Annuul Printed

Rev. of Fish Diseases, in the USA. All rights

pp. 225-240, reserved.

1993 Copyright 0

0959.8030/93 $24.00 + .OO 1993 Pergamon Press Ltd.

VIRAL

VACCINES
J.C. Leong*

FOR AQUACULTURE
and J.L. Fryer

Department of Microbiology and Laboratory for Fish Diseases Research, Oregon State University, Corvallis, Oregon 9733 l-3804, USA

Abstract. A description of the types of viral vaccines that are being developed for fish is presented in this review. All three types of vaccines, i.e. killed, attenuated, and subunit vaccines, have worked to some extent in fish under controlled laboratory conditions. However, with the exception of a killed vaccine for spring viremia of carp, there are no commercially available viral vaccines for fish. The introduction of these vaccines to aquaculture will require an understanding of immune recognition in fish and the development of cost-effective ways of producing a safe, immunogenic vaccine. A review of available information on vaccines for infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, spring viremia of carp virus, and channel catfish virus is presented. These are the main viruses that have been investigated for vaccine development. Very little information is available on vaccines for other viral diseases of fish. Keywords. Infectious disease, Salmon, Carp, Vaccines

INTRODUCTION

occurred in China where the artificial hatching and rearing of carp (Cyprinus carpio) dates back to 2000 B.C. (1). Since that time, man has extensively cultured species such as the milkfish (Chanos chanos), tilapia (Sarotherodon mossambica), yellowtail (Seriola quinqueradiata), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo s&r), and many other salmonid species. Aquaculture was first carried out in Europe by the Romans with oysters and now the culture of shellfish such as clams, oysters, and prawns is commonplace. All of these species are subject to infectious diseases and their impact on the economic viability of the aquaculture industry has become increasingly important. Now, as the natural fisheries provided by the open seas have declined and the world population has grown, aquaculture products are in great demand. Global aquaculture production is estimated at nearly 10 million metric tons (MT) annually and contributes over 12% of the total consumed fish and shellfish (Aquaculture Magazine, 1991 Buyer s Guide). The industry is now projected to grow at an annual rate of 8% through the year 2000 by the United Nations Food and Agriculture Organization (FAO). This industry growth will only come from increased cultural efficiency in existing facilities because there will be restrictions on water use, and increased production from increased acreage will be
The earliest record of aquaculture

unlikely. One of the primary means for increasing cultural efficiency will be the improvement of animal health and the control of infectious diseases. To this end, the development of vaccines is vital for the continued growth and development of the aquaculture industry.
IMPACT OF VIRAL DISEASES

ON AQUACULTURE

*To whom correspondence

should

be addressed. 225

While viral diseases can cause problems among natural fish stocks, these infectious agents are devastating and costly in aquaculture where fish are confined and intensively reared. It has been estimated that 10% of all cultured aquatic animals are lost as a result of infectious diseases, and this may be a conservative estimate. Highly pathogenic viruses and bacteria are capable of killing over 90% of hatchery-reared populations. The U.S. Trout Farmers Board of Directors survey in 1991 indicated that 52% (30.8 million fish of a total of 68.9 million trout produced) died as a result of disease. For catfish, 77% of the fish were lost between egg hatch and market size between the years 1988 to 1991. The majority of the losses were due to infectious diseases (J. Plumb and M. Johnson, personal communication). In the United States, it has been estimated that about 50% of the oyster crop valued at $32 million is lost due to infectious diseases. Thus, the health of fish in aquaculture is critical to the well-being of the industry and effective vaccines are desperately needed. For the purposes of this review, possible vaccines for viral diseases of fish are considered.

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The losses due to disease are startling and emphasize the need for development of effective, inexpensive vaccines for aquaculture. In analyzing these mortality values, it has been difficult to identify losses caused by disease agents and in particular, those resulting from viral diseases. However, it is reasonable to suggest that the losses of trout may have in part been due to infectious hematopoietic necrosis virus (IHNV) since losses in trout poundage were only 7% and losses in trout numbers were 52%. The difference indicates that a majority of the fish were lost when they were very young and low in weight, the size when these fish are most susceptible to IHNV. For channel catfish production in the United States, the majority of the losses are probably due to winterkill, a generic term used to describe the catfish mortality that occurs when water temperatures are below 55 F (12.8 C) (M. Johnson, Stoneville, Miss., personal communication). Most fish pathologists have attributed winterkill to a reduced immune response in these fish as a result of the lower temperatures or to chronic virus infections, e.g. channel catfish virus (CCV). If the latter is true, then a majority of the catfish mortalities may also be due to virus infection. Recently, there has been an increased association of the Sp strain of infectious pancreatic necrosis virus (IPNV-Sp) with exocrine pancreas disease in pen-reared Atlantic salmon (2) and it appears that the control of viral diseases for this species will also be important. The culture of shrimp in Taiwan, Thailand, and the Philippines has been severely curtailed as a result of monodon baculovirus (MBV) infections of the grass shrimp (Penueus mono&n). In 1986, Taiwan production of grass shrimp was 45,817 MT (Taiwan Agricultural Yearbook, 1986) with an export of 30,227 MT. In 1987, export levels reached 42,618 MT. However, in 1988 production decreased dramatically to 8,474 MT and by 1989 production reached a low level of 914 MT (Y.-L. Hsu and J.-L. Wu, personal communication). The shrimp under culture were dying in large numbers and the deaths were attributed to MBV (3,4). There was a strong correlation between increasing mortality rate and increasing incidence of MBV infection in postlarval P. monodon. The infection rate increased from 17% in 1984-1986 to 85% in 1988. Although secondary bacterial infections may have actually killed these animals, the viral infection appeared to predispose them to the bacterial infections. The MBV infection of shrimp in Taiwan is an example of the most severe impact a viral disease can have

on an industry because P. monodon culture has been virtually abandoned in Taiwan today.
IDEAL VIRAL VACCINE

