Fish 3

57
CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
2
Infectious Haematopoietic Necrosis Virus
L.M. Bootland and J.C. Leong
Department of Microbiology and Center for Salmon Disease Research, Oregon
State University, Corvallis, Oregon 97331-3804, USA.
INTRODUCTION
Infectious haematopoietic necrosis virus (IHNV) is a rhabdovirus that causes an
economically significant disease in Pacific salmon (Oncorhynchus spp.),
Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). The
disease was first observed in cultured sockeye salmon (Oncorhynchus nerka) on
the west coast of North America (Rucker et al., 1953; Watson et al., 1954) and
IHNV was first isolated in cell culture by Wingfield et al. (1969). Typically,
IHNV causes necrosis of the haematopoietic tissues, and the disease was named
infectious haematopoietic necrosis (IHN) by Amend et al. (1969). Economic
losses from IHNV can be a direct consequence of fish mortality, or indirect from
regulations restricting the movement of IHNV-infected fish or the destruction of
infected fish stocks to control the spread of the virus. Infectious haematopoietic
necrosis is the most important constraint to the profitability and continued
growth of the US commercial salmonid aquaculture industry, with lost revenue
in the Idaho trout industry estimated at $3 million annually (Congleton, 1988).
At government hatcheries along the Columbia River, upwards of 70 million fish
and eggs have been destroyed as a result of IHNV infection since 1981. The
value of these fish has been conservatively estimated at $350 million (Leong et
al., 1995). In addition to the Pacific North-West, IHNV is also endemic in
salmonid fish in Japan and has been isolated in several countries in Asia and
Europe.
Several excellent reviews of IHN and IHNV have been published (Pilcher
and Fryer, 1980a,b; Nicholson, 1982; Wolf, 1988). This chapter will review all
aspects of IHNV and IHN.
58
THE DISEASE AND AGENT
History and geographical distribution
Infectious haematopoietic necrosis virus primarily causes disease in the genus
Oncorhynchus and was first identified in the Pacific North-West of the USA.
During the 1950s, IHNV caused severe losses of sockeye salmon (O. nerka) at
hatcheries in Washington (Rucker et al., 1953) and Oregon (Wingfield et al.,
1969). The virus might have been introduced into Oregon from Washington in
unpasteurized sockeye salmon viscera that were fed to young fish, a practice that
was soon stopped (Amend and Wood, 1972). A similar disease was next reported
in hatchery-reared chinook salmon (Oncorhynchus tshawytscha) in California
(Ross et al., 1960; Wingfield and Chan, 1970). In 1967, IHN occurred in young
sockeye salmon in British Columbia and was reported for the first time in
rainbow trout (Amend et al., 1969).
With the realization that IHNV could devastate hatchery populations of
young sockeye salmon, chinook salmon and rainbow trout, several surveys were
carried out to determine the distribution and prevalence of IHNV in the Pacific
North-West. Prior to the 1970s, IHNV was not a problem in Washington stocks
of resident chinook or coho salmon (Oncorhynchus kisutch), but two stocks of
sockeye salmon were infected (Amend and Wood, 1972). During the 1970s, IHN
epizootics increased in Oregon stocks of rainbow trout, steelhead trout
(anadromous O. mykiss), chinook salmon and kokanee salmon (land-locked O.
nerka) (Groberg et al., 1980; Mulcahy et al., 1980). The source of the virus was
not clear but it was suggested that the import of infected eggs from Washington
and wild adult kokanee salmon or rainbow trout harbouring the virus might have
introduced the virus into the area. In 1977, IHNV began causing severe losses of
rainbow trout in Idaho (Busch, 1983). During the early 1980s, IHNV was
isolated from additional salmonid species and fish losses due to IHN increased
dramatically. By 1982, IHNV had been isolated in cutthroat trout (Onco-
rhynchus clarki) and there were very high IHN losses in rainbow and steelhead
trout in the Columbia River basin (Groberg, 1983; Groberg and Fryer, 1983).
The first reported natural IHN epizootic in Atlantic salmon fry occurred in 1984
in Washington (Mulcahy and Wood, 1986) and, in 1986, IHNV was isolated for
the first time from adult chum salmon (Oncorhynchus keta) with no clinical
disease (Hopper, 1987). In Idaho, IHNV became enzootic in rainbow trout
(Busch, 1983) and was isolated from adult steelhead trout and kokanee salmon
(Groberg, 1983). Currently, IHNV can be isolated from salmonid fish in Oregon,
Washington, Idaho, California, Alaska and British Columbia and is considered
endemic in the Pacific North-West (Pilcher and Fryer, 1980a,b; Wolf, 1988).
In Alaska, IHNV began causing epizootics in sockeye salmon in 1973 and is
now enzootic (Grischkowsky and Amend, 1976). Infectious haematopoietic
necrosis was a major cause of failure in the culture of sockeye salmon until 1981,
when a statewide policy devised by the Fisheries Rehabilitation, Enhancement
and Development Division (FRED) proved successful in minimizing losses
(Meyers et al., 1990). However, outbreaks of IHN are still occurring in enhanced
populations of sockeye salmon smolts in at least one lake system (Follett and
59 Infectious Haematopoietic Necrosis Virus
Burton, 1995). The virus has also been isolated in Alaska from diseased chinook
salmon and chum salmon (Follett et al., 1987).
Several other states have had sporadic IHN outbreaks in rainbow trout, and
this was usually associated with the import of infected eggs or fry. The first
epizootics outside the Pacific North-West occurred in 1969 in Minnesota
(Plumb, 1972) and South Dakota (Wolf et al., 1973). Disease outbreaks have
also occurred in West Virginia (Wolf et al., 1973), Montana (Holway and Smith,
1973), New York (Carlisle et al., 1979), Colorado (Janeke, 1984) and Utah
(Amos, 1985).
Infectious haematopoietic necrosis virus has spread by the movement of
infected eggs and/or fry outside North America (Hill, 1992). In Japan, IHNV
was apparently introduced in 1968 with eggs imported from Alaska (Sano et al.,
1977) and yearly IHN outbreaks occur in nearly all areas that rear salmonid fish
(Fukuda et al., 1992). Rainbow trout, yamame (Oncorhynchus masou), and
amago trout (O. masou macrostomus) are most severely affected but sporadic
outbreaks have been reported in sockeye and kokanee salmon, masu salmon (O.
masou masou), chum salmon, cherry salmon (Oncorhynchus rhodurus), Atlantic
salmon, brown trout (Salmo trutta), brook trout (Salvelinus fontinalis) and
Japanese char (Salvelinus leucomaenis) (Kimura and Awakura, 1977; Sano et
al., 1977; Yamazaki and Motonishi, 1992). In 1985, IHNV was spread to north-
east China by importation of infected eggs from Japan (Niu and Zhao, 1988;
Zhao and Niu, 1994). Importation of infected rainbow trout eggs into Italy
resulted in the first diagnosis of IHNV in 1987 (Bovo et al., 1987) and IHNV has
spread to at least six regions within the country (Bovo et al., 1991). Other
countries in which IHNV has been identified include Taiwan (Chen et al., 1985;
Wang et al., 1996), France (Laurencin, 1987; Hattenberger-Baudouy et al., 1989,
1995a,b), Belgium (Hill, 1992) and Korea (John et al., 1992; Park et al., 1993).
It is likely that the geographical range of IHNV will continue to increase.
The migration of infected anadromous fish species may spread IHNV (Hill
1992) and the movement of infected eggs and fish within and between countries
will probably continue to occur, despite attempts in several countries to avoid
this type of transfer by strict fish health certification requirements.
Host range
During the last 10 years, IHNV has been isolated from several new fish species,
and the host range of the virus has enlarged. Initially, the host range of IHNV
was thought to be limited to the genus Oncorhynchus (Table 2.1). Infectious
haematopoietic necrosis is often reported in rainbow trout, in steelhead trout and
in the Pacific salmon sockeye, kokanee, chinook and chum salmon (Wolf,
1988). Cutthroat trout are also susceptible to IHNV infection and disease
(Parisot, 1962; Groberg, 1983). Caution must be used before concluding
whether a fish species is susceptible to IHNV infection and mortality. There are
several examples where a fish species is not considered susceptible to IHNV, but
later studies have demonstrated susceptibility under different experimental
conditions. For example, the coho salmon (O. kisutch) and pink salmon
60
(Oncorhynchus gorbuscha) are considered to be refractory to IHN. No natural
epizootics have been reported and experimental infection with a high viral dose
resulted in only low coho fry mortalities (Wingfield and Chan, 1970; Wingfield
et al., 1970; Chen et al., 1990) and no pink salmon fry mortalities (Follett et al.,
1997). However, coho salmon are susceptible to IHNV infection virus has been
isolated from adult fish (LaPatra et al., 1989b; Eaton et al., 1991) and from fry
naturally or experimentally exposed to IHNV (Hedrick et al., 1987; LaPatra
et al., 1989b; Chen et al., 1990).
Several species of Salmo have been shown to be susceptible to IHNV
infection and disease. Atlantic salmon fry have suffered at least one epizootic in
North America (Mulcahy and Wood, 1986), and sporadic disease outbreaks have
occurred in Japan (Yamazaki and Motonishi, 1992). Traxler et al. (1991, 1993)
have demonstrated that Atlantic salmon can be infected with IHNV in salt water
by immersion, by injection or, most importantly, by cohabitation with infected
sockeye salmon. Recently, natural IHNV infections have been diagnosed in
Atlantic salmon adults during the marine phase of their life cycle (Traxler et al.,
1997). Brown trout are moderately susceptible to experimentally induced IHN
(LaPatra and Fryer, 1990), but natural infections and disease outbreaks have
occurred in Oregon (Engelking and Kaufman, 1994a,b) and occasional IHN
outbreaks have been reported in Japan (Yamazaki and Motonishi, 1992). In
France, brown trout may be refractory to IHN (Hill, 1992). Since brown trout
and Atlantic salmon are important species in aquaculture and sports fisheries in
several countries, and Atlantic salmon is also a valuable commercial species, the
Table 2.1. Host range of IHNV.
Common name Scientific name Reference
Fish species in which natural IHN epizootics have occurred
Sockeye salmon Oncorhynchus nerka Rucker et al., 1953
Kokanee salmon Land-locked O. nerka Sano et al., 1977
Chinook salmon O. tshawytscha Ross et al., 1960
Chum salmon O. keta Sano et al., 1977
Cherry salmon O. masou masou Sano et al., 1977
Biwa salmon O. masou rhodurus Sano et al., 1977
Yamame trout O. masou Sano et al., 1977
Amago trout O. masou macrostomus Sano et al., 1977
Rainbow trout O. mykiss Amend et al., 1969
Steelhead trout Anadromous O. mykiss Amend et al., 1969
Cutthroat trout O. clarki Parisot, 1962
Atlantic salmon Salmo salar Mulcahy and Wood, 1986
Brown trout S. trutta Yamazaki and Motonishi, 1992
Brook trout Salvelinus fontinalis Yamazaki and Motonishi, 1992
J apanese char S. leucomaenis Kimura and Awakura, 1977
Salmonid species of low susceptibility to IHN disease
Coho salmon O. kisutch Wingfield and Chan, 1970
Arctic grayling Thymallus arcticus Follett et al., 1997
Pink salmon O. gorbuscha Follett et al., 1997
Lake trout Salvelinus namaycush Yamamoto and Clermont 1990
Arctic char S. alpinus Follett et al., 1997
potential for IHNV to cause devastating disease in these two species is of serious
concern (Hill, 1992).
Salvelinus spp. vary in their susceptibility to IHNV. Lake trout (Salvelinus
namaycush) can be experimentally infected but appear to be relatively resistant
to disease (Yamamoto and Clermont, 1990) and Arctic char (Salvelinus alpinus)
may be completely resistant to IHNV (T.R. Meyers as cited in LaPatra et al.,
1992; Follett et al., 1997). Brook trout (S. fontinalis) have had sporadic
outbreaks of IHN in Japan (Yamazaki and Motonishi, 1992) and the virus has
been isolated from apparently healthy adult brook trout in Oregon (Pilcher and
Fryer, 1980a,b). The success of experimentally infecting brook trout fry with the
induction of disease is variable and depends on the water temperature, viral
isolate and viral dose. Yamamoto and Clermont (1990) stated that 1-month-old
brook trout were infected after immersion in IHNV, but unfortunately no data
were presented. In a second study, brook trout fry appeared to be completely
resistant to infection after immersion in a type 2 Idaho IHNV isolate (LaPatra et
al., 1992). The 15C water temperature used in the latter study may have
enhanced resistance of the brook trout to disease since several researchers have
shown that temperatures above the optimum for IHN epizootics (1012C)
resulted in decreased fish mortalities (Watson et al., 1954; Amend, 1970a).
Goldes and Mead (1992) found that 2-month-old brook trout immersed at 4C in
a type 1 IHNV isolate had a combined morbiditymortality of 6.7% and 93% of
the dead fish were infected. The virus was isolated from surviving fish 2 months
postimmersion. A higher mortality (35%) occurred in brook trout fry immersed
at 12C in a type 1 IHNV isolate and the virus was isolated for 3 weeks
postimmersion (Bootland et al., 1994). A similar immersion of brook trout in a
type 2 IHNV resulted in only a 5% mortality. Thus, brook trout fry are at least
moderately susceptible to IHN disease and infection, but the viral isolate and
dose influence the degree of susceptibility. The studies with brook trout clearly
demonstrate that, when examining the susceptibility of a fish species to IHNV, it
is important to test several viral doses and isolates at the optimal water
temperature for IHN disease. Resistance to one IHNV isolate does not guarantee
that the fish will not be susceptible to other IHNV isolates.
The susceptibility of non-salmonid fishes to IHNV has not been studied in
depth. Castric and Jeffroy (1991) found two marine fish species, sea bream
(Sparus aurata) and turbot (Scophthalmus maximus), suffered mortalities of
43% and 87%, respectively, after intraperitoneal (i.p.) injection of 10
6
plaque-
forming units (pfu) of a French IHNV isolate. Sea bass (Morone labrax) were
refractory to disease but were infected. LaPatra et al. (1995) examined the
susceptibility of white sturgeon (Acipenser transmontanus) cell lines, larvae,
juveniles and adults to IHNV. Only one of three cell lines was susceptible, and
the viral titre was only 17% of that produced in chinook salmon embryo
(CHSE-214) cells. Infectious haematopoietic necrosis virus replicates in larval
fish for at least 9 days, but juveniles fed infected rainbow trout or immersed in
virus are refractory to mortality and infection. Adult fish that cohabit with
infected rainbow trout are not infected, but there are neutralizing antibodies. It
was suggested that sturgeon should be considered as potential sources of virus
(LaPatra et al., 1995).
62
In Oregon, adult northern squawfish (Ptychocheilus oregonensis), adult
largescale suckers (Catostomus columbianus) and lamprey (Entosphenus
tridentatus) ammocoetes appear to be refractory to disease and infection after
injection of a type 1 IHNV isolate. However, adult mountain whitefish (Proso-
pium williamsoni), a member of the Salmonidae family, can be infected by
injection and the virus persists for at least 3 weeks (L.M. Bootland, H.V. Lorz
and J.C. Leong, unpublished data). A cell line derived from embryonic tissue of
the inconnu or sheefish (Stenodus leucichthys), a member of the Salmonidae, is
susceptible to IHNV (Follett and Schmitt, 1990), but it appears that in vivo
susceptibility tests have not been done. Arctic grayling (Thymallus arcticus) fry
are refractory to infection with a type 1 IHNV isolate, and this species is not
likely to be a reservoir of IHNV (Follett et al., 1997).
Infectious haematopoietic necrosis virus has been isolated from a few
invertebrates. The salmon leech (Piscicola salmositica) (Mulcahy, 1986;
Yamamoto et al., 1989; Mulcahy et al., 1990) and ectoparasitic copepods
(Salmincola sp.) removed from sockeye salmon (Mulcahy et al., 1990) harbour
IHNV. The third invertebrate from which IHNV has been isolated is the common
mayfly (Callibaetis sp.). Virus was detected in adult mayflies in Idaho by growth
in cell culture after two blind passages and by Western blots (Shors and Winston,
1989b). Invertebrate species may play a role in the life cycle of IHNV by acting
as vectors or reservoirs of infection, but this has to be clarified.
