Immunological and pathological responses of salmonids to infectious pancreatic necrosis virus (IPNV)

Annual Review of Fish Diseases, Vol 5, pp. 209-223, 1995 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0959-8030195 $29.00 + .OO

Pergamon

0959-8030(95)00001-1

IMMUNOLOGICAL AND PATHOLOGICAL RESPONSES OF SALMONIDS TO INFECTIOUS PANCREATIC NECROSIS VIRUS (IPNV) Eileen C. Saahsiv Department

of Fisheries,

Animal and Veterinary Science, The University Kingston, Rhode Island, 02881-0804, USA

of Rhode Island,

Abstract The aquatic bimavims IPNV is commonly found in association with apparently healthy, mature salmonids. Some bimaviruses cause lethal diseases in fry; however, many of those discovered in fish may not be pathogenic for the species from which they were isolated. More pathogenic virus may be produced from the acinar cells of the pancreas; less pathogenic virus by skin and gut cells. Skin infection could explain the lack of virus clearance after development of circulating antibody; the less pathogenic form of the virus may not induce protective antibodies. True vertical transmission to progeny fish would seem not to occur, but virus may adhere to egg cases and spread to fry which ingest them at the time of first feeding. When viruses are found with moribund mature fish, alternative causes of death must be considered. Atlantic salmon Salmo salar smelts, which have high levels of virus during pre-smoking, can develop pancreatic lesions (possibly mediated by the immune system) on transfer to salt water. IPNV infection may be linked to immunosuppression; possible controlling genes have been found. Keywords: Bimavirus,

Persistent

infection,

Aquaculture,

Salmo salar

INTRODUCTION

One of the important diseases of farmed salmonids is infectious pancreatic necrosis (IPN) which is caused by a birnavirus, infectious pancreatic necrosis virus (IPNV). Multiple strains of the virus exist that differ in virulence and in evoking variable serologic responses (1). Although the virus can be a highly destructive pathogen of hatchery-reared salmonids, it has been reported to be carried and possibly replicated in hosts for long periods without causing clinical disease. Broodstock carriage has been considered a likely source of virus for the lethal infection of progeny fish. IPN disease happens only with the coming together of susceptible fish and virus able to attach to, enter and replicate in cells of those fish. Salmonid defensive systems are either not present or are overcome. Each cell infected with virus can produce up to 1000 new infectious particles within a few hours, depending upon the temperature and cell type (2). Numerous studies of the disease processes caused by IPNV have been conducted. This paper will be limited to a discussion of IPNV infection of three species of salmonids: brook trout (Salvelinus fintinalis), rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Brook trout have been found to be the most sensitive to IPN and almost all survivors become carriers (3, 4). They are followed in sensitivity by rainbow trout, which are better antibody producers and have a lower and more rapidly declining carrier rate, and by Atlantic salmon which seem to be the least sensitive of the three species to the lethal effects of the virus. With the improvements in virus detection methods (5-8), it is apparent that IPNV, or IPNV-like viruses, are quite ubiquitous (9-14). Most hatchery efforts to date have been directed towards eliminating virus during juvenile development. An integral part of avoiding 209

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juvenile disease has been to select broodstock which have never encountered the virus. Control of IPN by vaccination has not been achieved and serological identification of the virus able to cause lethal disease has not been reliable (14). The purpose of this review is to explore how fish can have an antiviral immune response which does not result in elimination of virus from the fish. THE VIRUS

