infectious pancreatic necrosis

Pictures

Picture	Title	Caption	Copyright
Title Isolation of aquatic birnaviruses in the USA Caption Frequency of isolation of aquatic birnaviruses in the USA. Copyright P. W. Reno	Isolation of aquatic birnaviruses in the USA	Frequency of isolation of aquatic birnaviruses in the USA.	P. W. Reno
Title Frequency of isolation of aquatic birnaviruses in Canadian Provinces Caption Frequency of isolation of aquatic birnaviruses in Canadian Provinces. Copyright P. W. Reno	Frequency of isolation of aquatic birnaviruses in Canadian Provinces	Frequency of isolation of aquatic birnaviruses in Canadian Provinces.	P. W. Reno
Title Frequency of isolation of aquatic birnaviruses in other countries Caption Frequency of isolation of aquatic birnaviruses in other countries. Copyright P. W. Reno	Frequency of isolation of aquatic birnaviruses in other countries	Frequency of isolation of aquatic birnaviruses in other countries.	P. W. Reno

Identity

Preferred Scientific Name

• infectious pancreatic necrosis

International Common Names

• English: aquabirnavirus infection; aquatic birnavirus infection; infectious pancreatic necrosis and associated aquatic birnaviruses

English acronym

• IPN

Pathogen/s

Overview

Infectious pancreatic necrosis (IPN) is a highly contagious viral disease of young fish of salmonid species held under intensive rearing conditions (Hill, 1982; Wolf, 1988; Wolf et al., 1960). The disease most characteristically occurs in rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), brown trout (Salmo trutta), Atlantic salmon (Salmo salar), and several Pacific salmon species (Oncorhynchus spp.). Susceptibility generally decreases with age, with resistance to clinical disease in salmonid fish usually being reached at about 1500 degree-days (value obtained by multiplying the age in days by the average temperature in degrees centigrade during the lifespan) (Dorson and Torchy, 1981), except for Atlantic salmon smolts, which can be affected normally within the first weeks after transfer from fresh water to seawater, but also before seawater transfer (Smail et al., 1989). The first sign of an outbreak in salmonid fry is frequently a sudden and usually progressive increase in daily mortality, particularly in the faster growing individuals. Clinical signs include darkening pigmentation, a pronounced distended abdomen and a corkscrewing/spiral swimming motion. Cumulative mortalities in a tank may vary from less than 10% to more than 90% depending on the combination of several factors, such as virus strain (McAllister and Owens, 1995) and infectious dose (Okamoto et al., 1984), host and environment (Dobos and Roberts, 1983). For further details see reviews by Hill (1982), Reno (1999) and Wolf (1988).

The disease is transmitted horizontally via the water route. Vertical transmission has been demonstrated for brook trout and rainbow trout, but this has never been successfully demonstrated in Atlantic salmon (Ahne et al., 1989; Ahne and Negele, 1985; Dorson and Torchy, 1981). Surface disinfection of eggs is not entirely effective in preventing vertical transmission (Bullock et al., 1976).

The disease has a wide geographical distribution, occurring in most major salmonid-farming countries of North and South America, Europe, Asia and Oceania (Anderson, 1996; Crane et al., 2000).

IPN virus (IPNV), or viruses showing serological relatedness to IPNV, have been reported to cause diseases in some farmed marine fish species, such as yellowtail (Seriola quinqueradiata) (Nakajima et al., 1993), turbot (Scophthalmus maximus) (Castric et al., 1987; Mortensen et al., 1990; Novoa et al., 1993b), dab (Limanda limanda) (Olesen et al., 1988), halibut (Hippoglossus hippoglossus) (Mortensen et al., 1990, Rodger and Frerichs, 1997) and Atlantic cod (Gadus morhua). Subclinical covert infections have been detected in a wide range of estuarine and freshwater fish species, such as loach (Misgurnus anguillicaudatus) (Chou et al., 1993), pike (Esox lucius) (Ahne, 1978) and numerous other species in the families Anguillidae, Atherinidae, Bothidae, Carangidae, Catostomidae, Cichlidae, Clupeidae, Cobitidae, Coregonidae, Cyprinidae, Esocidae, Moronidae, Paralichthydae, Percidae, Poecilidae, Sciaenidae, Soleidae and Thymallidae (Reno, 1999). It has been suggested to reserve the term IPNV for when the virus is isolated from a salmonid fish or has been shown to be able to produce IPN in salmonids through challenge experiments. Otherwise the term aquatic birnaviruses or aquabirnavirus is used.

The causative agent, IPNV, is the type species of the genus Aquabirnavirus of the Birnaviridae virus family. Birnaviruses have a bi-segmented (called segment A and B), double-stranded RNA genome, which is contained within a medium-sized, non-enveloped, single-shelled, icosahedral capsid (see review by Dobos 1995). Isolates display wide antigenic diversity (Dorson and Torchy, 1981; Hill and Way, 1995; Melby et al., 1994; Okamoto et al., 1983) and fall into two serogroups that do not cross-react in serum neutralisation tests (Ahne et al., 1989; Olesen et al., 1988; Underwood et al., 1977), with the majority of strains belonging to serogroup A, which comprises at least nine serotypes (Hill and Way, 1995). Isolates also show marked differences in degrees of virulence (Hill, 1982; Hill, 1992; McAllister and Owens, 1995).

Control methods rely on the implementation of control policies and of hygiene practices in salmonid husbandry, through the avoidance of the introduction of fertilised eggs originating from IPNV-carrier broodstock, and the use of a protected water supply (e.g. spring or borehole pond) where the ingress of fish, particularly possible virus carriers, is prevented. In outbreaks, a reduction in the population density (‘thinning out’) may help to reduce the overall mortality. There are vaccines available aimed to induce protection of Atlantic salmon smolts after seatransfer (Christie 1997) but there are few publication of results allowing for reliable judgement about vaccine efficacy, and little information is provided to validate the protection under field conditions.

Infectious pancreatic necrosis (IPN) is a disease which causes high mortalities in young salmonid fishes; the aetiological agent of the disease (IPNV) was the first virus to be isolated from teleosts (Wolf et al., 1960). In the time since that work, agents biochemically and serologically identical to IPNV - the aquatic birnaviruses - have been detected in a wide variety of both diseased and non-diseased fishes and other aquatic animals.

The early work on IPNV was mainly responsible for the development of fish virology. Techniques for the isolation of viruses from teleosts were modified from methods used for the isolation of mammalian viruses.

The virus itself has sufficiently unusual biophysical characteristics to warrant its place as the archetype of a new family of viruses, the birnaviruses (Dobos et al., 1979).

Serologically and biochemically related viruses induce a variety of disease syndromes in taxonomically diverse host groups, which also include aquatic invertebrates. The worldwide distribution of these viruses in cultured as well as wild fishes and shellfish provides significant potential for economic damage.

Infectious pancreatic necrosis and aquatic birnavirus-induced diseases can cause a significant economic impact on cultured fishes, especially salmonids. The cost can be as a consequence of lethal disease in young-of-the-year fish, or the destruction of infected stocks even in the absence of disease, or in Atlantic salmon smolts after sea transfer. The virus may be vertically transmitted; therefore, the detection of virus in broodstock often means the destruction of these animals. In the late 1980s, there were instances in the USA where 7 million young salmonids were destroyed to eliminate IPNV from a single hatchery (P. Walker, Colorado, 1988, personal communication) and others where detection of the virus in returning coho salmon resulted in the destruction of 2 million eggs (Olson et al., 1994); similar occurrences were also reported in Canada (B. Larson, Alberta, 1988, personal communication). In addition, regulations currently in place to reduce the dissemination of diseases of salmonids often result in restriction of the movement of IPNV carrier fish between local, regional and/or national boundaries. These restrictions have attendant adverse economic consequences to aquaculturists. In many instances, the slaughter of infected fish and/or the restriction of movement of fish may be more economically detrimental than the loss associated with direct mortalities.

In some countries there are mandatory destruction of salmonid stocks and restriction of movement after detection of IPNV. This is controversial. Since swim-up fry and fingerlings are generally affected, the impact of the disease can be ameliorated by spawning additional brood fish to compensate for the potential loss caused by the disease. Another perspective is the dissemination into watersheds of a virus unresponsive to therapeutic agents

which can readily infect susceptible native or cultured stocks in adjacent drainages. Given the myriad of variables which come into play in the generation of IPN and the potential for extensive, irreparable damage to stocks, it would seem prudent to adhere to more stringent rather than lax requirements. Resolution of the problem of IPNV management will be difficult and achieved only through a thorough knowledge of the epizootiology of the disease and an adequate database on both the disease and the virus.

Infectious pancreatic necrosis virus may also have an economic impact on aquaculture because of fish health inspections mandated prior to the movement or sale of fish across local, regional and national boundaries. Tests required for the detection of IPNV and other teleost viruses often represent an expense for aquaculturists. Again, this can be a contentious issue because the regulations are not often applied uniformly, even within regions regulated by one agency.

Control of the disease worldwide will also be difficult, given the ubiquitous nature of the agent.

Table. Natural diseases caused by, or associated with, aquatic birnaviruses. Included are isolations of aquatic birnaviruses in the absence of substantive proof of causal association with disease.

Diseases for which Rivers' postulates have been fulfilled

Diseases for which birnaviruses have been isolated from moribund animals

1, Wood et al., 1995; 2, Castric et al., 1987; 3, Sorimachi and Hara, 1985; 4, Sano et al., 1981; 5, Stephens et al., 1981; 6, Kusuda et al., 1989; 7, Bonami et al., 1988; 8, Schutz et al., 1984; 9, Chen et al., 1990; 10, Diamant et al., 1988; 11, McAllister et al., 1984; 12, Nagabayashi and Wolf, 1979; 13, Giorgetti, 1990; 14, Lo et al., 1991; 15, Hill et al., 1982.

Host Animals

Hosts/Species Affected

Early reports of IPNV were limited to epizootics in cultured brook trout, Salvelinus fontinalis (Wood et al., 1955; Snieszko et al., 1957, 1959; Wolf et al., 1960). With the development of several continuous cell lines from teleost fishes (Wolf and Quimby, 1962; Wolf and Mann, 1980) and as more laboratories became more proficient in the use of cell lines, it was found that IPNV was responsible for disease in a variety of salmonid species, including members of the genera Salmo, Salvelinus and Oncorhynchus. During the early 1970s, IPN caused high mortalities in European and Japanese rainbow and brown trout (Ball et al., 1971; Sano, 1971; Vestergård-Jørgensen and Kehlet, 1971).

The first report of birnavirus-induced disease in non-salmonids was in Japanese eels (Anguilla japonica), first sampled in 1969 (Sano et al., 1981). This disease of cultured eels was characterized as branchionephritis. Experimental infection with the virus (eel virus European (EVE)) indicated that the branchial lesions were probably due to supervening bacterial infections and that the disease should be referred to as eel nephritis. Neutralization studies indicated that the aetiological agent was a birnavirus which is serologically related to the Sp (from Spjarup, Denmark) serotype of IPNV. Another birnavirus-induced disease which caused mortalities in Japanese fish caused ascites in yellowtail, Seriola quinqueradiata (Sorimachi and Hara, 1985). The condition was in feral fry used as seed stock for mariculture. Because clinical signs occurred in fry, the disease is similar to IPN. In addition, mortalities occurred in another flat-fish species from Japan, the Japanese flounder (Paralichthys olivaceus). Fish held in culture facilities died from ascites and cranial haemorrhage. An aquatic birnavirus was isolated from moribund fish and an ascitic disease was experimentally induced by intraperitoneal injection of the virus from moribund fish showing either disease syndrome (Kusuda et al., 1989).

Stephens et al. (1983) isolated an aquatic birnavirus from the brain and other tissues of menhaden (Brevoortia tyrranus) with ‘spinning disease’, a perennial high-mortality disease in the Chesapeake Bay region of the eastern USA. The virus was isolated in a cell line from the same species (Stephens, 1981). The disease was experimentally induced in susceptible menhaden after intraperitoneal injection of the virus. Subsequent attempts to isolate the same virus from diseased menhaden in other teleost continuous cell lines were unsuccessful. Since the original menhaden cell line was lost, there have been no further isolations of the virus from diseased or non-diseased fish.

In each of these cases, Rivers’ postulates have been fulfilled, indicating that the aetiological agents of the diseases were aquatic birnaviruses which cross-reacted serologically with IPNV. In several other instances, birnaviruses have been isolated from aquatic animals undergoing epizootic disease, but as yet there has not been irrefutable evidence that the isolated birnaviruses were causal agents.

A birnavirus infection was associated with haematopoietic necrosis and caused high mortalities in turbot (Scophthalmus maximus) held at 18°C in sea water on the coast of France (Castric et al., 1987). Within 15 days after transfer from fresh water to sea water, cumulative mortalities reach 25%, but no mortalities occurred in the same population of fish held at the freshwater site. No definitive studies have been done to confirm Rivers’ postulates for this disease and a birnavirus is suspected.

In addition to a birnavirus isolated from Japanese eels with nephritis (Sano et al., 1981), a birnavirus was also isolated in Japan from eels exhibiting stomatopapillomas (Nagabayashi and Wolf, 1979). Although the virus was isolated from moribund fish, there was no indication that the virus was capable of producing the papillomatous lesions in homologous animals.

Transmitting papillomas in homeotherms is often difficult, and the strong association of the papilloma group of viruses with this disease in other animals makes it unlikely that a birnavirus was the causal agent in eels. In retrospect, it is possible that the eels affected with the stomatopapillomas were adventitious carriers of the virus (Sano et al., 1981) and the isolated aquatic birnavirus had no involvement in the development of papillomas.

An aquatic birnavirus belonging to the Sp (European) serotype was isolated from visceral organs of snakehead fish (Ophicephalus striatus) and eyespot barb (Hampala dispar) undergoing episodes of ulcerative disease in Thailand and Laos (Wattanavijarn et al., 1988). Again, there is no further information to indicate that this agent was responsible for the syndrome.

Another birnavirus was isolated from sea bass (Dicentrarchus labrax) off the coast of France (Bonami et al., 1983) during a severe epizootic, in which mortalities reached 95%. There was no evidence of bacterial infection or parasites in the moribund fish, but again it is uncertain whether the virus is merely associated with the mortality or was the aetiological agent.