Clinical signs and histopathology
The clinical signs and general histopathology of IHN in young salmonid fish are
well documented (Amend et al.,1969; Amend, 1970b, 1974; Yasutake, 1970,
1975; Pilcher and Fryer, 1980a,b; Nicholson, 1982; Wolf, 1988). In acute disease
there is a sudden increase in fish mortality, but the fish may not show clinical
signs and may die without apparent cause. More typically, at the start of an
epizootic, there are moribund fish that are lethargic, with periods of sporadic
whirling or hyperactivity. Moribund fry also may have a dark coloration, a
distended abdomen, exophthalmia, pale gills and mucoid, opaque faecal casts
(Fig. 2.1). Petechial haemorrhages may be observed at the base of the fins and
vent and occasionally in the gills, mouth, eye, skin and muscle. In chinook
salmon, but not in sockeye salmon, the fry may have a subdermal haemorrhagic
area immediately behind the head (Yasutake, 1970; Amend, 1974). Many of the
above clinical signs are similar to those of infectious pancreatic necrosis (IPN)
and viral haemorrhagic septicaemia (VHS). In older fish there are fewer external
clinical signs. Two-year-old kokanee salmon have erratic swimming and
haemorrhages near the base of the fins (Traxler, 1986), and sockeye salmon
smolts have gill and eye haemorrhages, clubbed and fused lamellae and
cutaneous lesions (Burke and Grischkowsky, 1984).
The liver, spleen and kidney of fry are pale due to anaemia; there may be
ascites; and the stomach is filled with a milky fluid but no food. The intestine
contains a watery, yellowish fluid and there may be petechial haemorrhages in
the visceral mesenteries, adipose tissue, swim-bladder, peritoneum, meninges
and pericardium (Rucker et al., 1953; Ross et al., 1960; Wolf, 1988). Older fish
may have empty stomachs, intestines filled with yellowish mucus and lesions in
the musculature near the kidney (Traxler, 1986).
The haematopoietic tissues of the kidney and spleen of young fish are the
most severely affected and are the first tissues to show extensive necrosis
(Amend et al., 1969; Yasutake, 1970, 1975). In recent studies, the gills,
oesophagus/cardiac stomach region (OCSR), small intestine and pyloric caeca
of rainbow trout and coho salmon fry were examined within 24 h after
immersion in IHNV (Helmick et al., 1995a, b). No pathological changes were
observed in the gills, small intestine or pancreatic acinar cells. However,
pathological changes were observed in the OCSR mucus-secreting serous
cardiac glands (MSSG) and epithelial cells of both species. In a study with
steelhead trout fry, IHNV was transiently detected using immunohistochemistry
in the epithelial cells of most organs, but it had a propensity for the connective
tissue (Drolet et al., 1994). Virus was detected in the anterior kidney 1 day prior
to detection in the posterior kidney. This was also observed by Yasutake and
Amend (1972) in sockeye salmon. The first changes in the anterior kidney are
Fig. 2.1. Clinical signs of IHN infection. The diseased fish show darkening of the dorsal body
with petechial haemorrhaging on the body near the operculum and at the vent (diseased fish at
top). Exophthalmia is common (diseased fish in middle) and scoliosis is seen in some of the fish
(diseased fish at bottom).
64
small, lightly stained, focal areas, consisting of what appear to be macrophages
and degenerating lymphoid cells. As the disease progresses, degenerative
changes throughout the kidney become more noticeable. Macrophages increase
in number and may have a vacuolated cytoplasm and chromatin margination of
the nuclei (Klontz et al., 1965; Yasutake and Amend, 1972). There may also be a
decrease in the number of non-differentiated blast cells, and pyknotic and
necrotic lymphoid cells may be present. Focal areas of cells in the spleen,
pancreas, liver, adrenal cortex and intestine show nuclear polymorphism and
margination of the chromatin, with eventual necrosis (Amend et al., 1969;
Yasutake, 1975; Wolf, 1988). Necrosis may be so severe that the kidney tissue
consists primarily of necrotic debris (Yasutake, 1975; Wolf, 1988). Extensive
necrosis in all organs is accompanied by pyknosis, karyorrhexis and karyolysis
(Yasutake and Amend, 1972). A pathognomonic feature of IHN is degeneration
and necrosis of granular cells in the lamina propria, stratum compactum and
stratum granulosum of the alimentary tract (Yasutake, 1970; Wolf, 1988) and it
is postulated that sloughing of intestinal mucosa may give rise to faecal casts
(Amend et al., 1969; Yasutake, 1970, 1975). In chinook and sockeye salmon fry
there may also be hepatic deposits of ceroid (Wood and Yasutake, 1956;
Yasutake, 1970). In the final stages of disease, necrosis is not only in the
haematopoietic tissue of the kidney, but also in the glomeruli and kidney tubules.
There is little effect on the corpuscles of Stannius tissue (Yasutake, 1975; Wolf,
1988). Smolts and yearlings tend to show less severe histopathological changes.
The kidney, spleen, pancreas and liver may show necrosis, but there is only
moderate sloughing of the intestinal mucosa and no faecal casts (Yasutake,
1978; Burke and Grischkowsky, 1984; Traxler, 1986).
In spawning sockeye salmon, Yamamoto et al. (1989) found that the spleen
and kidney did not show the massive necrosis observed in fry. Instead, the
kidney had no tissue destruction and the spleen had small focal infections near
the periphery or at the outer cell layer. The connective tissues surrounding the
spleen were also infected. Gill tissue had highly localized lesions, with some gill
filaments having infection limited to the non-differentiating serosal cell layer,
which lies internal to the epithelial pavement cell layer.
The number of monocytes is not affected, but there is leucopenia, with
degenerating leucocytes and thrombocytes. Neutrophils are decreased or absent,
the number of immature erythrocytes is increased and the cell may be bilobed
(Watson et al., 1954; Wood and Yasutake, 1956; Holway and Smith, 1973;
Amend, 1974; Amend and Smith, 1975). Amend (1973) suggested that the fish
had a normocytic aplastic anaemia. Cellular debris, termed necrobiotic bodies,
observed in blood smears or kidney imprints is pathognomonic for IHN (Holway
and Smith, 1973; Amend and Smith, 1974, 1975; Wolf, 1988). The blood
haematocrit, haemoglobin content, osmolarity and levels of bicarbonate,
calcium, phosphorus, chlorides and bilirubin are decreased in infected fish.
Lactic acid dehydrogenase levels may increase (Amend, 1974). However, there
are no changes in kidney ascorbate levels, total plasma protein, mean
corpuscular volume, mean corpuscular haemoglobin or levels of plasma
glucose, esterase, glutamic oxylate transaminase or peptidase isozymes (Amend
and Smith, 1974, 1975).
Factors affecting transmission and virulence
Fish typically become more resistant to IHNV as they increase in age and weight
(Amend, 1974; Amend and Nelson, 1977; Leong and Turner, 1979; Wolf, 1988;
LaPatra et al. 1994a). Yolk-sac fry and fish up to 2 months of age are highly
susceptible, with mortality often over 90%. Older fish up to 6 months of age
typically have less than 50% mortality. After experimental IHNV exposures, fry
mortality generally increases as the viral dose increases from 10
2
to 10
56
pfu
ml
l
(Chen et al., 1990; LaPatra et al., 1990a, 1993a). Infectious haematopoietic
necrosis virus can kill juvenile to 2-year-old sockeye salmon (Yasutake, 1978;
Burke and Grischkowsky, 1984), kokanee salmon (Traxler, 1986; Banner et al.,
1991) and rainbow trout (Busch, 1983; Roberts, 1986), but mortality is often
very low.
The viral electropherotype and the host fish species are not definite
indicators of IHNV virulence (LaPatra et al., 1989a, 1990a; Chen et al., 1990;
Traxler et al., 1993). In general, type 1 IHNV isolates are most virulent in
kokanee and sockeye salmon and type 2 isolates are more virulent in rainbow
trout and steelhead trout than types 1 or 3 (LaPatra et al., 1990a,b, 1993a). For
type 2 IHNV, susceptibility of kokanee and sockeye salmon increases, but
susceptibility of rainbow trout decreases, with increasing fish age and weight
(Amend and Nelson, 1977; LaPatra et al., 1990a, 1991a). Chinook salmon are
more susceptible to type 3 isolates, but virulence of isolates within the same
electropherotype is variable (Chen et al., 1990; LaPatra et al., 1993a). Viral
virulence for a fish species appears to be dependent on the geographical location
of virus isolation. Type 3 isolates from California and southern Oregon are more
virulent for chinook salmon than for steelhead trout, but type 3 isolates from the
Columbia River are more virulent for steelhead trout than for chinook salmon
(LaPatra et al., 1993a). In addition to electropherotyping of IHNV, isolates can
be divided into antigenic groups, based on reactivity with monoclonal antibodies
(MAbs) (Winton et al., 1988; LaPatra et al., 1991a; Ristow and Arnzen-de Avila,
1991). LaPatra et al. (1994a) divided 106 type 2 isolates from the Hagerman
Valley, Idaho, into ten antigenic groups. Virulence of isolates from the first seven
antigenic types induced a wide range (1492%) of mortality in rainbow trout.
Forecasting the virulence of an isolate based on IHNV type or geographical
location of isolation would not be completely accurate. Each IHNV isolate
should be typed and evaluated for virulence in different fish species.
Fish genetics has a large influence on IHNV susceptibility. Wertheimer and
Winton (1982) demonstrated that chinook salmon fry from a Washington stock
were more susceptible to two IHNV isolates than two stocks from Alaska.
Amend and Nelson (1977) also noted distinct differences in IHNV susceptibility
in families of sockeye salmon fry. In highly susceptible families, mortality was
98% or more, while in other families, the mortality was 52%. McIntyre and
Amend (1978) found that the heritability of resistance to IHNV was about 30%.
Family-level differences in IHNV susceptibility have also been observed in
rainbow trout (Yamamoto et al., 1991; Kasai et al., 1993).
Interspecific hybrids have been made between resistant and susceptible
species to enhance resistance to IHNV. Hybrids of coho salmon rainbow trout
66
(Parsons et al., 1986; Chen et al., 1990; LaPatra et al., 1993c), brook trout
rainbow trout (Dorson et al., 1991; LaPatra et al., 1993c) and cutthroat trout
rainbow trout (LaPatra et al., 1994b) have increased resistance to IHNV.
However, it is also possible to obtain hybrids that have equal or higher
susceptibility than the parental species. Such was the case for coho salmon
chinook salmon (Hedrick et al., 1987). The most recent cross of brown trout
females lake trout males produced brake trout and these triploid hybrid
progeny were significantly more resistant to IHNV than rainbow trout after
immersion or injection of IHNV (LaPatra et al., 1996). Brake trout immersed in
IHNV had a weaker antibody response than rainbow trout, and it was postulated
that protection was occurring at the cellular level. After i.p. injection of IHNV,
the hybrids mounted a strong antibody response and had less extensive and
severe necrosis of the haematopoietic tissue compared with rainbow trout.
Hybrids generally have poorer performance in culture compared with parental
species; however, hybrids can provide a model system for examining resistance
mechanisms (LaPatra et al., 1996).
High fish density is correlated with outbreaks of IHN, possibly due to a
rapid horizontal spread of the virus. The density of adult fish in a spawning
channel may be a better predictor of outbreaks in fry than finding IHNV in adults
(Traxler and Rankin, 1989). When artificial spawning beaches are stocked with
two densities of sockeye salmon, IHN occurs only in the fry which have the
higher density of adult spawners (Olson and Thomas, 1994). In addition to adult
density, the egg density is also an important determinant of IHN outbreaks.
Sockeye salmon eggs at a medium or high density in incubation boxes can result
in IHN epizootics, but fry from the low-egg-density boxes are not infected
(Mulcahy and Bauersfeld, 1983). In natural spawning, egg and alevin densities
in the gravel are very low. There is one report of IHN epizootics in emergent
sockeye salmon fry in Chilko Lake, British Columbia (Williams and Amend,
1976), which suggests that factors other than fish density may be involved.
In Oregon and Washington, fry, smolts and adult salmonids are often moved
around dams by truck or barge. The high fish density during transport would
provide an excellent opportunity for horizontal virus transmission. There
appears to be only one study where water in transport trucks was tested for
IHNV. Using tangential flow filtration, an IHNV concentration of 1000 pfu ml
1
was found in the water from a truck transporting sockeye salmon fry (Batts and
Winton, 1989b), compared with 20 pfu ml
1
in the spawning beach water in
which the fry hatched (Olson and Thomas, 1994). These fry were infected with
IHNV prior to shipping and, 1014 days after shipping, fry mortality reached
100%. It is not certain whether fish density in the transport truck or IHNV in the
water exacerbated fish mortality. Under experimental conditions, small
differences in fry density do not affect IHN mortality. Groups of 25 or 50
rainbow trout fry immersed in IHNV did not have a significantly different
mortality or mean day to death (LaPatra et al., 1991b). However, a wider range
of fish densities must be evaluated to clarify the importance of fry density on
mortality.
The most important environmental factor affecting IHNV virulence is
temperature. Infectious haematopoietic necrosis epizootics most frequently
occur during the spring and autumn when water temperatures are 1012C
(Nicholson, 1982). Although it is generally accepted that disease outbreaks do
not occur above 15C, IHN killed rainbow trout fry from 3C to 18C. The mean
day to death was directly related to temperature in that the higher the
temperature, the shorter the survival of the fish (Hetrick et al., 1979a). In
contrast, rainbow trout and sockeye salmon held at temperatures above 15.5C
before infection or moved to a higher temperature within 24 h after exposure had
significantly reduced mortality. However, if virus was detectable in the fish, then
a shift to a higher water temperature had little effect on mortality (Amend,
1970a, 1976). Results may have varied between studies because of differences in
experimental design and fish size.
The nutritional status of the host and other environmental factors, such as
water salinity and the presence of pollutants, may influence susceptibility to
IHNV. Although the majority of IHN outbreaks occur in fresh water, IHN
epizootics have been reported in Atlantic salmon held in ocean net pens (Traxler
et al., 1991). Stress, caused either by physical handling or acute environmental
changes, is known to decrease fish resistance to disease. Exposure of rainbow
trout to copper increases IHN mortality. The copper may cause an increase in
cortisol levels, and this in turn may cause a depression of the immune response
(Hetrick et al., 1979b). In contrast, exposure of rainbow trout to polychlorinated
biphenyls or 2,3,7,8-tetrachlorodibenzo-p-dioxin, which are immunosuppres-
sive in mammals, did not significantly affect mortality or mean day to death due
to IHNV (Spitsbergen et al., 1988).
One factor that does not appear to affect IHNV virulence is the cell line used
for viral propagation. A type 2 IHNV isolate grown in rainbow trout gonad
(RTG-2), chinook salmon embryo (CHSE-214) or epithelioma papulosum
cyprini (EPC) cells had equivalent virulence for rainbow trout (LaPatra et al.,
1991b). It was also suggested that the IHNV glycoprotein is not responsible for
virulence, because the nucleic acid sequence is highly conserved (LaPatra et al.,
1993a). However, small differences in the amino acid sequence of the
glycoprotein can affect virulence. Neutralization-resistant (escape) mutants,
selected by incubation in neutralizing anti-glycoprotein MAb, often had
decreased virulence and changes in tissue tropism in comparison with the
homologous wild-type virus (Roberti et al., 1991; Kim et al., 1994). In addition
to detecting differences in the glycoprotein, MAbs have detected heterogeneity
in the nucleoprotein (Ristow and Arnzen, 1989; Ristow and de Avila, 1991). The
relation between virulence and heterogeneity in the nucleoprotein has not been
tested.
Infectious haematopoietic necrosis virus growth in vitro
and physical properties
There are several excellent reviews on work prior to 1991 that describe the
biophysical properties of IHNV and its stability to various chemical and physical
agents (Pilcher and Fryer, 1980a; Wolf, 1988; Winton, 1991).
68
Infectious haematopoietic necrosis virus in cell culture
Cell culture of IHNV was first made using salmonid fish cell lines, such as
CHSE-214 (Fryer et al., 1965) and RTG-2 (Wolf and Quimby, 1962). These cells
were used to isolate the virus from infected fish (Wingfield et al., 1969). The
virus has since been found to grow well in fish cell lines such as EPC, fathead
minnow (FHM), bluegill fry (BF-2) and steelhead trout embryo (STE-137) cells
at temperatures ranging from 4 to 20C. Other cell lines that are susceptible to
IHNV cytopathogenicity include CHSE-114, sockeye salmon embryo (SSE-5),
SSE-30, kokanee salmon ovary (KO-6), chum salmon heart (CHH-1), rainbow trout
hepatoma (RTH-149), guppy embryo (GE-4), coho salmon embryo (CSE-119),
rainbow trout spleen (RBS), rainbow trout fry (RTF-1) and Atlantic salmon (AS)
cells (Wolf and Mann, 1980; Lannan et al., 1984; Wolf, 1988). The optimum
temperature for growth is approximately 15C (Mulcahy et al., 1984a) and 2325C
does not support viral replication. The virus has been replicated in baby hamster
kidney (BHK/21), reptilian (Clark and Soriano, 1974), Drosophila melanogaster
(Bussereau, 1975) and Aedes albopictus cells at 16C (Scott et al., 1980).