IPNV is an aquatic birnavirus having at least four different proteins which are coded for by two segments of double-stranded RNA (15, 16). It is a simple and remarkably stable virus. It can be added to silage and pass through the digestive system of cows (17) or it can be retained within scallops for almost a year without being degraded (18). Pathogenicity for the host and tropism, or the likelihood that the virus will attach to and enter particular types of cells where it can be replicated, is determined by the interaction of viral proteins with host proteins (19, 20). The largest of the viral proteins, VP-I, is the polymerase which is coded for by the smaller RNA segment (21). It is not associated with virulence (19-22). The larger RNA segment encodes three proteins which are produced as a polyprotein and cleaved to yield the major capsid protein VP-2, a non-structural protein NS or VP-4, and an interior structural protein VP-3 (23). An exact cleavage site between VP-2 and NS has not been determined (24). VP-2 is the outermost protein and not surprisingly is involved in attachment to cells. Estay et al. (25) have reported that VP-2 is glycoslyated. VP-3 is associated with VP-2, but does not contain neutralizing epitopes and thus would be a less important target of the host’s defensive mechanisms. According to the work of Manning, Mason, and Leong in 1990 (26) and Heppell et d. in 1993 (24), NS is likely to function as a co-enzyme, pointing towards the importance of cellular enzymes for viral formation. The invaded host can respond to infection with a number of defensive strategies including neutralization by the production of specific antibodies against the virus surface. The viral protein epitope important in its neutralization would appear to be either a conformational arrangement of VP-2 or a sequential-conformational epitope (27-29). IPNV strains have outer proteins differing in molecular weight between 63K and 54K. This variability in length of the surface covering would be one way in which their ability to be neutralized would vary. The surface pattern formed by different length VP-2s would certainly present an antigenically altered antibody attachment surface. In 1986, Wolski, Roberson, and Hetrick (28) reported the production of monoclonal antibodies (MAB) to the Sp strain of IPNV Ten of the MAB were found to be broadly reactive against representatives of the 3 major serotypes of IPNV. Two others were specific for the Sp strain which was the immunogen. One of the specific MAB’s was shown to be directed against the major capsid protein while the other specific one, and the broadly reacting ones, reacted with the low molecular weight viral polypeptides. &swell-Reno and her associates (29-34) have used a panel of MAB to 11 aquatic birnavirus epitopes to group IPNV viral stains. Lecomte, Arella, and Bertiaume (31, 35) have described a MAB to VP-3 that can interact with the virus surface without neutralizing it and were able to show some differences between pathogenic and non-pathogenic virus. DNA probes have been developed to detect the virus (32-37). Probes, coupled with polymerase chain reaction (PCR) amplification techniques, can greatly increase the sensitivity of viral detection, but they have not performed well to date in field tests with infected fish. Some probes can detect areas of the viral genome not concerned with producing structures which form the virus neutralization epitope (38).

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211

IPN virus has been found to propagate in several lines of cultured cells, generally of epithelial or fibroblastic origin, in which cytopathic effects are produced or a persistent infection is achieved (3). Kidney cells have been noted to be infected at similar rates (O-OS%) as are persistently infected cultured cells (O.l-1.0%) (39). In the case of some smaller RNA viruses such as poliomyelitis, expression of viral proteins is altered during a persistent infection (4m2), but this has not yet been reported for birnaviruses. Kennedy and MacDonald (43) postulated that persistent infection with IPNV could be mediated by virus particles having altered properties, which might allow those cells to survive. Pathogenic properties of IPNV can be changed by culture conditions; cell-culture passage may produce a less pathogenic virus which in some cases may have an altered reaction with antiserum (44, 45). In susceptible fish, more highly pathogenic strains (Buhl, VR299), have been observed by Sano and associates to be produced in all tissues; whereas the avirulent EVE (Ab strain) was produced from epidermis only (19). They also agreed that the virulent strain becomes avirulent by passage in cell culture, but they did not suggest how these two observations might be related. Viral attenuation can occur in a number of ways. One is by accumulation of genetic mutations; these would not revert to the original form in a single step. Another way is by change due to non-hereditary causes (14) such as trimming the size of VP-2 or varying the sugar residues or their placement on the viral surface (gylcosylation). Each of these processes could yield an altered virus which might evade the immune system and attach to different cells. The attenuation of virus by a single passage in cell culture could be an example of a phenotypic alteration.

SALMONID

DISEASE

AND VIRUS

CARRIAGE

Brook trout

Yamamoto (46) reported that IPNV could be isolated from the kidneys of carrier brook trout but their antibody level could not be correlated with virus yield. Swanson (47) reported changes in the pathogenicity of IPNV due to its passage in cell culture. He used virus after it had been repeatedly passed in culture and contrasted its pathologic effects with those of a field isolate which had undergone only one culture pass. The high passage virus replicated in the intestinal cells while the field isolate replicated in the pancreas. Brook trout infected several months after first feeding were not found to have either virus-caused lesions or significant growth retardation. He first reported the presence of mononuclear white blood cells around damaged pancreatic acini. Swanson and Gillespie (48) reported that kidneys did not show evidence of spreading foci of viral infections, although they contained macrophages carrying cell debris and necrotic cells. Virus replicated in the intestine and was detected in the kidney, with the viral titer peaking at 7 days post-inoculation (49). It is also replicated in the digestive tract of yearlings without being found in the internal organs. They concluded that virus was picked up by phagocytic cells and transported to the kidney following intraperitoneal injection. Virus titers in blood cells were lower and variable compared to samples of kidneys, spleens or sexual excretions (50). It is notable that replication in older fish did not involve disease or viremia, which would release the virus to other organs. Around this same time Yu, MacDonald, and Moore (51) reported that they could recover IPNV consistently from the leukocytes of asymptomatic year-old brook trout. Although birnaviruses are good antigens, an immune response does not result in the elimination of the virus. Bootland, Stevenson, and Dobos (52) documented the puzzling