There are a number of factors that are important in the formulation of an ideal viral vaccine for fish. The primary consideration for any vaccine for aquaculture is, of course, its cost-effectiveness. The vacine must provide a clear economic benefit to the fish farmer. To accomplish this, the vaccine must have the following attributes: 1. provide adequate immunoprotection for the disease under the intensive rearing conditions found at commercial hatcheries and grow out sites; 2. provide protection at a time when the animal is most susceptible to the disease; 3. provide protection of long duration; 4. protect against all serotypic variants of the disease agent; 5. be easily administered, preferably by oral feed or immersion; 6. be safe; and 7. be inexpensive to produce and license. All of these attributes have been met in large measure by the commercially available bacterial vaccines. The success of these vaccines for controlling vibriosis and enteric redmouth disease (ERM) has raised the expectations of the aquaculture industry that similar successes should be forthcoming with the viral vaccines. However, those expectations were unrealistic and the very real differences between immunity to viral versus bacterial infections have been found to be important in developing viral vaccines. For many bacterial vaccines, the polysaccharide component of the organisms is important for immunogenicity. In mammals, the carbohydrate antigens induce antibody synthesis directly without required T lymphocyte activation. For example, formation of antibody to pneumococcal vaccine is T cell independent. Also, antibody is critically important in the immune response to bacterial pathogens such as pneumococci (5). The killing of bacterial cells is carried out by phagocytic cells in response to coating of the bacterium with antibody. The immunogenic component(s) for viruses, on the other hand, are largely protein antigens that induce T-dependent antibody responses and T cell immunity. In viral infections, both antibody and effector T cells play important roles in the ultimate resolution of the infection.

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Vaccines that are now available for commercial use have not had specific components that induce protective immunity identified (6). The very success of these vaccines had, in fact, made it appear unnecessary to understand the basis for the immune protection. Correlation between antibody levels and resistance had not been made; nor had there been any characterization of the cellular immune response other than early adoptive immunization studies. No information characterizing the fish antibody responses as T-independent or T-dependent was available and the reactivity of these antibodies with specific carbohydrate or protein antigens was not determined. It is now clear to investigators trying to develop vaccines for different bacterial and viral pathogens that the immunogenic components of a vaccine must be characterized (7). The attainment of all the attributes of an ideal viral vaccine for aquaculture will require more extensive knowledge of both the T and B cell arms of the immune response in these animals.
TYPES OF VIRAL VACCINES

The history of modern vaccination dates back to Edward Jenner first inoculation s of material from the cowpox lesions on the hand of a milkmaid into an eight year old boy to induce protection against smallpox in 1796. The success of this vaccine led to the purposeful eradication of smallpox from the world population in 1979 and therein lies s one of the greatest triumphs of man efforts to s conquer disease. There are now over 50 vaccines in use in the medical and veterinary areas and many more are under development. The most prevalent type is like the cowpox virus that retained the ability to induce protective immunity but did not cause disease. It was live and attenuated, i.e. avirulent or nonpathogenic. Other viral vaccines are composed of killed whole virus, such as those used to protect humans against rabies, influenza, hepatitis A, and polio (Salk). More recently, subunit vaccines have been developed for hepatitis B and influenza. The subunit vaccines are composed of a part of the virus particle, that part which is responsible for inducing protective immunity. Killed virus vaccines are prepared by growing virus in large amounts on cells in tissue culture or in the actual animal species. The virus is then harvested and inactivated with formalin, tri-N-butyl phosphate, urea, or /3-propiolactone, under conditions that ensure the retention of the immunogenic activity of the protective antigens. These vaccines

are generally thought to be safer because the virus is killed and the immunogenic virion proteins of the wild-type virus are uncompromised. However, extreme care is required in their manufacture to make certain that no virulent virus remains in the vaccine. Also, the immunity conferred is often brief and must be boosted. Booster vaccinations in fish farms may be logistically difficult to deliver and repeated immunization may increase the possibility that adverse reactions will be induced in the animals. Attenuated vaccines have the advantage of behaving like the natural infections in their effect on immunity. They multiply in the host and tend to stimulate longer-lasting antibody production. Most importantly, live vaccines induce antibody production and resistance at the normal entry site in the host. For example, the live, attenuated polio virus vaccine induces immunity in the gastrointestinal tract (IgA) as well as humoral immunity of the IgM and IgG type. The formalin-killed polio vaccine (Salk) requires booster immunization and induces humoral immunity, as the virus is not able to multiply in the person gastrointestinal s tract. There are obvious disadvantages in live vaccines, particularly for the aquaculture industry. There is always the risk that the virus will revert to greater virulence during multiplication in the vaccinated animals. Similarly, the attenuated virus may be avirulent in the species being purposefully vaccinated but extremely virulent in other fish species in the watershed. Unless steps are taken to confine the live virus to fish farms, live viruses are easily spread in the effluent water. Another disadvantage of live vaccines is that they may be contaminated with latent, alien viruses that are present in the tissue culture cells or the animal host used to grow the vaccine preparation. In addition, live vaccines must be stored carefully to preserve viability and the limited shelf life of these agents may make distribution of the product more difficult. There is an additional problem that makes commercial firms reluctant to license attenuated viral vaccines and that is that retaining proprietary rights over such a vaccine may be difficult without specific genetic markers built into the vaccine strain. The subunit viral vaccine consists of the viral antigens that will induce protective immunity in the animal. The subunit vaccines developed for humans include the HA/NA surface antigens for influenza and the 22 nm surface antigen for type B hepatitis. The influenza subunit vaccines are less toxic than inactivated whole virus vaccines and that is a distinctly advantageous property, but they provide no