A one-step growth curve in CHSE-214 cells at 18C, reported by McAllister
et al. (1974), showed new virus production by 4 h postinfection, followed by an
exponential release of virus until approximately 16 h postinfection, when the
maximum viral titre reached about 10
7
pfu ml
1
. Different cell lines may produce
viral titres of 10
8
10
8.5
pfu ml
1
, but the majority of cell lines produce viral titres
of approximately 10
7
pfu ml
1
.
Cell cytopathology is evident at 1015 h postinfection and is character-
istically one of cells looking like balloon-shaped cells with nuclei pushed to one
side of the balloon (Engelking and Leong, 1981). Surrounding a region destined
to become a plaque, the affected cells appear like grapes clustered at the
periphery of a cleared region where the infected cells have pulled away from the
surface substrate.
Infectious haematopoietic necrosis virus genome structure and
transcription
VIRAL STRUCTURE
The virions of IHNV are typically bullet-shaped, with measurements of 110 nm
70 nm in fixed and thin-sectioned preparations of the virus in infected cell
cultures, and 111 nm 11 nm in negatively stained preparations of purified virus
(Hill et al., 1975).
Infectious haematopoietic necrosis virus is one of the first fish rhabdo-
viruses to be characterized biochemically, and it has five structural proteins
(McAllister and Wagner, 1975). Each of these proteins was analysed using
SDS-polyacrylamide gel electrophoresis (PAGE), and the relative migration
pattern of the virion proteins tentatively placed IHNV in the lyssavirus genus of
the family Rhabdoviridae (Wunner and Peters, 1991). In 1995, it was listed as an
unassigned member of the family (Wunner et al., 1995). The five virion proteins
include a high-molecular-mass L or polymerase protein (150225.2 kDa), a
glycoprotein G (6770 kDa), a phosphorylated nucleoprotein N (40.544 kDa),
a phosphorylated Por M1 protein (22.527 kDa) and a matrix protein M or M2
(17.521.8 kDa). A non-virion protein NV of 12 kDa has also been identified in
infected cells (Kurath and Leong, 1985; Schutze et al., 1996). This protein is
missing in the prototype rhabdoviruses that infect mammals and distinguishes
IHNV from other members of the lyssavirus genus (Wunner et al., 1995).
An estimate of the number of molecules per virion for each protein was
made for IHNV (Leong et al., 1983a). The ratio of viral protein to ribonucleic
acid (RNA) is 21 : 1, a very low figure in comparison with vesicular stomatitis
virus (VSV) and rabies virus (RV), which have ratios of 92 : 1 to 72 : 1,
respectively (Bishop and Roy, 1972; Coslett et al., 1980). The low protein-to-
RNA ratio for IHNV is unusual and may reflect differences in the membrane
structure of fish and mammalian cells (Moore et al., 1976). It is similar to that
obtained for La Crosse virus, a bunyavirus, which has a ratio of 30 : 1 in BHK/21
cells (Obijeski et al., 1976).
The relative contribution of each protein to the total molecular weight of the
virion was estimated from densitometer tracings of SDS-polyacrylamide gels of
purified virus. For both silver- and Coomassie blue-stained gels, the relative
proportion of each virion protein was different from that reported by McAllister
and Wagner (1975) for
14
C-amino acid-labelled virus (Table 2.2). These
differences may be a result of differences in the virus strain. More probably it
reflects differences in the methods for determining the proportion of each virion
protein. The numbers of molecules of IHNV virion proteins are 2340 L, 198
290 G, 560774 N, 391514 P, and 8741044 M (Table 2.2). These are strikingly
Table 2.2. Estimated number and molecular mass of the IHN virion proteins
(modified from Leong et al., 1983b).
Percentage of Mol. mass of protein No. molecules
total virus proteins per virion 10
6
* per virion
Protein Mol. mass

species (kDa)
Incorp.
Stained
||
Incorp. Stained Incorp. Stained
L 150.0 4.6 8.1 3.4 6.1 23 40
G 67.0 17.8 26.1 13.3 19.5 198 290
N 40.5 42.0 30.4 31.3 22.7 774 560
P 22.5 11.8 15.5 8.8 11.6 391 514
M 17.0 23.8 19.9 17.8 14.9 1044 874
*Total virion protein was derived from a 21 : 1 ratio of virus protein to RNA
and the estimate of the viral genome molecular mass as 3.57 10
6
(G. Kurath
and J .C. Leong, unpublished observation).
Number of protein molecules per virion was calculated by dividing the

daltons of protein per virion by their molecular mass.
Molecular mass estimates were determined by electrophoretic migration in

SDS-polyacrylamide gels.
Percentage of each protein determined by using virus labelled with

14
C
amino acids (from McAllister and Wagner, 1975).
||
Percentage of each protein was calculated for four separate determinations
of the area under each peak of gels, stained with Coomassie blue or silver
and scanned at 620 nm. The average percentage of each protein for silver
staining and for Coomassie blue staining was again averaged for the final
result.
70
different from those for RV which are 79 L, 1723 G, 1975 N, 402 P and 1156 M
(Coslett et al., 1980). The remarkable difference is in the number of G molecules
per virion. It is difficult to produce high-titre neutralizing antibody to IHNV in
warm-blooded animals. The poor immunogenicity of G or the poor neutralizing
activity of the antisera may be a direct result of the low numbers of G molecules
on the surface of the IHNV virion.
VIRAL GENOME
Molecular analysis of RNA from purified IHNV (Round Butte (RB-1) strain
isolated from an Oregon steelhead trout) indicates a genome of approximately
11 kb, a size very similar to that of other rhabdoviruses (Kurath and Leong,
1985). However, unlike the prototype rhabdoviruses, VSV or RV, there appear to
be six instead of five viral genes. A complementary deoxyribonucleic acid
(cDNA) library constructed to the viral messenger RNAs (mRNAs) contains
clones for six viral genes. The sixth gene encodes a previously unknown viral
protein, NV or non-virion protein. These genes were identified by hybridization
to specific mRNA sequences in Northern blots and then used in R-loop mapping
studies to determine the viral genome gene order from 3 to 5 as NPMG
NVL (Kurath et al., 1985). Since these initial findings, the viral genes for G and
N for the RB-1 strain have been sequenced (Koener et al., 1987; Gilmore and
Leong, 1988) and, recently, the complete genome sequence of IHNV was
determined by Morzunov et al. (1995) on a Western Regional Aquaculture
Consortium (WRAC) IHNV isolate and by Schutze et al. (1995) on a KS-1
IHNV isolate. The viral genome is 11,131 nucleotides (nt) in length and contains
a 60 nt leader sequence at its 3 end and a 101 nt trailer sequence at its 5 end. The
3 and 5 ends of the IHNV genome are complementary, just like the ends of the
VSV and RV genome (Fig. 2.2), and are thought to provide the means for the
viral RNA to form panhandle structures for priming the initiation of RNA
synthesis (Banerjee and Barik, 1992).
The internal gene junctions of IHNV are different from those of other
rhabdoviruses. Typically, the gene junctions for the VSV and RV contain the
sequence, UC(U)
7
NNUUGU (Fig. 2.3). In contrast, the consensus intergenic
region for the fish rhabdoviruses is UC(U)
7
RCCGUG, where R is a T or C. Thus,
during mRNA synthesis, the polymerase, which initiates transcription at the
AACA sequence for both VSV and RV, initiates transcription at T/
C
GGCAC for
IHNV (P.-W.P. Chiou, unpublished data).
The viral sequences have been the basis for a phylogenetic analysis of IHNV
and its evolutionary relationship to other rhabdoviruses (Morzunov et al., 1995;
Bjorklund et al., 1996). A comparison of the G, M and L genes indicates that
there are three distinct clades in the Rhabdoviridae family tree. The members of
the vesiculovirus genus (VSV, Piry and Chandipura viruses) form one clade and
a second clade is composed of members of the lyssavirus genus (RV and Mokola
viruses). In addition, there is a third distinct and well supported clade which is
composed of the fish rhabdoviruses (IHNV, VHS virus (VHSV) and hirame
rhabdovirus (HIRRV) (Basurco and Benmansour, 1995; Kurath et al., 1997).
These findings and the discovery of a new viral gene encoding a non-virion
protein have prompted investigators to seek a new genus grouping for the fish
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72
rhabdoviruses. The new genus has been approved by the International
Committee on the Taxonomy of Viruses and named novirhabdovirus (Kurath et
al., 1997).
MESSENGER RIBONUCLEIC ACID TRANSCRIPTION
Six viral mRNA species have been identified in cells infected with IHNV, and
their molecular weights have been estimated in denaturing glyoxal or
methylmercury agarose gels (Kurath and Leong, 1985). The six species are: L
mRNA, 2.26 10
6
Da; G mRNA, 5.63 10
6
Da; N mRNA, 4.48 10
5
Da; P
mRNA, 3.00 10
5
Da; M mRNA, 3.00 10
5
Da; and NV mRNA, 1.95 10
5
Da.
The P and M mRNAs comigrate in both glyoxal and methylmercury gels. The N
mRNA is presumably the first mRNA species produced during viral replication,
since N is the first viral protein that appears in infected cells. When the relative
concentration of each mRNA species is measured at the height of virus
replication, the N concentration is 1.00, P and M mRNAs are 2.52 0.40, G
mRNA is 0.49 0.03; NV mRNA is 0.41 0.14 and L mRNA is 0.02 0.01
(Kurath and Leong, 1985).
The mRNAs of rhabdoviruses are considered to be monocistronic although
read-through transcription products have been identified for VSV (Masters and
Samuel, 1984), RV (Ravkov et al., 1995), and the ephemeroviruses (Wang et al.,
1995). No read-through transcripts have been identified for IHNV (P.P.-W.
Chiou, unpublished data). However, there is a possibility that a single IHNV
mRNA species might encode more than one translation product. Since there
are many examples of different translation products either encoded in different
reading frames, initiated at a different start codon or resulting from an internal
frame shift that leads to a hybrid protein product, the possibility that there are
additional proteins encoded by the IHNV genome is very high. For example,
the paramyxoviruses and rhabdoviruses have encoded in their P mRNA
transcripts for more than one protein (Lamb et al., 1976; Curran et al., 1992;
Spiropoulou and Nichol, 1993). In fact, a seventh protein, S for small protein,
at 6.5 kDa, has been described in IHNV-infected cells (Chiou, 1996). Whether
this S protein is encoded by one of the IHNV mRNA transcripts has not been
determined.
An in vitro transcription system for purified virions of IHNV was first
described by McAllister and Wagner in 1977. They described a heterogeneous
array of IHNV-specific transcripts, ranging in size from 9S to 17S with no
discrete species identified. Later studies by Kurath and Leong (1987)
demonstrated that optimal polymerase activity required HEPES buffer
supplemented with S-adenosyl-L-methionine. In this case, RNA transcripts
contained polyadenylated species, which comigrated with the IHNV N, P, M, G
and NV mRNAs from IHNV-infected cells. These transcripts were fully
functional in translation reactions with rabbit reticulocyte extracts to produce N,
P and M proteins.
The process by which IHNV replicates its viral genome is unknown. If the
process is similar to that of VSV, then the viral N protein provides the trigger that
switches the RNA transcription process from mRNA synthesis to progeny
genome plus synthesis. The viral N protein is present in such large quantities in
the late phase of viral replication that it binds to the nascent RNA transcript and
prevents the viral polymerase from recognizing the mRNA transcription
termination signals. This results in the synthesis of a full length plus strand
template for the production of progeny minus strand viral genomes.
VIRAL GENE EXPRESSION
Infection of salmon or trout cells with IHNV results in the inhibition of cellular
protein synthesis. In this characteristic, IHNV differs from other members of the
RV group of the Rhabdoviridae. Cellular protein synthesis is not inhibited after
infection with RV (Coslett et al., 1980) and any studies of RV protein synthesis
in the cell require exposure to hypertonic shock to reduce the background of host
protein synthesis. However, it has been possible to examine the synthesis of
IHNV proteins in the cell without resorting to this drastic treatment. In
pulse-labeling studies with
35
S-methionine, the first protein of IHNV to appear in
the course of infection is the N, or nucleocapsid, protein, at 23 h after infection
(Hsu et al., 1985). At 67 h after infection, the P and M proteins can be identified
in autoradiograms. The two forms of the glycoprotein, G1 and G2, representing
different glycosylation states of the protein, are found at 910 h after infection. It
is not possible to distinguish the virion L protein from other host proteins in the
gel until 15 h postinfection, when cellular host protein synthesis is completely
inhibited. Virus production begins 12 h after infection (Leong et al., 1981a).
The migration of the structural proteins of IHNV varies among different
isolates of the virus and the differences in migration patterns form the basis for
distinguishing different IHNV isolates by their electropherotype. The molecular
weights of the N and G proteins for the different isolates were used to type
serologically similar strains of IHNV (Table 2.2).
VIRAL PROTEINS
Nucleocapsid protein. The N protein is the most abundant viral protein in the
IHN virion and in virus-infected cells (Table 2.2). It is present as a phosphory-
lated protein in the virion (McAllister and Wagner, 1974; Hsu et al., 1985) and is
found tightly associated with the viral genome (Engelking and Leong, 1989a,b).
The complete nucleotide sequence of the N gene has been determined for three
different isolates of IHNV (RB-1 isolate, Gilmore and Leong, 1988; WRAC
isolate, Morzunov et al., 1995; KS-1 isolate, Schutze et al., 1995) from cDNA
clones to the viral N mRNA. The N gene encodes a protein of 413 amino acids
which is highly hydrophilic at its amino and carboxyl terminal ends, with a
hydrophobic region in the middle third of the protein (Fig. 2.3). It has been
speculated that the carboxyl terminal end of the IHNV N protein may be
involved in binding of the N protein to the viral RNA (Gilmore and Leong,
1988). For the prototype rhabdovirus VSV, the N protein has been shown to play
a crucial role in regulating the balance between viral transcription and
replication.
Diagnostic tests have been developed for the viral N protein and nucleic acid
sequence because the N protein is the first viral protein synthesized, as well as
the most abundant viral protein (Rose and Schubert, 1987).
74
Phosphoprotein. The P or M1 protein is a phosphorylated protein of 230 amino
acids with a calculated molecular weight of 25.6 kDa (McAllister and Wagner,
1975; Hsu et al., 1985; Ormonde, 1995). Sequence analysis of the gene encoding
the P protein for IHNV RB-1 and KS-1 strains indicate that the deduced amino
acid sequence contains 45 potential phosphorylation sites, SXXD/E, which are
found predominantly in the first half of the protein (Ormonde, 1995). The M1
protein is a very basic protein with an estimated isoelectric point (pI) of 8.4,
similar to that reported for the VHSV (Makah strain) M1 protein (Benmansour et
al., 1994; Ormonde, 1995). The basic nature of these proteins contrasts sharply
with the RV and VSV phosphoproteins, which have pIs of 4.36 and 4.84,
respectively. The M1 or P proteins of the RB-1 strain and the European KS-1
strain of IHNV share a high degree of similarity, with 94% identity at the amino
acid level (Ormonde, 1995; Morzunov et al., 1995). When compared with the
M1 proteins for HIRRV and VHSV, a 63% and 38% similarity was observed
between these proteins and the IHNV M1 protein (Ormonde, 1995).
Sequence analysis of the M1 genes of different IHNV strains indicates that
there might be a second overlapping open reading frame (ORF) encoding a
highly basic, arginine-rich protein, with an estimated pI of 10.112.8. Located at
position 121 from the end of the polyadenylated sequence of the N gene, this
second ORF is 146 nucleotides in length and has the potential to encode a 42
amino acid protein with an estimated molecular weight of 4.8 kDa. The protein
is similar in size to the 55 amino acid, arginine-rich C protein reported for VSV
(Spiropoulou and Nichol, 1993) and RV (Chenik et al., 1995). The VSV C
protein is also encoded by the P mRNA transcript in a second overlapping ORF
within the gene. These proteins are found only in infected cells and their function
is currently not known.
