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**Fish Disease Leaflet 83****Viral Hemorrhagic Septicemia of Fishes****Philip E. McAllister**
U.S. Fish and Wildlife Service, National Fisheries Research Center-Leetown, National Fish Health Research Laboratory,  Box 700,  Kearneysville, West Virginia.   25430.

UNITED STATES DEPARTMENT OF THE INTERIOR, Fish and Wildlife Service.  1990.

*(Revision of Fish Disease Leaflet 6 (1966), "Viral hemorrhagicsepticemia of rainbow trout."  by Ken Wolf.)*- **Introduction**  **Geographic Distribution****Host Susceptibility**
- **Clinical and Histopathologic Signs of Disease**    - ***Clinical Signs***  ***Histopathological Changes*****Etiology**
***Virus Detection and Identification***
- **Epizootiology**    - ***Transmission***  ***Factors Affecting the Disease******Disease Control***
- **Bibiolography**     **Introduction**  Viral hemorrhagic septicemia (VHS), a systemic infection of varioussalmonid and a few nonsalmonid fishes, is caused by a rhabdovirusdesignated as the viral hemorrhagic septicemia virus (Office Internationaldes Epizooties 1963). The virus infection occurs in salmonidsand certain other fishes of any age and may result in significantcumulative mortality. Fish that survive may become carriers. Viralhemorrhagic septicemia has been reported under various names,of which "Egtved disease" and "Infektiose Nierenschwellungund Leberdegeneration" are the best known. The viral etiologyof the disease was established by Jensen (1965), and several serotypesof the virus (also known as "Egtved virus") are currentlyrecognized.  Comprehensive reviews of the disease and the virus have been publishedby de Kinkelin (1983), McAllister (1979), Pilcher and Fryer (1980),Roberts (1978), and Wolf (1988).  **Geographical Distribution**Viral hemorrhagic septicemia is enzootic in most countries ofcontinental Eastern and Western Europe, and the virus has beenisolated in the Puget Sound area of Washington in the United States.No outbreaks of VHS or isolations of VHS virus have been reportedelsewhere.
**Host Susceptibility**
In Europe, epizootics of VHS occur primarily in rainbow trout,*Oncorhynchus mykiss*; brown trout, *Salmo trutta*;and to a lesser extent in northern pike, *Esox lucius* (Jorgensen1980; Meier and Jorgensen 1980). Natural infections have alsooccurred in grayling, *Thymallus thymallus*, and whitefish*Coregonus* sp. (Wizigmann et al. 1980; Ahne and Thomsen1985; Meier et al. 1986). Outbreaks of VHS have been suspectedin pollan, *Coregonus lavaretus*, and lake trout, *Salvelinusnamaycush*. In the United States, natural infections have beendiagnosed in chinook salmon, *O*. *tshawytscha*; cohosalmon, *O*. *kisutch*; and steelhead (searun rainbowtrout).