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increase in protective efficacy (8). In fact, in unprimed individuals, the influenza virus subunit vaccine appears to be less antigenic than inactivated whole virus vaccine. This difference is possibly due to the subunit vaccine failure to stimulate T-helper s cell responses that are normally activated by the nonstructural antigens. This activation leads to an amplification of the B cell responses to the viral surface glycoproteins which, in turn, increases the protective immune response (9). The phenomenon of reduced immunogenicity in subunit vaccines is not the case for the subunit vaccine preparation for hepatitis B virus (HBV) (10). The initial version of this vaccine was purified from the blood of persons persistently infected with HBV. Although the HBV vaccine prepared in this way was effective, it was not widely accepted because it was too expensive to use for routine vaccination procedures. The cost of the vaccine was high because it had to be purified from human sources and extreme precautions had to be taken to ensure proper safety tests for certification. A second generation HBV vaccine consisting of the HBV surface antigen (HBsAg) was produced by expressing this protein in yeast by way of a recombinant plasmid containing the HBsAg gene (11). The yeast-produced HBsAg was not glycosylated, but it self-assembled into typical HBsAg particles and was immunogenic in humans (12). A careful analysis of the T and B cell epitopes on the HBsAg provided an explanation for the efficacy of this vaccine since both types of epitopes were located on the same protein (13). The gene encoding the HBV surface antigen has been cloned into vectors for a variety of expression systems including bacteria, insect, and mammalian cells (14). The success of these endeavors has made the hepatitis vaccine a prime example of how the advanced techniques of gene cloning, sequencing genes, and synthesizing synthetic oligonucleotides are making biotech vaccines. The use of monoclonal antibodies to delineate epitopes in the cloned protein, to identify neutralizing and serotypic epitopes, and T cell epitopes, has made it possible to develop synthetic peptides as vaccines. A cogent argument for the use of synthetic peptides as vaccines for fish has been made by Vallejo et al. (7), who argue that such vaccines were considered possibilities by the finding that T and B cell epitopes were localized to small regions of native proteins. Also, peptide vaccines offer several advantages such as being more stable, noninfectious, and simpler to produce by recombinant DNA technology or peptide synthesizers. The most intriguing advantage was that these peptides lend themselves

to engineering, i.e. they can be designed for greater stability and stronger binding to the T or B cell receptor. If T and B cell epitopes are distantly located on the same protein or are on different proteins of the virus, it is now possible to engineer a vaccine with the T and B cell epitopes residing on the same peptide. The technology is now available for the construction of potent fish viral vaccines once the T and B cell epitopes recognized by fish are identified.
FISH VIRAL VACCINES UNDER DEVELOPMENT

Infectious pancreatic necrosis virus Several laboratories have active research programs to develop an effective vaccine for infectious pancreatic necrosis virus (IPNV) and there is an excellent review of the literature on this subject by Dorson (15). In the review presented here, the authors would like to summarize the pertinent findings regarding the problems in developing an IPNV vaccine and provide a synopsis of work published since 1988. IPNV is a very serious pathogen of farm-reared brook trout (Salvelinus fontinalis), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and cutthroat trout (Salmo clarki). It can also kill amago salmon (Oncorhynchus rhodurus), sockeye salmon (Oncorhynchus nerka), and Artic char (Salvelinus alpinus) (15). The virus kills these animals when they are very young and survivors, now lifelong carriers, maintain the virus in the population by continuously shedding and transmitting it to their progeny or other susceptible species. In addition, the virus is serotypically heterogeneous with two major serogroups: serogroup I is composed of nine serotypes which are pathogenic for salmonid fish, and serogroup II has only one member isolated from molluscs (16). The ideal vaccine for IPNV must induce early protection, prevent carrier formation, and induce protection against a large number of serotypes. Several inactivated IPNV vaccines have been tested and found to be efficacious only after vaccination by intraperitoneal inoculation (17). The protection was observed in rainbow trout vaccinated at four to eight weeks posthatching. Since these fish are very small and vaccination by injection for fish of that size was not practical, immersion vaccination was also used. All attempts to immunize fish with inactivated virus (formalin or @-propiolactone treated) by immersion vaccination did not result in protective immunity. In fact, when brook trout fry were immunized by immersion in three very high doses of inactivated IPNV (up to

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187 pg viral protein per ml), the immunized fish died at a higher rate than the control fish after challenge with live virus (18). When individual virion polypeptides were used to immunize fish either by immersion or intraperitoneal injection, no protective immunity was observed (1.5). The virion proteins had lost their antigenicity following disruption with sodium dodecyl sulphate, urea, and acetic acid (19). Recent studies by Bootland et al. (20) suggest that the age and size of the fish at the time of immunization are important determinants in the development of protective immunity. Brook trout fry in eight age groups of 1-8 weeks after hatching were immunized by a single direct immersion in formalin-inactivated IPNV and then maintained at 10 C. At four weeks after immunization, the fry were challenged with 10 pfu/ml IPNV. Only fry immunized at two and three weeks posthatching were protectively immunized. Analysis of the growth rate of the fry suggested that protection against IPNV required immunization in the eleutheroembryo phase, a time of slow weight gain in salmonid fish development. This finding may be very important because it indicates that very young fish are capable of responding to vaccination and suggests that investigators may have to consider the growth rate of young fish in their vaccination trials with killed viral vaccines. Since the Ab serotype of IPNV has been found to be less virulent than the other IPNV serotypes, e.g. Sp, West Buxton, or Jasper, Dorson and colleagues attempted to create an attenuated IPNV strain from the Ab isolate by successive passage in RTG-2 (rainbow trout gonad) cells (21). Neither the Ab strain nor the serially passaged Ab variant conferred protection, however, successful vaccination was obtained with an avirulent IPNV strain isolated from perch (Perca fluviatilis) when this virus was delivered by immersion vaccination after hyperosmotic treatment. McAllister (22) also reported that IO-day posthatching brook trout fry could be protected against subsequent IPNV challenge by vaccination with a live, avirulent IPNV isolate as early as three weeks after vaccination. Subunit vaccines produced by recombinant DNA techniques for IPNV are currently under development in a number of laboratories (23-26). For many of these studies, the major capsid protein, VP2, has been identified as the virion protein responsible for inducing protective immunity in fish. Thus, these investigators have cloned the IPNV gene encoding VP2 and expressed it in Escherichia co/i as part of a fusion protein with either fl-galactosidase (23-25) or trpE anthranilate synthetase