Matrix protein. The IHNV matrix protein (M), previously called M2, is similar
to all reported rhabdovirus matrix proteins (Ormonde, 1995). The M protein is
encoded by a sequence of 585 nucleotides to yield a protein of 195 amino acids,
with 21.8 kDa estimated molecular weight. It is very basic, with an estimated pI
of 10.08, similar to the M proteins of the other rhabdoviruses, and contains a
high concentration of charged amino acids in the amino terminal half of the
protein. Highly charged amino termini have also been found in the
vesiculoviruses and the lyssaviruses (Rose and Gallione, 1981; Bourhy et al.,
1993) and in paramyxoviruses (Chambers et al., 1986). In VSV, the first 51
amino acids were required for stable interaction with the plasma membrane
(Chong and Rose, 1994), for viral assembly (Black et al., 1993) and possibly in
the inhibition of RNA transcription (Ogden et al., 1986). This region is also
conserved among IHNV, HIRRV and VHSV matrix proteins and whether this
region is involved with viral assembly, transcription inhibition and membrane
interaction is unknown.
The deduced IHNV M2 amino acid sequence does not share significant
homology with either VSV, RV or the fish vesiculovirus, spring viraemia of carp
virus (SVCV). However, it shares a 74% amino acid identity with HIRRV M2
protein and a 37% identity with the VHSV M2 protein at three localized regions
of homology (Fig. 2.4) (Ormonde, 1995). Sequence conservation is highest in
the N terminal region of the first 29 amino acids, which contains a large number
of basic residues. This feature has also been described for the amino termini of
RV and VSV.
Glycoprotein. The IHNV glycoprotein G is a membrane-associated protein
which forms spike-like projections on the surface of the mature virion
(McAllister and Wagner, 1975). Antiglycoprotein serum neutralizes viral infect-
ivity, and immunization with purified glycoprotein prevents subsequent lethal
infection with IHNV (Engelking and Leong, 1989b). Immunological studies
with polyvalent antiglycoprotein sera have indicated that the glycoproteins are
conserved among different geographical isolates of IHNV (Hsu and Leong,
1985) and that there is only one major serotype with several serovariants
(Engelking and Leong, 1989a). This finding has been confirmed by studies with
MAb (Huang et al., 1996) and sequence analysis of the G and NV genes (Nichol
et al., 1995).
The IHNV glycoprotein has many of the features characteristic of
membrane associated glycoproteins of negative-stranded RNA viruses. The
predicted translation product of the IHNV G gene is a protein of 508 amino
acids, with a hydrophobic domain of 20 amino acids at the N terminus forming
the signal peptide. This includes a central core of hydrophobic amino acid
residues, in the form of three repeated pairs of LeuIle (Koener et al., 1987) and
a consensus signal peptide cleavage site, AlaAsnSer, at position 18. The
arrangement of the amino acids in this region is consistent with the signal
Fig. 2.4. Alignment of the deduced amino acid sequences of IHNV (RB-1), hirame rhabdovirus
(HIRRV) and viral haemorrhagic septicaemia virus (VHSV) matrix proteins (M2). Sequences were
aligned with the Clustal IV alignment option of the Genetic Data Environment (GDE) computer
analysis software package. Asterisks identify identical amino acid residues.
76
peptidase cleavage sequences identified by Perlman and Halvorson (1983).
There is an additional hydrophobic domain near the C terminus, which is the
presumed transmembrane domain of the protein.
A comparison of the predicted amino acid sequences of the glycoproteins of
the vertebrate rhabdoviruses suggests that these proteins are highly similar in
structure. The cysteine residues of the IHNV G protein are highly conserved
amino acids, whose positions in the G proteins of VSV-New Jersey (VSV-NJ),
VSV-Indiana (VSV-Ind) and RV are largely identical. In the case of VSV-Ind,
12 of 15 cysteine residues are aligned with IHNV at 12 of its 16 cysteine
residues. For RV, nine were aligned with the 17 cysteine residues in the IHNV G
protein. When the positions of the proline residues were aligned, approximately
one third of the proline positions between RV and IHNV, as well as between
VSV and IHNV, were identical. However, a hydropathy plot of the G proteins
did reveal a striking difference between them. The profiles revealed a very
hydrophilic domain extending from IHNV G amino acid 365 to 450, which was
not evident in either VSV or RV. Within this region were two possible
glycosylation sites and no cysteine residues. The importance of this region as a
possible antigenic domain was first suggested by Koener et al. (1987).
A more recent study by Bjorklund et al. (1996) expanded the comparison of
the fish rhabdovirus glycoproteins to the glycoproteins of other rhabdoviruses.
The study included IHNV, SVCV, HIRRV, Chandipura virus, Adelaide River
virus, bovine ephemeral fever virus, VHSV-07-71, VHSV-DK, Mokola virus,
sigma virus, Sonchus yellow net virus, VSV-NJ, VSV-Ind, RV-ERA and RV-PV.
The analysis found that the amino acids cysteine, proline, glycine and leucine
were conserved in the aligned glycoprotein sequences.
Non-virion gene. The NV gene and its encoded protein was first identified in
1985 (Kurath and Leong, 1985; Kurath et al., 1985). Located between the G and
L genes, NV is 371 nt in length and encodes a protein of 111 amino acids. The
overall pI of the deduced protein is approximately 7, despite the fact that the
protein has a high content of charged amino acids (38%) (Nichol et al., 1995).
An NV protein has also been identified at the GL junctions of HIRRV and
VHSV, a finding that has prompted scientists to propose a new taxonomic
classification for the salmonid fish rhabdoviruses (Schutze et al., 1996; Kurath
et al., 1997). The gene is highly conserved. Among 14 different IHNV isolates,
comprising 13 isolates from North America and one from Europe, more than
97% identity at the amino acid level was observed (Nichol et al., 1995; Schutze
et al., 1995; Chiou, 1996; Kurath et al., 1997). The NV amino acid identity/
similarity values between IHNV and VHSV or HIRRV indicate that this protein
is highly conserved, with identity/similarity values of 23.3%/47.6% and 16.5%/
40.4%, respectively (Kurath et al., 1997). There are sufficient differences in the
NV genes among the different IHNV isolates for ribonuclease (RNAse)
protection assays to have been used to distinguish the isolates (Kurath et al.,
1995).
Characterizing the expression of the NV protein in infected cells has been
problematic. In the original report by Kurath and Leong (1985), the NV protein
was clearly observed in autoradiograms of SDS-PAGE gels containing lysates of
infected cells labelled with
35
S-methionine or the in vitro translation products of
mRNA from infected cells. Subsequent reports have shown that the expression
of NV is very low or below detection limits for IHNV by radiolabelling (Chiou,
1996), VHSV (Basurco and Benmansour, 1995) and HIRRV (Nishizawa et al.,
1991a, b). More recently, NV protein expression was detected in cells infected
by either IHNV or VHSV by immunofluorescence and Western immunoblot
(Schutze et al., 1996).
Although there has been much speculation concerning the function of NV
(Kurath and Leong, 1985; Nichol et al., 1995; Chiou, 1996), the role NV plays in
the replication of the virus remains unknown. No functional motifs have been
identified in the protein sequence and no significant homology to other protein
sequences in the GenBank database has been uncovered (Basurco and
Benmansour, 1995; Nichol et al., 1995). Thus, until investigators are able to
knock out NV gene function during replication in vivo, it will not be possible to
determine what role NV plays in the IHNV life cycle.
Polymerase. The L gene (nt 5016 to nt 10,976) of IHNV encodes a protein of
1986 amino acids with an estimated molecular mass of 225.2 kDa (Bjorklund et
al., 1995; Morzunov et al., 1995; Schutze et al., 1995). The IHNV polymerase is
similar to the VSV polymerase, which has been shown to be solely responsible
for all transcriptional activities. Thus, the IHNV polymerase should also be
capable of RNA-dependent RNA polymerization, cap methylation and poly-A
polymerization. The deduced amino acid sequence contains the six conserved
blocks of amino acids identified as conserved sequences in negative stranded
RNA polymerases (Poch et al., 1990). Any dissection of polymerase function
will require the isolation of a functional polymerase cDNA clone, a feat that has
not yet been accomplished.
Serotype analysis and strain characterization
Early work on the characterization of different isolates of IHNV with polyclonal
antisera indicated that there was only one serotype of the virus and thus there
was no means of distinguishing the different viral isolates (McCain et al., 1971).
It was not until the work of Hsu et al. (1985, 1986) that a means of distinguishing
the different isolates was developed. Based on the migration patterns of the IHN
viral proteins on SDS-polyacrylamide gels, IHNV typing resulted in the
identification of five electropherotypes (Table 2.3). This provided the first
evidence that the viral type was geographically defined and all host species in
the area would carry the same viral type.
Further verification that there was only one serotype of IHNV was carried
out with antisera to the viral glycoprotein. The IHNV glycoprotein had been
shown to be the only viral protein capable of eliciting a neutralizing antibody
response in rabbits and providing protective immunity in young fish (Engelking
and Leong, 1989a,b). Purified glycoprotein from IHNV-RB-1 protected the fish
against challenge with a potentially lethal IHNV infection (Engelking and
Leong, 1989a). The work was extended to show that the purified glycoprotein
from IHNV-RB-1 also induced protective immunity against the five IHNV
electropherotypes by immersion vaccination (Engelking and Leong, 1989b).
78
These findings suggested that there was only one serotype of IHNV. A
comparison of the neutralization indices with ten different isolates of IHNV
representing all five electropherotypes against anti-IHNV-RB-1, anti-
IHNV-CO-2, anti-G protein of IHNV-RB-1, and anti-G protein of IHNV-CO-2
sera indicated that there was only serotype. However, the four antisera did define
two groups: a group of readily neutralized IHNV isolates, including RB-1(type
1), CO-2 (type 4), Elk River (ER) (type 3), CD-2 (type 5), HA-1 (type 2) and TA-
1 (type 1), and a group of less readily neutralized virus isolates, including DW-2
(type 3), DW-3 (type 3), LE (type 2) and NS (type 3).
Monoclonal antibodies have replaced electropherotyping for characterizing
isolates of IHNV. Winton et al. (1988) separated 12 IHNV strains into four
groups, using three MAbs. Further characterization of 12 different IHNV
isolates with seven MAbs produced ten different reactivity patterns (Nichol et
al., 1995). The atypical IHNV isolate, HO-7, was not neutralized by any of the
seven MAbs. S. Ristows group at Washington State University examined 17
isolates of IHNV with a bank of five glycoprotein-reactive MAbs (Ristow and
Arnzen-de Avila, 1991). Twelve of the 17 isolates were neutralized by two MAbs
(3GH127B and 3GH92A) and, of the 12, one was the only isolate neutralized by
a single MAb (1GH131A). Five isolates were not neutralized by any of the
MAbs. Two MAbs did not neutralize any of the virus isolates (3GH135L and
2GH5F). When the same 17 isolates were examined with a bank of 15 MAbs
reactive with either the N or G proteins, 15 distinct reaction patterns were
observed. All of these studies supported the conclusion that strains of IHNV
were distributed geographically. Different species in a geographical location
harboured the same type of IHNV.
Kurath and her colleagues are now utilizing RNAse protection assays to
evaluate the differences in strains of IHNV (Kurath et al., 1995). Oshima et al.
(1995) used two-dimensional oligonucleotide patterns to assess genetic diversity
among different IHNV isolates. All of these techniques have confirmed the
finding that IHNV strains are geographically confined and the appearance of a
new strain in an area might indicate the introduction of IHNV-infected fish or
eggs.
Table 2.3. Molecular mass characteristics of proteins used to type IHNV.
Virion
protein 1 2 3 4 5
L 150 150 150 150 150
G 67 67 67 70 67
N 40.5 42.8 43.25 40.541.0 41.044.0
P 27 27 27 27 27
M 21 21 21 21 21
Type strain RB-1 RA ER CO CO-3
RB-1, Round Butte 1; RA, Rangen; ER, Elk River; CO, Coleman Hatchery, 1980;
CO-3, Coleman Hatchery, 1979.
Mol. masses (kDa) in type
DIAGNOSIS OF INFECTIOUS HAEMATOPOIETIC
NECROSIS DISEASE AND VIRUS
If there is a previous history of IHN at the facility and the fish are showing
characteristic clinical signs, IHNV may be the cause of a disease outbreak.
However, the preliminary diagnosis should be confirmed by isolation and
identification of IHNV. Recommendations for fish sampling, processing and
diagnosis have been outlined by fish health officials in the USA and Canada
(Department of Fisheries and Oceans, 1984; Ganzhorn and LaPatra, 1994;
Thoesen, 1994). Fish tissues to be tested are dependent on fish size and disease
status. The preferred tissues are the kidney and spleen; mucus has also been used
in non-lethal sampling (LaPatra et al., 1989c). For testing brood stock, the
ovarian fluid is preferred, since the virus is less frequently detected in the milt.
Sampling of postspawning females, storage of ovarian fluid or incubation of
ovarian fluid cells enhances the sensitivity of viral detection (Mulcahy et al.
1984b; LaPatra and Groberg, 1985; Mulcahy and Pascho, 1986; Mulcahy and
Batts, 1987; LaPatra et al., 1990c). If milt is tested for virus, it should be
centrifuged and, after the pellet is incubated in water, the water is assayed for
virus. This procedure enhanced IHNV detection by at least fourfold (Batts, 1987;
Meyers et al.,1990).
Methods used for diagnosing IHNV should be rapid, simple, sensitive,
specific, inexpensive and able to test a large number of samples. Ideally, they
should also be suitable for field use. The only method initially available for the
diagnosis of IHNV was to detect virus in cell culture and then identify the virus
by a serum neutralization test (Amend, 1970b). This classical method is widely
accepted and is still commonly used. The discovery that rabbit antiserum has
higher levels of IHNV-binding antibodies than IHNV-neutralizing antibodies
(Hsu and Leong, 1985) allowed the development of several more rapid
serological methods. There are also non-serological methods available for
diagnosing IHNV. Each of the available methods are discussed below.
Virus isolation by cell culture
Samples are typically added to susceptible cell lines, such as EPC, CHSE-214 or
FHM. Methods used for detecting and determining the quantity of IHNV in the
sample are end-point titrations and plaque assays. The plaque assay is more
sensitive than end-point titrations (Fendrick et al., 1982; Leong et al., 1983a).
The American Fisheries Society Blue Book (Amos, 1985; Ganzhorn and
LaPatra, 1994) and the Manual of Compliance of the Department of Fisheries
and Oceans (1984) in Canada recommend that two cell lines (pH 7.6) are
inoculated when 8090% confluent and incubated at 15C for a minimum of 14
days for end point dilutions or 7 days for plaque assays.
For plaque assays, the inoculum volume should be adjusted to reflect the
size of the cell-culture well, since using too high a volume can reduce the
efficiency of plaquing (Burke and Mulcahy, 1980; Batts et al., 1991). Viral
adsorption is optimal after 1 h; shorter times reduce the detectable viral titre and
80
longer times have no effect on the number of plaques (Burke and Mulcahy, 1980;
Brunson et al., 1988; Batts and Winton, 1989a,b; Batts et al., 1991). Plaques are
larger when cells are overlaid with methyl cellulose, but gum tragacanth or
agarose at a pH of 7.28.0 can be used (Wolf and Quimby, 1973; Burke and
Mulcahy, 1980; Okamoto et al., 1985; Batts et al., 1991).
Cells should be examined daily for cytopathic effect (CPE). If no CPE
occurs, then the sample is virus negative, especially if the sample has been
passed in tissue culture several times (blind passaged). If there is CPE
characteristic of IHNV, then a presumptive diagnosis of IHNV is made. Typical
CPE in cell cultures consists of grape-like clusters of rounded cells, with
margination of the chromatin of the nuclear membrane (Wolf, 1988). Typical
IHNV plaques consist of a cell sheet that retracts or piles up at the inner margins
of the opening and the centre contains granular debris (Wolf and Quimby, 1973).
Attempts have been made to enhance the sensitivity of viral detection in cell
cultures. Pretreatment of cell monolayers with polybrene, a polycation that
enhances the ionic attraction of viruses to cells, increased titres of a laboratory
strain of IHNV by five- to tenfold (Leong et al., 1981b) but did not affect titres of
field isolates of IHNV assayed by plaque assays or end-point dilution (Fendrick
et al., 1982). Pretreatment of cell monolayers with 7% polyethylene glycol
(PEG, 20,000 M
r
) improved the speed and sensitivity of plaque assays for the
quantification of low levels of IHNV and resulted in the production of larger
plaques (Batts and Winton, 1989a,b; Batts et al., 1991). Polyethylene glycol can
also be added directly to the antibiotic solution that samples are incubated in,
prior to inoculation on to cells (Brunson et al., 1988). The discovery of the
effectiveness of PEG pretreatment is an important advance in the detection of
IHNV in cell culture. Once a virus is detected in cell culture, its identity must be
confirmed.