With the rather large exception of IPN disease, the disease syndromes that have been strongly associated with aquatic birnaviruses (yellowtail ascites, Japanese flounder disease syndromes, eel nephritis, spinning disease of menhaden and turbot renal necrosis) occur in marine animals or in those that spend the bulk of their life in seawater. In the intensive marine farming of Atlantic salmon there has likewise been an increase of the occurrence of IPN-virus and IPN outbreaks in post-smolts in sea farms through the second half of the 1980s (Willumsen 1988, Bruheim 1991). Both Jarp (1999) and Brun (2001) have shown that the median time from sea transfer to IPN outbreak in post smolts is about 35 days (range 3-75, Brun 2001). Melby (1991) found that 63.7% out of 1939 individual post-smolts from 608 sea farms of Atlantic salmon in Norway were positive for IPNV.

It is likely that diseases caused by aquatic birnaviruses are more extensive in nature because reports of this family of viruses are primarily in cultured animals, which have economic importance. Relatively little information is available regarding birnavirus infections in wild populations of aquatic animals that are not of commercial importance and this is unlikely to change unless some of these animals develop commercial importance.

Table. Species of aquatic animals from which birnaviruses have been isolated.

1, Lo et al., 1991; 2, Hill et al., 1982; 3, Hill, 1976; 4, Bovo et al., 1984; 5, Munro et al., 1976; 6, Stephens et al., 1983; 7, Sano et al., 1981; 8, Hudson et al., 1981; 9, Ahne, 1979; 10, Ahne et al., 1989b; 11, Parisot et al., 1963; 12, Wolf, 1988; 13, Wolf and Pettijohn, 1970; 14, Sano, 1973; 15, Hedrick et al., 1986a; 16, MacKelvie and Artsob, 1969; 17, Wolf et al., 1960; 18, Wattanavijarn et al., 1988; 19, Ahne, 1980; 20, Hill, 1982; 21, Chen et al., 1984; 22, Sorimachi and Hara, 1985; 23, Adair and Ferguson, 1981; 24, Wechsler et al., 1986b; 25, McAllister et al., 1984; 26, Castric et al., 1987; 27, Comps et al., 1991; 28, Yamamoto and Kilistoff, 1979; 29, Bonami et al., 1983; 30, T. Håstein, personal communication.

Distribution

Disease

While the disease was evidently first described from the Maritime Provinces, Canada (M’Gonigle, 1940), and thereafter in the eastern USA (Wood et al., 1955), it was later noted in many areas of the western USA (Yasutake et al., 1965), especially in rainbow trout and brook trout transferred from the eastern USA. Epizootics in rainbow trout with signs of IPN disease reported in France (Besse and de Kinkelin, 1965) were caused by an aetiological agent which was serologically distinct from the North American serotype (Wolf and Quimby, 1971). The detection of IPN in France was followed by diagnosis of disease in rainbow trout in Denmark (Vestergård-Jørgensen and Bregnballe, 1969), Norway (Håstein and Krogsrud, 1976), Sweden (Ljungberg and Vestergård-Jørgensen, 1973), the UK (Ball et al., 1971), Germany (Schlotfeldt et al., 1975) and Italy (Ghittino, 1972). Outside Europe, the disease has been documented in the Far East, usually in fish imported from enzootic areas or in fish cohabiting the same water source as infected imported fish. The virus has been isolated from several countries like Japan (Sans, 1971), Korea (Hah et al., 1984), Taiwan (Hedrick et al., 1983), China (Jiang et al., 1989), Thailand and Laos (Wattanavijarn et al., 1988), Australia (Crane et al., 2000), New Zealand (Anderson, 1996) and Turkey (Candan, 2002).

Virus

The isolation of aquatic birnaviruses from various geographical areas has increased markedly since the isolation of IPNV from the eastern USA (Wolf et al., 1960). In a few instances, there has been sufficient epizootiological evidence to support the orderly ‘spread’ of the virus from region to region. When the viruses were serotyped, they have in most cases proved to be serologically identical to serotypes of IPN from epizootics (Caswell-Reno et al., 1986; Hill and Way, 1995).

The aquatic birnaviruses have been isolated from regions where salmonids are reared and from areas without salmonids. The data are often difficult to access and summarize easily because of the inconsistent nature of the published literature and the variety of sources from which samples have been taken. The presence of these viruses in feral animals is a confounding factor. Since little current information about the distribution of aquatic birnaviruses is available in the literature, an attempt has been made in this review to update the information.

Prior to the late 1980s, the virus was found to be present in most areas of North America. In many areas, the prevalence levels were low, but in some areas (Maritime Canada, Pennsylvania, West Virginia and some north-eastern US states) a large number of hatcheries were contaminated. However, because of increased awareness of vertical transmission of the virus and regulations on movement of fish eggs, the majority of states and provinces have significantly reduced or eliminated the virus from hatcheries. There has been a marked reduction in reports of the virus, especially in the western USA and eastern Canada. This may be due to the reduction in rearing of brook trout, which serve as reservoirs for the virus in the western states.

Unfortunately, the situation in Europe and Asia is not as positive. The virus has been found in virtually every European country (de Kinkelin, 1989, personal communication), with almost total contamination of facilities in some countries (Melby et al., 1991). The pervasive dispersal of the virus in Europe may be due to the transfer of contaminated stocks and an inability to start with virus-free stocks without destroying the industry. The presence of IPNV in trout is often considered an impediment to the industry, not because of the consequences of disease but rather because of the restriction of fish movements (Ghittino, 1982).

The virus and epizootic outbreaks of the disease are widespread in salmonids in Asia, where importations from other countries, especially the USA and Europe, have been extensive. Most probably, the increased detection of IPNV in this region reflects an increased dispersal of the virus, an increased ability of fish health workers to isolate the viruses, and the examination of a wider variety of aquatic animals for birnavirus infection.

Iceland is one of the few countries which raise salmonids on a large scale and where extensive fish health inspections are routinely performed that have no reports of IPN. The country has very strict importation policy for fin-fish. However, birnavirus serogroup B has been isolated from Icelandic farmed halibut (Hippoglossus hippoglossus) (Skall et al., 2000).

The serotypes of aquatic birnavirus serogroup A are called A1 to A9, but are also known as West Buxton, Sp, Ab, He, Te, Can 1, Can 2, Can 3 and Jasper. There are antigenic cross-reactions between the viruses in serogroup A, and the criterion for each of these 9 serotypes being a serotype is suggestions based on calculations of results in neutralization tests (Hill and Way, 1995).The serotype A1 (WB archetype from brook trout in West Buxton, Maine, USA) is found extensively throughout the USA and on many occasions in Canada. However, the Canadian serotypes have rarely been isolated in the USA (Caswell-Reno et al., 1990). The isolations of A1 viruses from other countries have almost always been traced to the importation of specific batches of contaminated eggs or fish from the USA (Sano, 1971; McAllister and Reyes, 1984; Bragg and Combrink, 1987a). Interestingly, the A1 serotype has only been isolated from Europe in a single instance - from turbot in Spain (Novoa et al., 1993a) - although trout have been extensively transferred to Europe from the USA. Serotypes A2 (Sp) and A3 (Ab from Abild, Denmark) have been disseminated widely not only throughout Europe, but also to many countries in Asia. Serotype A2 has been detected only once in feral fish in marine waters off the coast of the USA (McAllister et al., 1984). The four serotypes Canada 1, 2 and 3 and Jasper, Alberta, are restricted to Canada, except for a few recent isolations from facilities in watersheds that are contiguous in the USA and Canada (Caswell-Reno et al., 1990). Although fish have been exported from Canada to many other countries, none of the Canadian 1, 2 or 3 serotypes have been found outside of Canada or its borders with the USA. Thus, the distribution of serotypes does not seem to support the hypothesis that the aquatic birnaviruses are spread from continent to continent with the importation of fish or fish eggs. It is likely that the virus had a global distribution prior to the widespread dissemination of salmonids in the nineteenth and the early twentieth century.

Distribution Table

The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.

Diagnosis

Clinical Diagnosis

Infectious pancreatic necrosis virus causes mortality of fry and fingerling salmonids and Atlantic salmon smolts, and is characterized by behavioural changes and gross external, internal and histopathological lesions. There are no specific pathognomonic signs of IPN disease. Behavioural changes (Wood et al., 1955) include anorexia and an agonal corkscrew swimming motion interspersed with ataxia. Nonspecific external signs include hyperpigmentation, exophthalmia and petechial haemorrhage on the ventral surfaces. Internal gross lesions are visceral petechia and an empty gut containing a yellow exudate. The disease may be manifested with only a few, or even none, of these signs. Microscopically, there is focal necrosis of the acinar and islet cells of the pancreas and of the haematopoietic cells of the kidney. There are typical icosahedral virus particles in the cytoplasm of pancreatic acinar cells (Lightner and Post, 1969; Hedrick et al., 1985). Viral titres in the tissues of infected fish are usually quite high, usually in the range of 10⁷-10¹⁰ (TCID₅₀) g^-1 (Wolf et al., 1969; Castric et al.,1987).

Survivors of epizootics or those fish with no disease have few effects of residual infection, although high titres of virus can be isolated from their viscera. In carrier Atlantic salmon there are interstitial cells in the lamina propria of the gut which appear necrotic, the so-called ‘McKnight cells’ (McKnight and Roberts, 1976). Studies examining the effect of IPNV infection on Atlantic salmon which evince no clinical signs of IPN indicate that there is no adverse effect on growth rate or susceptibility to other diseases (Smail et al., 1986).

Several studies have demonstrated that there are markedly variable virus titres in carrier fish. Yamamoto (1975b) found that rainbow trout had IPNV titres in the viscera ranging from 10^0.85 to 10^4.2 TCID₅₀ g^-1, while they ranged from 10^0.85 to 10^6.5 TCID₅₀ g^-1 in brook char (Reno, 1976).

The virulence of IPNV isolates is quite variable. Most have been isolated from aquatic animals with no evidence of disease. Only a few isolates from nondiseased animals have been tested for virulence.

Table. Virulence of aquatic birnaviruses in experimental infections of homologous and heterologous hosts. Serotype of virus, if reported, in parentheses.

NR, not reported; IMM, immersion; IP, intraperitoneal; SC, subcutaneous; d, days; w, weeks; mo, months; y, years.

1, Wolf and Quimby, 1969; 2, Reno et al., 1978; 3, Swanson et al., 1982; 4, Sano et al., 1981; 5, Vestergård-Jørgensen and Kehlet, 1971; 6, Kohlmeyer et al., 1986; 7, Smail et al., 1986; 8, Stephens et al., 1983; 9, Kusuda et al., 1989; 10, McAllister et al., 1984; 11, Chen et al., 1984; 12, Wechsler et al., 1987b; 13, Klontz et al., 1965; 14, Parisot et al., 1963; 15, Silim et al., 1982; 16, McAllister and McAllister, 1988; 17, Hill, 1982; 18, Hill et al., 1982; 19, Hill, 1976; 20, Moewus-Kobb, 1965.

Wolf et al. (1969) demonstrated differences in virulence with 15 serotype A1 (WB) isolates by infecting brook trout fry. Mortalities ranged from 7 to 98%. The viral titre in the mortalities was approximately 10⁹ g^-1; there was no correlation between the titre and mortality levels (regression analysis of data of Wolf et al., 1969). Unfortunately, the titre of virus in the more resistant or in surviving fish was not reported in rainbow trout infected with three European strains: Sp, Ab and Hecht (He). Hecht was avirulent, while the two trout isolates were causing significant mortality, albeit at different levels; virulence was directly correlated with plaque size (Kohlmeyer et al., 1986). Only a few passages of virulent Sp serotype virus in cell culture may cause attenuation of the virus (Santi et al., 2004) This variation in virulence is a reflection of the complex nature of the disease, which is not well understood. The two European serotypes were originally classified as virulent for trout (Sp) or avirulent for trout (Ab) (Vestergård-Jørgensen and Kehlet, 1971).

Predisposing factors for IPN include temperature, age and stress (Frantsi and Savan, 1971a; LaPierre et al., 1988). Six-month-old brook trout at 3 g are resistant to clinical IPN disease; however, it is not known whether the host had cleared the virus entirely or whether residual virus was present, as in typical IPN carriers. Temperatures at which maximum mortalities occur vary, but temperature effects may be obscured by the strain of virus and host species used: e.g. brook trout infected with a Canadian isolate had maximal mortality at 10°C, while those infected with IPNV VR-299 succumbed most readily at 15.5°C (Frantsi and Savan, 1971a). In the epizootic affecting turbot, mortalities were markedly higher in juvenile fish held in sea water at 18°C than at 11°C (Castric et al., 1987). Similar results were obtained by Okamoto et al. (1987b) in rainbow trout fry.

Nephritis in eels, caused by a birnavirus termed eel virus European (EVE), occurs primarily during the winter, when water temperatures are lowest (Sano et al., 1981). The compromised physiological condition of the fish during this period may be a factor in the disease process (Egusa et al., 1971; Oka et al., 1976).

The severity of the disease is dependent not only on the species of fish, but also on the strain of fish within a species. Silim et al. (1982) demonstrated that strains of brook trout from four genetic pools varied in their susceptibility to IPN, with mortalities ranging from 40 to 85%. Similarly, Okamoto et al. (1987a) found that rainbow trout from one hatchery (strain RT-101) were consistently more susceptible than fish from two other hatcheries to lethal IPNV infection over a course of 6 years. Additionally, these authors demonstrated that the quantity of virus to which fish were exposed influenced mortalites. No mortalities occurred in 0.26 g fish exposed to less than 10² TCID₅₀ ml^-1 of IPNV-Buhl, whereas 61% of fish died when exposed to 1000-fold higher virus concentrations. The relationship between infection of salmonids and severity of clinical disease is complicated and the IPNV carrier state has been difficult to elucidate.