(24). The only successful report of induction of protective immunity with a recombinant DNA-derived subunit vaccine for IPNV was made with a vaccine containing the proteins encoded by the entire A segment of IPNV, Sp serotype. The A segment encodes the viral proteins VP2, VP3, and NS; a crude bacterial lysate containing these proteins was used to immunize rainbow trout fry by immersion vaccination. After four weeks, the fry were challenged with lo4 pfu/ml IPNV (Buhl strain) for 18 h at 20 C. The relative percent survival (RPS) at 30 days postchallenge was 91% for the vaccinated fish versus fish that received a bacterial lysate containing no viral proteins (24). This finding was very encouraging because it demonstrated that a recombinant DNA-derived subunit vaccine in the form of a crude bacterial lysate was capable of inducing protective immunity against IPNV by immersion vaccination. It also showed that the cloned cDNA taken from the Sp serotype of IPNV would produce a protein in bacterial cells containing neutralizing epitopes that cross-reacted with those of the Buhl strain of IPNV, a member of the West Buxton serotype (16). The sequence of the entire A segment of IPNV has been determined for at least four strains of IPNV: Sp (27); Jasper (28); N-l, a member of the SP serotype (23); and DRT, an isolate from South Korea (Lee, personal communication). A comparison of the derived amino acid sequence from the cDNA clones of all four strains indicates that there are conserved amino acid regions in the VP2 protein from amino acid 86 to 210. Only four amino acid differences between the IPNV strains Sp, Jasper, N-l, and DRT were observed in this region (Leong, unpublished observation). The neutralizing monoclonal antibody, AS-l, was found to react with this polypeptide region of VP2 in radioimmune precipitation assays (27). The AS-l reagent is broadly reactive with 75 different isolates of IPNV representing all serotypes from Group I. This result would suggest that there is a conserved neutralizing epitope in this region and that a subunit vaccine that contained this region might be effective as a vaccine for all serotypes of IPNV. Although the results of studies with the IPNV subunit vaccine appear promising, more extensive evaluation of its efficacy for IPNV in fish must be continued. Not only must the vaccine be tested against the IPNV strain from which the recombinant DNA clone was derived, but it must be tested against representative virulent isolates of all nine serotypes of the serogroup I. For many vaccine developers, this has been a real problem. A reliable challenge test for IPNV vaccines in fish has to be

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developed before any vaccine will be accepted by the industry. Many investigators have had to contend with maintaining the virulence of an IPNV isolate in culture, defining appropriate challenge conditions in terms of water temperature, feeding regimen, chemical composition of the water, gas saturation, the adaptation of the fish to these conditions before challenge, and obtaining rainbow trout or brook trout fry at a susceptible stage of life. This problem has been studied by several investigators who found that the virulence of IPNV is not only correlated with virus isolate (serotype), but with a multitude of other factors such as virus concentration, the length of time fish are exposed to the virus, cell culture passage number, the age/weight of the fish at the time of virus exposure, and the adaptation of the fish to the challenge conditions (29-32). There were even differences in the degree of IPNV susceptibility within a given fish species. Different genetic strains of brook trout varied in mortality rates when exposed to the same IPNV isolate (33,34) and a rainbow trout strain that was highly susceptible to IPNV had to be developed in Japan for IPNV challenge studies (35). The basis for the differences in susceptibility to the lethal effects of IPNV infection has not been determined. Nevertheless, the importance of developing a reliable challenge procedure to assess immunization for IPNV has prompted investigators to begin to identify the important factors that determine virulence for IPNV (36). Viral hemorrhagic septicemia virus The disease caused by viral hemorrhagic septicemia virus (VHSV) is considered one of the most serious viral diseases in salmonid fish species in Europe (for review of VHSV vaccines, see de Kinkelin [37]), and has been estimated to cause losses in Europe of 40 million pounds of fish per year valued at approximately $80 million (38). The rhabdovirus, which was once thought to be only pathogenic for rainbow trout, will produce clinical disease in lake trout (Salvelinus namaycush), brown trout (Salmo trutta), grayling (Thymallus thymallus), white fish (Coregonus sp.), turbot (Scophthalmus maximus), sea bass (Dicentrarchus labrax), sea bream (Chrysophris aurata), and pike (Esox Lucius) as well. It is an economically devastating disease for the aquaculture industry because it may occur in all age groups of rainbow trout, from young feeding fry to mature adults. In general, however, the susceptibility to disease decreases with age. The disease usually occurs in water temperatures below 15 C and is most severe in the range 8-12 C. This ac-

counts for the majority of outbreaks that occur in the months of late autumn, winter, and early spring. Some cases do occur in the summer in marine rearing units or in farms fed by spring water. The virus was once confined to continental Europe where it was frequently found in trout farms in France, Germany, Denmark, and Italy. Recently, the virus was discovered in North America in coho salmon (Oncorhynchus kisutch), chinook salmon (0. tshawytscha), steelhead trout (0. mykiss), and Pacific Cod (Gadus macrocephalus) (39-43). Its appearance in North America led to an extensive eradication program in the state of Washington because the possibility existed that the establishment of VHSV in the Pacific Northwest would have a negative impact on its salmonid fish rearing industry. Fortunately, the North American VHSV isolates were subsequently shown to be avirulent in rainbow trout and coho salmon (44). Despite these findings, the appearance of VHSV in North America among marine free-living fish species should be monitored carefully. Virulent strains of VHSV may evolve as the virus becomes adapted to the fish species in the area. The ideal vaccine for VHSV, in addition to all the other necessary attributes for fish viral vaccines, must protect the fish prior to 1500 C degree x days when maximum susceptibility to the virus in fish has been observed. It also must be polyvalent to protect against the five serotypes of the virus (37). There are at least four laboratories that are currently engaged in developing vaccines for VHSV: de Kinkelin and colleagues in France; VestergaardJorgensen group in Denmark; Thiery group in s s Belgium; and Coil group in Spain. Much of the s early work on anti-VHSV vaccines was carried out by P. de Kinkelin and his colleagues. They found that P-propiolactone inactivated virus was immunogenic only after inoculation vaccination (37); no protective immunity was induced in fish following bath immersion vaccination. Since a VHSV vaccine must protect very young fish when individual inoculation was impractical, the pursuit of a killed vaccine was dropped and this group went on to develop several live viral vaccines. These vaccines strains were attenuated by successive passage at progressively increasing temperatures to 25 C in EPC (cyprinid) cells (45-47). The first vaccine strain F25 (21), which was derived from the Fl serotype of VHSV, was found to induce protective immunity against the three VHSV serotypes 07.71,23.75, and a wild strain belonging to serotype I. The final survival rates of the immunized groups ranged from 3958%, while survival of nonimmunized controls was