Fish shown by experimental challenge to be susceptible to VHSvirus infection are Atlantic salmon, *Salmo salar*; brooktrout, *Salvelinus fontinalis*; golden trout, *O*. *aguabonita*;rainbow trout x coho salmon hybrids; giebel, *Carassius auratusgibelio*; sea bass, *Dicentrarchus labrax*; and turbot,*Scophthalmus maximus* (de Kinkelin and Castric 1982; Castricand de Kinkelin 1984; Wolf 1988). Fish shown by experimental challengesto be refractory to VHS virus infection are common carp, *Cyprinuscarpio*; chub, *Leuciscus cephalus*; Eurasian perch, *Percafluviatilis*; roach, *L*. *rutilus*; and tench, *Tincatinca*.**Clinical and Histopathologic Signs of Disease**
A variety of clinical signs and histopathologic changes may beapparent in fish infected with VHS virus. Some fish show frankclinical manifestations of disease, whereas others look normal.Historically, clinical and pathologic signs of VHS have been cataloguedinto acute, chronic, and latent forms. Such descriptions representdegrees of severity rather than progressive stages of the disease.***Clinical Signs***
The clinical signs of VHS vary with the severity of infection(Yasutake 1970; Wolf 1988). Acute signs are typically accompanied
by a rapid onset of heavy mortality. Fish are lethargic, darkin color, exophthalmic, and anemic. Hemorrhages are evident inthe eyes, skin, and gills and at the bases of the fins. Internally,punctiform hemorrhages are evident in periocular tissues, skeletalmuscle, and viscera; the liver appears mottled and hyperemic andthe kidneys are red and thin. In chronically infected fish, significantcumulative mortality occurs, but is protracted. Fish are lethargic,dark in color, exophthalmic, and severely anemic, but not grosslyhemorrhagic. The abdomen is markedly distended due to edema ofthe liver, kidneys, and spleen. The liver appears pale and petechiated,and the kidneys are ashen. In a latent infection, mortality islow, and the fish seem nearly normal, but may be hyperactive.Inapparent virus carriers show no clinical signs of VHS.***Histopathological Changes***
Histopathologic changes are generally confined to the liver, kidneys,spleen, and skeletal muscle (Ghittino 1965; Yasutake 1970; Amlacheret al. 1980; Wolf 1988). In acutely affected fish, the liver sinusoidsare engorged with blood, and hepatocytes show focal to extensivenecrobiotic changes-cytoplasmic vacuoles, pyknosis, karyolysis,Iymphocytic invasion, and occasionally intracytoplasmic and intranuclearinclusions. Similar changes occur in the spleen and in the hematopoieticand renal elements of the kidneys. In skeletal muscle, erythrocytessometimes accumulate in muscle bundles and fibers, but littletissue damage occurs. In chronically infected fish, liver sinusoids
remain enlarged and contain coagulated plasma, and kidney andsplenic hematopoietic tissues and mononuclear lymphoid cells arehyperplastic. No remarkable histopathologic changes have beenreported in inapparent virus carriers.**Etiology**
The VHS rhabdovirion is an enveloped, bulletshaped particle about180 nm long and 60 nm in diameter. Arrayed over the surface ofthe virion envelope are peplomers 5 to 15 nm long. The envelopeis acquired as the virus matures, by budding from cell surfacemembranes or into cytoplasmic vacuoles. The intact virion, whichsediments to a density of 1.141.15 g/cm3, is composed of fivestructural proteins and contains one segment of singlestrandedRNA (de Kinkelin and Scherrer 1970; McAllister 1979; de Kinkelin1983).
The virus is moderately stable in cell culture medium and canbe preserved for years by freezing at 20°C or lower and bylyophilization. It is inactivated by exposure to ether, chloroform,glycerol, formalin, sodium hypochlorite, iodophors, ultravioletirradiation, or heat (5660°C). The virus is stable at pH5.010.4, but is inactivated at pH 3.5 (McAllister 1979; de Kinkelin1983).
In cell culture, the VHS virus replicates to a titer of 107108plaqueforming units per milliliter. The optimum temperature forvirus replication is 1415°C; virus yield is reduced at 6°C,and little replication occurs above 20°C (de Kinkelin andScherrer 1970). Optimum virus replication in cell culture requiresthat the pH of the medium be maintained in the range of pH 7.47.8(Campbell and Wolf 1969). Interferon can be produced in cell culturesinfected by VHS virus, and nascent or exogenous interferon reducesvirus yield (de Kinkelin and Le Berre 1977).