Viral identification
Serum neutralization test
An approved method of viral identification is the serum neutralization test, using
either rabbit antisera or MAbs (Department of Fisheries and Oceans, 1984;
Amos, 1985; LaPatra, 1994). Rabbit anti-IHNV serum typically has low
neutralizing antibody titres (1 : 250 or less), but binding antibody titres as high
as 1 : 50,000 have been reported (Leong et al., 1983a; Hsu and Leong, 1985;
McAllister and Schill, 1986). The best schedule for producing polyclonal serum
is to give rabbits an intramuscular injection of concentrated virus in Freunds
complete adjuvant, followed by intravenous boosts at 4 and 5 weeks with
purified virus (Hill et al., 1981). Monoclonal antibodies are less toxic than rabbit
antiserum, have neutralization titres of up to 1 : 1000, and can be used to
distinguish IHNV variants (Roberti, 1987; Winton et al., 1988; Eaton et al.,
1991; Roberti et al., 1991; Kamei et al., 1991; Huang et al., 1996).
Accurate detection of IHNV in cell cultures and identification by serum
neutralization tests is expensive and time consuming. A 50% plaque reduction
assay is more sensitive than a 50% end-point titration (Ahne, 1981), but neither
assay is rapid. It can take from 7 to 10 days before results are obtained from
neutralization tests and the total time to make a diagnosis can take from 2 to 8
weeks. Since IHN can kill the majority of fish within 23 weeks, it becomes
moot to determine whether the fish died from IHN (Mulcahy et al., 1980; Leong
et al., 1983a). Obviously, more rapid diagnostic methods had to be developed so
that infected fish could be quickly destroyed or quarantined to prevent further
spread of the virus.
In addition to the serum neutralization test, the fish health Blue Book
(LaPatra, 1994) lists immunofluorescence, staphylocoocal coagglutination,
enzyme-linked immunosorbent assay (ELISA), immunoblots and DNA probes
as acceptable methods of identifying IHNV.
Immunofluorescence
Immunofluorescence has been used to study fish viruses since 1972 (Jrgensen
and Meyling, 1972) and is widely accepted for the detection and identification of
IHNV in cell cultures. Either direct (DFAT) or indirect (IFAT) fluorescent
antibody tests can be used. Routinely, DFAT uses fluorescein isothiocyanate
(FITC)-labelled anti-IHNV polyclonal antibody or MAb and IFAT uses
unlabelled primary antibody, which is detected using FITC-conjugated
secondary antibody. Leong et al. (1983a) first reported IFAT for detecting IHNV
in cell cultures and this method is rapid, specific and sensitive and can detect all
five electropherotypes of IHNV (LaPatra et al., 1989a). A diagnosis can be made
in 24 h using 50% end-point titration, if ovarian fluid inoculated on to cells has a
virus titre of at least 10
2.9
pfu ml
1
, and pretreatment of the cells with PEG
improves the limit of detection to 10
2.5
pfu ml
1
. A plaque assay is slightly more
sensitive, detecting as low as 10 pfu ml
1
, but requires up to 8 days to observe
plaques and only provides a presumptive diagnosis (LaPatra et al., 1989a).
Polyclonal and monoclonal antinucleoprotein and antiglycoprotein antibodies
used in IFAT have equivalent sensitivity and time requirements for making a
diagnosis, but preadsorption of polyclonal serum is required to prevent
non-specific fluorescence (LaPatra et al., 1989b; Arnzen et al., 1991).
Used on cell culture samples, IFAT and DFAT have similar sensitivity
(LaPatra et al., 1989a; Arnzen et al., 1991), but DFAT may be preferred because
it is more rapid (Ristow and Arnzen, 1989; Arnzen et al., 1991). Cells should
ideally be incubated for 1624 h before using IFAT or DFAT, but, when rapid
decisions are required, DFAT can be performed after only 1012 h incubation
(LaPatra et al., 1989b; Arnzen et al., 1991). Monoclonal antibody-based IFAT or
DFAT can be used to type viral isolates, identify new viral types and study viral
variants (Ristow and Arnzen, 1989; Ristow and Arnzen-de Avila, 1991; Roberti
et al., 1991).
The two potential disadvantages of using fluorescent antibody tests (FATs)
on cell cultures have been the use of cover glasses and detachment of cells after
acetone fixation (LaPatra et al., 1989a). However, these problems have been
overcome since LaPatra et al. (1989a) demonstrated that IFAT could be used
directly on plaque assay plates and cells could be fixed with other fixatives.
Kamei et al. (1991) confirmed that cover glasses were not needed by showing
that IHNV could be detected in formalin-fixed cells in 96-well plates. Other
82
disadvantages of FATs are that trained personnel are required and fluorescent
microscopes are fairly expensive.
Direct detection of IHNV in fish tissues using IFAT or DFAT could
substantially shorten the time for a diagnosis from several days to a few hours.
Indirect FAT detected IHNV in blood smears and kidney imprints from clinically
infected juvenile fish, but was unsuccessful in detecting virus in smears of
seminal fluid (LaPatra et al., 1989a). The use of DFAT on fish tissues has yet to
be investigated.
Staphylococcal coagglutination
Staphylococcal coagglutination can specifically detect and identify IHNV
grown in cell cultures or in infected fish tissue (Bootland and Leong, 1992). This
test is simple and, since it takes only 15 min, it is one of the most rapid methods
of diagnosing IHNV. It has the added benefit of being suitable for field use,
because it only requires a light microscope, glass slides and one reagent. The test
uses formalin-fixed Staphylococcus aureus cells sensitized with unadsorbed
polyclonal rabbit anti-IHNV antiserum. When the antibody-coated cells are
mixed with samples containing IHNV, the antibody specifically binds to the
virus and causes the bacterial cells to coagglutinate.
For viruses grown in cell culture, the assay is specific for IHNV isolates
belonging to all five electropherotypes, but the test is not very sensitive
10
6
pfu ml
1
is generally required. However, this should not affect its use in
diagnosing IHNV, since cell cultures showing complete CPE typically have
virus titres greater than 10
6
pfu ml
1
. The test is not suitable for use in cell
cultures pretreated with PEG, due to non-specific coagglutination.
The coagglutination test identified IHNV directly from fry homogenates,
adult organs and ovarian fluids, as long as the virus titre was at least 10
6
pfu g
1
.
No false positives were observed. Detection of IHNV in adult carriers may not
be as effective due to the low sensitivity of the coagglutination test. Samples
with a low viral titre would have to be passaged in cell culture to amplify the
virus before a coagglutination test could be used.
The binding of MAbs to staphylococcal cells or latex beads, although not
yet successful, may increase the sensitivity of the assay and allow differentiation
between different types of IHNV.
Immunoassays (enzyme-linked immunosorbent assay, immunoblots and
Western blots)
Several different types of immunoassays have been developed for the
identification of IHNV. They all utilize the binding of antigen or antibody to a
solid support and detection of the virus directly with labelled anti-IHNV
antibodies or indirectly with labelled secondary antibody. These assays are not
as rapid as coagglutination, IFAT or DFAT, but they are simple, are usually
specific and can be quantitative. As for IFAT, rabbit polyclonal serum has to be
preadsorbed with fetal bovine serum and cell lysates to make the assays more
specific (Dixon and Hill, 1984). The limit of IHNV detection is generally 10
3
10
6
pfu ml
1
for ELISA and dot blots, and virus can be detected once cells begin
to show CPE. This level of sensitivity is greater than that of the coagglutination
test, but not as high as for IFAT and DFAT, which can detect IHNV prior to cells
showing CPE. Unlike IFAT or the coagglutination test, none of the immuno-
assays are recommended for detecting IHNV directly from fish tissues.
ENZYME-LINKED IMMUNOSORBENT ASSAY
Using polyclonal antibodies or MAbs, ELISA techniques can detect and identify
IHNV in cell cultures and have shown some success in detecting IHNV in fish
tissues. Two different ELISA systems have been developed: a sandwich or
capture ELISA and a direct ELISA. Both assays require 622 h to complete, and
antibody concentrations, plate type, blocking agent, buffers, incubation times
and temperatures each have to be optimized.
The initial sandwich ELISAs used plates coated for 6 h with rabbit
anti-IHNV serum (Dixon and Hill, 1984) or 18 h with purified anti-IHNV
immunoglobulin (Ig) (Way and Dixon, 1988), and IHNV antigen was detected
using labelled rabbit anti-IHNV Ig. The assays were specific and detected IHNV
in cell cultures once the virus titre had reached 10
6
10
7
pfu ml
1
. The above two
assays were improved by Medina et al. (1992) by use of a double-antibody
sandwich ELISA. Plates were coated with chicken anti-IHNV serum and virus
was detected by incubation with rabbit anti-IHNV serum, followed by labelled
goat antirabbit serum. This assay was very sensitive, since it could detect 500
pfu ml
1
, could be completed in 1 day and detected virus belonging to the four
types tested.
A sandwich ELISA using biotinylated anti-N MAbs was developed and
optimized by Ristow and de Avila (1991) to detect IHNV in cell cultures.
Monoclonal antibodies had to be carefully chosen because they can recognize
the same epitope, lose virus binding ability or have altered reactivity after
biotinylation. The sandwich ELISA recognized many virus isolates belonging to
the five IHNV electropherotypes and 10
3
pfu ml
1
could be detected with some
IHNV isolates, but other isolates at high titres did not react. Using a mixture of
MAbs or polyclonal antibodies combined with MAbs may improve the
sandwich ELISA technique (Ristow and de Avila, 1991). This method is still not
ideal, nor is it as sensitive and reliable as plaque assays, IFAT or dot blots for
detecting IHNV in cell culture.
In the direct ELISA, plates are coated with purified virus instead of
antibody. Using this method, with unlabelled MAbs to G, N and M2 IHNV
proteins, Kamei et al. (1991) detected several isolates of purified IHNV, but the
specificity depended on which MAb was used. There were no cross reactions
between the IHNV N- or M2-specific MAbs and HIRRV, but one of three IHNV
G-specific MAbs did cross-react, suggesting that IHNV and HIRRV have a
common epitope. Before using G-specific MAbs in ELISA to detect IHNV, it
must be ensured that cross-reactions with HIRRV do not occur.
Identification of IHNV in fish tissues using ELISA is not yet optimal. With a
sandwich ELISA, Dixon and Hill (1984) and Way and Dixon (1988) found that
whole fry homogenates or abdominal extracts gave a high background and weak
cross reactions between heterologous viruses. To decrease background and make
the assay more specific, Dixon and Hill (1984) suggested using concentrated Ig
for plate coating, a more dilute affinity-purified, labelled Ig and diluted fish
84
viscera. In the double antibody sandwich ELISA of Medina et al. (1992), there
were low background reactions and IHNV was detectable directly from Atlantic
salmon tissue homogenates if the titre exceeded 70 pfu ml
1
, but only six fish
were tested. The use of MAbs in ELISA to detect IHNV in fish tissues has not yet
been investigated. Monoclonal antibodies could increase the specificity and
sensitivity of the assay, thus improving the ability to detect virus in fish tissues
without background. The techniques of ELISA may be well suited for field use,
because visual results can be obtained without the need for specialized
equipment and the assay is relatively rapid, especially if plates used in sandwich
ELISA are precoated (Dixon and Hill, 1984).
IMMUNOBLOTS (DOT BLOTS)
The immunoblot assay or dot blot is a variation of the ELISA technique. Instead
of binding antigen to wells, antigen is bound directly to a nitrocellulose or nylon
membrane and detected with labelled primary antibody or indirectly with
labelled secondary antibody. Immunoblots are faster than ELISA, requiring less
than 4 h to complete, and may be more specific for identifying IHNV in cell
cultures. Cell incubation time depends on the input multiplicity of infection
(MOI), but ranges from 36 to 72 h or until CPE is observed and the virus titre has
reached 10
3
10
6
pfu ml
1
(McAllister and Schill, 1986; Schultz et al., 1989;
Ristow et al., 1991). Dot blots have detected as little as 4 ng of purified virus,
and, unlike ELISA, no cross-reactions were observed with heterologous viruses
and the primary antibody can be reused several times (McAllister and Schill,
1986; Eaton et al., 1991). Blot assays with labelled MAbs did not significantly
enhance the sensitivity of the assay, but could distinguish between IHNV and
VHSV, detect IHNV at titres as low as 10
3
pfu ml
1
, recognize all five
electropherotypes of IHNV and be completed in as little as 2.5 h (Schultz et al.,
1989; Ristow et al., 1991).
Immunoblots have not been successful for identifying IHNV in fish
samples, because the high protein content of tissues or reproductive products
clogged the filters (McAllister and Schill, 1986). Also, an unknown component
in ovarian fluids, which had a migration rate similar to the IHNV G and M2
proteins, cross reacted when biotinylated anti-IHNV MAb was used (Schultz et
al., 1989). The cross-reacting components in ovarian fluids were not identified
and cross-reactions were not due to endogenous peroxidase activity (Schultz et
al., 1989). Further work is required to adapt immunoblots for detecting IHNV in
fish samples.
WESTERN BLOTS
In this method, cell culture proteins are separated by SDS-PAGE and transferred
on to a membrane. Specific IHNV proteins are detected directly with labelled
anti-IHNV antibodies or indirectly with labelled secondary antibody. The
indirect method was first used to identify IHNV in cell cultures by Hsu and
Leong (1985) and Leong et al. (1985). Using adsorbed polyclonal rabbit serum
and
125
I-protein A or secondary antibody labelled with horseradish peroxidase,
all IHNV proteins were visible when the total viral protein was 2 g. The viral G,
N and M1 proteins were detectable at 10 ng total viral protein. Cell growth and
the Western blot required a total of 51.566 h, which included a 9 h cell
incubation and 4 h for cell lysis and SDS-PAGE. Radioactivity is no longer
required and several steps in the method of Hsu and Leong (1985) have been
shortened. When anti-N antibody is used, cell incubation time can be decreased
from 9 h to 3 h because viral N protein has already been synthesized in infected
cells (Leong et al., 1983b). SDS-PAGE still takes 12 h, but protein transfer and
the Western blot can now be completed in less than 6 h (Kamei et al., 1991; L.M.
Bootland and J.C. Leong, unpublished observations). Western blots are of value
in typing IHNV isolates and for comparing immunological relatedness among
IHNV isolates or to other viruses. Kamei et al. (1991) used N-, M2- and
G-specific MAbs in Western blots to study the antigenic relationship between
IHNV and HIRRV and suggested that the two viruses have at least one G epitope
in common. Chou et al. (1993) showed by Western blots that Japanese IHNV
isolates had an N epitope that was only faintly visible in the North American
strains tested. This may be of relevance in monitoring the spread of IHNV from
area to area.
Western blots have not been commonly used in diagnostic laboratories
because they are slower than other assays and specialized equipment is needed.
However, Western blots are useful in determining the specificity of other assays
(Schultz et al., 1989), provided that the antibodies have been proved to be
IHNV-specific. Reports of using a direct technique with labelled primary
antibody in Western blots have not been made, but this method may further
decrease the time required to diagnose IHNV.
IMMUNOPEROXIDASE AND ALKALINE PHOSPHATASE CELL STAINING
These two methods are similar to FAT but use horseradish peroxidase- or
alkaline phosphatase-labelled antibodies instead of FITC-labelled antibodies.
Direct and indirect methods are rapid, specific and simple, do not require
specialized equipment (Ahne, 1981) and may be suitable for field use.
For identification of IHNV in cell cultures, immunoperoxidase staining was
first suggested by Leong et al. (1983a), but this method is not routinely used.
Kamei et al. (1991) used indirect immunoperoxidase staining with MAbs to
IHNV N and M2 proteins to distinguish between IHNV and HIRRV. However,
one of the three anti-G MAbs cross-reacted with HIRRV.
Alkaline phosphatase staining of cells has recently been adapted for
confirming IHNV in plaque assays (Drolet et al., 1993). Plaque assay plates
showing CPE were formalin-fixed, blocked overnight with 5% non-fat dry milk
in PBS, incubated with unlabelled anti-N MAb and then biotinylated secondary
antibody and avidinalkaline phosphatase. If the plaques were caused by IHNV,
then the cells around the margin of the plaque were coloured after the addition of
substrate. If plates were initially stained with crystal violet, they could easily be
destained by washes with 70% ethanol prior to blocking. The test was specific
for IHNV, detected all five electropherotypes and often resulted in a positive
reaction prior to visible CPE.