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between 815%. Protection was demonstrable as early as day 1 postimmunization and suggested that interference was responsible for the early protection. Later, neutralizing antibody did develop in the immunized fish. There was still significant mortality associated with this attenuated strain of VHSV where 2-13% of the fish died as a result of vaccination alone (45,46). More recently, a subunit vaccine for VHSV has been developed using recombinant DNA technology. Direct and indirect evidence had shown that the protective epitopes of VHSV were located on the surface glycoprotein of the virus and that antibody directed against the viral glycoprotein neutralized virus infectivity (48,49). Thus, the VHSV glycoprotein gene was cloned and expressed in a bacterium (Escherichia co/i), yeast (Saccharomyces cerevisiae), and insect cell cultures to produce the glycoprotein inexpensively and in large quantities (50,5 1, P. de Kinkelin, personal communication). The recombinant proteins produced in bacteria were nonglycosylated, whereas the proteins produced in yeast and insect cells contained glycosylation profiles different from the viral glycoprotein produced in fish cells (M. Thiery, personal communication). These recombinant proteins are currently being tested for immunogenicity in trout in the laboratory. Recently, Jorgensen reported that the central part of the VHSV glycoprotein gene was able to induce virus neutralizing antibodies in rainbow trout. The protein was expressed in E. cofi as a fusion protein and then purified. After the fusion protein was denatured, cleaved from the fusion partner, and refolded, the VHSV glycoprotein was able to induce virus neutralizing antibodies as well as antibodies detectable by immunofluorescence and Western blots in injected trout (51). Preliminary results indicated that the recombinant protein induced protection following injection vaccination, but no studies on immersion vaccination were reported (52). Thus, for VHSV, a recombinant subunit vaccine may be a promising way of producing an inexpensive and safe vaccine for fish. Infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis (IHN) is a disease of some salmonid fish species native to countries of the North Pacific Rim. The disease was first reported in 1953 as the cause of mortality among sockeye salmon (0. nerka) in the state of Washington (53) and since that time, it has been detected in susceptible salmonid fish species from Alaska to Northern California. It is now also found in Japan (54), Korea (M.A. Park, personal communication),

Taiwan (55), and has recently appeared in the European countries of France, Belgium, and Italy (56). The disease is caused by a rhabdovirus, IHNV, which produces a severe disease among fry and juveniles of susceptible species of salmonid fish. The animals usually die as a result of the massive destruction of the hematopoietic tissue in the anterior kidney. The primary means for transmission of the virus in nature is unknown. However, the disease can be transmitted horizontally from infected animals via the water and there is evidence that the disease can be transmitted vertically as well. The strongest evidence for vertical transmission comes from the association made between the appearance of the disease for the first time in an area and the shipment of eggs from infected adults. This transmission appears to be dependent upon surface virus contamination of the eggs since iodophore treatment eliminates the subsequent appearance of the virus in the newly hatched fry (57). The finding that iodophore treatment prevented IHNV transmission led hatcheries to adopt a practice of routinely treating eggs with iodophore before raising the fry in specific pathogen-free well water. When the fish were sufficiently mature to have a fully developed immune system, they were placed in ponds with water taken from surface sources. This practice did reduce the incidence of IHN in the Columbia River Basin, but it did not completely prevent the appearance of the disease in the hatcheries. Epizootics of IHN were still a problem and fish pathologists have accounted for the IHN continued presence of the s virus by the failure of the iodophore treatment or by sequestered virus inside the fish egg. Although vertical transmission via sequestered virus has not been demonstrated in the laboratory, there are cases where virus has appeared in fish hatched from eggs which have been treated with iodophore under strictly regulated conditions. These observations do suggest that internal vertical transmission is possible, albeit at a very low frequency. When fish are infected with IHNV in the laboratory so that 30-50% succumb to the infection, virus can be isolated from the survivors up to 45 days postinfection. After that period, infectious virus can no longer be detected in any of the fish organs. Yet, if these fish are kept until they reach sexual maturity, they will produce virus in the milt or ovarian fluids (58). On the basis of these results, an IHNV carrier state was postulated as a model for persistence of the virus in nature. However, these observations have not been repeated and several investigators have questioned the findings. Did the

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sexually mature fish express a latent virus or did viral reinfection occur from some other source/ reservoir in the laboratory? The answer to this question is important because the nature of any carrier state and the identification of potential reservoirs are essential factors in the development of an IHNV vaccine. Several vaccines for IHNV have been developed and the relative merits of these vaccines have been described (59). The killed vaccine consisting of IHNV inactivated by either ,&propiolactone (60) or formalin (61) was most effective when delivered by intraperitoneal injection as described for VHSV. Hyperosmotic immersion with the formalin-killed vaccine was also effective in stimulating some limited immunity. An attenuated IHN vaccine was developed at Oregon State University by multiple passage of a rainbow trout isolate on steelhead trout cell cultures (62,63). The vaccine did induce excellent protective immunity (5% mortality in vaccinated vs. 90% mortality in control, unvaccinated fish) in kokanee and chinook salmon. These results were very encouraging; however, when rainbow trout were vaccinated, significant residual virulence was detected (64). No further attenuation or testing of this attenuated strain was carried out because the commercial vaccine industry expressed concern about licensing requirements for attenuated vaccines. Also, fish pathologists, who rely on viral detection methods for certification of fish stocks, were reluctant to accept a live vaccine that would make it difficult to identify fish stocks infected with a virulent strain of IHNV. The drawbacks of killed and attenuated vaccines led to research efforts to develop an effective subunit vaccine for IHNV through recombinant DNA technology (65). Studies had shown that the viral glycoprotein purified from one isolate would induce protective immunity in fish to a wide variety of IHNV isolates from different geographic areas and different fish species (66). This finding indicated that it might be possible to construct a vaccine for IHNV which consisted of the viral glycoprotein from just one virus isolate. Therefore, the viral glycoprotein gene from the Round Butte strain of IHNV was cloned and genetically manipulated so that it was expressed as a trpE fusion protein in E. coli. When a crude lysate of bacterial cells that contained the fusion protein were used to immunize fish
by immersion, protection was observed in fish subsequently challenged with a lethal dose of IHNV (67). In many of the studies, the mortality ranged from O-19070 in the vaccinated fish group and 6492% in the control fish group (Tables 1 and 2). This