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difficulty of vaccination, showing that immunization by immersion could increase the percentage of fry succumbing to IPN. Their work confirmed that IPNV could not be found in fertilized eggs from brook trout carrier broodstock (53). Transmission from broodstock to progeny was demonstrated, with progeny having low mortality, low prevalence and low titers. They concluded that the carrier state and vertical transmission of IPNV was unpredictable. The mechanism of transmission was not determined. IPNV in yearling brook trout induced an asymptomatic chronic virus presence that persisted for at least 76 weeks in 95% of the fish. They confirmed that the fish have an immune response, but that it did not result in clearance of virus from the fish (54). The prevalence of “shedders” and their viral titers varied over time. The infection did not affect growth rate, which argued against an ongoing infection, and a wide variability was noted in the humoral immune responses of individual fish. In his continuing efforts to explain long-term virus carriage, McAllister et al. (55) confirmed that those brook trout which survive can remain lifelong asymptomatic reservoirs of infection, shedding virus in urine, feces and sexual fluids. They reported that although virus could be detected in the leukocyte cell fraction from a small portion of asymptomatic 2 year old brook trout it was present in the majority of 4 year old fish, with up tolO infectious doses being detected in an infected cell. The virus was universally detectable with high titers in the kidney-spleen, pancreas-pyloric cecum and in the cell fraction of ovarian fluid from heavily infected brook trout. Chemical immunosuppression accompanied by elevated water temperatures increased both the percentage of fingerlings which were IPNV positive, as well as their virus titers. The offspring of infected broodstock were not infected, but could become infected by exposure (56).

Rainbow

trout

Although rainbow and brook trout are genetically similar, their susceptibility to IPNV is not identical. McKnight and Roberts (57) noted that the extent of pancreatic damage was variable in moribund fish, but there was always severe enteritis. Sano, Tanaka and Fukuzaki (58) felt that they could at least show virus clearance in fish with high antibody titers. They, like Swanson, also noted that older fish which had survived infection later developed pancreatic damage. Yu, MacDonald and Moore (51) found that rainbow trout leukocytes rarely harbored IPNV or supported virus replication. They recovered up to 400 infectious units of virus per cell from a very few (0.01%) adherent leukocytes (which they suggested could have been from a subpopulation of cells) of asymptomatically infected three and four year old fish. They found no IPNV-specific fluorescence, suggesting little or no viral replication. Swanson and Gillespie (49) recovered IPNV from white blood cells for up to forty days after experimental infection, but found that the virus localized in the kidneys thereafter. They found a persistent viremia in yearling rainbow trout, and destruction of the pancreatic acinar cells with necrosis. Macrophages appeared in the affected tissue. Swanson and Gillespie (49) then followed the effects produced by the virus and found IPNV primarily in the gasterointestinal tract, using IPNV strain VR-229 and a field isolate. Although virus titers rose, VR-229 did not cause mortalities in fry or yearlings. The field isolate replicated in the pancreatic acinar cells, Kidney and spleen samples have been consistently noted to have higher virus titers than pyloric cecum (59, 60). Again, it was noted that there appeared to be no correlation between antibody levels and virus isolation. Okamoto and Sano (61) were able to delineate more of the pathogenic effects of IPNV on fry. Using high and low virulent strains of IPNV and resistant and sensitive rainbow trout,