The diseases known as yellowtail ascites, Japanese flounder ascites and eel nephritis are caused by aquatic birnaviruses. All are severe and cause high mortalities in cultured flat-fish and eels in Japan. In addition, three more diseases have been found to be associated with birnaviruses, although unequivocal proof of their involvement in the promulgation of the diseases is lacking. These diseases cause sea-bass mortalities (Bonami et al., 1983), turbot haematopoietic necrosis (Castric et al., 1987) and spinning disease of menhaden (Stephens et al., 1983).

Diagnostic methods

The diagnosis of IPN has historically been predicated on clinical signs of the disease, isolation and identification of the aetiological agent by cell culture methods and confirmation using serological methods. Consistent detection of aquatic birnaviruses has proved to be simple using cell culture systems. There are at least five reasons for this.

1. The virus is usually present in relatively high titres in tissues.

2. Unlike other fish viruses, such as infectious haematopoietic necrosis (IHN) and channel catfish virus (CCV), isolations of aquatic birnaviruses are made most frequently from non-diseased animals.

3. There is no latent stage, in which the virus cannot be isolated in cell culture.

4. The time required for isolation and identification of the agent is usually 2-3 weeks.

5. Easily available continuous teleost cell lines are generally highly sensitive to the virus and undergo a characteristic, readily observable cytopathic effects (CPE) when infected.

Thus, isolation of virus from visceral samples in standardized teleost cell lines has remained the regular diagnostic method.

Cell culture methods: primary isolation

Many teleost cell lines are susceptible to IPNV infection, with the consequent production of characteristic CPE, usually manifested as a ‘stringy’ appearance of the cells.

Table Continuous teleost cell lines in which aquatic birnaviruses have been propagated.

1, Kelly et al., 1978; 2, Wolf and Mann, 1980; 3, Lannan et al., 1984; 4, Wolf and Quimby, 1969; 5, Piper et al., 1973; 6, Chen et al., 1990; 7, Follett and Schmitt, 1990; 8, Kelly and Loh, 1972; 9, Moewus-Kobb, 1965;10, Lee and Loh, 1973; 11, Wolf and Quimby, 1966; 12, Ahne, 1978; 13, Lee and Loh, 1975; 14, Wattanavijarn et al., 1988; 15, Kelly et al., 1980; 16, Stephens et al., 1981; 17, Fijan et al., 1983; 18, Kusuda et al., 1989.

The ‘standard’ cell lines for aquatic birnavirus isolation are: RTG-2 (rainbow trout gonad; Wolf and Quimby, 1962), CHSE-214 (chinook salmon embryo; Lannan et al., 1984), FHM (fathead minnow; Gravell and Marlsberger, 1965), BF-2 (bluegill fry; Wolf and Quimby, 1966) and EPC (epithelioma papulosum cyprini; Fijan et al., 1983). For diagnostic purposes, variations in the susceptibility of the teleost cell lines to infection become important and, for this reason, attempts at the primary isolation of virus are usually made on at least two different, well characterized continuous cell lines (Hill, 1976; Gillespie et al., 1977; Amos, 1985). Hill suggested that the BF-2 cell line, derived from tissues of bluegill (Lepomis macrochirus), is the most sensitive for the isolation of aquatic birnaviruses in molluscs and invertebrates (Hill, 1982). Kelly et al. (1978) compared the sensitivities of three cell lines to infection with IPNV from fish tissues and found that EPC was the most sensitive. In many instances, if the fish or shellfish to be examined is a non-salmonid, it is prudent to include a cell line from the homologous or closely related species. For example, in attempting primary isolation of the aetiological agent of spinning disease of menhaden (B. tyrranus), the birnavirus could only be isolated in cells from this fish species (Stevens, 1981).

While the in vitro host range of aquatic birnaviruses is wide, it is also erratic. In one instance, only one of four cell lines derived from carp was susceptible to a birnavirus from milkfish (Chanos chanos) (Chen et al., 1990). In vitro host range variants of IPNV also occur: some strains do not replicate in FHM or EPC cells, while others produce a high yield (Scherrer and Cohen, 1975; Kelly et al., 1978; Nicholson et al., 1979; Castric et al., 1987). Furthermore, Kelly et al., (1978) found that the RTG-2 cell line was more sensitive than the FHM cell line in detecting IPNV from tissues of carrier fish (50% vs. 12.5% of carriers detected). The use of multiple cell lines is recommended and adopted by national and international regulatory agencies for the detection of IPNV (Hill, 1976; Gillespie et al., 1977; Amos, 1985).

Other methods which have been developed to enhance the sensitivity of birnavirus detection in cell cultures are based on increasing the efficiency of the sample preparation rather than on increasing the susceptibility of cells. Two main methods of sample preparation are utilized. In both, homogenization of the sample is followed by low speed centrifugation. In one method, the supernatant was filtered through a bacteria-retaining filter, while in the others antibiotics were added to inhibit bacterial growth (Gillespie et al., 1977; Okamoto and Sano, 1984; Amos, 1985; Hedrick et al., 1986); the two methods yield comparable results. Cocultivation is another method; the separated host cells (usually kidney or spleen) are allowed to settle on a preformed monolayer of continuous cells, thereby infecting them by direct contact. This method has been reported to provide greater sensitivity than homogenization techniques (Agius et al., 1982, 1983).

The screening procedure for IPN is based on IPNV isolation tests in cell culture (Agius et al., 1982) followed by immunological identification, either by serum neutralisation (Hill and Way, 1995) or enzyme-linked immunosorbent assay (ELISA) (Dixon and Hill, 1983), of virus isolated. Diagnosis of clinical cases is normally based on histology (McKnight and Roberts, 1976) and/or immunological demonstration of IPNV antigen in infected tissues (Evensen and Rimstad, 1990), confirmed by isolation and immunological identification of IPNV in tissue culture, as for screening.

Serological and biochemical methods: detection and identification

Serological techniques have been used extensively in the identification and classification of aquatic birnaviruses. The serological identification methods have increased in sensitivity over the years, but the neutralization test remains the benchmark for other tests due to its inherent sensitivity and its use for classifying viruses. Neutralization tests utilizing polyvalent antisera are used to identify serogroup A aquatic birnaviruses (Lientz and Springer, 1973; Amos, 1985). A cautionary note, however, was raised by Vestergård-Jørgensen and Grauballe (1971), who found that the method used to immunize rabbits in the production of anti-IPNV antiserum could affect the specificity of the reaction.

The most frequently employed neutralization test uses a constant viral titre (approximately 100 TCID₅₀) and varies the antibody concentration. The test is confirmatory for the identification of the virus and requires up to 1 week to complete. Other serological techniques used include complement fixation (Finlay and Hill, 1975), fluorescent antibody (Nicholson and Dunn, 1974; Tu et al., 1974; Vestergård-Jørgensen, 1974), immunoperoxidase tests (Reno, 1976; Nicholson and Henchal, 1978), neutralization kinetics (Nicholson and Pochebit, 1981), Staphylococcus coagglutination (Kimura et al., 1984; Bragg and Combrick, 1987b), counterimmunoelectrophoresis (Dea and Elazhary, 1983), enzyme-linked immunosorbent assay (ELISA) (Nicholson and Caswell, 1982; Dixon and Hill, 1983; Hattori et al., 1984), immunodot (McAllister and Schill, 1986; Ramsey et al., 1986; Caswell-Reno et al., 1989; Babin et al., 1991; Ross et al., 1991), and immunoprecipitation (Lipipun, 1988) tests. The tests vary in sensitivity, as well as in specificity and in the complexity and efficiency of the performance of the test.

Table Serological tests used for the detection and identification of aquatic birnaviruses, their sensitivities and time required for assay.

1, Wolf and Quimby, 1969; 2, Finlay and Hill, 1975; 3, Piper et al., 1973; 4, Elazhary et al., 1982; 5, Vestergård-Jørgensen, 1974; 6, Nicholson and Henchal, 1978; 7, Reno, 1976; 8, Hedrick and Fryer, 1981; 9, Nicholson and Caswell, 1982; 10, Caswell-Reno et al., 1989; 11, Ross et al., 1991; 12, Lipipun, 1988; 13, Kimura et al., 1984.

The most commonly used tests, ELISA and immunodot, have sensitivity levels that are sufficient to detect viral antigen in the 10-100 ng range (Nicholson and Caswell, 1982; Dixon and Hill, 1983; Caswell-Reno et al., 1986), which corresponds to approximately 10⁶ infectious doses of virus ml^-1 of cell culture fluid or organ homogenate (Hattori et al., 1984; Babin et al., 1991). However, viral titres in carrier animals are frequently more than 10,000-fold lower than this (Wolf et al., 1968b; Yamamoto, 1975a; Reno, 1976). The sensitivity of these tests is, therefore, inadequate for confidently detecting virus in carrier animals; hence cell cultures are still used for the primary isolation of the virus. However, it has been demonstrated that viral antigen is widespread in the tissues of carrier fish, even in the absence of high viral titres (Reno, 1976, 1988; Hedrick and Fryer, 1982).

Serological techniques are frequently used to confirm the identity of virus following isolation in cell culture.Rodriguez Saint-Jean et al. (1991) reported the use of flow cytometry to detect IPNV in the blood leucocytes in an IPN epizootic. It was determined that at least 70% of the leucocytes must be infected before a positive could be confirmed, although only small numbers of cells (approximately 10,000) are needed for the test. The test is rapid and can be performed more rapidly than enzyme linked assays. A cocultivation method was used to increase the percentage of leucocytes that were infected, but this increased the time required for the assay.

Vestergård-Jørgenson and Grauballe (1971) reported that the adjuvant used for the immunization of rabbits with IPNV affected the specificity of the response to different strains of the virus: Freund’s complete adjuvant promoted the generation of more specific antibodies than did Freund’s incomplete adjuvant. As with other viral systems, the assay that is used to determine cross-reactivity of antibodies can play a role in the results obtained. For example, Evensen and Rimstad (1990) determined that rabbit antisera against strains Sp, Ab and VR-299 cross-reacted in neutralization and Western blotting assays; however, in paraffin-embedded tissues from fish infected with strain Sp, only the homologous antiserum reacted. Small differences in reaction patterns were noted with neutralization reactions versus solid-phase immunoassays, even when MAbs were used (Caswell-Reno et al., 1986).

An in situ hybridization (ISH) technique has been developed to detect aquatic birnavirus in infected BF-2, EPC, and CHSE-214 cells (Alonso et al., 2004).

Many prototcols for the use of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay have been developed for the detection of aquatic birnaviruses, both in cell culture (Rimstad et al., 1990; Cepica et al., 1991; McAllister et al., 1992; Shankar and Yamamoto, 1994; Wang et al., 1997) and in fish (Taksdal et al., 2001; Candan, 2002). The sensitivity and specificity of the RT-PCR method has improved accordingly with the general technical development and present RT-PCR protocols detect a higher number of IPNV-positive samples than standardized cell culture methods. The RT-PCR methods have also been developed to be able to detect several fish viruses simultaneously (multiplex-RT-PCR) (Williams et al., 1999).

In a comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fish it was concluded that RT-PCR and flow cytometry were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish (Rodriguez Saint-Jean et al., 2001).

There is a continuous improvement of laboratory diagnosis and detection of IPN/IPNV along with the development of new and more sophisticated methods. Detection of IPNV with special properties (i.e. virulent types/strains) requires that the molecular basis for these properties are well and unequivocally described.

An evaluation of the ability of a test to detect IPNV includes evaluation of both the specificity and sensitivity. The gold standard for IPNV detection is virus isolation in cell culture, which is thoroughly described in the OIE manual. It is advisable to select a method with regard to the information required. Is it solely to detect the presence of IPNV or are quantitative measurements wanted? Is it required to relate eventual virus presence to pathological changes? Are qualitative properties of the detected IPNV, i.e. possible virulence markers, present?

List of Symptoms/Signs

Disease Course

Little has been done with natural IPN disease to determine the portal of entry, dissemination of virus to target organs or effects on the target cells, except histopathologically.

In experimental infections, the disease is manifest by 1 week postinfection and generally has run its course within another week or so (Wood et al., 1955; Reno, 1976; Reno et al., 1978). The acute nature of the disease is characteristic of all aquatic birnavirus-induced diseases, including Japanese flounder and yellowtail ascites, turbot disease, nephritis of eels and spinning disease of menhaden (Sano et al., 1981; Bonami et al., 1983; Stephens et al., 1983; Sorimachi and Hara, 1985).

Swanson and Gillespie (1981, 1982) experimentally infected 6-month-old or yearling brook trout with IPN (strain VR-299) by intraperitoneal injection and followed the distribution of virus in the gastrointestinal tract, kidney and blood. While no clinical disease was produced , high viral titres were found in the gut and kidney, and focal necrotic lesions were detected in the pyloric caeca within 3 days postinfection and in the kidney within 5 days postinfection. Indirect fluorescent antibody tests indicated the presence of foci of IPNV antigen in pyloric caeca, kidney and liver and the virus was detected in both the serum and the mononuclear cell fractions of the blood. Hornich et al. (1986) indicated that fry undergoing acute IPN had the most intense fluorescence in the gut epithelial cells. In contrast, in natural carrier brook trout no virus was detected in the plasma, although virus was detected in the leucocytic fraction (Yu et al., 1982). In addition, McAllister et al. (1987) found that the cellular phase of the ovarian fluid of carriers contained up to 400 times more virus than the fluid portion. The distribution of virus within the viscera is not uniform, with distinct tissue tropisms.

Epidemiology

Transmission of the disease and epidemiology

In the classic Culture and Diseases of Game Fishes, Davis (1953) makes the following reference to M’Gonigle’s information (1940) on alleviating acute catarrhal enteritis, now assumed to be IPN: "M’Gonigle recommends that affected fish be planted in small streams where they can get natural food such as insect larvae. Since the disease is not due to infection by animal parasites or bacteria, liberation of the fish in natural waters can do no harm." It is unknown how many fish farmers adhered to this practice, which was promulgated before the discovery of virus infections in teleosts. It is possible that the panzootic nature of aquatic birnaviruses may be due in part to this type of remedy for a disease of unknown aetiology.

The epizootiology of IPN is complex and is not thoroughly understood. The disease affects young fish and postsmolts, the virus may be retained in the body after primary infection without causing clinical signs in older fish, and the virus may be transmitted vertically.