At least three VHS virus serotypes (designated F1, F2, 23.75)can be distinguished by infectivity neutralization assays (LeBerre et al. 1977; de Kinkelin 1983). Hyperimmune sera for virusidentification are generally prepared in rabbits, but virusneutralizingantibody titers are generally low because VHS virus is a weakimmunogen (Ahne 1981; Hill et al. 1981). Hybridoma cell lineshave been developed that secrete binding and neutralizing monoclonalantibody specific to VHS virus (Lorenzen et al. 1988; P. de Kinkelin,unpublished data; P. E. McAllister, unpublished data).***Virus Detection and Identification***
The VHS virus can be recovered from homogenates of internal organs,sex products, or urine. Concentrations of virus are higher inthe anterior kidney and spleen than in liver, heart, or muscle(de Kinkelin 1983). Brain samples should be included when oneis assaying for inapparent virus carriers (Castric and de Kinkelin1980). Little virus can be recovered from feces. Although infectedfish can develop an immune response to VHS virus, the detectionof antiVHS antibody is not a reliable indicator of the presenceor absence of current infection (Dorson and Torchy 1979; Jorgensen1982a,b). Nevertheless, the detection of VHS virusspecific neutralizingantibody in fish can be a useful tool for VHS surveillance (Olesenand Vestergard Jorgensen 1986). Mixed virus infections have beenreported in which VHS virus was isolated from fish concurrentlyinfected with infectious pancreatic necrosis virus.
The isolation of VHS virus in cell culture is the standard fordiagnosis. The virus replicates in a variety of piscine cell lines-forexample, BF2, CaPi, CHSE214, EPC, FHM, PG, RTG2, RTM, STE, andsea bass-as well as in primary cultures of tench, carp ovary,and goldfish, *Carassius auratus* (McAllister 1979; de Kinkelin1983). In addition, VHS virus replicates in cell lines of mammalian(BHK21 and WI38) and reptilian (GL1 and TH1) origin. The optimumtemperature for virus replication in cell culture is 1415°C(de Kinkelin and Scherrer 1970). Replication is greatly influencedby hydrogen ion concentration; a pH of 7.4 to 7.8 must be maintainedin diagnostic assays (Campbell and Wolf 1969; Wolf and Quimby1973). The detection of VHS virus in cell culture can be enhancedby pretreating cells with a 7% solution ofpolyethylene glycol(molecular weight = 20,000) or by including DEAEdextran (50mg/mL)or polyethylene glycol with the sample during virus adsorption(Campbell and Wolf 1969; W. N. Batts and J. R. Winton, unpublisheddata). Detection of virus in tissue sections by fluorescent antibodystaining is less sensitive than virus isolation in cell culture(Jorgensen and Meyling 1972).
A variety of serological techniques can be used to identify VHSvirus: infectivity neutralization, fluorescent antibody, immunoperoxidase,complement fixation, immune precipitation, countercurrent immunoelectophoresis,and immunoblot and plate enzymelinked immunosorbent assays (Meierand Vestergard Jorgensen 1975; Ahne 1981; de Kinkelin 1983; McAllisterand Schill 1986; Way and Dixon 1988). The three currently recognizedserotypes of VHS virus (F1, F2, and 23.75) are distinguished byinfectivity neutralization; therefore, three antisera (or a compositepolyvalent antiserum) are used for virus identification by infectivityneutralization assay. Viral antigen can be detected in inoculatedcell cultures by indirect fluorescent antibody and immunoperoxidasestaining (Meier and Jorgensen 1975; Faisal and Ahne 1980), andimmunoblot and plate ELISA systems are used for detection andidentification of VHS virus in cell culture fluids (McAllisterand Schill 1986; Way and Dixon 1988).
Cross-reactivity is seen among the VHS virus serotypes in indirectfluorescent antibody assays and in some instances in infectivityneutralization assays (Jorgensen 1972, 1980; Meier and Jorgensen1980). Similarly, one can identify all three VHS virus serotypesby immunoblot ELISA, using antiserum solely to VHS serotype F1(McAllister and Owens 1987).**Epizootiology*****Transmission***
The VHS virus is readily transmissible to fish of all ages, andsurvivors of infection can become lifelong carriers that shedvirus with urine and sex products. The virus ostensibly gainsaccess to the fish through the secondary gill lamellae. Virusshed with sex products appears to be solely a surface contaminantof the egg and is readily dissipated. Although virus can be isolatedfrom eggs for 3 to 4h after spawning, true vertical transmissionhas not been demonstrated. Experimentally, fish can be infectedby cohabitation, immersion, intraperitoneal and intramuscularinjection, brushing virus on the gills, and feeding (Jorgensen1980; de Kinkelin and Castric 1982; de Kinkelin 1983; Castricand de Kinkelin 1984).