Table 1. Immunization of rainbow trout with a subunit vaccine against infectious hematopoietic necrosis virus (IHNV)a
Treatment Inoculatedb ImmersedC Nonimmunized controls Challenge Water-borne Water-borne Water-borne Percent mortality 9 36 60

Y3ee Gilmore et al. (91). bRainbow trout fry (0.4 g) were injected with 0.01 ml of a bacterial lysate (3 mg/ml) containing 8% expressed G protein. Rainbow trout fry (0.4 g) were immersed in the same bacterial lysate (50 fish per 10 ml of solution) for 3 min. After 1 month, fish were challenged by water-borne exposure to various concentrations of virus. Data shown are for a challenge with 3.2 x lo3 TCID,c/ml. Each challenge was done in replicate.

type of protection was observed in rainbow trout, sockeye salmon (Table 3), and chinook salmon fry as small as 0.4 g at the time of immunization. The use of a cloned viral gene in the vaccine made it possible to dissect the viral glycoprotein and determine which regions were immunogenically important. When different regions of the viral gene

Table 2. Immunization of rainbow trout and chinook salmon with subunit vaccine against infectious hematopoietic necrosis virus (IHNV) Homologous vs. heterologous challengea Treatment Immersed Control Immersed Control Immersed Control Challenge Round Butte IHNVb Round Butte IHNVb Elk River IHNVC Elk River IHNV Dworshak IHNVd Dworshak IHNVd Percent mortality 19 92 0 64 11 85

YSee Gilmore et al. (91). bRainbow trout (0.4 g) were immersed in a bacterial lysate (3 mg/ml) containing 8% expressed G protein for 3 min. After 1 month, the fish were challenged by water-borne exposure to various concentrations of virus. Data shown are for a challenge of 7.2 x 10TCIDsc/ml. Each challenge was done in replicate. CChinook salmon (0.4 g) were immunized by immersion as above. The data shown here are for a challenge of 1.93 x IO6 TCID,c/ml. Each challenge was done in replicate. dRainbow trout (0.4 g) were immunized by immersion as above. The data shown here are for a challenge of 2.85 x lo3 TCIDSo/ml. Each challenge was done in replicate.

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Table 3. Immunization of sockeye salmon with a subunit vaccine against infectious hematopoietic necrosis virus (IHNV) Treatment groupb Immersed Control Immersed Control Immersed Control Challenge dose (TCID&ml) 100 100 1,000 1,000 10,000 10,000 Percent mortality 0 50 45 53 41 65

See Engelking et al. (92). bSockeye salmon fry (0.5 g) were lysate (3 mg/ml) containing 8% the groups marked immersed group received a bacterial lysate ral protein.

immersed in a bacterial expressed G protein for for 1 min. The control which contained no vi-

The fish were challenged after 1 month with differing doses of virulent IHNV isolated from Packer Creek, s
Alaska in 1990. Each virus challenge replicate. level was done in

A field trial of the IHNV subunit vaccine was conducted in Idaho where 100,000 rainbow trout at 1 .O g average weight were immunized by immersion in the bacterial lysate in lots of 20,000 fish for 2 min. The control, unvaccinated group also contained 100,000 fish. After three weeks in pathogenfree well water, the fish were moved to ponds that received water from a source containing resident IHNV-infected fish. Virus related mortalities were observed in the control fish after 18 days and reached a maximum at day 23 after transfer to the outside ponds. No significant mortalities were observed in the vaccinated fish until day 33 after transfer (Fig. 1). After 80 days postponding, the percent cumulative mortality in the control fish was 27%; in the vaccinated fish the percent cumulative mortality was 4% (65) (Fig. 2). Thus, the IHNV subunit vaccine has been very effective in inducing protective immunity in both laboratory and field trials.

Rhabdovirus carpio
Spring viremia of carp (WC) is caused by Rhabdovirus carpio or, WC virus, as it is more often referred to in the virology literature. The disease was first described by Fijan et al. (69) in carp suffering from dropsy in Yugoslavia. Since that report, the disease has been recognized in virtually every European country having carp culture. The virus is widespread and serological surveys indicate that

were expressed as trpE fusion proteins in bacteria and the subsequent bacterial lysate used to immunize fish, an immunodominant domain in the middle of the viral glycoprotein was identified (67,68). This region also contained epitopes recognized by neutralizing and binding glycoprotein-specific monoclonal antibodies (67).

1000

CONTROLS VACCINATES

500

10

15 20

25 30 35 DAYS

40

45

50

55

60

Fig. 1. Daily mortalities for control and vaccinated rainbow trout populations exposed to infectious hematopoietic necrosis virus (IHNV) under field conditions. Control and vaccinated groups each contained 100,000 fish with an average weight of 1 g. Three weeks after immunization the fish were moved to ponds that received water from a source containing resident IHNV-infected fish.

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z
1 8 8 9 i= s g u3 G 8

30 25 20 15 10 5 0

CONTROLS

VACCINATES

lo

20

30 DAYS

40 POST

50

60

70

80

PONDING

Fig. 2. Cumulative mortalities for control and vaccinated rainbow trout populations exposed to infectious hematopoietic necrosis virus (IHNV) under field conditions.