IPNV may be introduced to a hatchery by eggs, fry, human, fomites, different animals, and water. The impacts of these possible introductory alternatives may vary.

The prevalence of IPNV and IPN disease is not easily appraised, although the virus appears to be present at rather high levels in some instances.

Table. Reported IPNV carrier rates in various aquatic animals.

ATS, Atlantic salmon; BKT, brook trout; BNT, brown trout; RBT, rainbow trout. S, spleen; K, kidney; P, pyloric caeca; G, gonads; I, intestine; L, liver; F, faeces; PW, peritoneal wash; OF, ovarian fluid; B, blood.

1, Yamamoto, 1975b; 2, Wolf et al., 1961; 3, Reno, 1976; 4, Wolf et al., 1968b; 5, Frantsi and Savan, 1971b; 6, Hedrick and Fryer, 1982; 7, Billi and Wolf, 1969; 8, Munro et al., 1976; 9, Dorson, unpublished; 10, Dorson 1980, 1982; 11, Dorson, 1981, 1982; 12, Bucke et al., 1979.

While there is considerable information in the literature on prevalence levels, substantial variations exist within even small geographical locales and, temporally, even at the same facility. This diversity precludes easy analysis or compilation of information. It is generally accepted that, if the virus is present in a facility, the infection will remain for long periods in the absence of active measures to eradicate the agent. IPNV seems to be persistent once introduced into a hatchery.

For example, in one hatchery, the disease was present in the 1940s and mortalities were in excess of 90% in brook trout; by the late 1970s, the disease was still present, but mortality had dropped to around 40% (L. McCullogh, personal communication). Conversely, a brook trout hatchery known to have IPNV present at enzootic levels for many years has had no virus-induced mortalities for more than 25 years (P.W. Reno, personal observations). It is interesting to note that this hatchery has distributed IPNV-infected brook trout to many other aquaculture facilities, but the virus has not necessarily remained with all of the fish that were moved. For instance, one fish farm received fish from the facility in 1975 and inspection of the stocks indicated the presence of the virus. The only measures taken were to house the fish at the tail-end of the impoundments. When these fish were tested annually, starting in 1987, no virus was detected in them. This is one of only a few instances in which IPNV has been eliminated from a population of carrier fish. Bucke et al. (1979) report that in 1975 IPNV was detected in grayling at the outfall of an IPNV-contaminated hatchery, but there was no evidence of virus within 2 years in the same population of fish.

There is accumulating evidence that the mode of transmission within a hatchery may be a combination of vertical and horizontal transmission. It is most likely that the initial source of infection is from previously infected stocks or from homeothermic vertebrates or invertebrates. Horizontal transmission in hatcheries is most probably due to contact with the virus in faeces and urine of infected fish (Billi and Wolf, 1969; Frantsi and Savan, 1971b).

Little is known of the epizootiology of the other diseases caused by aquatic birnaviruses (branchionephritis of eels, whirling disease of menhaden and yamabe ascites of flat-fish). Much more work in this area is needed.

The aquatic birnaviruses are not restricted to hatchery environments or only associated with fish released from contaminated facilities. The virus is in the viscera of wild stocks in remote regions that have had no known plantings or contacts with hatchery-reared fish. Souter et al. (1986) isolated IPNV from nearly half of 229 anadromous Arctic char, Salvelinus alpinus, prespawners collected from the Mackenzie river delta and rivers in the Yukon Territory, where there was no known contact with hatchery-reared fish. The high level enzootic nature of the virus in this location indicates that natural infections may be widespread and that the maintenance of the carrier state in fish is efficient in the absence of the high viral densities found in rearing facilities. In another remote region of Canada (Hudson Bay), IPNV was enzootic in brook trout during the late 1970s and early 1980s (A.J. Sippel, personal communication); likewise, the virus was detected in wild Atlantic salmon in Loch Awe in Scotland (Munro et al., 1976). In addition, aquatic birnaviruses have been isolated from wild brown trout at the outfalls of IPNV-contaminated hatcheries in England and Wales

(Bucke et al., 1979). In Japan, yellowtail ascites virus was also detected in 15% of wild yellowtail fingerlings some distance from mariculture sites; nearly half of infected fish reared in the laboratory developed clinical disease (Isshiki et al., 1989). However, it cannot be assured that these populations were isolated from potential virus carriers in the sea or from fish upstream of the mouth of the bay. Routine culture of yellowtail in Japan employs feral fingerlings as mariculture stocks and these fish are probably the source of virus which causes the epizootics in cultured yamabe. Thus, it appears that the aquatic birnaviruses are indigenous to some fish populations, even wild stocks.

It appears that IPN disease is extremely contagious in hatchery facilities. Fomites and humans are apparently capable of transmitting the virus within and between fish-rearing facilities. The virus is transmitted both vertically and horizontally. In most instances, it appears that if no recourse is taken to eliminate IPNV from a trout culture facility, the virus will remain at the facility for years. Many of the facilities from which IPNV was originally isolated still have virus present (P.W. Reno, unpublished observations). The perennial pattern of infection and disease in facilities that harbour IPNV led to early suspicion that the virus was transmitted vertically. Wolf et al. (1968a) provided evidence that this was the case with brook trout. This was later confirmed (Sano, 1971; Yamamoto, 1975a; Reno, 1976; Hedrick and Fryer, 1982; Luqi and Zhizhuang, 1988). The exact location of the virus during embryonation is unknown, but it is extremely difficult to isolate virus from the egg. Fijan and Georgetti (1978) detected the virus at low levels in the chorion of the fertilized ovum. However, the virus is readily demonstrable after hatching (Reno, 1976; Hedrick and Fryer, 1982).

Other animals may also be involved in transmission of the virus within and between hatcheries. Sonstegård et al. (1972) infected several mammalian and avian species being able to vector IPNV and found infectious virus in the faeces of mink (Mustela sp.), great horned owl (Bubo virginianus) and chickens (Gallus domesticus) after 1 week. The virus was not detected in the faeces of great blue herons, common eider, or common merganser. Similarly, Eskildsen and Vestergård-Jørgensen (1973) isolated IPNV from gulls (Larus ridibundus) administered virus per os. Peters and Neukirch (1986) demonstrated that herons (Ardea cinerea) which were fed virus either by direct intubation or by ingestion of IPNV-injected trout harboured the virus at fairly high levels for about 30 days after the last feeding. While these findings lend credence to the possibility of transferring the virus by other animals, the consensus is that transfer in hatcheries most often occurs either horizontally within the same facility or watershed or through dissemination of contaminated eggs. It was also demonstrated that freshwater crayfish (Astacus astacus) were susceptible to IPNV, retained the virus in their tissues and haemolymph and shed virus constantly into the water (Halder and Ahne, 1988). Transmission from crayfish to trout eggs occurred, as did the infection of the crayfish from ingestion of IPNV-infected trout. There appeared to be no diminution of virus titre in the crayfish haemolymph or tissues for at least 6 months, but there was no indication of viral replication in the crayfish. These findings indicate that crustaceans and other aquatic invertebrates may be involved in the transmission of aquatic birnaviruses. Trout-rearing facilities whose water-supply is from open sources or sources that are not treated with ultraviolet irradiation or ozonation could have aquatic birnaviruses disseminated by both fish and crustacean vehicles, such as crayfish or copepods.

Mixing populations from several hatcheries, mode of transportation, and size of smolts at sea transfer are associated with increased risk of IPN outbreaks in seawater (Jarp, 1999). Intensified rearing conditions in the freshwater phase (superoxygenation, low water supply and high density) are shown to increase risk of IPN outbreaks in seawater (Jarp, 1999).

The movement of fish and especially eggs, in the international market must have contributed to the dissemination of the virus worldwide, although no thorough retrospective analysis has been done. Widespread movement of infected fish may have spread the virus to non-salmonid animals. However, it must be pointed out that non salmonids, especially invertebrates, show little or no evidence of disease when infected with aquatic birnaviruses and it is possible that the virus can be transmitted from these animals to salmonids, which under artificial rearing conditions were susceptible to disease. In this context, it has been reported by Hill (1982) that at least eight of the aquatic birnaviruses isolated from marine shellfish species were capable of producing characteristic signs of clinical IPN in experimentally infected rainbow trout.

Retrospective epizootiological studies of the diseases and infections caused by aquatic birnaviruses are difficult, due to a lack of consistency and accuracy in record keeping. Molecular markers for different virulence variants of IPNV within different serotypes can be revealed by RT-PCR and nucleotide sequencing (Bruslind et al., 2000; Santi et al., 2004; Shivappa et al., 2004). This implies that more precise characterization of IPNV isolates and the distribution of different variants in the cultivated and wild fish populations are needed. Such information is important for further understanding of IPN, risk factors for spread of the infection and for the implementation of proper control measurements.

Viruses belonging to the He and Tellina serotypes (types A4 and A5 of Hill and Way) have been detected exclusively in Europe, and Canada 1, 2, and 3, and Jasper (types A6-A9) have been confined to North America (Hill and Way, 1988b). This is interesting because it is known that trout infected with the WB serotype (A1) were often transferred to Europe from North America in the 1960s, but there is a single report of a WB serotype virus isolated in Europe (Novoa et al., 1993a). There are, however, some instances where the presence of disease in areas previously free from virus could be traced directly to the importation of infected eggs. Such importations occurred in South Africa (Bragg and Combrick, 1987a) and in Japan (Sano, 1971). In both instances, virus accompanied eggs imported from the USA. In other instances, there was circumstantial temporal evidence that the disease arrived with eggs imported into France from the USA (Besse and de Kinkelin, 1965), but subsequent serological evidence indicated that it was probably an indigenous strain (Wolf and Quimby, 1971). A heightened awareness of the disease in France by fish health workers, rather than the importation of virus in fish, was probably responsible for the detection of these strains. In other instances, there have also been indications that aquatic birnaviruses were transmitted to indigenous and cultured species from imported fish or eggs: to Chile from the USA (McAllister and Reyes, 1984; Espinoza et al., 1985), to Japan from Europe with European eels (Sano et al., 1981) and to Taiwan from trout imported from Japan (Hedrick et al., 1983). It is very difficult, however, to accurately differentiate between ‘indiginous’ aquatic birnaviruses and those that were brought into a country or region. The lack of efficient record-keeping at facilities, the lack of universally applied inspection regimes for viruses and the limited usefulness of the serological classification make epidemiological studies less than optimal.

Except for ‘classic’ IPN disease in freshwater trout, all of the diseases associated with aquatic birnaviruses affect either stenohaline or euryhaline animals: yellowtail and Japanese flounder ascites, sea bass, menhaden, eels and possibly clams.

The virus is quite stable, even under conditions that inactivate most other fish pathogens. The infectivity of the virus survives for nearly a year at 4°C and nearly 2 months at 15°C in buffer (Dorson, 1982) and also for long periods in seawater, brackish water and unsterilized fresh water (Moewus-Kobb, 1965; Toranzo and Hetrick, 1982). This stability increases transmission of the virus from carrier or clinically ill fish in rearing facilities to susceptible fish upstream or downstream in the watershed, or even to susceptible hosts in seawater. The ‘spread’ of the virus among freshwater, brackish and seawater environments is facilitated by the stability of the virus, the migration of anadromous and catadromous fishes and the predator-prey relationships. The latter is supported by the finding of a birnavirus-like agent in mass cultures of rotifers, Branchionus plicatilis (Comps et al., 1991), which could potentially be transmitted to larval sea bass (D. labrax), since rotifers are the primary food source for this species in culture. It was suggested that rotifers could be responsible for the epizootics of the disease in maricultured sea bass in France (Bonami et al., 1983).

Vertical transmission and the carrier state

Vertical transmission is usually defined as the transmission of virus from one generation to the next. The virus can either be within the contents of the gametes, often referred to as true vertical transmission, or alternatively on the surface of gametes or in ovarian and seminal fluids and mucus.

Vertical transmission of the virus via the reproductive products of salmonids has been substantiated (Wolf et al., 1968a; Sano, 1971; Yamamoto, 1975a; Reno, 1976; Hedrick and Fryer, 1982; Luqi and Zhizhuang, 1988); however, the actual method by which this occurs remains obscure. The perennial nature of the disease in salmonid hatcheries facilities indicates that this is an important transmission mechanism. This makes sense with trout which are reared in freshwater facilities, often with a well-water source, which would preclude infection via a water-borne route, although the possibility of contamination by birds or transfer by aquatic mammals cannot be ruled out. However, this cannot explain the recurring disease problems in salmonid seawater culture, which indicate that transmission from an environmental source is important. This can be compared to infectious haematopoietic necrosis, which was originally postulated to be vertically transmitted but now appears to be horizontally transmitted (Leong and Munn, 1991).

Wolf et al. (1968a) demonstrated that the virus was transmitted via reproductive products. Eggs and milt were collected from IPNV-positive fish and three pairings were made. The virus was isolated from two matings and frank disease was noted in one batch of the offspring. Bullock et al. (1976) found that the virus was transmitted to an uninfected facility via fertilized brook trout eggs transferred from a contaminated hatchery. However, the presence of the virus in the parent’s reproductive products or progeny does not invariably lead to the development of clinical disease. Reno (1976) found low levels (10^1.4 TCID₅₀ g^-1) of IPNV in all eight groups of progeny from matings of naturally infected brook trout which harboured fewer than 10^2.4 TCID₅₀ ml^-1 of IPNV in milt or ovarian fluid. Virus was not detectable during the embryonation period or until feeding commenced, but IPNV was detected in postfeeding fish for only 1 month. Similarly, in other species, vertical infection has been transient and sporadic. In Atlantic salmon, Smail and Munro (1989) detected virus for less than 1 h in eggs from brood fish exposed to IPNV at titres of 10⁶-10⁹ plaque-forming units (pfu) kg^-1 (injection) or 10⁷ pfu kg^-1 (immersion), and the resultant fry were negative for virus. However, when fry were infected experimentally, the virus was isolated even after 2 years.