The immunoperoxidase and alkaline phosphatase cell-staining techniques
have been frequently used in immunohistochemistry to study the pathogenesis
of IHNV (Yamamoto et al., 1989, 1990, 1992; Drolet et al., 1994; Kim et al.,
86
1994). Thin sections of fish were incubated with unlabelled primary monoclonal
anti-G or anti-N antibody and horseradish peroxidase or biotin-labelled
secondary antibody. The colour of the IHNV-infected cells depended on the
substrate employed. Non-specific reactions have been observed, but could be
removed by pretreatment of tissues with hydrogen peroxidase for horseradish
peroxidase-based assays (Yamamoto et al., 1989) or by including levamisole in
the substrate solution in alkaline phosphatase-based assays (Drolet et al., 1994).
It is unlikely that staining of fish tissues will be adapted for the routine diagnosis
of IHNV. The fixing, embedding and sectioning of fish tissues is the most time
consuming part, with the actual antibody portion of the test being rapid, taking
less than 6 h.
Identification of infectious haematopoietic necrosis virus by
non-serological methods
In most of the above serological methods of diagnosing IHNV, the virus must be
amplified in cell culture until protein antigen concentrations reach detectable
levels. Instead of detecting antigens, it is possible to detect viral particles by
electron microscopy (EM). Nucleic acids can be detected using the polymerase
chain reaction (PCR) and nucleic acid probes. Since viral mRNA is synthesized
before proteins in infected cells, detection of viral RNA may result in an earlier
diagnosis of IHNV.
ELECTRON MICROSCOPY
Electron microscopy was used for identifying IHNV in water and reproductive
products (Leong et al., 1983a). The method is relatively cheap and very rapid,
taking only 34 h, and detects 10
2
10
3
particles IHNV ml
1
of water, or 1 10
4
tissue culture infectious dose at 50% end-point (TCID
50
) ml
1
. Diagnosis is based
on visualizing viral particles having the characteristic bullet shape of
rhabdoviruses. However, EM does not allow easy differentiation of viruses
within the rhabdovirus family, especially between IHNV and VHSV, since the
two viruses are of similar size. This technique provides only a presumptive
diagnosis and is of little value in areas where both IHNV and VHSV occur. It
also has the disadvantages of requiring trained personnel and an electron
microscope.
Immunoelectron microscopy has the advantage of being able to specifically
identify IHNV. It was first used by Helmick et al. (1991) to detect IHNV G and
N proteins on the surface of infected cells. Using biotinylated MAbs,
streptavidin-gold, scanning EM and X-ray analysis, viral proteins can be
detected on cells at 8 h postinfection. Immunogold-labelling and EM has also
been used to identify IHNV N protein in the kidney tissue of rainbow trout 1 year
postinfection (Drolet et al., 1995). Although the method is sensitive and allows
differentiation between IHNV and other fish rhabdoviruses, it required over 16 h
to complete. It is unlikely that this technique will be used in diagnostic
laboratories, since more rapid, simpler and less expensive serological methods
have been developed. However, this method is of value for studying the life
cycle of IHNV, since viral proteins can be detected in fish when infectious virus
is not present.
POLYMERASE CHAIN REACTION AND NUCLEIC ACID PROBES
Polymerase chain reaction is a powerful technique used to amplify specific
regions of DNA or RNA to easily detectable levels on agarose gels or with
nucleic acid probes. This technique uses two primers that span the nucleic acid
sequence of interest and a thermostable polymerase to exponentially increase the
amount of nucleic acid through repeated cycles of synthesis. Nucleic acid
probes consist of pieces of labelled RNA or DNA that specifically bind to
complementary sequences in nucleic acids (Winton, 1991).
The N and G mRNA of IHNV have been completely sequenced (Koener
et al., 1987; Gilmore and Leong, 1988), making it possible to develop PCR
techniques and design specific probes to either IHNV gene. Initially, PCR and
probes were developed for detecting N RNA, since the N mRNA is the first and
most abundant mRNA produced in infected cells (Rose and Schubert, 1987) and
is probably the best target for amplification and detection. Polymerase chain
reaction has been used to detect IHNV N RNA in cell cultures, fresh fish tissue
and paraffin-embedded fish kidney tissues.
Arakawa et al. (1990) were the first to use PCR for detecting IHNV N RNA
in cell cultures. Using 20 mer (position 319338) and 19 mer (position 570552)
primers, they detected a 252 bp PCR product from RNA extracted from cells
infected with the five IHNV electropherotypes. No bands were evident after
PCR of uninfected cells or with VHSV-infected cells, but two bands were
observed from HIRRV-infected cells, suggesting that HIRRV and IHNV have
some homology at the nucleotide level. Southern blots and dot blots, using a 30
mer (position 427456) biotinylated antisense oligonucleotide probe, detected
only IHNV. Cell cultures were harvested prior to CPE at 24 h and, as long as the
extracted RNA template was 1 pg, PCR successfully amplified the target to
detectable levels in blots. Deering et al. (1991) confirmed that the probe was
specific for IHNV and showed that the cell incubation time was dependent on the
input MOI. At a high MOI of 10, IHNV mRNA was detected by the probe at 6
9 h, but, at a low MOI of 0.0002, the cells had to be incubated 4 days or until
small foci of CPE were evident. More recently, reverse transcriptase-dependent
PCR (RT-PCR) has been developed for differentiating between VHSV and
IHNV, using primer pairs that amplify viral glycoprotein gene fragments
(Bruchhof et al., 1995). By amplifying an IHNV 548 bp fragment and a VHSV
443 bp fragment from total RNA extracts of virus-infected RTG-2 cells, these
researchers could differentiate the two viruses within 8 h.
Polymerase chain reaction has been used to amplify IHNV RNA extracted
from fresh fish tissues (Arakawa et al., 1990). Ribonucleic acid extracted from
kidney/spleen and leucocytes of rainbow trout killed at 3 days postinfection
produced an N gene PCR product when the fish were infected with 10
4
or
10
3
pfu ml
1
, but not when the viral dose was 10
2
pfu ml
1
. All samples yielding a
PCR product were also positive in plaque assays, but only the plaque assay
identified IHNV in one fish infected with the lowest virus dose. Polymerase
chain reaction is not as sensitive as plaque assays for detecting IHNV in fish
tissues, but it is much more sensitive, more rapid and cheaper than several other
techniques. At least 400 pfu ml
1
was required before PCR was able to amplify
viral RNA to detectable levels in gels or blots.
88
Polymerase chain reaction has also been used to amplify RNA from
formalin-fixed and paraffin-embedded fish tissues (Chiou et al., 1995; Drolet
et al., 1995). Tissues could be stored in formalin for up to 2 years before
embedding in paraffin, and PCR-amplifiable RNA template was obtained when
RNAzol (phenol and guanidinium thiocyanate) was used to extract RNA.
However, tissues could not be subjected to proteinase K digestion. Using the
same N gene primers as Arakawa et al. (1990) and RT-PCR, a 252 bp product
was identified in the kidneys of steelhead trout fry sampled at 7 days
postinfection (Chiou et al., 1995) and in 1-year-old survivors (Drolet et al.,
1995). Specificity was confirmed using Southern blots and the N-gene-specific
probe of Arakawa et al. (1990).
Polymerase chain reaction and probes to IHNV mRNA are relatively rapid,
very sensitive and specific. Twenty-five PCR cycles require approximately 2 h,
gels take 14 h and dot blots with the probe require over 6 h (Arakawa et al., 1990;
Deering et al., 1991). For identifying IHNV in cell cultures infected with a high
MOI, there is no clear advantage in using PCR and probes over other methods,
when consideration is given to the time required and the need for specialized
equipment, primers and oligonucleotide probe. Eventually, use of automated PCR
and probes may result in methods that are faster and less costly than the present
techniques (Arakawa et al., 1990), but further optimization is required. The real
advantage of PCR is its ability to amplify very low amounts of IHNV RNA more
than a millionfold in a few hours to levels detectable with probes. This would be
useful in detecting very low levels of virus in samples such as water, sediment, fish
or other animals. Another area where PCR is very useful is the direct detection of
IHNV in fish tissues, especially when infectious virus is no longer present.
Although oligonucleotide probes are quite specific, their use for diagnosing
IHNV in fish sections is not feasible, because of the amount of time involved
approximately 24 days. However, probes should prove valuable in studying the
progression of virus within fish during and after an epizootic and for
determining whether the virus enters a latent state.
Detection of anti-infectious haematopoietic necrosis virus antibodies in
fish serum
For all of the above diagnostic methods, fish are usually sacrificed to obtain
samples. A serological diagnosis of IHNV, based on the detection of anti-IHNV
antibodies in fish serum, could determine the IHNV status of a fish stock without
killing the fish.
Anti-IHNV antibodies were first demonstrated in fish serum using a serum
neutralization test, in which heat-inactivated rainbow trout serum was incubated
for 1 h with a low dose of virus (50100 TCID
50
per well) (Amend and Smith,
1974). Hattenberger-Baudouy et al. (1989) then showed that most fish sera
would be negative for anti-IHNV antibodies if complement was absent and
incubation was for only an hour. Infectious haematopoietic necrosis virus
neutralization by trout antibodies is complement-dependent and is more evident
after 16 h incubation. Heat-inactivation or one freezethaw of serum readily
inactivates complement and the serum neutralization test is inconsistent and
inaccurate unless serum containing complement is added.
In the improved serum neutralization technique of Hattenberger-Baudouy et
al. (1989), heat-inactivated fish serum is diluted in two steps, mixed with 5%
virus-free trout serum as a complement source and IHNV (1250 pfu per well)
and incubated for 16 h at 4C in 96-well plates. After adding EPC cells, the
plates are incubated for 34 days at 15C, fixed and stained with crystal violet.
The antibody titre is defined as the highest serum dilution giving obvious
protection of the cell monolayer.
In plaque reduction assays, the antibody titre is the serum dilution that
results in a 50% reduction in the number of plaques compared with plates
containing only a complement source. Background neutralization varies with the
batch of complement and was observed at serum dilutions of less than 64 and
with haemolysed serum (Hattenberger-Baudouy et al., 1989). These researchers
determined that the plaque reduction technique was approximately ten times
more sensitive than the microtechnique, but the latter method was preferred
because a large number of samples could be easily analysed. Plaque
neutralization tests were used to compare the antibody titre of trout injected with
the five electropherotypes of IHNV (Basurco et al., 1993). Jrgensen et al.
(1991) determined that IFAT was the most sensitive technique for detecting
anti-IHNV antibodies in IHN survivors, and that a sandwich ELISA and
neutralization tests had equivalent sensitivity. For IFAT, a double-labelling
technique allowed detection of both IHNV and anti-IHNV antibody. Cross-
reactions of fish serum with VHSV and IHNV were more evident with ELISA
than with IFAT, but did not occur in neutralization tests. Ristow et al. (1993)
found that a simpified ELISA was more rapid (18 h) and detected a higher
number of fish with anti-IHNV antibodies than a plaque neutralization test,
which requires over 6 days. Western blots did not detect anti-IHNV antibodies in
the majority of the fish, possibly because the conformation of the viral epitopes
had been changed, due to denaturing conditions, but this assay is valuable in
determining which viral proteins are inducing a humoral immune response.
For determining the virus-free status of a hatchery fish population,
Jrgensen et al. (1991) recommended using neutralization assays, because of the
specificity of the test. However, this method requires proper storage of the serum
used as a complement source, is time-consuming and requires technical
expertise. Ristow et al. (1993) suggested that ELISA could replace the plaque
neutralization test for detecting anti-IHNV antibodies, because the two assays
were of similar sensitivity but ELISA required less time to complete. The ELISA
is valuable for screening large numbers of samples, is relatively specific and
reproducible and is of low cost in time and materials.
CONTROL AND TREATMENT
Transmission and pathogenesis
The epizootiology of IHNV is not completely understood and the source of virus
infecting salmonids is still unknown. However, survival of IHNV in a fish
population depends on a close association of the virus with the life cycle of the
90
fish host (Fig. 2.5). Similarly to other salmonid viruses, IHNV typically causes
an acute disease in young salmon and trout. Mortalities of fry and fingerlings can
be as high as 90%, but occasional epizootics have been reported in smolts and
older fish (Yasutake, 1978; Busch, 1983; Burke and Grischkowsky, 1984;
Roberts, 1986; Traxler, 1986). The source of virus infecting young salmonids
may be from vertical transmission or by direct water-borne exposure to IHNV
from an unknown reservoir or host. During an epizootic in young fish, the virus
is transmitted horizontally from fish to fish and infectious virus is readily
isolated up to approximately 50 days after viral exposure (Amend, 1975; Busch,
1984; Bootland et al., 1995; Drolet et al., 1995). Thereafter, infectious virus is
usually not isolated again until the fish near or reach sexual maturity (Amend,
1975; Busch, 1984; Meyers et al., 1990; Hattenberger-Baudouy et al., 1995b).
Two mechanisms may explain why infectious virus is isolated at only two stages
of the salmonid life cycle. First, IHNV may be entering a latent state in fish
surviving an IHN epizootic, with virus reactivation occurring only as fish mature
sexually (Amend, 1975). Alternatively, the fish may completely clear the virus
but become reinfected prior to or during their spawning migration (Elston et al.,
1989; Traxler et al., 1997). Although non-piscine reservoirs have not been
identified, there is evidence to support the suggestion that both mechanisms
exist.
Fig. 2.5. Life cycle of IHNV in salmonid fish.
Infectious haematopoietic necrosis virus in spawning salmonid fish
Natural IHNV infections have been reported in sexually mature kokanee
salmon, chinook salmon, chum salmon, coho salmon, rainbow trout and
steelhead trout from feral and hatchery populations (Busch, 1984; Groberg,
1985; Goldes et al., 1986; Follett et al., 1987; LaPatra et al., 1987, 1989b;
Engelking and Kaufman, 1994a). However, the majority of studies on IHNV in
sexually mature fish have been on female sockeye salmon because of their high
susceptibility and because IHNV is enzootic in this species in the Pacific North-
West of North America. Populations containing spawning sockeye salmon,
chinook salmon and steelhead trout have an IHNV infection rate that varies from
0 to 100% and females have a higher prevalence of infection than males
(Wingfield and Chan, 1970; Mulcahy et al., 1983a, 1984b; LaPatra et al., 1989b;
Yamamoto et al., 1989; LaPatra, 1990; Meyers et al., 1990). The differences in
prevalence between the sexes may be related to hormone levels or difficulties in
detecting IHNV in milt (Meyers et al., 1990). Infectious haematopoietic necrosis
virus should be eluted from sperm using water to enhance detection (Batts,
1987), but many studies do not appear to have used this method.
In naturally spawning sockeye salmon, IHNV was not isolated from fish
during their migration from the ocean to the spawning area, but virus suddenly
appeared in a low percentage of prespawning females in the spawning grounds.
As the spawning migration progressed and fish density increased, viral
prevalence and titres increased (Mulcahy et al., 1982, 1984b; Mulcahy and
Pascho, 1986). A recent study by Traxler et al. (1997) showed that IHNV could
be isolated from adult sockeye salmon during the ocean phase of their life cycle.
It was postulated, based on the presence of neutralizing anti-IHNV antibodies in
the fish at approximately 2 months prior to spawning, that the fish could have
been exposed to IHNV 56 months prior to sexual maturation. Postspawning
fish have a high viral prevalence and titres in the ovarian fluid, spleen, kidney
and gill, but results vary from year to year (Meyers et al., 1990). When Meyers et
al. (1990) analysed the data from 96 wild and hatchery populations taken over 14
years, there was no significant difference between spawning and postspawning
fish for the prevalence of IHNV infection or viral titres. It is evident that
infection rates cannot be predicted from year to year for different fish
populations and that fish do not necessarily need to be on their spawning
grounds to become infected with IHNV.
How adult salmonid fish are becoming infected
What has intrigued biologists most about the life cycle of IHNV is that virus
typically can not be reisolated from adult fish until they approach sexual
maturity. There are two hypotheses to explain this phenomenon. The first is that
fish may become reinfected by a new exposure to IHNV during the spawning
migration. This implies that the virus is present in the environment or in another
host. The second hypothesis is that fish surviving IHNV as fry may develop a
latent infection, which is reactivated when the fish reach sexual maturity. Fish
returning to fresh water to spawn are undergoing stress, which may weaken the
immune system so that the fish cannot control the virus (Mulcahy et al., 1984b).
