A newly recognized virus, lethal for fry and fingerlings, has been isolated from brood stock rainbow trout (Salmo gairdneri) at the Winthrop National Fish Hatchery, Washington. A similar if not identical virus has been found in natural epizootics occurring annually since 1970 among fry of landlocked sockeye salmon (Oncorhynchus nerka) on Honshu Island, Japan. More recently, this agent was isolated from moribund and dead adult O. nerka on Hokkaido Island; Japan, but it remains to be determined whether or not the virus causes death in adult salmonids. The purpose of this leaflet is to bring the new agent to the attention of diagnosticians and researchers. It is recognized that some aspects of the virus and its effect upon host species are still incompletely known

Kimura, Takahisa

Sano, Tokuo

Wolf, Ken

English

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University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln US Fish & Wildlife Publications US Fish & Wildlife Service 1975 HERPESVIRUS DISEASE OF SALMONIDS Ken Wolf Eastern Fish Disease Laboratory Tokuo Sano Tokyo University of Fisheries Takahisa Kimura Hokkaido University Follow this and additional works at: https://digitalcommons.unl.edu/usfwspubs  Part of the Aquaculture and Fisheries Commons Wolf, Ken; Sano, Tokuo; and Kimura, Takahisa, "HERPESVIRUS DISEASE OF SALMONIDS" (1975). US Fish & Wildlife Publications. 151. https://digitalcommons.unl.edu/usfwspubs/151 This Article is brought to you for free and open access by the US Fish & Wildlife Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in US Fish & Wildlife Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. HERPESVIRUS DISEASE OF SALMONIDS Ken Wolf Eastern Fish Disease Laboratory Route #1, Box l7A Kearneysville, West Virginia 25430 Tokuo Sano Tokyo University of Fisheries Konan 4-5-7 Minato-ku, Tokyo Takahisa Kimura Hokkaido University Hakodate, Hokkaido, Japan FISH DISEASE LEAFLET 44 December 1975 UNITED STATES DEPARTMENT OF THE INTERIOR Fish and Wildlife Service Division of Cultural Methods Research Washington, D. C. 20240 For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C., 20402-Price $1.00 Stock Number 024-010-00412-6 A newly recognized virus, lethal for fry and finger-lings, has been isolated from brood stock rainbow trout (Salmo gairdneri) at the Win-throp National Fish Hatchery, Washington. A similar if not identical virus has been found in natural epizootics occurring annually since 1970 among fry of landlocked sockeye salmon (Onco-rhynchus nerka) on Honshu Island, Japan. More recently, this agent was isolated from moribund and dead adult O. nerka on Hokkaido Island; Japan, but it remains to be determined whether or not the virus causes death in adult salmonids. The purpose of this leaf-let is to bring the new agent to the attention of diagnosticians and researchers. It is recog-nized that some aspects of the virus and its effect upon host species are still incompletely known. DIAGNOSIS AND IDENTIFICATION Behavior Changes Most affected fry become lethargic as death approaches. During terminal stages some swim erratically, apparently losing motor control, and others are hyperactive. External Signs Various degrees of abnormal darkening are common, and exoph-thalmia--at times extreme--is frequent (Fig. 1). Hemorrhage may be present in the orbit of fish with extreme exophthalmia. Similarly, abdominal distention is common. Gills are abnormally pale, but other external signs are lacking. 2 Figure 1. Rainbow trout fry with extreme exophthalmia, prominent distention of abdomen, and darkening associated with infection by Herpesvirus salmonis. The scale is in millimeters. Internal Signs Ascitic fluid is abundant, and anemia and edema are evident in the visceral mass. The liver, spleen, and digestive tract are flaccid and the vascular organs are mottled with areas of hyper-emia. Kidneys are abnormally pale, though not noticeably swollen. The digestive tract is devoid of food. Clinical Findings Stained blood films show 10% or more immature erythro-cytes, and some of the cells appear to be dividing. Hema-tocrit value is greatly de-pressed and may be as little as 30% of normal. Histopathological Findings Blood in infected speci-mens contains abnormally high numbers of immature erythro-cytes and leucocytes. Kidney hematopoietic tissue is greatly increased and mitotically hyperactive. Kidney tubules are filled with serous mate-rial; renal tissue is edem-atous but necrosis is focal and mild where it occurs. Serous deposits in the orbits are undoubtedly the cause of exophthalmia. Skel-etal muscle is edematous and also shows accumulation of serous material. Cardiac mus-cle is similarly edematous, and hematopoietic activity is present in heart tissue, an organ in which blood cell for-mation does not normally occur. The liver is perhaps the target organ for viral effects for it is edematous and shows fatty infiltration and vascu-lar stasis. Hepatic tissue shows areas of mild necrosis--as does pancreatic acinar tis-sue, but to a lesser degree. Pancreatic necrosis is never as severe or as extensive, how-ever, as it is in cases of infectious hematopoietic necro-sis (IHN) and infectious pan-creatic necrosis (IPN). The spleen lacks red pulp. Identification Thus far, antiserum has not been developed for this herpes-virus, and diagnosis and virus identification must be based on internal signs, histopatholog-ical changes, and presumptive tests of the agent itself. The virus is the only salmonid pathogen demonstrably capable of inducing syncytium formation in rainbow trout gonad cells (RTG-2). The rain-bow trout fry (RTF-I) cell line is similarly susceptible; but