Dorson and Torchy (1985) experimentally infected rainbow trout 4.5 months prior to spawning. In addition, at spawning, they infected milt and ova with high doses of IPNV Sp. Three of six infected females had detectable IPNV in ovarian fluid at spawning, whereas none of the males had virus in the milt. There was no evidence of IPN disease in the progeny. When milt was infected, however, IPN disease developed in the fry. Similarly, Ahne and Negele (1985) demonstrated that experimental infection of rainbow trout and Arctic char resulted in consistent isolation of virus from the chorion of the eggs during embryonation and posthatch. In fry, virus was isolated after only 1 month posthatch, but there was no evidence of disease in these fish. This indicates that eggshells may be involved in transmission of virus from one generation to the next and important for the horizontal transmission of IPNV to first feeding fry. The variations in results between the different experiments may be due to the species differences; brook trout are regarded as more susceptible to disease than other salmonids. However, different virulence variants of viruses, especially in experimental infections using cell culture-derived virus may explain differences in disease development. Only a few passages of virus in cell culture may cause attenuation of the virus (Santi et al., 2004).

Survivors of IPN epizootics remain carriers for up to 6 years (Wolf, 1966; Yamamoto,1975a; Reno, 1976). In a given population, the proportion of fish from which virus can be isolated varies drastically. In naturally infected brook trout, the prevalence ranged from 99% to less than 5% (Reno, 1976; unpublished observations). The latter is the prevalence used in fish health inspections for the detection of viruses in salmonids (Amos, 1985). The potential for the dissemination of the virus through the faeces has been demonstrated (Billi and Wolf, 1969; Frantsi and Savan, 1971b; Reno, 1976) and this ‘shedding’ of virus, albeit at low levels, may be responsible for the maintenance of the virus in susceptible fish. Carrier states have also been reported in survivors of epizootics associated with other aquatic birnaviruses: striped bass, turbot, yellowtail, eels and menhaden (Sano et al., 1981; Stephens et al., 1983; Sorimachi and Hara, 1985; Wechsler et al., 1987a).

There are no reported instances of zoonotic infections contracted from any aquatic birnaviruses.

Impact Summary

Impact: Economic

Traditionally, IPNV was a disease of fry and fingerlings, with brook and rainbow trout being considered to be more susceptible than brown trout, lake trout (Salvelinus namaycush), coho and Atlantic salmon (Salmo salar). However, mortality varies considerably between outbreaks and has been related genetic susceptibility of the host (Ozaki et al., 2001 and Silim et al., 1982) and differing level of environmental stress (Frantsi and Savan, 1971 and McAllister and Owens, 1986). IPNV also infects non-salmonid species including eels, molluscs and crustaceans, all of which may act as carriers. The economic impact of IPN mortality of marine-reared Atlantic salmon has been of increasing concern, partly because of the size of the fish (Jarp et al., 1995).

Infection of susceptible species in fresh water can result in high mortality. Virus-associated mortality is rapid between 10 and 14°C, and at lower temperatures is prolonged. Water temperature, fish age and the virus strain affect the severity of the disease, as well as the establishment of covert infections in fish. IPNV replicates in kidney, pancreas, gonad, spleen and intestinal epithelium and may be shed from carrier fish via faeces, as well as through the seminal and ovarian fluids. Virus shedding appears to be dependent on stress. Although stress may induce a recurrence of IPN in fish 6-11 months old, older fish show no clinical signs.

The apparent increased incidence of IPN is considered to be the result of widespread fish and egg movements between countries, and because of increased sensitivity of diagnostic methods, resulting in improved surveillance practices.

IPNV is transmitted horizontally and vertically (Reno, 1999). However, vertical transmission has been confirmed only in brook trout and rainbow trout (Ahne et al., 1989; Ahne and Negele, 1985; Dorson and Torchy, 1981). The exact mechanism of egg entry or location of the virus within the egg is still unclear. Bebak et al. (1988) infected rainbow trout with IPNV by immersion challenge. The fish started to excrete the virus within 2 days of infection and shedding increased and then declined in less than 12 days post-exposure. From this study it was estimated that within 14 days, more than 75% of the population can be infected (Bebak et al., 1988). This gives rise to a rapid spread of IPNV.

Ethanol, methanol, iodophore and chlorine inactivate IPAV (Inouye et al., 1990), but it retains more than 90% of its infectivity after treatment with chloroform or ethyl ether, at pH 3.0 for 60 min. IPNV has been shown to be infective for several years at -70°C and for several months at 4°C. Smail et al., (1993) found that IPNV was not deactivated by an acidic pH unless the sample (silage) was heated for at least 2 h at 60°C. This confirms that IPNV is a robust virus with long survival in the environment.

Zoonoses and Food Safety

This disease is not a zoonosis.

References

Agius C; Mangunwiryo H; Johnson RH; Smail DA, 1982. A more sensitive technique for isolating infectious pancreatic necrosis virus from asymptomatic carrier rainbow trout, Salmo gairdneri Richardson. Journal of Fish Diseases, 5(4):285-292.

Agius C; Richardson A; Walker W, 1983. Further observations on the co-cultivation method for isolating infectious pancreatic necrosis virus from asymptomatic carrier rainbow trout, Salmo gairdneri Richardson. Journal of Fish Diseases, 6(5):477-480.

Ahne W, 1978. Isolation and characterization of infectious pancreatic necrosis virus from pike (Esox lucius). Brief report. Archives of Virology, 58(1):65-69.

Ahne W; Jorgensen PEV; Olesen NJ; Fischer-Scherl T; Hoffmann R, 1989. Aquatic birnaviruses: virus of the serogroup II isolated from an IPN outbreak in brook trout (Salvelinus fontinalis). Bulletin of the European Association of Fish Pathologists, 9(1):14-16.

Ahne W; Negele RD, 1985. Studies on the transmission of infectious pancreatic necrosis virus via eyed eggs and sexual products of salmonid fish. In: Ellis A, ed. Fish and Shellfish Pathology. London, UK: Academic Press, 262-270.

Alonso MC; Cano I; Castro D; Perez-Prieto SI; Borrego JJ, 2004. Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures. Journal of Virological Methods, 116(2):133-138.

Amend DF; Pietsch JP, 1972. Virucidal activity of two iodophors to salmonid viruses. Journal of the Fisheries Research Board of Canada, 29(No.1):61-65.

Amos K, 1985. Procedures for the Detection and Identification of Certain Fish Pathogens. Corvallis, Oregon, USA: Fish Health Section, American Fisheries Society, 6-13.

Anderson C, 1996. Distribution of salmonid diseases in New Zealand. Surveillance (Wellington), 23(4):23-24; 11 ref.

Archetti I; Horsfall FL, 1951. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ova with heterologous immune sera. Journal of Experimental Medicine, 92:441-462.

Azumi K; Yoshimizu M; Suzuki S; Ezura Y; Yokosawa H, 1990. Inhibitory effect of halocyamine, an antimicrobial substance from ascidian hemocytes, on the growth of fish viruses and marine bacteria. Experientia, 46(10):1066-1068.

Babin M; Hernandez C; Sanchez C; Dominguez J, 1991. Immunodot assay for detection of IPN [infectious pancreatic necrosis] virus in organ homogenates. Bulletin of the European Association of Fish Pathologists, 11(2):65-67.

Ball HJ; Munro ALS; Ellis A; Elson KGR; Hodgkiss W; McFarlane IS, 1971. Infectious pancreatic necrosis in rainbow trout in Scotland. Nature, 234(No. 5329):417-418.

Bebak J; McAllister PE; Smith G, 1998. Infectious pancreatic necrosis virus: transmission from infectious to susceptible rainbow trout fry. Journal of Aquatic Animal Health, 10(3):287-293.

Besse P; de Kinkelin P, 1965. Sur l’existence en France de la nécrose pancréatique de la truite arc en ciel (Salmo gairdneri). Bulletin de l’Académie Vétérinaire, 38:185-190.

Billi JL; Wolf K, 1969. Quantitative comparison of peritoneal washes and faeces for detecting infectious pancreatic necrosis (IPN) virus in carrier brook trout. Journal of the Fisheries Research Board of Canada, 26:1459-1465.

Blake S; Ma JY; Caporale DA; Jairath S; Nicholson BL, 2001. Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA. Diseases of Aquatic Organisms, 45(2):89-102.

Bonami JR; Cousserans F; Weppe M; Hill BJ, 1983. Mortalities in hatchery-reared sea bass fry associated with a birnavirus. Bulletin of the European Association of Fish Pathologists, 3(3):41.

Bragg RR; Combrink ME, 1987. Immunostaphylococcus-protein-A (ISPA) test for the identification of infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus. Bulletin of the European Association of Fish Pathologists, 7(2):32-34; [2 fig.].

Bragg RR; Combrink ME, 1987. Isolation and identification of infectious pancreatic necrosis (IPN) virus from rainbow trout in South Africa. Bulletin of the European Association of Fish Pathologists, 7(5):118-120.

Brown LL; Bruno DW, 2002. Infectious diseases of coldwater fish in fresh water. In: Diseases and disorders of finfish in cage culture [ed. by Woo, P. T. K.\Bruno, D. W.\Lim, L. H. S.]. Wallingford, UK: CAB International, 107-169. http://www.cabi.org/CABeBooks/default.aspx?site=107&page=45&LoadModule=PDFHier&BookID=147

Bruheim T, 1991. Smoltdødelighet - erfaringer fra Trødelagskysten (Mortality in smolts - experience from the coast of Troendelag). Nor Fiskeoppdr 3: 44-45.

Brun E, 2001. A Field Study to Evaluate Vaccination against Infectious Pancreatic Necrosis in Sea Farmed Atlantic Salmon (Salmo salar L.) in Norway. MSc -thesis. National Veterinary Institute, Norway.

Bruslind LD; Reno PW, 2000. Virulence comparison of three Buhl-subtype isolates of infectious pancreatic necrosis virus in brook trout fry. Journal of Aquatic Animal Health, 12(4):301-315.

Bucke D; Finlay J; McGregor D; Seagrave C, 1979. Infectious pancreatic necrosis (IPN) virus: its occurrence in captive and wild fish in England and Wales. Journal of Fish Diseases, 2(6):549-553.

Bullock GL; Rucker RR; Amend D; Wolf K; Stuckey HM, 1976. Infectious pancreatic necrosis : transmission with iodine-treated and nontreated eggs of brook trout (Salvelinus fontinalis). Journal of the Fisheries Research Board of Canada, 33(5):1197-1198.

Calvert JG; Nagy E; Soler M; Dobos P, 1991. Characterization of the VPg-dsRNA linkage of infectious pancreatic necrosis virus. Journal of General Virology, 72:2563-2567.

Candan A, 2002. First report on the diagnosis of infectious pancreatic necrosis (IPN) based on reverse transcription polymerase chain reaction (RT-PCR) in Turkey. Bulletin of the European Association of Fish Pathologists, 22(1):45-48.

Castric J; Baudin-Laurencin F; Coustans MF; Auffret M, 1987. Isolation of infectious pancreatic necrosis virus, Ab serotype, from an epizootic in farmed turbot, Scophthalmus maximus.. Aquaculture, 67(1/2):117-126.

Caswell-Reno P; Lipipun V; Reno PW; Nicholson BL, 1989. Use of a group-reactive and other monoclonal antibodies in an enzyme-immunodot assay for identification and presumptive serotyping of aquatic birnaviruses. Journal of Clinical Microbiology, 27(9):1924-1929.

Caswell-Reno P; Reno PW; Nicholson BL, 1986. Monoclonal antibodies to infectious pancreatic necrosis virus: analysis of viral epitopes and comparison of different isolates. Journal of General Virology, 67:2193-2205.

Caswell-Reno PA; Carlin LJ; Bouchard DA; Parker R; Nicholson BL; Reno PW, 1990. Serological analysis of infectious pancreatic necrosis virus (IPNV) isolates from eastern North America using monoclonal antibodies. In: Proceedings of the 19th Annual Eastern Fish Health Workshop, University of Prince Edward Island, Charlottetown, PEI:7.

Cepica A; Zhou C; Qian B, 1991. Detection of infectious pancreatic necrosis virus by polymerase chain reaction. In: Abstracts 14th Annual American Fisheries Society/Fish Health Section Meeting, Newport, Oregon, 50.

Chen MM; Chi SC; Guu JJ; Chen JC; Kou GH, 1990. Characteristics of a birnavirus isolated from Chanos chanos. COA Fisheries Series No. 10, Fish Disease Research, 6:6-11.

Chi SC; Chen SN; Kou GH, 1991. Establishment, characterization and application of monoclonal antibodies against eel virus European (EVE). Gyobyo Kenkyu = Fish Pathology, 26(1):1-7.

Chou HY; Lo CF; Tung MC; Wang CH; Fukuda H; Sano T, 1993. The general characteristics of a birnavirus isolated from cultured loach (Misgurnus anguillicaudatus) in Taiwan. Gyobyo Kenkyu = Fish Pathology, 28(1):1-7.

Christie KE, 1997. Immunization with viral antigens: infectious pancreatic necrosis. Developments in Biological Standardization, 90:191-199; 54 ref.

Christie KE; Håvarstein LS; Djupvik HO; Ness S; Endresen C, 1988. Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon. Archives of Virology, 103(3-4):167-177.

Cohen J; Poinsard A; Scherrer R, 1973. Physico-chemical and morphological features of infectious pancreatic necrosis virus. Journal of General Virology, 21(Part 3):485-498.

Comps M; Menu B; Breuil G; Bonami JR, 1991. Viral infection associated with rotifer mortalities in mass culture. Aquaculture, 93(1):1-7; 16 ref.

Crane MStJ; Hardy-Smith P; Williams LM; Hyatt AD; Eaton LM; Gould A; Handlinger J; Kattenbelt J; Gudkovs N, 2000. First isolation of an aquatic birnavirus from farmed and wild fish species in Australia. Diseases of Aquatic Organisms, 43(1):1-14.

Davis L, 1953. Culture and Diseases of Game Fishes. Sacramento, California, USA: California Division of Fish and Game, 292.

Dea S; Elazhary MASY, 1983. Counterimmunoelectrophoresis for identification of infectious pancreatic necrosis virus after isolation in cell culture. Canadian Journal of Fisheries and Aquatic Sciences, 40(12):2200-2203.