In Europe, the gray heron, *Ardea cinerea*, is known to bea mechanical vector of VHS virus (Olesen and Jorgensen 1982; Petersand Neukirch 1986), but the virus is inactivated in the gastrointestionaltract of birds (Eskildsen and Jorgensen 1973). The virus is apparentlynot transmitted by parasitic vectors and, as judged from a studywith the fruit fly *Drosophila melanogaster*, it does notreplicate in insects (Bussereau et al. 1975).
In the hatchery environment, mechanical transfer of VHS viruson the surface of animate or inanimate objects presents a substantialhazard. The virus has been isolated from feral fish in watersreceiving hatchery effluent, and can persist in water for severaldays (de Kinkelin and Scherrer 1970).
***Factors Affecting the Disease***
Fish of any age are susceptible to infection, although sac fryand fish older than 6 months are sometimes resistant, (Ghittino1965). Epizootic losses occur at temperatures of 3° to 12°C(mortality is greatest at 3° to 5°C); mortality andthe proportion of virus carriers decrease at higher temperatures(de Kinkelin 1983). Deaths from VHS rarely occur at temperaturesabove 15°C.
Infected fish mount a strong interferon response, but may ultimatelysuccumb to infection (Dorson et al. 1975; de Kinkelin 1983). Increasedinterferon synthesis may play a role in transiently mitigatingthe effects of VHS at relatively high temperatures (de Kinkelinet al. 1982). Virusneutralizing antibody has also been demonstratedin infected fish, but the antibody response in survivors of epizooticand inapparent infections varies with the fish and the seasonof the year (Jorgensen 1971; Dorson and Torchy 1979; Dorson etal. 1979).
In captive fish, culture and environmental stress seemingly increasesusceptibility and recurrence of infection (Ghittino 1965; deKinkelin 1983). In feral fish, inapparent infection is more commonthan disease (Jorgensen 1982b) .***Disease Control***
Prevention of contact between the virus and the host is the mosteffective method for controlling VHS. A systematic program of
hatchery disinfection, combined with restocking with specificpathogenfreefish and eggs, has been used successfully (Kehlet 1973; Jorgensen1974a, 1980). Eggs used for restocking are decontaminated by iodophortreatment. The water supply should ideally be controlled and virusfree,although ultraviolet irradiation has been used to inactivate VHSvirus in the water supply (Maisse et al. 1980). Conditions thatpromote physiological stress should be alleviated. Although VHSrarely occurs above 15°C, disease control by temperaturemanipulation has not been described. Selective breeding to increasehost resistance to VHS has not been successful.
The potential for vaccinating fish against VHS has been demonstrated,and several avirulent VHS virus vaccines are under development(Jorgensen 1976; de Kinkelin and Le Berre 1977). One vaccine candidate,a VHS virus variant of low pathogenicity (F25), was selected byserial passage in EPC cells at progressively increasing temperature.This F25 variant replicates at 25°C, but the wildtype virusdoes not (de Kinkelin et al. 1980; de Kinkelin and Bearzotti 1981).A second vaccine candidate, a low-pathogenicity virus strain (Reva),was selected by serial passage in RTG2 cells (Jorgensen 1982b).Although both the F25 and the Reva strains induced a protectiveresponse, they also retained residual pathogenicity. The mechanismof the protective response is unclear. Some mechanism occurringwithin 48h after immunization, such as interferon stimulation,might be responsible for early protection, whereas the antibodyresponse occurs later (Jorgensen 1982b; Bernard et al. 1985).Fry can be protected by injection of interferon (de Kinkelin etal. 1982). Although the protective effects of avirulent vaccineshave been demonstrated in the laboratory, their efficacy underproduction conditions has not been proven, and their value forcontrolling VHS in healthy fish populations has been questioned(Erlzmann 1983).**Bibliography**
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