IPNV in salmonids

213

they showed that virus propagated in the pancreas of healthy and moribund fry. However, only in the moribund fry was the virus found to multiply in the intestines. Water temperature at the time of viral exposure did not affect the likelihood of illness, but the temperature of the rearing water (5-20°C) was related to disease outbreaks. Mortalities at 5°C were minimal, whereas at 10, 15, and 2O”C, 80-100% of the fish died. Later, Okamoto and Kamon (62) confirmed the protective effects of low temperatures in IPN. Fry infected and held at 5°C suffered no mortalities; however, similar fry succumbed when moved to 15°C even after 93 days. Their work confirmed that the cause of death in fry is intestinal virus, as had been shown by McKnight and Roberts in 1976 (57) and that viral replication in the pancreas was not, at least immediately, lethal. More recently, Okamoto et al. (63) have shown that stocks of rainbow trout can develop a genetically stable resistance to IPN. Ahne and Negele (64) reported that eyed eggs of rainbow trout and Arctic char could be infected via water and that the egg cases remained infectious even after hatching. Sperm and eggs rapidly lost virus and hatched fry were free of virus. The source of infection, at least in the case of the Arctic char, appeared to be ingestion of discarded virus-coated egg cases. Dorson (45) noted that virus could be found in the skin mucus of rainbow trout for long periods of time, along with specific antibody in the mucus. This antibody was described as being elicited independently of the circulating serum antibody. Virus has been repeatedly found in leukocytes, although its ability to replicate in them would appear limited. Mangunwiryo and Agius (65) described the isolation of IPNV from leukocytes of carrier fish. They found no IPNV (strain Sp) in juvenile hatchery-held rainbow trout but virus and antibody were detected in healthy larger fish in open ponds, implying that fish beyond the stage of disease sensitivity could mount an antibody response should they become exposed. Virus titers peaked as waters warmed in the spring while antibody titers peaked as waters cooled in the fall. Rodriguez et al. (6) also described the presence and possible multiplication of IPNV in leukocytes. Ledo et al. (3) ascribed adult rainbow trout mortalities to the concurrent presence of bacterial pathogens with IPNV. Dorson, DeKinkelin and Torchy (66) showed interferon synthesis in fry infected with pathogenic IPNV-Sp. Adult rainbow trout injected with the virus did not produce interferon, suggesting that the virus did not replicate in adult fish as it did in fry.

Atlantic salmon Virus has been isolated from disease-free Atlantic salmon (67). Dorson (68) reported that salmon infected at 3 weeks of age could still be carriers of IPNV after 6 months, along with detectable antibody. Although he tried several different isolates, Dorson (4) was unable to reproduce IPN disease in salmon fry. He pointed out that although virus had frequently been isolated, no clinical cases were seen. An atypical finding of IPNV-related late pathology in Atlantic salmon was reported by Smail and Munro (69) who found IPNV-Sp associated with subclinical pancreatic necrosis in Atlantic salmon post-smolts, and correlated mortality with rising virus titers in the pre-smolting fish. They suggested that this late response might be due to immune recognition of viral antigens on the acinar cells of the pancreas. This deduction would support the earlier findings with older surviving rainbow trout (49, 58) showing pancreatic damage. All populations of survivors had a significant number of individuals with life-long infections. In the great majority of the fish (141/143), virus could be detected yet only 65% of the fish developed antibody, whether infected with the more pathogenic Sp or the less pathogenic Ab strains. The kidneys

214

E. C. Sadasiv

of smolts with pancreatic lesions were normal; the presence of virus did not seem to compromise the digestive function of the pancreas. In 1986, Knott and Munro (70) isolated virus from what had been considered virus-negative fish. They pointed out that only one case of clinical IPN had been found in Scottish Atlantic salmon, despite numerous isolations of IPNV from carriers. Smail et al. (71) confirmed that a high level of IPNV-Sp was associated with the mortality of post-smolts. Moribund fish were thin, with virus in the intestinal cells and necrosis in the pancreatic acinar cells. Disease was noted as Atlantic salmon culture increased. In 1989, epizootics of farmed salmon smolts were reported by Krogsrod, Hastein, and Ronningten (72). More than 50% of the farmed fish in Norway were IPNV carriers (73). Ledo et al. (3) found that, in Spain, IPNV-Sp but not the disease could be detected in juvenile Atlantic salmon and rainbow trout. They concluded that virus had been acquired after the fish had been placed on fish farms, rather than being vertically passed. It has been difficult to account for the quantity of virus found in apparently healthy fish. Evensen and Rimstad (74) pointed out that relatively high virus titers (up tolO infectious doses) can be found in normal fish, but titers of lo6 to 10’ infectious doses per gram of tissue would indicate IPNV as a cause of death. Using virus-negative smolts (110 g, 4-month, post-transfer to sea water), Rimstad et al. (75) found that when these fish were inoculated with a cell-culture-grown Sp strain of IPNV and kept at 11°C none died, no clinical signs developed, and no pathological or immunohistological evidence for virus replication was noted. Any pancreatic damage noted was due to the more pathogenic strain Sp rather than the less pathogenic Ab strain. Small amounts of virus were isolated only sporadically from the kidneys (but not the pyloric ceca or gills) of presumptively virus-negative fish. However, virus was consistently isolated from fish inoculated with cell-culture-propagated virus. This virus also readily spread to contact control fish from which it could be isolated. Virus could be reisolated from inoculated groups of fish for 80 days even though they were immuno-histochemically negative for virus replication and without disease symptoms. Rimstad’s work (75) emphasized the fact that cell-culture grown virus differs in some way from virus passed either in pancreatic cells or sensitive fry, certainly in its ability to spread to other fish without appearing to replicate in them. In order to demonstrate such a viral difference in fry, sensitive fry were exposed to a highly passed strain of IPNV (VR-229). There was 80% mortality, peaking at 6 days post-exposure. One month later, the survivors were exposed to a field isolate of IPNV (passed once in cell culture), and again 80% mortality was produced, with deaths peaking at 6 days (R.E. Wolke and E.C. Sadasiv, unpublished data). Virus was reisolated from both epizootics, but in agreement with Swanson and Gillespie’s (67) work no histological lesions were found in the fish. The full interpretation of this finding has not yet been developed. McAllister et al. (76) have reported that older Atlantic salmon exposed to IPNV rarely suffer mortalities due to IPN and their antibody response is variable. Disease seemed to occur only under conditions of virus exposure accompanied by additional stress, such as might be found when the fish were undergoing a concurrent bacterial infection. In 1993, Smail and Munro (77) reported on vertical transmission. Virus could bind to sperm and enter the eggs of Atlantic salmon, yet the progeny were not infected. Even when ovarian fluid was carrying virus, the progeny fish were not infected.