Hormonal changes may play a role, but injection of sex hormones did not induce
92
or suppress virus infection in spawning fish (Grischkowsky and Mulcahy, 1982).
It is possible that a combination of the two hypotheses accounts for the infection
in sexually mature fish. A portion of fish may have reactivated latent infections
and virus shed from these fish into the water may spread horizontally to other
fish.
Water-borne IHNV can result in horizontal transmission between adult
salmonids. Field observations showed that yearling fish cohabiting with sockeye
salmon adults became infected with IHNV (Mulcahy et al., 1983a) and water-
borne virus potentially spread from adult chinook salmon to adult coho salmon
at the Trinity River hatchery in California (LaPatra et al., 1987, 1989b).
Laboratory data have confirmed that spawning fish can be infected by water-
borne IHNV. Sexually mature kokanee salmon were infected after only 30 min
immersion in a low dose of IHNV and viral titres in pooled spleen and gill
increased over an 8-day period (Yamamoto et al., 1989). Immersion of sexually
mature kokanee salmon in 10
3
pfu ml
1
of RB-1 (type 1) or 10
4
pfu ml
1
of
WRAC (type 2) IHNV isolates for 1 h resulted in infection of 11 organs per fish
and the reproductive products, with mortalities reaching 94% and 72%,
respectively. Control fish had a mortality of 60%, attributable to natural causes
(H.V. Lorz, L.M. Bootland, J.S. Rohovec and J.C. Leong, unpublished data).
Thus, exposure of adult kokanee to IHNV in the water for only a short time can
result in a rapid and widespread infection of the organs and cause premature fish
mortality. Water taken from the spawning grounds has viral titres that may be
sufficient to infect salmonids, but titres vary during the spawning season and
from year to year, and IHNV probably does not survive in water over the winter
(Mulcahy et al., 1983a). This suggests that there must be an alternate source of
virus. Based on the presence of IHNV in adult sockeye salmon and Atlantic
salmon in salt water, there may be a marine reservoir of infection (Traxler et al.,
1991, 1993, 1997; Armstrong et al., 1993). However, virus reservoirs, except for
the fish themselves, have not been identified.
It is now clear that IHN survivors can become latently infected with IHNV.
The initial evidence that IHNV enters a latent state was presented by Amend
(1975). In his study, IHNV could not be reisolated from rainbow trout fry within
3 weeks after an IHN epizootic or over the next 2 years, but 33% of the sexually
mature fish had IHNV in the reproductive products. Similar results were
obtained by Busch (1984), who found that virus was not reisolated from rainbow
trout after 60 days postinfection or over the next 2 years from fish kept in
virus-free water, but, when the fish reached sexual maturity, infectious IHNV
was isolated from 2.56% of the males and 2.84% of the females. In contract,
field data from sockeye salmon in Washington, USA, suggested that fish did not
have a latent infection but became reinfected upon reaching the spawning
grounds. Fish captured over 3 years from a population which normally had a 98
100% incidence of IHNV did not become infected when held to sexual maturity
in pathogen-free water (Amos et al., 1989). Similarly, it was initially reported
that sockeye salmon held in freshwater net pens before the fish reached their
spawning grounds did not become infected with IHNV, even though 89% of fish
removed from the spawning ground were infected (Kent et al., 1988; Elston et
al., 1989). However, when this study was continued, at least 25% of the fish held
in net pens became infected, and yet groups of spawning fish held in two
locations upstream of the net pens were uninfected or had an infection rate of
17% (Elston et al., 1989). These results demonstrate that it is not possible to
predict infection rates from one year to the next and that fish do not need to be at
the spawning grounds to become infected with IHNV. The failure to detect a
reactivation of a latent state may have been due to a small sample size. Typical
sample sizes will detect a 5% prevalence of infection, but, based on the results of
Busch (1984), this presumed incidence may be too high.
In a long-term laboratory study, over 2800 rainbow trout fry were
experimentally infected by immersion and the surviving fish were held in
pathogen-free water and sampled for up to 4 years (Bootland et al., 1995; Drolet
et al., 1995). Infectious haematopoietic necrosis virus was isolated from fry for
50 days postinfection and, even though 30 fish were sampled every 6 months,
including during two spawning cycles, in which over 600 reproductive samples
were tested by plaque assays, infectious virus was not reisolated from organs or
from reproductive products. These results indicate that IHNV did not enter a
latent state that was reactivated as the fish reached sexual maturity. However,
examination of the tissues of the survivors proved that IHNV had entered a latent
state. Kidney tissues taken from 30 surviving fish at 3, 6, 12 and 24 months
postinfection contained both the N and G IHNV proteins by immuno-
histochemistry. Viral proteins were not seen in any of the other ten tissues,
except for the liver of one fish sampled at 90 days. Examination of the kidneys of
1-year-old survivors using PCR indicated that viral N RNA was present (Chiou
et al., 1995), and immunogold EM showed that the renal epithelium of four fish
had intracytoplasmic inclusions containing truncated IHNV particles that
resembled the defective interfering particles (DIPs) of other rhabdoviruses
(Drolet et al., 1995). It was subsequently demonstrated that livers of surviving
fish produce large quantities of DIPs and that these particles have biological
activity (C.H. Kim and J.C. Leong, unpublished data). When explant liver and
kidney tissues from survivor or control fish were re-exposed to IHNV, the viral
titres in the liver tissues, but not in the kidneys, of survivor fish were
significantly decreased compared with control fish tissues. By immunosorbent
EM, survivor tissues had truncated IHNV particles, while control fish tissues
had only standard IHNV particles. It was postulated that superinfection of
survivor liver tissues resulted in the release of a large number of DIPs, with
subsequent reduction in standard viral production. Other factors, such as
interferon and cell-mediated immunity, might also have played a role in
decreasing tissue viral titres, but there is strong evidence that DIPs are involved.
It is now clear that at least some IHNV survivors harbour a latent infection,
but to date it has not been possible to show a reactivation of the latent state.
Conditions that trigger reactivation still have to be identified.
Humoral immune response to infectious haematopoietic necrosis virus
Amend and Smith (1974) showed that fish hyperimmunized with IHNV
mounted a humoral immune response, with the production of neutralizing
antibodies. It was later shown that viral neutralization by antibodies was
complement-dependent (Hattenberger-Baudouy et al., 1989). The humoral
94
immune responses to IHNV and other fish viruses are very similar. Young
rainbow trout during an IHN epizootic did not have detectable neutralizing
antibody titres, but by 4.56 months after infection, when the virus was no
longer detectable, over half of the surviving fingerlings tested had a significant
humoral immune response. By 8 months, the number of antibody-positive fish
and the antibody titre had decreased (Hattenberger-Baudouy et al., 1989).
Similar results were obtained by LaPatra et al. (1993b). Juvenile rainbow trout
immersed in IHNV had a low prevalence and titre of serum anti-IHNV
antibodies at 1 week postexposure, but by 6 weeks over 50% of the fish had high
neutralizing antibody titres. Neutralizing activity was also observed in the
mucus. Cain et al. (1996) noted that juvenile rainbow trout injected or immersed
in IHNV produced serum neutralizing antibodies by 21 and 28 days. Mucus from
the skin and gastrointestinal (GI) tract had viral neutralizing activity, but no
antibodies were detected using ELISA. They suggested that there are innate
mechanisms of viral resistance which may play an important role as the first line
of defence.
Antibodies are readily detected in fish exposed to IHNV using FATs, ELISA
and Western blots (Jrgensen et al., 1991; Zhuang et al., 1992; Ristow et al.,
1993; Engelking and LaPatra, 1996; Traxler et al., 1997). Ristow et al. (1993)
examined the serum from juvenile rainbow trout that had survived from one to
five natural exposures to IHNV. They reported that 92% of the fish had
anti-IHNV antibodies and, by Western blot, some fish reacted to all IHNV
proteins, but the majority of the sera reacted with only the IHNV M1 and G
proteins. Similarly, rainbow trout injected with virus in adjuvant (Mourich and
Leong, 1991) or immersed in IHNV (LaPatra et al., 1993b) only responded to
the M1 and G proteins by Western blots. It is possible that fish are reacting to
other IHNV proteins, but the epitopes may be destroyed by denaturing
conditions used in Western blots.
Adult salmonids may have detectable serum IHNV neutralizing antibodies
after a natural or experimental exposure to the virus (Hattenberger-Baudouy
et al., 1989, 1995a,b; LaPatra et al., 1993b; Bootland et al., 1995). Basurco et al.
(1993) found that individual adult rainbow trout varied in their ability to produce
antibody after injection with the five IHNV electropherotypes. Rainbow trout
distinguished the type 2 isolate from the other isolates, but it was concluded that
the fish did not discriminate with high fidelity between most types of the virus.
In Oregon, a survey of anadromous adult salmonids showed that the fish
mounted a similar humoral immune response to IHNV to that of rainbow trout
(Engelking and LaPatra, 1996). Antibody titres tended to increase with time in
chinook salmon, and, in some cases, chinook salmon and steelhead trout with
antibodies were also infected with IHNV. Traxler et al. (1997) found that
sexually maturing sockeye salmon mounted a humoral immune response to
IHNV. In one group of fish captured in the autumn, antibody titres increased
over time, although IHNV was only detected in one in 19 fish. In a second group,
none of the 21 fish had increased antibody titres, but 42% were virus-positive.
Similar results were found with fish removed from the spawning grounds
virus was only found in fish that had no detectable antibodies. That sexually
mature fish produce antibodies against IHNV indicates that these fish are
immunocompetent. Although adults have now been shown to produce
anti-IHNV antibodies, an unresolved question is whether there is maternal
transfer of antibodies to the eggs. There is one report of isolating
IHNV-neutralizing antibodies in steelhead trout eggs (Shors and Winston,
1989b), suggesting that maternal transfer of antibody occurs. However, this
study did not indicate if the parents were infected with IHNV or whether the
antibody was specific to IHNV. Transfer of antibodies against Vibrio
anguillarum to eggs was reported for coho salmon, but protection against the
bacteria did not last after the yolk-sac was absorbed (Brown et al., 1994).
Whether antibodies transferred from the female to the eggs will be effective in
protecting fry against IHNV has not been determined.
Vertical transmission infectious haematopoetic necrosis virus in the
reproductive products
Vertical virus transmission is the transmission of virus from one generation to its
offspring, regardless of whether the virus is located within or external to the egg
(Fenner and White, 1976; Pilcher and Fryer, 1980a,b). An alternate definition is
the transmission of a virus within the egg (Nicholson, 1982) and this narrower
definition has caused confusion.
The most frequent evidence for vertical transmission is the association
between the shipment of infected eggs into new geographical areas and resultant
outbreaks of IHN (Plumb, 1972; Holway and Smith, 1973; Sano et al., 1977; Niu
and Zhao, 1988). Further evidence of vertical transmission is that IHN has
occurred in progeny from eggs disinfected with iodophor and raised in virus-free
water (Wingfield and Chan, 1970; Ratliff, 1982; Mulcahy and Bauersfeld, 1983;
Mulcahy and Pascho, 1985; Meyers et al., 1990; Roberts, 1993). However,
vertical transmission may be an infrequent event. There are several reports
where IHNV-infected parents did not produce IHNV-infected progeny when the
eggs were raised in virus-free water (Amend, 1975; LaPatra, 1990; Engelking et
al., 1991; LaPatra et al., 1991b; Yamazaki and Motonishi, 1992; Traxler et al.,
1997). This has raised doubts as to whether vertical transmission really occurs.
However, negative results do not eliminate the possibility of vertical
transmission of IHNV. Mulcahy and Pascho (1985) indicated it was difficult to
demonstrate vertical transmission and they were only successful three times in
isolating IHNV from live and dead eggs and fry of infected adult sockeye
salmon. They found that only a small portion of sockeye eggs and fry contained
IHNV and that not all parents transmitted the virus to their progeny and not
every egg from infected females contained the virus. These observations would
explain why studies to demonstrate vertical transmission have failed when only
a small number of eggs were used, and why IHN epizootics appear intermittently
within hatcheries.
It is accepted that salmonid reproductive products can harbour IHNV, but it
is controversial whether the virus is on the egg surface or within the egg.
Observations that disinfection of eggs does not always prevent IHNV infection
of progeny suggests that the virus is within the egg (Amend, 1975). However,
the virus may be external. Another fish virus, IPN virus (IPNV), adheres
strongly to the chorion of water-hardened eggs, but not to unfertilized eggs
96
(Ahne and Negele, 1985). Infectious pancreatic necrosis virus was isolated from
eggshells after fry hatched and the fry may become infected by eating the
eggshells. The chorion of unfertilized eggs is smooth, but hardened eggs have a
rough, lobed and porous texture, which would provide anchorage for the virus
and may prevent disinfectants from reaching the virus. A similar situation may
be occurring with IHNV. Examination of eggshells for IHNV has not yet been
done.
Fish species may be an important determinant for vertical transmission.
When eggs of masu salmon and chum salmon were exposed to IHNV and then
fertilized, the eggs and resulting fry were not infected (Yoshimizu et al., 1989).
The stage of egg development influenced the susceptibility to IHNV replication.
Exposure of unfertilized eggs to virus did not result in viral replication, but when
eyed eggs were injected with IHNV, the virus replicated and the resulting
fry suffered IHN-induced mortality. Egg-yolk components inhibited viral
replication. An increase in IHNV susceptibility as the egg matured was
correlated with a decrease in yolk components. Yoshimizu et al. (1989)
concluded that direct vertical transmission of IHNV within the egg is doubtful.
The anti-IHNV action of the yolk-sac components may be species-specific.
Rainbow trout may have similar viral-inhibiting components in the yolk-sac,
since fry developed IHN when eyed eggs were immersed in IHNV (Amend,
1975). However, sockeye salmon do not appear to have viral-inhibiting egg
components, since Burke and Mulcahy (1983) found that IHNV infectivity was
stable in egg homogenates and Mulcahy and Pascho (1985) isolated IHNV from
eyed eggs reared in virus-free water. Inhibition of IHNV by egg components of
other species does not appear to have been tested.
Infectious haematopoietic necrosis virus is frequently isolated from milt
(Wingfield and Chan, 1970; Meyers et al., 1990; Yamazaki and Motonishi,
1992) and male salmonid fish may have a role in vertical transmission. Sockeye
salmon and steelhead trout have a lower prevalence of infection and viral titres
in milt than in the kidney and spleen (Mulcahy et al., 1987). The prevalence of
infection in milt is lower than that in ovarian fluid, but the proportion of males
with high milt viral titres may be equivalent to that of females with high virus
concentrations in the ovarian fluid (Meyers et al., 1990). Virus strongly and
quickly adsorbs to the surface membrane of steelhead trout and chinook salmon
sperm (Mulcahy and Pascho, 1984). Sperm which has adsorbed IHNV from
male fish or from infected ovarian fluid could deliver the virus directly into the
egg during fertilization (Mulcahy and Pascho, 1984; Meyers et al., 1990).
However, the role of sperm in IHNV transmission is still unknown. Contam-
ination of masu salmon or chum salmon milt with IHNV did not result in
infection of eggs or fry (Yoshimizu et al., 1989) and there is no direct evidence
that sperm can transmit IHNV into eggs.
Additional factors such as hatchery practices, genetics and environmental
conditions may influence vertical transmission. Large-scale hatchery operations
raising over 20 million eggs annually may have an increased probability of
vertical transmission. However, hatcheries rearing less than 7 million eggs may
have no IHNV losses, despite females having a 100% prevalence of infection
and high viral titres (Meyers et al., 1990). Infectious haematopoietic necrosis
epizootics can be prevented by not mixing eggs from several females and by
incubating the eggs in virus-free water. Mixing of eggs from many females may
contaminate the eggs from females that had a low viral titre with a lethal dose of
virus from a few females with high virus titres (Mulcahy et al., 1983b). The roles
of fish genetics and environmental factors in vertical transmission are unclear.
Fry to juvenile susceptibility to infectious haematopoietic necrosis virus
Fry and fingerlings are most susceptible to IHNV infection and mortality.