95% of carp farms in Austria (70) and 28% of carp farms in France (71) contain animals which have been infected with SVCV. The virus will infect several species of carp including crucian carp (Curussius carassius), silver carp (Aristichthys nobilis), grass carp (Ctenopharyngodon idella), bighead carp (Hypophthalmichthys molitrix), and the fry of sheatfish (Sifurus glanis) and pike (Esox Lucius) (72). Outbreaks of the disease are usually found during April-June when the water temperatures are commonly between 11 and 17 C. Thus, the SVCV outbreaks occur in fish at 9-12 and 21-24 months of age. Mortality rates in such outbreaks are usually 30-40% of the population but it can reach as high as 70%. The affected fish become sluggish and uncoordinated with petechial hemorrhages at the bases of fins, in the skin, eyes, and gill. There is exophthalmia, abdominal distension, and trailing pseudofecal casts. The virus does persist in carriers who may maintam the virus once fish are infected in a pond. These fish transmit the disease horizontally to uninfected fish in the pond. Vertical transmission of the virus is not well-established since the virus is not often found in the sexual products of carrier fish (73). Carp lice and leeches are also suspected vectors of the disease (74). The virus will remain infective in water at 10 C for 35 days and in mud at 4 C for 42 days. If the mud is dried at constant tempera-

tures between 4 and 20 C, the virus will maintain viability for more than 4 weeks (75). These observations suggest that any vaccination program for SVCV will require rearing of naive animals in sterilized ponds in pathogen-free water during the vaccination regimen. There appear to be two serotypes of SVCV isolated from carp: a type that could be regarded as a SVCV subtype and another type more closely related to pike fry rhabdovirus (PFRV). An antigenic analysis of the G, N, and M proteins of SVCV and PFRV has suggested that these two viruses were related but serotypically different. The study indicated that it was very likely that SVCV and PFRV were serotypic variants of the same virus agent (76). The effect of this viral heterogeneity on vaccine development has not been determined. There is one commercially available SVCV vaccine, Bioveta, which has been in use since 1981 (for review, see Fijan [77]). The vaccine consists of inactivated virus of two different strains and is administered by intraperitoneal injection in an oil emulsion. Field trials of the vaccine indicated that losses in the vaccinated populations were lower and serum neutralizing antibodies were present. A live, attenuated vaccine has been tested by Fijan et al. (78). The SVCV was attenuated by multiple passage on human diploid, baby hamster kidney (BHK-21), and fathead minnow (FHM) cells. When fish were

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vaccinated by intraperitoneal (i.p.) injection of 6.3 x lo4 TCIDsc per fish of 40-100 g, only 5% of 60 fish died after challenge with i.p. injected virulent SVCV. In the control, unvaccinated group of fish, 55% of 40 fish died. Fish were also orally vaccinated with 3.1 x 10 TCID,, of virus in food containing 30% protein and delivered during 2 consecutive days as a dough, stable in water for 4 h. In this group of fish, 22% of 60 fish died after challenge with virulent SVCV. Fish were also tested for neutralizing antibodies after vaccination. Fijan and colleagues found that l-2 months after vaccination, serum neutralizing antibodies were present in 7.5% of orally vaccinated fish and 38.2% of i.p. vaccinated fish. In control fish, virus neutralizing antibodies were not present at l-2 months but did appear in 23% of the fish sera taken after 2 months. No explanation for the appearance of virus-specific antibodies in the control fish was offered. Although the live vaccine did provide excellent protection against the lethal effects of a virulent SVCV, the vaccinated fish apparently became asymptomatic carriers of the virus. When these fish were raised with unvaccinated stock, a SVC outbreak occurred in the unvaccinated carp. This outbreak might have resulted from the shedding of virus by the vaccinated fish and the subsequent infection of the naive fish. Since there was no other outbreak of SVC on that farm, this explanation was very compelling. The development of a subunit vaccine for SVCV has been considered (72), but research laboratories who might undertake this project have not yet been identified. The work that has been carried out indicates strongly that vaccination will control SVCV in carp culture. Since live vaccines may generate carrier fish, making it difficult to license a live vaccine in many countries, a subunit vaccine becomes an attractive alternative for SVCV. However, before any further vaccine development can take place, it is very important to develop models for vaccination trials in the laboratory and standards for vaccine potency testing. Herpesvirus ictaluri Herpesvirus ictaluri or channel catfish virus (CCV) is the etiologic agent of a highly virulent disease of young, intensively cultured channel catfish, Ictaluruspunctatus. The CCV disease was first described by Fijan (79) in channel catfish fingerlings in Alabama and since that report, it has been found throughout the southern United States where channel catfish are cultured (80). An outbreak of the dis-

ease is associated with an abrupt rise in mortality of young-of-the-year channel catfish when the water temperatures are 25 C or higher. The onset of the disease is sudden and can claim up to 100% of the population within 7 to 10 days. Most often, the high mortalities are correlated with stress (high density rearing conditions with poor water quality) and secondary bacterial infections. These fish die of a hemorrhagic viremia with marked necrosis of the kidneys, liver, gastrointestinal tract, spleen, muscle, neural tissue, and pancreas (81). The virus infection is usually a problem in fish between 1 week and 6 months of age; rarely has CCV been isolated from diseased fish older than 6 months (80). Also, the disease occurs during the warm summer or early fall months when the water temperature is 25 to 34 C. At temperatures of 18-25 C, mortalities range from 40 to 70% and below 18 C, very few fish die. Once the fish are infected with the virus, survivors become carriers for their entire life. A recombinant DNA probe has been used to detect CCV DNA in broodstock and in adults which had no history of CCV disease (82). These fish can transmit the disease horizontally and there is considerable circumstantial evidence that they can transmit the disease vertically as well. In a series of studies carried out by J.A. Plumb and his colleagues, it was found that channel catfish is the primary species affected by CCV. Blue catfish (Zctalurusfurcatus) may be infected by injection but not by immersion or oral exposure (83). The brown bullheads (I. nebulosus) and yellow bullheads (I. natalis) are resistant to CCV even by injection exposure. Galla and Hartmann (84) have reported that the walking catfish (Clarias batrachus L.) is susceptible to injected CCV but that observation has not been confirmed. Plumb and colleagues have also shown that the European catfish or sheatfish (Sihrus glanis) is resistant to injected CCV (85). Thus, CCV is remarkably similar to other mammalian herpesviruses in showing a high degree of host specificity. At present, CCV epizootics are prevented by management strategies. There are resistant strains of channel catfish and regional fish health personnel recommend virus-free broodstock for supplying fish farms. Avoidance is now the only control method available. However, vaccination holds great promise for protecting young catfish from the disease in areas where CCV is endemic (86). An attenuated live vaccine has been developed and granted U.S. Patent No. 040,108 (87). The vaccine was attenuated by serial passage in a tissue culture cell line of the walking catfish. In laboratory testing of the