IPNV in salmonids ANTIBODY

215

AND VACCINATION

Antigenic relationships among IPNV isolates have been studied by a number of groups, but IPNV does not fit well into classification schemes. It has been categorized by Hill and Way (78) in 1988; Christie, Ness, and Djupvik (79) in 1990; Lecomte, Arella, and Bertiaume (31) in 1992; and Tarrab et al. (80) in 1993. Genotype does not equal serotype (81) and non-pathogenic viral forms can be serologically identical to pathogenic forms (82, 83). Antibody response in salmonids is inherently variable (84), but the majority unquestionably develop antibody. Whether the antibody is protective is not easily demonstrated, for it must be tested by exposing susceptible fry to virus. In trying to interpret virus-antibody reactions, one must bear in mind that both the virus and the antibody, as well as the strain of fish or the cells being used as the test system, may vary. It is not even certain that antibody is produced only in response to infection, as opposed to viral exposure. In Norway, Havarstein et al. (73) found that adult Atlantic salmon produced variable levels of IPNV-specific immunoglobins in response to intraperitoneal injection with cell-culture-passed IPNV. In our laboratory, we confirmed that Atlantic salmon exposed to IPNV by immersion can produce neutralizing antibodies following exposure to virus with a history of many passages in cell culture. We tested a limited number of mature returning Atlantic salmon and found that specific IPNV antibody levels were higher in spawning fish (presumptively virus-free, hatchery-raised and released) than in similar, hatchery-held fish. Our conclusion is that during their maturation in the oceans, they could have been exposed to virus and developed antibodies. These antibodies were detected by ELISA, so they were not necessarily neutralizing antibodies. The failure of fish to develop protective immunity after exposure or vaccination has been ascribed to tolerance or to immune suppression. Tolerance was expected to result from an encounter with virus before immunological competence had developed. Immune suppression would have been due to low temperatures. In 1990, Bootland, Dobos, and Stevenson (53), did a careful study to address the first point. They reported that immunization with inactivated virus reduced brook trout mortalities in some cases but did not prevent infection of any age group of fry. Immunization could lessen mortalities of 2 and 3 week eleutheroembryos and 6 week alevins but not 1, 4, 5, 7, or 8 week old fish. They proposed that lymphoid organs are not only important in the immune response but that they could also be involved in IPNV pathogenesis. This supported the earlier suggestion made by Hedrick and Fryer (39) that, upon IPNV introduction, the virus initiated widespread infection of the visceral organs and then became sequestered from antibody exposure before an adequate immune response could be mounted. Temperature affects virus production as well as immunity. Telost immune function in relation to temperature has been reviewed by Bly and Clem (85). The production of antibody has been found to be faster and of a higher magnitude at higher temperatures, with lower temperatures tending to inhibit immune responses. Bly and Clem believe that the adaptive immune responses of virgin T-cells, rather than memory T-cells, B-cells or accessory cells (at least of channel catfish), are particularly susceptible to low temperatures (85). Immunization at low temperatures does not induce antigen-specific tolerance, but rather specifically inhibits T-helper cells, with the immunologically non-permissive temperature for salmonids being 4°C. Thus, based upon Bly and Clem’s summary, fish exposed to IPNV supposedly would not develop an immunological tolerance to the virus and argues against any role for it in virus persistence. Low temperature would also not explain the mechanism of IPNV persistence. In our laboratory, we have shown that Atlantic salmon kept at 6°C can develop a good antibody response to IPNV, although somewhat more slowly than those held at higher temperatures (86).