Desautels D; MacKelvie RM, 1975. Practical aspects of survival and destruction of infectious pancreatic necrosis virus. Journal of the Fisheries Research Board of Canada, 32(4):523-531.

Dixon PF; Hill BJ, 1983. Rapid detection of infectious pancreatic necrosis virus (IPNV) by the enzyme-linked immunosorbent assay (ELISA). Journal of General Virology, 64(2):321-330.

Dobos P, 1976. Size and structure of the genome of infectious pancreatic necrosis virus. Nucleic Acids Research, 3:1903-1924.

Dobos P, 1995. The molecular biology of infectious pancreatic necrosis virus (IPNV). Annual Review of Fish Diseases, 5:25-54.

Dobos P; Hallett R; Kells DTC; Sorensen O; Rowe D, 1977. Biophysical studies of infectious pancreatic necrosis virus. Journal of Virology, 22(1):150-159.

Dobos P; Hill BJ; Hallet R; Kells DTC; Becht H; Teninges D, 1979. Biopyhsical and biochemical characterisation of five animal viruses with bisegmented double-stranded RNA genomes. Journal of Virology, 32:593-605.

Dobos P; Roberts TE, 1983. The molecular biology of infectious pancreatic necrosis virus: a review. Canadian Journal of Genetic Microbiology, 29:377-384.

Dobos P; Rowe D, 1977. Peptide map comparison of infectious pancreatic necrosis virus-specific polypeptides. Journal of Virology, 24:805-820.

Dorson M, 1977. Vaccination trials of rainbow trout fry against infectious pancreatic necrosis. (Summary). Bulletin de l'Office International des Epizooties, 87(5/6):405.

Dorson M, 1982. Infectious pancreatic necrosis of salmonids: present status of knowledge concerning the viruses and the possibilities of controlling the disease. Bulletin Français de Pisciculture, 285:195-209.

Dorson M, 1988. Vaccination against infectious pancreatic necrosis. Fish vaccination., 162-171.

Dorson M; Michel C, 1987. An evaluation of the activity of five quaternary ammonium compounds on main viruses and bacteria pathogenic for salmonids. Bulletin Français de Pisciculture, 305:61-66.

Dorson M; Torchy C, 1981. The influence of fish age and water temperature on mortalities of rainbow trout, Salmo gairdneri Richardson, caused by a European strain of infectious pancreatic necrosis virus. Journal of Fish Diseases, 4(3):213-221.

Dorson M; Torchy C, 1985. Experimental transmission of infectious pancreatic necrosis virus via the sexual products. [Spermatozoa]. Fish and shellfish pathology. Proceedings of the European Association of Fish Pathologists, Plymouth, September 1983., 251-260.

Duncan R; Mason CL; Nagy E; Leong JA; Dobos P, 1991. Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: a putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif. Virology, 181:541-552.

Duncan R; Nagy E; Krell PJ; Dobos P, 1987. Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a virus-encoded protease and fine structure mapping of genome segment A coding regions. Journal of Virology, 61:3655-3664.

Economon P, 1963. Experimental treatment of infectious pancreatic necrosis of brook trout with polyvinylpyrrolidone-iodine. Transactions of the American Fisheries Society, 92:180-182.

Egusa S; Hirose H; Wakabayashi H, 1971. A report of investigations on branchionephritis of cultured eel. II. Conditions of the gills and serum ion concentrations. Fish Pathology, 6:57-61.

Elazhary MASY; Lagace A; Cousineau G; Roy RS; Berthiaume L; Paulhus P; Frechette JL, 1976. Outbreak of infectious pancreatic necrosis in yearling brook trout (Salvelinus fontinalis). Journal of the Fisheries Research Board of Canada, 33(11):2621-2625.

Eskildsen UK; Vestergård-Jørgensen PE, 1973. On the possible transfer of trout pathogenic viruses by gulls. Rivista Italiana di Piscicoltura e Ittiopatologia, A8:104-106.

Espinoza E; Farías G; Soler M; Kuznar J, 1985. Identity between infectious pancreatic necrosis virus VR-299 and a Chilean isolate. Intervirology, 24(1):58-60.

Estay A; Farias G; Soler M; Kuznar J, 1990. Further analysis of the structural proteins of infectious pancreatic necrosis virus. Virus Research, 15:85-95.

Etchberger KJ, 1987. The effect of infectious pancreatic necrosis infection on the immune response of salmonids. PhD dissertation. Orono, Maine, USA: University of Maine, 134 pp.

Evensen O; Rimstad E, 1990. Immunohistochemical identification of infectious pancreatic necrosis virus in paraffin-embedded tissues of Atlantic salmon (Salmo salar).. Journal of Veterinary Diagnostic Investigation, 2(4):288-293.

Farias G; Navarrete E; Kiss J; Kuznar J, 1988. Effect of ammonium chloride on the multiplication of infectious pancreatic necrosis virus. Archives of Virology, 98:155-162.

Fijan N; Sulimanovic D; Bearzotti M; Muzinic D; Zwillenberg LO; Chilmonczyk S; Vautherot JF; DeKinkelin P, 1983. Some properties of the epithelioma papulosum Cyprini (EPC) cell line. Annales de Virologie, Institut Pasteur, 134E:207-220.

Fijan NN; Giorgetti G, 1978. Infectious pancreatic necrosis: isolation of virus from eyed eggs of rainbow trout Salmo gairdneri Richardson. Journal of Fish Diseases, 1(3):269-270.

Finlay J; Hill BJ, 1975. The use of the complement fixation test for rapid typing of infectious pancreatic necrosis virus. Aquaculture, 3:305.

Frantsi C; Savan M, 1971. Infecious pancreatic necrosis virus - temperature and age factors in mortality. Journal of Wildlife Diseases, 7(No.4):249-255.

Frantsi C; Savan M, 1971. Infectious pancreatic necrosis virus: comparative frequencies of isolation from feces and organs of brook trout (Salvelinus fontinalis). Journal of the Fisheries Research Board of Canada, 28(No.7):1064-1065.

Frost P; Havarstein LS; Lygren B; Stahl S; Endresen C; Christie; KE, 1995. Mapping of neutralization epitopes on infectious pancreatic necrosis viruses. Journal of General Virology, 76:1165-1172.

Frost P; Ness A, 1997. Vaccination of Atlantic salmon with recombinant VP2 of infectious pancreatic necrosis virus (IPNV), added to a multivalent vaccine, suppresses viral replication following IPNV challenge. Fish & Shellfish Immunology, 7(8):609-619.

Ghittino P, 1972. Malattie esotoche dei pesci che minacciano troticultura e carpicultura italiane. Rivista Italiana di Piscicoltura e Ittiopatologia, 7:53-62.

Ghittino P, 1982. INP, from a joke fish disease may be converted into an obstacle in trout trades. Rivista Italiana di Piscicoltura e Ittiopatologia, 17:1-2.

Gillespie DC; Evelyn TPT; Frantsi C; MacKelvie RM; Neufeld N, 1977. Fish Health Protection Regulations Manual of Compliance. Miscellaneous Special Publication No. 31. Ottawa, Canada: Fisheries and Environment Canada, Fisheries and Marine Service.

Giorgetti G, 1990. Disease problems in farmed penaeids in Italy. Actes Colloquia Ifremerea, 9:75-87.

Gravell M; Marlsberger RG, 1965. A permanent cell line from the fathead minnow (Pimephales promelas). Annals of the New York Academy of Sciences, 126:555-565.

Grinnell B; Leong JC, 1979. Recovery and concentration of infectious pancreatic necrosis (IPN) virus in water. Journal of the Fisheries Research Board of Canada, 36(11):1405-1408.

Hah YC; Hang SW; Kim MH; Fryer JL; Winton JR, 1984. Isolation of infectious pancreatic necrosis virus from goldfish (Carassius auratus) and chum salmon (Oncorhynchus keta) in Korea. Korean Journal of Microbiology, 22:85-90.

Halder M; Ahne W, 1988. Freshwater crayfish Astacus astacus - a vector for infectious pancreatic necrosis virus (IPNV). Diseases of Aquatic Organisms, 4:205- 209.

Hastein T; Krogsrud J, 1976. Infectious pancreatic necrosis. First isolation of virus from fish in Norway. Acta Veterinaria Scandinavica, 17(1):109-111.

Hattori M; Kokama H; Ishiguro S; Honda A; Mikami T; Izawa H, 1984. In vitro and in vivo detection of infectious pancreatic necrosis virus in fish by enzyme-linked immunosorbant assay. American Journal of Veterinary Research, 45:1876-1879.

Hedrick RP; Eaton WD; Fryer JL; Hah YC; Park JW; Hong SW, 1985. Biochemical and serological properties of birnaviruses isolated from fish in Korea. Fish Pathology, 20(4):463-468.

Hedrick RP; Fryer JL, 1982. Persistent infections of salmonid cell lines with infectious pancreatic necrosis virus (IPNV): a model for the carrier state in trout. Fish Pathology, 16(4):163-172.

Hedrick RP; Fryer JL; Chen SN; Kou GH, 1983. Characteristics of four birnaviruses isolated from fish in Taiwan. Fish Pathology, 18(2):91-97.

Hedrick RP; McDowell T; Rosemark R; Aronstein D; Chan L, 1986. A comparison of four apparatuses for recovering infectious pancreatic necrosis virus from rainbow trout. Progressive Fish-Culturist, 48(1):47-51.

Heppell J; Berthiaume L; Corbin F; Tarrab E; Lecomte J; Arella M, 1993. Comparison of amino acid sequences deduced from a cDNA fragment obtained from infectious pancreatic necrosis virus (IPNV) strains of different serotypes. Virology (New York), 195(2):840-844.

Heppell J; Tarrab E; Berthiaume L; Lecomte J; Arella M, 1995. Characterization of the small open reading frame on genome segment A of infectious pancreatic necrosis virus. Journal of General Virology, 76(8):2091-2096.

Heppell J; Tarrab E; Lecomte J; Berthiaume L; Arella M, 1995. Strain variability and localization of important epitopes on the major structural protein (VP2) of infectious pancreatic necrosis virus. Virology, 214:40-49.

Hill BJ, 1976. Procedures for the isolation and identification of IPN, VHS, IHN and SVC viruses from diseased fish. Fisheries Research Technical Report, No.27:13pp.; [ISSN0309-5589].

Hill BJ, 1982. Infectious pancreatic necrosis virus and its virulence. Microbial diseases of fish, 91-114; [Special Publication of the Society of General Microbiology No. 9].

Hill BJ, 1992. Impact of viral diseases of salmonid fish in the European Community. In: Kimura T, ed. Proceedings of the OJI International Symposium on Salmonid Diseases. Sapporo, Japan: Hokkaido University Press, 48-59.

Hill BJ; Dixon PF, 1977. Studies on IPN [infectious pancreatic necrosis] virulence and immunization. Bulletin de l'Office International des Epizooties, 87(5/6):425-427.

Hill BJ; Way K, 1983. Serological classification of fish and shellfish birnaviruses. In: Proceedings of the First Annual Meeting of the European Association of Fish Pathologists, Plymouth, UK, 10.

Hill BJ; Way K, 1988. Epidemiological aspects of the proposed serological classification of aquatic birnaviruses. In: Proceedings of the International Fish Health Conference, Vancouver, British Columbia, 163.

Hill BJ; Way K, 1988. Proposed standardisation of the serological classification of aquatic birnaviruses. In: Proceedings of the International Fish Health Conference, Vancouver, British Columbia, 151.

Hill BJ; Way K, 1995. Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Annual Review of Fish Diseases, 5:55-77.

Hill BJ; Way K; Alderman DJ, 1982. Further investigations into the pathogenicity of IPN-like viruses for oysters. In: Invertebrate Pathology and Microbial Control. Proceedings of the 3rd International Conference on Invertebrate Pathology, September 1982, Brighton, UK, 273-274.

Hjalmarsson A; Carlemalm E; Everitt E, 1999. Infectious pancreatic necrosis virus: identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2. Journal of Virology, 73:3484-3490.

Hong JiannRuey; Gong HongYi; Wu JenLeih, 2002. IPNV VP5, a novel anti-apoptosis gene of the Bcl-2 family, regulates Mcl-1 and viral protein expression. Virology, 295(2):217-229.

Hong JR; Hsu YL; Wu JL, 1999. Infectious pancreatic necrosis virus induces apoptosis due to down-regulation of survival factor MCL-1 protein expression in a fish cell line. Virus Research, 63:75-83.

Hornich M; Pospísil Z; Tománek J, 1986. Diagnosis of infectious pancreatic necrosis (IPN) by immunofluorescence. Veterinární Medicína, 31(1):55-64; 29 ref.

Huang MTF; Manning SD; Warner M; Stephens EB; Leong JC, 1986. A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived form viral cDNA clones. Journal of Virology, 60:1002-1011.

Hudson JB; Graham EA; Simpson MF, 1988. The efficacy of amantadine and other antiviral compounds against two salmonid viruses in vitro. Antiviral Research, 9:379-385.

Inouye K; Ikeya F; Yamazaki T; Hara T, 1990. Virucidal activities of various germicides against infectious pancreatic necrosis virus (IPNV). Fish Pathology, 25(2):81-86.

Isshiki T; Kawai K; Kusuda R, 1989. Incidence of yellowtail ascites virus (YAV) in wild yellowtail fingerling. Nippon Suisan Gakkaishi = Bulletin of the Japanese Society of Scientific Fisheries, 55(4):633-637; 16 ref.

Jarp J, 1999. Epidemiological aspects of viral diseases in the Norwegian farmed Atlantic salmon (Salmo salar L.). Bulletin of the European Association of Fish Pathologists, 19(6):240-244.

Jarp J; Gjevre AG; Olsen AB; Bruheim T, 1995. Risk factors for furunculosis, infectious pancreatic necrosis and mortality in post-smolt of Atlantic salmon, Salmo salar L. Journal of Fish Diseases, 18(1):67-78.

Jiang Y; Xu B; Li W; Li Z; Hu Q, 1989. Isolation and identification of infectious pancreatic necrosis virus (IPNV) from imported rainbow trout (Salmo gairdneri) in PR China. Acta Hydrobiologia Sinica, 13:353-358.

