Biotin Complex (ABC) solution
10 ml PBS
2 drops of A, mix
2 drops of B, mix
Vectastain (Vector Laboratories, Burlingame, CA), Anti-mouse IgG (H+L)
Alkaline Phosphatase Kit
Let sit at room temp for 30 min before use
Fast-Red Phosphate Substrate
1 drop Levamisoletm (Vector, Laboratories), mix
2 drops #1, mix
2 drops #2, mix
2 drops #3, mix
5 ml 100 mM Tris-HC1
Vectastain (Vector Laboratories, Burlingame, CA), Vector - Red Alkaline
Phosphate Substrate Kit
Make and use immediately