216

E. C. Sadasiv

There has been question of whether IPNV can replicate in white blood cells or is simply carried by them. Both Yu, MacDonald, and Moore (51) and McAllister et al. (55) have found virus-positive brook trout leukocytes, with each cell containing up to 400 virus infectious doses, which suggests virus multiplication in the cells. Johansen and Sommer (87) found that Atlantic salmon adherent leukocytes (primarily macrophages) from head kidney could carry IPNV. Adherent leukocytes were described by Tate, Kodoma, and Izawa (88) as possessing surface immunoglobulins and corresponding to avian and mammalian B-cells. The number of virus-specific fluorescent cells increased from lo2 to 106, during one week in culture which suggests replication (87). These studies do not, however, conclusively prove that virus or virus-infected cells could not have been ingested by scavenging macrophages, from which the virus could be released undegraded at a later time. Dannevig et al. (89) demonstrated that the sinusoidal endothelium of the head kidney can take up particles the size of IPNV. Koumans-vanDiepen et al. (90) deduced that rainbow trout immunoglobulin-bearing cells first appear in the kidney 4-5 days after hatching, while those of salmon were noted by Ellis (91) at first feeding, times that coincide with IPN sensitivity. All of these points taken together suggest that IPNV infects leukocytes. There have been several reports that IPNV could cause immunosuppression, as avian birnaviruses do. Tate, Kodama, and Izawa (88) proposed that IPNV could have an immunosuppressive effect on those rainbow trout mononuclear leukocytes which correspond in function to avian and mammalian B-cells, and that this could be involved with the establishment of a carrier state. These putative B-cells would appear to be the cells found by Johansen and Sommer (87) to allow replication of IPNV. Older salmonids exposed to IPNV were reported to have variable antibody responses to virus. The establishment of a carrier state was attributed to the ability of the virus to suppress mononuclear leukocytes. In birds, birnaviruses cause immunodeficiency (92). Avian birnaviruses have a predilection for actively dividing and differentiating lymphocytes of B lineage (93), particularly those which carry IgM, the immunoglobulin most similar to piscine immunoglobulins (94). Infected young birds experience a decline in the percentage of immunoglobulin-expressing cells, due to lysis of antibody-producing B-cells (95). All fish lymphocytes are similar to mammalian B-cells, in that they possess membrane immunoglobulins (91). Great advances in understanding the salmonid immune system are currently being made in laboratories around the world (96), but a discussion of them is beyond the scope of this review. However, a few points should be noted. Salmonids have been described as also possessing a T-cell-like immunity, possibly based in the non-adherent leukocytes (88). Viruses, such as IPNV, are described as T-dependent antigens (54). In mammals, carbohydrate antigens can directly induce antibody synthesis without T-lymphocyte activation (97). If IPNV is indeed glycosylated, would it then be capable of directly inducing antibody? At this time, not enough is known about the development of the salmonid immune system to accurately determine the processes which occur with IPNV, but these observations suggest plausible lines of investigation. However, if IPNV compromised the immunity of fish, it should be reflected in a decreased resistance to other pathogens. Bruno and Munro (98) have shown that is not so, at least for bacterial antigens. Vaccine development has not been easy despite great efforts on the part of many researchers. In 1982, Dorson (4) reported that protection could be produced with inactivated pathogenic virus, but when he (45) summarized the work on immunization against IPN in 1988, the production of an effective vaccine seemed more elusive. Avirulent cell-culture virus, capable of producing an active infection, has not proven to be of great value as a live vaccine. Fry infected with it have not been protected against subsequent challenge with virulent virus of

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the same serotype. Fish surviving IPN became poor neutralizing antibody producers, despite the fact that they reacted to injection of virus by producing high antibody titers. Viral antigen could be found in epithelial macrophages (99). Kelly and Nielsen (100) studied the neutralizing antibodies induced by vaccination of rainbow trout with different IPNV strains, using cell-culture adapted virus. By using methods designed to avoid the effects of non-specific viral inhibitors, they also found that the immune response was highly variable. Manning and Leong (38) have been the only researchers able to induce protective immunity, and they did it in rainbow trout fry using cloned viral proteins of the entire larger RNA segment. This indicates that the genome products produced by bacterial cells, and possibly insect cells (16), are different from the virus produced in fish-derived cell-culture. Lawrence et al. (lOl), Havarstein et al. (102), Hah, Park, and Jeong (103), Bootland (104) and Singer (105) have also had some recent success with subunit vaccines. Leong and Fryer (97) have summarized the problems and pointed out that there are still no commercially available IPN vaccines.