Joergensen PEV; Kehlet NP, 1971. Infectious pancreatic necrosis (IPN) viruses in Danish rainbow trout. Nordisk Veterinaermedicin, 23(No.11):568-575.

Kelly RK; Souter BW; Miller HR, 1978. Fish cell lines: comparisons of CHSE-214, FHM, and RTG-2 in assaying IHN and IPN viruses. Journal of the Fisheries Research Board of Canada, 35(7):1009-1011.

Kimura T; Yoshimizu M; Ezura Y; Kamei Y, 1990. An antiviral agent (46NW-04A) produced by Pseudomonas sp. and its activity against fish viruses. Journal of Aquatic Animal Health, 2(1):12-20.

Kimura T; Yoshimizu M; Yasuda H, 1984. Rapid, simple serological diagnosis of infectious pancreatic necrosis by coagglutination test using antibody-sensitized staphylococci. Fish Pathology, 19(1):25-33.

Kohlmeyer G; Ahne W; Thomsen I, 1986. Plaque size, virulence and immunogenicity of infectious pancreatic necrosis virus subtypes Sp, Ab and He. Tierärztliche Umschau, 41(8):532, 535-541.

Kusuda R; Kado K; Takeuchi Y; Kawai K, 1989. Characteristics of two virus strains isolated from young Japanese flounder, Paralichthys olivaceus. Suisanzoshoku, 37:115-120.

Lannan CN; Winton JR; Fryer JL, 1984. Fish cell lines: establishment and characterization of nine cell lines from salmonids. In Vitro, 20(9):671-676.

Lawrence WR; Nagy E; Duncan R; Krell P; Dobos P, 1989. Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus. Gene, 79(2):369-374.

Leong JC; Munn C, 1991. The use of molecular biological techniques in the development of fish vaccines. Bulletin of the European Association of Fish Pathologists, 11:34-36.

Liao L; Dobos P, 1995. Mapping of a serotype specific epitope of the major capsid protein VP2 of infectious pancreatic necrosis virus. Virology, 209:684-687.

Lientz JC; Springer JE, 1973. Neutralization tests of infectious pancreatic necrosis virus with polyvalent antiserum. Journal of Wildlife Diseases, 9(No.2):120-124.

Lightner D; Post G, 1969. Morphological characteristics of infectious pancreatic necrosis virus in trout pancreatic tissue. Journal of the Fisheries Research Board of Canada, 26:2247-2250.

Lipipun V, 1988. Antigenic characterisation and identification of aquatic birnaviruses using monoclonal antibodies. PhD dissertation. Orono, Maine, USA: University of Maine, 137 pp.

Ljungberg O; Vestergård-Jørgensen PE, 1973. Infectious Pancreatic Necrosis (IPN) of Salmonids in Swedish Fish Farms. European Inland Fisheries Advisory Committee, Technical Paper 17, supplement 2. Rome: FAO.

M’Gonigle RH, 1940. Acute catarrhal enteritis of salmonid fingerlings. Transactions of the American Fisheries Society, 70:297-302.

MacDonald RD; Dobos P, 1981. Identification of the proteins encoded by each genome segment of infectious pancreatic necrosis virus. Virology, 114(2):414-422.

Macdonald RD; Gower DA, 1981. Genomic and phenotypic divergence among three serotypes of aquatic birnaviruses (infectious pancreatic necrosis virus). Virology, 114(1):187-195.

MacKelvie RM; Artosob H, 1969. Infectious pancreatic necrosis virus in young salmonids of the Canadian Maritime Provinces. Journal of the Fisheries Research Board of Canada, 26:3259-3262.

Magyar G; Dobos P, 1994. Evidence for the detection of the infectious pancreatic necrosis virus polyprotein and the 17-kDa polypeptide in infected cells and of the NS protease in purified virus. Virology, 204:580-589.

Maheshkumar S; Goyal SM; Peterson RB; Economon PP, 1991. Method for the concentration of infectious pancreatic necrosis virus from hatchery water. Journal of Virological Methods, 31(23):211-218.

Maisse G; Dorson M; Torchy C, 1980. Ultraviolet inactivation of two viruses pathogenic for Salmonidae: infectious pancreatic necrosis and haemorrhagic septicaemia viruses. Bulletin Francais de Pisciculture, 53(No.278):34-40.

Manning DS; Leong JC, 1990. Expression in Escherichia coli of the large genomic segment of infectious pancreatic necrosis virus. Virology (New York), 179(1):16-25; 19 ref.

Manning DS; Mason C; Leong J, 1990. Cell-free translational analysis of the processing of infectious pancreatic necrosis virus polyprotein. Virology, 179:9-15.

Mason CL, 1992. Molecular characterization of the proteins or and RNA dependent RNA polymerase of infectious pancreatic necrosis virus, a fish birnavirus, PhD thesis. Corvallis, Oregon, USA: Oregon State University, 158 pp.

McAllister P; Schill WB; Owens WB; Hodge DL, 1991. Infectious pancreatic necrosis virus: a comparison of methods used to detect and identify virus in fluids and tissues of fish. In: Proceedings of the Second International Symposium on Viruses of Lower Vertebrates. Corvallis, Oregon, USA: Oregon State University Press, 191-202.

McAllister PE; Newman MW; Sauber JH; Owens WJ, 1984. Isolation of infectious pancreatic necrosis virus (serotype Ab) from diverse species of estuarine fish. Helgoländer Meeresuntersuchungen, 37(1/4):317-328.

McAllister PE; Owens WJ, 1986. Infectious pancreatic necrosis virus: protocol for a standard challenge to brook trout. Transactions of the American Fisheries Society, 115(3):466-470.

McAllister PE; Owens WJ, 1995. Assessment of the virulence of fish and molluscan isolates of infectious pancreatic necrosis virus for salmonid fish by challenge of brook trout, Salvelinus fontinalis (Mitchill). Journal of Fish Diseases, 18(1):97-103.

McAllister PE; Owens WJ; Ruppenthal TM, 1987. Detection of infectious pancreatic necrosis virus in pelleted cell and particulate components from ovarian fluid of brook trout Salvelinus fontinalis. Diseases of Aquatic Organisms, 2(3):235-237.

McAllister PE; Reyes X, 1984. Infectious pancreatic necrosis virus: isolation from rainbow trout, Salmo gairdneri Richardson, imported into Chile. Journal of Fish Diseases, 7(4):319-322.

McAllister PE; Schill WB, 1986. Immunoblot assay: a rapid and sensitive method for identification of salmonid fish viruses. Journal of Wildlife Diseases, 22(4):468-474.

McKnight IJ; Roberts RJ, 1976. The pathology of infectious pancreatic necrosis. I. The sequential histopathology of the naturally occurring condition. British Veterinary Journal, 132(1):76-85.

McMichael J; Fryer J; Pilcher KS, 1975. An antigenic comparison of three strains of infectious pancreatic necrosis virus of salmonid fishes. Aquaculture, 6(3):203-210.

Melby HP; Caswell-Reno P; Falk K, 1994. Antigenic analysis of Norwegian aquatic birnavirus isolates using monoclonal antibodies - with special reference to fish species, age and health status. Journal of Fish Diseases, 17(1):85-91.

Melby HP; Krogsrud J; Hastein T; Stenwig H, 1991. All commercial Atlantic salmon seawater farms in Norway harbour carriers of infectious pancreatic necrosis virus (IPNV). In: Proceedings of the Second International Symposium on Viruses of Lower Vertebrates. Corvallis, Oregon, USA: Oregon State University Press, 211-217.

Migus DO; Dobos P, 1979. Effect of ribavirin on the replication of infectious pancreatic necrosis virus in fish cell cultures. Journal of General Virology, 47:47-57.

Mikalsen AB; Torgersen J; Alestrom P; Hellemann AL; Koppang EO; Rimstad E, 2004. Protection of atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination. Diseases of Aquatic Organisms, 60:11-20.

Moewus-Kobb L, 1965. Studies with IPN virus in marine hosts. Annals of the New York Academy of Sciences, 165:328-342.

Mortensen SH; Hjeltnes B; Rdseth O; Krogsrud JK; Christie KE, 1990. Infectious pancreatic necrosis virus, serotype N1, isolated from Norwegian halibut (Hippoglossus hippoglossus), turbot (Scopthalmus maximus) and scallops (Pecten maximus). Bulletin of the European Association of Fish Pathologists, 10(2):42-43.

Munro ALS; Liversidge J; Elson KGR, 1976. The distribution and prevalence of infectious pancreatic necrosis virus in wild fish in Loch Awe. Proceedings of the Royal Society of Edinburgh, B., 75(4):223-232.

Nagabayashi T; Wolf K, 1979. Characterization of EV-2, a virus isolated from European eels (Anguilla anguilla) with stomatopapilloma. Journal of Virology, 30(1):358-364.

Nakajima K; Maeno Y; Arimoto M; Inouye K; Sorimachi M, 1993. Viral deformity of yellowtail fingerlings. Gyobyo Kenkyu = Fish Pathology, 28(3):125-129; [7 ref].

Nicholson BL; Caswell P, 1982. Enzyme-linked immunosorbent assay for identification of infectious pancreatic necrosis virus. Journal of Clinical Microbiology, 16(3):469-472.

Nicholson BL; Dunn J, 1974. Homologous viral interference in trout and Atlantic salmon cell cultures infected with infectious pancreatic necrosis virus. Journal of Virology, 14(No.1):180-182.

Nicholson BL; Henchal EA, 1978. Rapid identification of infectious pancreatic necrosis virus in infected cell cultures by immunoperoxidase techniques. Journal of Wildlife Diseases, 14(4):465-469.

Nicholson BL; Pochbit S, 1981. Antigenic analysis of infectious pancreatic necrosis viruses (IPNV) by neutralization kinetics. In: Karger S, International Symposium on Fish Biologics: Serodiagnostics and Vaccines, Basel, 35-41.

Nicholson BL; Thorne GW; Janicki C; Hanson A, 1979. Studies on a host range variant from different isolates of infectious pancreatic necrosis virus (IPNV). Journal of Fish Diseases, 2(5):367-379.

Novoa B; Figueras A; Puentes CF; Ledo A; Toranzo AE, 1993. Characterization of a birnavirus isolated from diseased turbot cultured in Spain. Diseases of Aquatic Organisms, 15(3):163-169.

Novoa B; Toranzo AE; Dopazo CP; Barja JL; Figueras A, 1993. Isolation of IPN virus serotype VR-299 from turbot in Europe. Diseases of Aquatic Organisms, 17(1):61-65.

OIE, 2003. Manual of Diagnostic Tests for Aquatic Animals, 4th Edition. Paris, France: Office International des Epizooties, 358 pp.

Okamoto N; Hirotani H; Sano T; Kobayashi M, 1988. Antiviral activity of the crude extracts of phototrophic bacteria to fish viruses. Bulletin of the Japanese Society of Scientific Fisheries, 54:2225-2231.

Okamoto N; Nasuya K; Sano T, 1987. IPN virus attenuation due to passages on RTG-2 cell line. Bulletin of the Japanese Society of Scientific Fisheries, 53:1897.

Okamoto N; Sano T, 1984. Comparison of the virus recovery from rainbow trout fry of infectious pancreatic necrosis by the two homogenization methods: a mortar-pestle and a Waring blender. Bulletin of the Japanese Society of Scientific Fisheries, 50(11):1959.

Okamoto N; Sano T; Hedrick RP; Fryer JL, 1983. Antigenic relationships of selected strains of infectious pancreatic necrosis virus and European eel virus. Journal of Fish Diseases, 6(1):19-25.

Okamoto N; Taniguchi N; Seno Y; Sano T, 1984. The relation between the change of quantities of infectious pancreatic necrosis virus in infected rainbow trout fry and the disease process. Fish Pathology, 19(1):1-4.

Okamoto N; Yasutomi R; Shibazaki H; Hanzawa S; Sano T, 1987. The influence of immersing temperature for inoculation with IPNV and/or rearing temperature on mortality of rainbow trout fry postinfection. Nippon Suisan Gakkaishi (Bulletin of the Japanese Society of Scientific Fisheries), 53(7):1125-1128.

Olesen NJ; Jorgensen V; Bloch B; Mellergaard S, 1988. Isolation of an IPN-like virus belonging to the serogroup II of the aquatic birnaviruses from dab, Limanda limanda L. Journal of Fish Diseases, 11(5):449-451.

Olson C; Stewart BL; Lutz S; Gleckler J; Reno PW; Dunphy G, 1994. Detection of an aquatic birnavirus in an anadromous coho broodstock, Oncorhynchus kisutch, in Puget Sound, Washington. Fish Health News, 22:5-6.

Oye AK; Rimstad E, 2001. Inactivation of infectious salmon anaemia virus, viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus in water using UVC irradiation. Diseases of Aquatic Organisms, 48(1):1-5.

Ozaki A; Sakamoto T; Khoo S; Nakamura K; Coimbra MRM; Akutsu T; Okamoto N, 2001. Quantitative trait loci (QTLs) associated with resistance/susceptibility to infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss). Molecular Genetics and Genomics, 265(1):23-31.

Perez L; Chiou PP; Leong JC, 1996. The structural proteins of infectious pancreatic virus are not glycosylated. Journal of Virology, 70:7247-7249.

Peters F; Neukirch M, 1986. Transmission of some fish pathogenic viruses by the heron, Ardea cinerea. Journal of Fish Diseases, 9(6):539-544.

Ramsey DW; Bright SW; Ludlow JW; Winston V, 1986. Detection of infectious pancreatic necrosis virus using an inhibition enzyme-linked immunosorbent assay. Journal of Immunoassay, 7(4):229-239.

Reno PW, 1976. Quantitative and qualitative aspects of the infectious pancreatic necrosis (IPN) carrier state. PhD dissertation. Guelph, Ontario: University of Guelph.

Reno PW, 1999. Infectious pancreatic necrosis and associated aquatic birnaviruses. Fish diseases and disorders. Volume 3: viral, bacterial and fungal infections., 1-55.