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vaccine where fry were vaccinated by hyperosmotic filtration, the vaccine provided very good protection against 5 LD,, of CCV administered by injection. Fish receiving only buffered salt solution in the vaccination procedure all died after injection of a lethal dose of CCV, whereas 90% of the vaccinated fish survived. A second study by Walczak et al. (88) demonstrated that booster vaccination increased the efficacy of the attenuated vaccine dramatically. In the fish group vaccinated by a single immersion, 30% survived versus the control fish with only 8% survival. When the fish were given a booster immersion vaccination, 95% survived and only 20% of the control fish survived. The vaccine is not available commercially and has not been licensed by the U.S. Department of Agriculture. The vaccine may not have been licensed because of concerns regarding the risk of reversion to virulence and the possible formation of carriers in the vaccinated fish. If carrier fish are generated as a result of vaccination, these fish may no longer be certified as virus-free broodstock. For these reasons, farmers are unlikely to utilize this vaccine. Attempts to develop a killed CCV vaccine have been hampered by its low immunogenicity (89). The poor immunogenicity of the preparation was credited to the heat lability of the CCV antigens since heat was used to kill the virus. More recently, a subunit vaccine was prepared for CCV (90). The soluble envelope proteins of CCV were separated from the virus by treatment with Triton X-100 (octoxynol) and centrifugation of the preparation. The supernatant fluid containing the envelope fraction was collected and frozen at -70 C. When 3-4 day old eggs and 1 week-old fry of channel catfish were vaccinated by immersion and challenged with viable CCV after 8 weeks, excellent protection was observed in the vaccinated fish. All challenged nonvaccinated control fry died whereas, in fish vaccinated as eggs or fry, 69% and 18% died, respectively. A subgroup of fry was given a booster dose of vaccine at 2 weeks after vaccination. When these fish were challenged with CCV 8 weeks later, 56% of the control fish died and only 19% of the egg vaccinated and 11% of the fry vaccinated died. Thus, envelope components of CCV were capable of inducing protective immunity in very young fish and eggs. These findings make it important to identify the major immunogenic components of the CCV envelope. Once accomplished, recombinant DNA technology might then be used to clone the immunodominant protein for large-scale production of a vaccine against CCV disease.

ECONOMIC

AND LICENSING

CONSIDERATIONS

In preparing this review, it has become clear that a vaccine for viral diseases in fish must meet the criteria of safety before it will be accepted by fish health personnel, the vaccine licensing agencies, and the fish farmer. This appeared to be the most important concern for all members of the aquaculture community. Coupled with that concern was the economic reality of using a vaccine on fish farms or at federal/state hatcheries. For years these facilities have operated by managing around disease problems and have tried to avoid viral epizootics by routine inspection/certification regimens and the use of the best standard animal husbandry health practices available. In the case of viral diseases, they have had to use these methods because there were no other control measures available. Now, however, viral vaccines are being developed that will meet the criteria of safety and economic viability. The fish rearing community must be made aware of these developments and a program developed to transfer this technology to the field. Water quality and contamination of water with infectious agents will be critically important factors in the decision to use fish vaccines. The issue is one that is now a priority for many fisheries agencies and will occupy the dockets of regulatory agencies in the coming decade. Hatcheries and other rearing facilities will have to pay careful attention to the quality of their effluents. In particular, the transmission of infectious agents from farmed to wild fish will become critical issues that may be resolved by the use of vaccines. An effective vaccination program should reduce the concentration of infectious agents that might be released from a fish facility. Moreover, the use of antimicrobials for disease control will be reduced if fish are immunized by bacterial vaccines, and the subsequent clearance of these drugs from the fish and the environment will become less of a problem. It is clear that an effective vaccination program is a more sound approach to disease control at fish rearing facilities. Economic viability of individual viral vaccines will depend upon the seriousness of the disease problem and the value of the vaccinated fish. Farmers are more willing to vaccinate large fish in which they have invested considerable time and money. Likewise, salmonid species have a higher economic value than tilapia or carp. A cost-effective vaccine must not only reduce economic losses through decreased mortalities, but it should also prevent the

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decreased growth rates and physical deformities which may result from virus infection. It should also reduce the need for chemotherapeutic agents that are costly added expenses for the farmer. For vaccines that must protect fish at a very early stage of life, the vaccine industry has estimated that the cost per dose should not exceed 0.5 cents. Many industry analysts are aware that it will be difficult to develop a viral vaccine that will meet this cost limitation. The cost of bacterial vaccines has been less than 0.5 cents per dose; but the very nature of bacterial vaccines makes them economical and easy to produce. This review has shown that viral vaccines for fish will not be easy to develop and they certainly will not be as inexpensive to produce as their bacterial counterparts. The aquaculture community must develop an appreciation for these difficulties and the vaccine industry must provide cost-benefit analyses that show clearly that viral vaccines will be profitable for aquaculture. The fish vaccine industry is still a relatively small industry. It will require federal assistance for licensing of fish vaccines in a timely manner and mounting an educational program for fish-rearing personnel. Because the size of the industry is small, the federal government must support research for vaccine development and promote the transfer of these technologies to private industry. Alternatively, the federal government may provide a means of producing these vaccines in the public sector for use by the aquaculture industry.
Acknowledgments-

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The authors wish to acknowledge the support of Bonneville Power Administration, Project Number 88-152, Contract Number DE-PS79-88BP92431 (G.R. Bouck and R. Morinaka served as the Contracting Office Technical Representatives on the project); United States Dept. of Agriculture to the Western Regional Aquaculture Consortium under Grant Number 92-38500-7195, Project Number 9208044-l; and Oregon Sea Grant with funds from the National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of Commerce, under Grant Number NA89AAD-X108, Project Number R/FSD-16, Oregon Agricultural Experiment Station Technical Paper Number 10,095. REFERENCES t. Avault, J. (1987). Aquaculture.
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