CONCLUSIONS

No single answer can yet be given as to the way in which IPNV is able to be carried in fish. A few points do seem to be clear, however. Late-occurring fish deaths, some noted to be related to increasing water temperatures, could be explained by antibody-mediated processes (4, 61, 69). Low temperature is found to have a sparing effect on fry. Cold temperatures would decrease the rate of virus production by infected cells, as well as slow the production of antibodies. The mechanism for such sparing has not been demonstrated, but would likely relate to the maturation of vulnerable cells to a stage where they either would not be sensitive to the virus or they would produce only non-pathogenic virus. The evidence for autoimmune cellular damage is circumstantial, but rather convincing. Although the vertical passage of virus inside sperm or eggs from brood stock to progeny does not seem to occur, virus can be carried on sperm and egg cases (6). Infection has been reported to coincide with time of first feeding (l), which would be logical if its occurrence were due to fry eating their virus-coated egg cases. But, if the chain of infection can be broken by protecting fry at their vulnerable stage, much of the concern about carriage and passage of IPNV becomes less important. This does not resolve the problem of whether any aquatic birnavirus carried over to fry might be lethal to them. Our single test with Atlantic salmon suggests that virus with low pathogenicity can become more pathogenic again by passage in susceptible fish. Not all virus detected fish indicates systemic infections. Virus found in surface mucus could result from an active infection of epidermal cells or it could be attributable to absorption from water. Similarly, virus found in the digestive system or feces could be ingested material. Aquatic bimaviruses have been isolated from numerous invertebrates and IPNV can be harbored by many non-salmonid species. Of these species, only a few have proved susceptible to IPN disease. Roberts (106) reported on the IPNV prevalence of steelhead trout broodstock, which do not develop lethal IPN disease although the virus is often found in association with them (P McAllister, personal communication). Roberts (106) noted that no pattern of prevalence was found (O-10% positive pooled samples over a period of 10 years). Virus did not easily spread to other broodfish, as would be expected in the course of a true infection. Although the virus is most often found in kidneys, the kidney has not been considered to be the prime site for virus replication (50). Histopathologic changes can be visualized there

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(1) and multiple tiny viral foci have been seen within its hematopoietic cells (48). Salmonid kidneys perform a blood filtration function, as well as being a hematopoietic organ. The kidney, with its reservoir of progenitor blood cells, could be the key to viral carriage. One might speculate that, if IPNV were to be sequestered in the hematopoietic kidney cells (in some yet-to-be-discovered manner), those cells might produce infected leukocytes in which the virus could be replicated. Viral pathogenicity is controlled by viral structural proteins, which can be altered phenotypically as well as genotypically (44). The mechanisms of alterations have not been unequivocally established. Ahne (107) pointed out the influence of host cells on the formation of VP-2 from preVP-2. The size of avian birnavirus VP-2 can correlate with pathogenicity. When the outer protein is produced in fibroblastic cells, it can be larger, less pathogenic, elicit antibody which is less protective and to have a different net charge from that produced from a hemopoietic organ. Cellular environments may not be capable of cooperating with the viral NS protein to suitably truncate VP-2. Additional changes in shape and weight of VP-2 would come from altered numbers or locations of carbohydrate moieties; cellular-mediated glycosylation might produce virus with altered glycosylation patterns (108). Glycosylation has been reported in IPNV by Estay et al. (25) but it has not been found in related avian birnavirus. Either or both of these mechanisms could account for the lack of coincidence between serotype and genotype. Structurally altered particles could play a role in modulating virus production, allowing for the different responses detected. If either the trimming of VP-2 or its glycosylation are critical, the differences should be detected after only a single passage in cultured cells. Since Sano et al. (19) reported pathogenic as well as non-pathogenic virus being produced in cell culture, this may be too simplistic an explanation. To my knowledge no work has been done using pathogenic virus directly taken from fish. No alteration of expression of IPNV proteins during persistent infection has been found; however, several groups have recently reported on additional proteins. Work from Dobos’ laboratory (101, 109) described one produced by a frame-shift reading of the end section of the larger RNA which encodes a 17K polypeptide found in infected cells. Manning and Leong (38) describe a protein expressed from the putative negative strand of one of the RNAs. Havarstein et al. (102) reported one specified by the N terminus of the larger RNA near the area coding for VP-2. These findings point towards additional gene products which could be involved in modulating persistence (or any other yet undiscovered function). Knott and Munro’s (70) finding of IPNV in leukocytes only after they were stimulated suggests that an altered form of the virus could have initially been present in those cells. This hypothesis is also supported by the work of Tate et al. (88), as well as the work of Rimstad et al. (75). However, it could also be explained by a low sensitivity of the detecting systems. Virus with a low potential for disease-causation, as well as a limited ability to induce protective antibody, could be produced from persistently infected epidermis or other epithelial-derived cells. This occurs with the avian birnavirus (110). For this birnavirus, vaccine produced from fibroblasts or whole embryos is not as protective as a vaccine in which the antigen is produced in cells of the hematopoietic organ. As Dorson (3) noted with kidney cells, epithelial-derived cells (skin and gut, including kidney cells) might sustain long-term infections, with low rates of production of non-pathogenic virus. This could be one possible explanation for virus production which can extend for years. Virus production from the skin could also account for the lack of clearance of virus from fish having circulating serum antibodies. Virus produced from the skin might be less affected by circulating antibodies; the locally-induced antibody from skin might not be directed at pathogenic virus. It is worth noting that those rare persistently infected cells of the kidney might, indeed, be hematopoietic cells.