Reno PW; Caswell-Reno PA; Nicholson BL, 1994. Epitope mapping of a worldwide collection of aquatic birnaviruses using a panel of monoclonal antibodies. In: Proceedings of the International Symposium on Aquatic Animal Health, Seattle, Washington State, USA, Abstract P-113.

Reno PW; Darley S; Savan M, 1978. Infectious pancreatic necrosis: experimental induction of a carrier state in trout. Journal of the Fisheries Research Board of Canada, 35(11):1451-1456.

Rimstad E; Hornes E; Olsvik O; Hyllseth B, 1990. Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized segments. Journal of Clinical Microbiology, 28:2275-2278.

Roberts RJ, 1975. Fish Pathology, 1st edition. London, UK: Baillière-Tindall.

Rodger HD; Frerichs GN, 1997. Clinical infectious pancreatic necrosis virus infection in farmed halibut in the United Kingdom. Veterinary Record, 140(15):401-402.

Rodriguez Saint-Jean S; Borrego JJ; Perez-Prieto SI, 2001. Comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fish. Journal of Virological Methods, 97(1/2):23-31.

Rodriguez Saint-Jean S; Pilar Vilas Minondo M; Angel Palacios M; Perez Prieto S, 1991. Detection of infectious pancreatic necrosis in a carrier population of rainbow trout, Oncorhynchus mykiss (Richardson), by flow cytometry. Journal of Fish Diseases, 14(5):545-553.

Ross K; Thomson AM; Melvin WT; Munro ALS, 1991. Sensitive confirmation of infectious pancreatic necrosis virus by dot blot using monoclonal antibodies. Bulletin of the European Association of Fish Pathologists, 11(4):137-139.

Sano T, 1971. Studies on viral diseases of Japanese fishes I. Infectious pancreatic necrosis of rainbow trout: first isolation from epizootics in Japan. Bulletin of the Japanese Society of Scientific Fisheries, 37:495-498.

Sano T; Okamoto N; Nishimura T, 1981. A new viral epizootic of Anguilla japonica Temminck and Schlegel. Journal of Fish Diseases, 4(2):127-139.

Santi N; Vakharia VN; Evensen O, 2004. Identification of putative motifs involved in the virulence of infectious pancreatic necrosis virus. Virology, 25(322):31-40.

Scherrer R; Cohen J, 1975. Studies on infectious pancreatic necrosis virus interactions with RTG-2 and FHM cells: selection of a variant virus-type in FHM cells. Journal of General Virology, 28:9-20.

Schlotfeldt HJ; Leiss B; Frost JW, 1975. Erst isoleirung und identifizierung des virus der infektiosen pancreasnekrosl (IPN) der salmoniden in der Bundesrepublik Deutschland. Berliner und Munchener Tierärtzliche Wochenschrift, 88:109-111.

Shankar K, 1991. Characterisation of infectious pancreatic necrosis virus associated with lake trout (Salvelinus namaykush) in Cornwall Lake, Alberta. PhD dissertation. Edmonton, Alberta, Canada: University of Alberta, 134 pp.

Shankar K; Yamamoto T, 1994. Epitope analysis of IPN virus isolates from feral and hatchery salmonid fishes by neutralizing monoclonal antibodies. Current Science, 67:95-98.

Shivappa RB; Song H; Yao K; Aas-Eng A; Evensen O; Vakharia VN, 2004. Molecular characterization of Sp serotype strains of infectious pancreatic necrosis virus exhibiting differences in virulence. Diseases of Aquatic Organisms, 61:23-32.

Shwed PS; Dobos P; Cameron LA; Vakharia VN; Duncan R, 2002. Birnavirus VP1 proteins form a distinct subgroup of RNA-dependent RNA polymerases lacking a GDD motif. Virology, 296(2):241-250; many ref.

Silim A; Elahary M; Lgace A, 1982. Susceptibility of trouts of different species and origins to various isolates of infectious pancreatic necrosis virus. Canadian Journal Fisheries and Aquatic Science, 39: 1580-1584.

Skall HF; Mellergaard S; Olesen NJ, 2000. Isolation of Birnavirus serogroup B in wild and aquacultured fish species. Bulletin of the European Association of Fish Pathologists, 20(6):229-236.

Smail DA; Bruno DW; Dear G; McFarlene LA; Ross K, 1992. Infectious pancreatic necrosis (IPN) virus Sp serotype in farmed Atlantic salmon, Salmo salar L., post-smolts associated with mortality and clinical disease. Journal of Fish Diseases, 15(1):77-83.

Smail DA; Grierson RJ; Munro ALS, 1986. Infectious pancreatic necrosis (IPN) virus in Atlantic salmon: Virulence studies and sub-clinical effects with respect to growth, smolting performance and condition. Copenhagen, Denmark: ICES Council Meeting, 10 pp.

Smail DA; Huntly PJ; Munro ALS, 1993. Fate of four fish pathogens after exposure to fish silage containing fish farm mortalities and conditions for the inactivation of infectious pancreatic necrosis virus. Aquaculture, 113(3):173-181; 6 ref.

Smail DA; Munro ALS, 1989. Infectious pancreatic necrosis virus in Atlantic salmon: transmission via the sexual products?. Viruses of lower vertebrates., 292-301; [1st International Symposium on Viruses of Lower Vertebrates, Munich, August 1988].

Snieszko SF; Wolf K; Camper JE; Pettijohn LL, 1959. Infectious nature of pancreatic necrosis. Transactions of the American Fisheries Society, 88:289-293.

Snieszko SF; Wood EM; Yasutake T, 1957. Infectious pancreatic necrosis in trout. American Medical Association Archives of Pathology, 63:229-233.

Sonstegard RA; McDermott LA; Sonstegard KS, 1972. Isolation of infectious pancreatic necrosis virus from white suckers (Catastomus commersoni). Nature, 236(No.5343):174-175.

Sorimachi M; Hara T, 1985. Characteristics and pathogenicity of a virus isolated from yellowtail fingerlings showing ascites. Fish Pathology, 19(4):231-238; 10 ref.

Souter BW; Dwilow AG; Knight K; Yamamoto T, 1986. Infectious Pancreatic Necrosis Virus in Adult Arctic Char, Salvelinus alpinus (L.), in Rivers in the Mackenzie Delta Region. Canadian Technical Report of Fisheries and Aquatic Sciences No 1441, 2. Toronto, Canada: Fisheries Research Board of Canada, 11 pp.

Stephens EB, 1981. Isolation and molecular characterisation of infectious pancreatic necrosis virus from ‘spinning’ menhaden in the Chesapeake Bay. PhD dissertation. College Park, Maryland, USA: University of Maryland, 132 pp.

Stephens EB; Newman MW; Zachary AL; Hetrick FM, 1980. A viral aetiology for the annual spring epizootics of Atlantic menhaden Brevoortia tyrannus (Latrobe) in Chesapeake Bay. Journal of Fish Diseases, 3(5):387-398.

Swanson RN; Gillespie JH, 1981. An indirect fluorescent antibody test for the rapid detection of infectious pancreatic necrosis virus in tissues. Journal of Fish Diseases, 4(4):309-315.

Swanson RN; Gillespie JH, 1982. Isolation of infectious pancreatic necrosis virus from the blood and blood components of experimentally infected trout. Canadian Journal of Fisheries and Aquatic Sciences, 39(1):225-228.

Taksdal T; Dannevig BH; Rimstad E, 2001. Detection of infectious pancreatic necrosis (IPN)-virus in experimentally infected Atlantic salmon parr by RT-PCR and cell culture isolation. Bulletin of the European Association of Fish Pathologists, 21(5):214-219.

Taksdal T; Thorud K, 1999. Evaluation of a rapid coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples of Atlantic salmon, Salmo salar L. Journal of Fish Diseases, 22(2):117-124.

Tisdall DJ; Phipps JC, 1987. Isolation and characterisation of a marine birnavirus from returning Quinnat salmon (Oncorhynchus tshawtscha) in the South Island of New Zealand. New Zealand Veterinary Journal, 35(12):217-218.

Toranzo AE; Hetrick FM, 1982. Comparative stability of two salmonid viruses pancreatic necrosis and haematopoietic necrosis viruses and poliovirus in fresh, estuarine and marine waters. Journal of Fish Diseases, 5(3):223-231.

Tu K-C; Spendlove RS; Goede RW, 1974. Immunofluorescent cell assay of infectious pancreatic necrosis virus. Applied Microbiology, 27(No.3):593-599.

Underwood BO; Smale CJ; Brown F; Hill BJ, 1977. Relationship of a virus from Tellina tenuis to infectious pancreatic necrosis virus. Journal of Genetic Virology, 36:93-109.

Vestergård-Jørgensen P; Bregnballe F, 1969. Infectious pancreatic necrosis in rainbow trout (Salmo gairdneri) in Denmark. Nordisk Veterinaermedicin, 21:142-148.

Vestergård-Jørgensen P; Grauballe PC, 1971. Problems in the serological typing of IPN virus. Acta Veterinaria Scandinavica, 12:115-117.

Vestergård-Jørgensen; PE, 1974. Indirect immunofluorescent antibody techniques for determination of trout viruses and corresponding antibody. Acta Veterinaria Scandinavica, 15:198-205.

Wang Way Shyan; Wi Yea Ling; Lee Jainn Shyan; Wang WS; Wi YL; Lee JS, 1997. Single-tube, non-interrupted reverse transcription PCR for detection of infectious pancreatic necrosis virus. Diseases of Aquatic Organisms, 28:229-233.

Watanabe RA; Fryer JL; Rohovec JS, 1988. Molecular filtration for recovery of waterborne viruses of fish. Applied and Environmental Microbiology, 54(6):1606-1609.

Wattanavijarn W; Torchy C; Tangtronpiros J; Kinkelin Pde, 1988. Isolation of a birnavirus belonging to Sp serotype, from South East Asia fishes. Bulletin of the European Association of Fish Pathologists, 8(5):106-108.

Wechsler SJ; Woods LC; Kraeuter JN; Hetrick FM; McAllister PE, 1987. Transmission of infectious pancreatic necrosis virus in striped bass, Morone saxatilis (Walbaum). Journal of Fish Diseases, 10(1):29-34; 16 ref.

Williams K; Blake S; Sweeney A; Singer JT; Nicholson BL, 1999. Multiplex reverse transcriptase PCR assay for simultaneous detection of three fish viruses. Journal of Clinical Microbiology, 37(12):4139-4141.

Willumsen B; Brun E, 1988. Infectious pancreatic necrosis in salmon spawn and smolt. Norsk Veterinaertidsskrift, 100(12):900-901.

Wolf K, 1966. Infectious Pancreatic Necrosis (IPN) of Salmonid Fishes. United States Fish and Wildlife Service, FDL-1:1-4.

Wolf K, 1988. Fish viruses and fish viral diseases. Fish viruses and fish viral diseases., xii + 476 pp.

Wolf K; Bullock GC; Dunbar CE; Quimby MC, 1969. Comparison Studies of IPN Viruses. Washington DC, USA: Division of Fishery Research, Bureau of Sport Fisheries and Wildlife Resource Publication No 77, 139-141.

Wolf K; Mann JA, 1980. Poikilotherm vertebrate cell lines and viruses: a current listing for fishes. In Vitro, 16(2):168-179.

Wolf K; Quimby MB; Bradford AD, 1968. Egg-associated transmission of IPN virus of trouts. Virology, 21:317-321.

Wolf K; Quimby MB; Carlson CP; Bullock GL, 1968. Infectious pancreatic necrosis: selection of virus-free stock from a population of carrier trout. Journal of the Fisheries Research Board of Canada, 25:383-391.

Wolf K; Quimby MC, 1962. Established eurythermic cell line of fish cells in vitro. Science, 135:1065-1066.

Wolf K; Quimby MC, 1966. Lymphocystis virus: its propagation in centrarchid cell lines. Science, 151:1004-1006.

Wolf K; Quimby MC, 1971. Salmonid viruses: infectious pancreatic necrosis virus: morphology, pathology and serology of first European isolations. Archiv für die gesamte Virusforschung, 34:144-156.

Wolf K; Snieszko SF; Dunbar CE; Pyle E, 1960. Virus nature of infectious pancreatic necrosis in trout. Proceedings of the Society for Experimental Medicine and Biology, 104:105-108.

Wolski SC; Roberson BS; Hetrick FM, 1986. Monoclonal antibodies to the Sp strain of infectious pancreatic necrosis virus. Veterinary Immunology and Immunopathology, 12:373-381; [International Meeting on Fish Immunology, Sandy Hook, September 1985].

Wood E; Snieszko SF; Yasutake WT, 1955. Infectious pancreatic necrosis in brood trout. American Medical Association Archives of Pathology, 60:26-28.

Xu HT; Si WD; Dobos P, 2004. Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis. Virology, 322:199-210.

Yamamoto T, 1975. Frequency of detection and survival of infectious pancreatic necrosis virus in a carrier population of brook trout (Salvelimus fontinalis) in a lake. Journal of the Fisheries Research Board of Canada, 32(4):568-570.

Yamamoto T, 1975. Infectious pancreatic necrosis (IPN) virus carriers and antibody production in a population of rainbow trout (Salmo gairdneri). Canadian Journal of Microbiology, 21(9):1343-1347.

Yamamoto T; Kilistoff J, 1979. Infectious pancreatic necrosis virus: quantification of carriers in lake populations during a 6-year period. Journal of the Fisheries Research Board of Canada, 36(5):563-567.

Yasutake WT; Parisot TJ; Klontz GW, 1965. Virus diseases of the Salmonidae in the western United States. II. Aspects of pathogenesis. Annals of the New York Academy of Sciences, 165:520-530.

Yu KK-Y; Macdonald RD; Moore AR, 1982. Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state. Journal of Fish Diseases, 5(5):401-410.

Zhang CX; Suzuki S, 2004. Aquabirnaviruses isolated from marine organisms form a distinct genogroup from other aquabirnaviruses. Journal of Fish Diseases, 27:633-43.

Links to Websites

Contributors

Main Author
Espen Rimstad
Norwegian School of Veterinary Science, Dept. of Food Safety & Infection Biology, POB 8146, dep 0033 Oslo, Norway

Distribution Maps