Vertical transmission may be the cause of infection in some young fish, but no
correlation was found between outbreaks of IHN in progeny and the infection
rate or IHNV titres in the parents (Mulcahy et al., 1983b). Horizontal virus
transmission from fish to fish through the water is the major mechanism of
spread between fry (Amend, 1974). Prior to the onset of an epizootic, viral titres
in water may be undetectable or very low (up to 0.07 pfu ml
1
), but during the
early stages of epizootics viral titres rise and may reach 50 pfu ml
1
to over 10
3
TCID
50
ml
1
during epizootics (Nishimura et al., 1988; Zhang and Congleton,
1991, 1994). Viral titres are progressively amplified by repeated cycles of
shedding and infection (Zhang and Congleton, 1994). Hatchery effluent water
has had a viral titre of 400 pfu ml
1
(Leong and Turner, 1979) and this is high
enough to infect fish.
Young fish show signs of IHN and mortalities within 510 days of exposure
(Wingfield and Chan, 1970; Amend, 1975; Nishimura et al., 1988: Yamamoto et
al., 1990). The general pathology and histopathology of IHNV infections are
well characterized, but it is only recently that the route of virus entry has been
investigated. Studies with rainbow trout, coho salmon and steelhead trout
immersed in IHNV indicated that the gills, the oral region, the OCSR and the
skin are the principal sites of virus entry (Yamamoto et al., 1990; Drolet
et al., 1994; Helmick et al., 1995a,b). No histological changes or virus was
apparent in the gill tissues within 24 h postinfection (Helmick et al., 1995a), but
virus was observed at 2 days (Drolet et al., 1994) and 9 days (Yamamoto and
Clermont, 1990) in rainbow trout fry. Yamamoto et al. (1990) indicated that the
skin was a major portal of entry and was more important than the gills. However,
skin epithelial cells were weakly positive by immunohistochemistry at 1 day
postinfection and it appeared that the epithelial cells were only transiently
infected in steelhead trout fry (Drolet et al., 1994). In coho salmon and rainbow
trout fry, another early target area for IHNV attachment and replication is the
OCSR, particularly the MSSG (Helmick et al., 1995b). Because of the proximity
of the MSSG to the swim-bladder duct, which is adjacent to the anterior kidney,
these researchers suggested that the MSSG may be a portal of entry for viral
infection. In steelhead trout fry, the progression of the virus was followed for 14
days postinfection (Drolet et al., 1994). It was found that virus infection
progressed from two major sites: from the gill into the circulatory system and
from the oral region into the GI tract and then the circulatory system. Once
IHNV was in the blood, the virus was disseminated to virtually every organ.
Although IHN frequently occurs in fry and fingerlings reared in hatcheries,
IHN also occurs in feral or wild salmon. There have been at least two instances
where sockeye salmon fry emerging from the gravel in spawning channels died
98
of IHN, and natural epizootics have occurred in sockeye salmon fry and smolts
(Williams and Amend, 1976; Traxler and Rankin, 1989; Olson and Thomas,
1994).
In older fish, yearling rainbow trout (Busch, 1983; Roberts, 1986), chinook
salmon (LaPatra et al., 1990d), sockeye salmon smolts (Yasutake, 1978; Burke
and Grishchkowsky, 1984; Olson and Thomas, 1994) and 23-year-old kokanee
salmon (Amend, 1974; Traxler, 1986; Banner et al., 1991) have succumbed to
IHNV in fresh water. In many cases, infected fish within the population or other
species of infected fish are thought to be the virus source, and horizontal
transmission through the water probably occurred (Burke and Grishchkowsky,
1984; LaPatra et al., 1990d; Olson and Thomas, 1994).
Potential sources of infectious haematopoietic necrosis virus
Sources of IHNV other than salmonid fish have not been identified, but potential
sources include freshwater and marine invertebrates, sediment and other fish
species. Depending on temperature, microflora and electrolyte concentration,
IHNV survives in fresh water for from 3 days to several months (Wedemeyer et
al., 1978; Toranzo and Hetrick, 1982; Yoshimizu et al., 1986; Kamei et al.,
1988b). Infectious haematopoietic necrosis virus survives longer in salt water
than in fresh water (Winton et al., 1991), and, although it is unlikely that fish
become infected through the ocean water, because of the high dilution factor,
this remains a possibility. Ingestion of marine organisms harbouring IHNV is
another possible mechanism of infection, but IHNV has not been isolated from
marine organisms (Traxler et al., 1997).
Salmon leeches (P. salmositica) can harbour IHNV; however, there is little
relationship between leech viral titres and those of the host sockeye salmon
(Mulcahy, 1986; Yamamoto et al., 1989; Mulcahy et al., 1990). Although
leeches can have high IHNV titres, it is not clear whether IHNV is actually
replicating in the leech or whether the leech is concentrating and storing the
virus from fish blood. Infectious haematopoietic necrosis virus has also been
isolated from copepods (Salmincola sp.) and the common mayfly (Callibaetis
sp.). Copepods always had lower viral titres than those of fish gill tissue
(Mulcahy et al., 1990). Transmission from invertebrates to salmonid fish has not
been demonstrated, but the role of invertebrate reservoirs cannot be ruled out.
Control strategies and vaccines
Disinfection practices and antiviral compounds
Present control of IHNV relies upon hatchery practices. Eggs and fry should be
from stocks with no previous history of the virus and reared in specific
pathogen-free (SPF) water. Eggs from fish stocks with a history of IHNV
outbreaks are treated with iodine-containing surfactant to inactivate virus
(Amend and Pietsch, 1972). Iodophor (100 mg l
1
) egg disinfection destroys at
least 99.98% of IHNV on the surface of green and eyed eggs (Goldes and Mead,
1995). In most cases this treatment has been successful in preventing the
transmission of IHNV; however, occasional outbreaks of disease still occur in
the progeny after treatment with iodophor and rearing in SPF water (Myers et
al., 1990).
Controlling the spread of IHNV by disinfection of water-supplies has had
limited success. Ozonation, ultraviolet light, chlorinationdechlorination and
the addition of elemental iodine or other germicides, such as ethanol, phenol,
cresol soap solution or methanol, have all been effective under laboratory
conditions (Winton, 1991; Inouye et al., 1992). In field trials, problems have
arisen because of mechanical and electrical failures. Nevertheless, the approach
has sufficient promise, and hatcheries with limited or no SPF waters are
beginning to incorporate water disinfection equipment into their facilities (J.R.
Winton, personal communication).
Twenty-four antiviral compounds were tested in vitro in CHSE-214 cells
and 11 were toxic for the virus (Hasobe and Saneyoshi, 1985). Of these, five
were tested in steelhead trout fry and there were more survivors in the treatment
groups (1434%) than in the control, untreated group (8%). The five compounds
(6-thioinosine, 5-hydroxyuridine, 9-(S)-(2,3-dihydroxypropyl) adenine, virazole
and chloroquine) were added to the water daily or on alternate days after
infection. Hudson et al. (1988) examined the antiviral compounds amantadine,
mitisazone, bis-benzimidazole and ribavirin. Amantadine was very effective
against IHNV in rainbow trout cells in culture. The other compounds were also
effective but had some associated cytotoxicity. It is likely that the costs of these
antiviral compounds make their use prohibitive for aquaculture. An antiviral
compound produced by Pseudomonas fluorescens has also been found to be
effective against IHNV and O. masou virus (OMV), a fish herpes virus (Kamei
et al., 1988a; Kimura et al., 1990). The compound, a cyclic peptide of 1126 Da,
has been shown to be very effective in vitro, producing a 94% plaque reduction
at concentrations of 12.5 mg ml
1
. The compound did not inhibit the fish viral
pathogen IPNV, a non-enveloped birnavirus, suggesting to the investigators that
the inhibitor is specific for enveloped viruses.
Vaccines
Several vaccines for IHNV have been developed and the relative merits of these
vaccines have been discussed (Leong et al., 1988, 1997; Winton, 1991; Leong,
1993; Leong and Fryer, 1993). There are five basic formulations used in vaccine
development: live attenuated vaccines, whole inactivated vaccines, purified
subunits (proteins or glycoproteins) of the pathogen, purified proteins from
cloned genes and DNA vaccines. These formulations have had some success
against IHNV in the laboratory, but a commercial vaccine is not available.
The killed vaccine is IHNV-inactivated by either -propiolactone (Amend,
1976) or formalin (Nishimura et al., 1985) and is most effective when delivered
by i.p. injection. The requirement for i.p. injection and the cost are the
limitations of killed vaccines. Attenuated vaccine for IHNV, although highly
effective, has not been adopted because of possible residual virulence and
present policies regarding certification of fish stocks. Thus, several investigators
have turned to recombinant DNA technology to develop a subunit vaccine for
IHNV that utilizes the viral glycoprotein gene (Leong et al., 1992).
Viral glycoprotein purified from one IHNV isolate induced protective
100
immunity against at least five different IHNV isolates, representing the different
electropherotypes defined by Hsu et al. (1986) (Engelking and Leong, 1989b).
Thus, a single glycoprotein gene from a single IHNV isolate could be used to
develop a recombinant-DNA-based vaccine. Using several different host/vector
expression systems, the IHNV glycoprotein has been produced in Escherichia
coli (Gilmore et al., 1988), Caulobacter crescentus (Bingle et al., 1997), insect
cells with a baculovirus expression system (Koener and Leong, 1990) and
Aeromonas salmonicida (Noonan et al., 1995). Efficacy of these vaccines has
only been proven for the E. coli- and C. crescentus-derived vaccines (Gilmore et
al., 1988; Oberg et al., 1991; B. Simon and J.C. Leong, unpublished data).
The latest approach in vaccine design is genetic immunization, using naked
DNA. This approach is based on the finding that skeletal muscle cells injected
with naked DNA are able to express the plasmid DNA-encoded proteins (Wolff
et al., 1990; Tang et al., 1992). The newly synthesized antigen can stimulate a
specific immune response, composed of cytotoxic T cells, T-helper cells and
antibodies. A DNA vaccine encoding the IHNV G protein, under the regulatory
control of the cytomegalovirus immediate early promoter/enhancer, has been
developed (Anderson et al., 1996a,b). In this study, 8385% of the animals
receiving the DNA (pCMV4-G or pCMV4 + pCMV4-N) injection survived an
IHNV challenge, whereas only 2530% of the animals in the control plasmid
and unvaccinated groups survived. The fish in the experimental group also
developed G-protein-specific and viral-neutralizing antibody responses after
DNA vaccination. Thus, the DNA vaccine for IHNV is very promising. The
licensing of this type of vaccine for commercial use in aquaculture will require
proof of safety, and the very expensive cost of producing and administering the
DNA is a problem, but the efficacy of the DNA vaccine is remarkable.
FUTURE DIRECTIONS FOR RESEARCH
Further research is required to fully understand how IHNV is maintained in fish
and how the virulence of the virus is altered for different fish species. Only a full
understanding of the entire life cycle of the virus will allow development of
effective measures to control the spread of IHNV and IHN.
Diagnosis
Since IHN is an untreatable disease and control is by avoidance, it is critical that
there be a rapid diagnostic technique for IHNV. Current diagnostic assays are too
slow to adequately prevent spread of the virus. To decrease the time required,
sensitive assays to detect IHNV directly from fish tissue need to be developed.
Assays such as coagglutination, DFAT and ELISA, which incorporate MAbs
may have improved sensitivity and specificity and ultimately allow detection of
IHNV without the use of cell culture.
Infectious haematopoietic necrosis virus pathogenicity and
epizootiology
Progress has been made in elucidating the life cycle of IHNV, but this remains a
critical area for future research. Studies are needed to fully characterize the
latent state in rainbow trout and determine if latent infections also occur in other
fish species. It must also be determined if viral reactivation occurs and, if so, the
conditions that trigger the production of infectious virus need to be elucidated.
Chronic IHNV infections in fish over 6 g often cause less than a 30%
mortality, but, because losses occur in market-sized fish and fish may also be
scoliotic, high economic losses can occur. It was estimated that discarding
scoliotic fish caused losses of $350,000 per year at one facility in Idaho (R.A.
Busch in Nicholson, 1982). Infectious haematopoietic necrosis virus isolates
show variations in tissue tropism (Kim et al., 1994), but the mechanism causing
scoliosis is unknown. Studies are required to elucidate the virulence factors of
IHNV, identify IHNV cell receptors and further characterize the correlations
between IHNV antigenic types and virulence.
It was recently shown that IHNV could be isolated from sexually immature
fish in the marine environment (Traxler et al., 1997). These fish, as well as
sexually mature fish in fresh water, may have a reactivation of a latent infection.
Alternatively, fish may be infected through contact with a marine or freshwater
reservoir or vector of IHNV. Although marine or freshwater organisms could
harbour IHNV, sources of IHNV other than salmonid fish have not been
identified. The susceptibility of non-salmonid fish to IHNV and the role of wild
fish in spreading IHNV have not received much attention.
The evidence for vertical transmission is primarily circumstantial.
Confirming that vertical transmission occurs will be difficult, but attempts
should continue. Another area that needs further investigation is the immune
response to IHNV and whether maternal transfer of antibodies to eggs occurs.
Vaccines
The promise of recombinant DNA techniques to produce inexpensive and safe
vaccines has been realized to some extent with several IHNV vaccines.
However, with few exceptions, most of the vaccines under development require
extensive purification and delivery by injection. For very small fish, injection
vaccination is not practical. One of the primary objectives of future vaccine
development will be to enable fish farmers to deliver an oral or immersion
vaccine that is safe and economically produced. The advantages of oral
vaccination are, of course, not only the ease of delivery, but also the stimulation
of mucosal immunity, which is very important for IHNV, since the virus enters
the body through the oral mucosa (Drolet et al., 1994).
Several methods of introducing an IHNV vaccine orally have been
considered and they include microencapsulation, delivery with chimerical viral
vectors, such as polio or adenovirus, and bacterial carriers which replicate in the
intestinal tract. These carriers can be used to deliver the DNA vaccine as well, a
102
procedure that has been demonstrated in warm-blooded vertebrates: an
attenuated Shigella flexneri that no longer induced apoptosis of macrophages but
was still able to enter epithelial cells was used to deliver plasmid DNA
(Sizemore et al., 1995). For fish vaccines, investigators are considering the
intracellular bacterial pathogens, Renibacterium salmoninarum, A. salmonicida,
and Edwardsiella ictaluri as potential vectors.
Two of the most serious constraints to evaluating the effect of IHNV
vaccines on the fish immune response are the lack of leucocyte cell lines and
reagents that can distinguish the different types of immunoreactive cells in
salmonid fish. The influence of different antigen delivery systems on the
antibody isotype and lymphokine profile has long been recognized in mammals
and this effect is well characterized in mice (Hocart et al., 1989; Nerome et al.,
1990; Brett et al., 1993). Murine antibody of the IgG2a isotype is associated
with humoral immunity to many viruses. It promotes phagocytosis, activates
complement and mediates antibody-dependent cellular cytotoxicity. When mice
are vaccinated with inactivated or live influenza virus, the predominant antibody
response is the IgG2a isotype. When isolated influenza viral components or a
recombinant nucleoprotein is used to vaccinate mice, the predominant antibody
response is the IgG1 isotype, which is much less protective (Hocart et al., 1989).
This type of study is not possible in fish, as we lack reagents to distinguish the
components of the fish immune system.
Adjuvants and the delivery vehicle are extremely important in stimulating a
protective immune response in animals. Scientists in academic institutions and
vaccine companies are beginning to examine the effect of delivery vehicles, such
as liposomes, and different adjuvants on aquaculture vaccines. Until we
understand how fish respond to different antigens, developing vaccines for
IHNV will be a serendipitous rather than a deliberate pursuit.
ACKNOWLEDGEMENTS
The authors thank Pinwen Chiou, Barbara Drolet, Eric Anderson and Patricia
Ormonde for their thesis material, which was used in this review. We also thank
Patrick Woo for his patience and apologize to all whose work we have not cited.
We acknowledge funding by the US Department of Agriculture (to the Western
Regional Aquaculture Consortium) under grant 92-38500-7195, project no.
92080441; Oregon Sea Grant with funds from the National Oceanic and
Atmospheric Administration, Office of Sea Grant, Department of Commerce,
under grant NA89AA-D-SG108, project R/FSD-16, and grant NA36RG451,
project R/FSD-23 and amendment no. 2; the National Oceanic and Atmospheric
Administration (SaltonstallKennedy funds), grant NA46FD0490; and the
Bonneville Power Administration, projects DE-FG79-88BP92431 and 88-152.
This is an Oregon State Agriculture Experiment Station Paper No. 11371.
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CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Protein Mol. mass

Number of protein molecules per virion was calculated by dividing the

Molecular mass estimates were determined by electrophoretic migration in

Percentage of each protein determined by using virus labelled with

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