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For virus to persist for years in a fish without being detected, it would have to either be sequestered and present in such low numbers that current systems do not detect it; or be present only as genetic material which would not interact with the immune system. One would not expect that such a simple virus as IPNV would be able to carry that quantity of genetic information which would allow persistence of the type seen, for example, with herpesviruses. With the DNA-containing herpesvirus, only the nucleic acid, rather than the complete virus with its proteins, is latent in cell nuclei of nerve and brain tissue. This would not likely be the case with a virus, like IPNV, which is not known to enter nuclei or produce a DNA intermediate which could integrate the cellular genome. Virus would not be expected to remain in circulating leukocytes for years; however, B-cell immortalization by a herpesvirus suggests that viruses can alter cell functioning (111). Could virus be sequestered in the renal hematopoietic stem or in blast cells, as suggested and discounted by Yu, MacDonald, and Moore (51), or could the virus influence the production/activation of leukocytes? There is currently no information to prove either hypothesis. If IPNV were able to suppress the immune system (70, SS), it would explain the variable immune responses, as well as being an alternative explanation for the difficulty in producing protective vaccines. Virus can be found in leukocytes and replication may occur in them. Is it possible that there could be some interaction of stable viral antigens with macrophages of the head kidney (112)? Investigation of this might also elucidate the mechanism of the long-term presence of putative pathogens. It is interesting to note that several other fish pathogens can persist for long periods of time including the rhabdoviruses infectious hematopoietic necrosis (IHNV) and viral hemorrhagic septicemia (VHS). These viruses are quite labile, in contrast to the birnaviruses. Since birnaviruses and rhabdoviruses are so different structurally, it seems unlikely that the two virus families would have evolved similar mechanisms for latency. More plausibly, similarities should be looked for in the response of the fish to these very different infections. Note added in proof: Some additional recently published work by Johansen and Sommer (Johansen L.-H. and A.-I Sommer, Aquaculture 132: 91-95. 1995; Johansen L.-H. and A.-I. Sommer, J. Fish Diseases 18: 147-156. 1995) has shown that IPNV replication was found in adherent head kidney leukocytes (macrophages). Viral specific fluorescence was detected in both carrier and in vitro infected macrophages, with the number of infected cells increasing during a week in culture. IPNV infected macrophages were found to have reduced respiratory burst activity. They theorize that non-cytolytic infected macrophages may protect virus from immune defenses of host, leading to a “carrier” condition. - The author wishes to thank Drs. Pei W. Chang, Richard E. Wolke, and Robert E. Carleson for critical readings of the manuscript; and for helpful discussion with Drs. Philip E. McAllister, David A. Smail, Sharon A. MacLean, Eva Nagy and Ahmed A. Azad. The author is also indebted to Ms. Carole Klawansky for gracious assistance in manuscript preparation. Contribution No. 2950 of the College of Resource Development, University of Rhode Island, with support from the Rhode Island Agricultural Experiment Station and the US Department of Agriculture. This work was also supported by a gift from Abbott Laboratories, in recognition of the achievements of Dr. George Dawson.

Acknowledgments

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