fathead minnow (FHM) cells are refractory, or at least do not 3 show cytopathic effects. In fixed and stained cultures, the syncytia are readily visible, and some nuclei show atypical Cowdry Type A inclusions. May-GrUnwald-Giemsa staining reveals prominent cytoplasmic basophilia in the infected cells. Rabbit antisera against IHN, lPN, and Egtved viruses do not neutralize this salmonid herpes-virus. The herpesvirus plaque type differs from those of the other salmonid viruses: cell rounding and syncytium development are readily recognizable, and also cellular debris is much reduce~ (Fig. 2). Herpesvirus involvement in infected tissues or cell cul-tures, as revealed by electron microscopy, is also presumptive identification of the agent. CAUSE OF THE DISEASE The etiologic agent is a member of the herpesvirus group. Electron microscopy shows that the replication cycle begins in cell nuclei which contain both empty capsids and capsids con-taining nucleoids. Typical of herpesviruses, mature virions are enveloped and may be found in vesicles in the cytoplasm and especially in extracellular aggregates. A peculiarity of this herpesvirus is the rather intense osmiophilic nature of the envelope or material with-in the envelope. Infected cells show paramyelin-like lamellae or fibrils within the nucleus similar to those caused by the channel catfish virus. Figure 2. Plaque characteristics of salmonid viruses in RTG-2 cells. A. IPN virus (all strains thus far isolated) characteristically produces a-5tellate plaque with very irregular margins. The cells become some-what pyknotic but plaque interiors often contain cells that have not been killed. There is little cellular debris. The plaque itself has a lace-like network of RTG-2 cells which, though killed, retain a semblance of normal shape even after death. Killed cells stain only slightly with crystal violet. B. IHN virus produces a well-defined plaque in which affected cells characteristically round up and accumulate by contraction at the plaque margins. Interiors of the plaque typically have prominently rounded cells and may also be filled with discontinuous, coarsely granular ~ debris. Under higher magnification, cells near the edges of the plaque show margination of chromatin, a diagnostic feature of IPN virus in cell cultures. C. Herpesvirus salmonis plaques tend to be elongate and to have relatively clean interiors. Plaques often follow whorl lines of typical RTG-2 growth, and living cellular processes extend into the open area. The key features are cellular fusion and syncytium formation. D. Egtved virus produces a plaque which characteristically has a well-defined margin. The plaques appear to be punched out of the cell sheet, and their interiors are filled with a uniformly distributed finely granular debris. Cells in the plaque interior are usually all dead. Herpesvirus salmonis, the name proposed for this agent, is heat-, chloroform-, ether-, and acid-labile, but some in-fectivity persists after stan-dard treatment at pH 10. With a suitable positive control, the agent does not agglutinate human Type 0+, rabbit, or rain-bow trout erythrocytes at pH 7.3 during incubation at 4 C. Differential filtration shows that infectivity readily passes a membrane filter of 220-nm mean porosity, but is retiined by a 100-nm membrane. Infectivity titers are comparatively low, attaining only about 1 X 105 plaque-forming units (PFU) per milliliter. SOURCE AND RESERVOIR OF INFECTION Since virus has been iso-laTed from ovarian fluids, it is safe to consider that adults are the prime source of the agent. In this conText, it also appears reasonable to assume that this feature en-sures generation-to-generation survival, because abundant young host fish are available after the spawning season. The histopathologic changes in renal tissues indicate that infecTed fry and fingerlings shed virus during the time dis-ease signs are present, and possibly earlier. MODE OF TRANSMISSION Fish-to-fish transmission is assumed--at least during epizootics. Also, one must consider the possibility of 6 vertical transmission--the virus being either in or on eggs Thus far, experimental transmis-sion by holding susceptible young fish in water with virus has not been reported. INCUBATION PERIOD After intraperitoneal in-jection of virus at 10 C, an incubation time of at least a month elapsed before the first death occurred. Fry 5 to 10 cm in total length sustained the first mortality at 33 days and fingerlings began to die at 38 days. Thus far, other methods of infection have not been reported. PERIOD OF COMMUNICABILITY It can only be postulated that the disease is communi-cable while clinical signs are present. A carrier state is obviously present in adults, be-cause Herpesvirus salmonis was originally isolated from ovarian fluid. SUSCEPTIBILITY AND RESISTANCE Rainbow trout lfry and fin-gerlings) and kokanee or land-locked sockeye salmon ~ry) are thus far the only known suscep-tible species. The effect on adults of these two species is not yet known. Atlantic salmon (Salmo salar), brown trout (S. trutta), and brook trout (SaI-velinus fontinalis) have been suitably tested by intraperi-toneal injection, but have proved to be refractory. Other species of Pacific salmon have not been tested. RANGE The newly reported infec-tion is known to occur in Japan and the United States. Because it is best isolated at 10 C, a temperature lower than that usually employed for salmonid virological examinations, the virus might be present else-where--having been missed dur-ing earlier work. OCCURRENCE In Japan, the virus has been implicated with problems that occurred only during the summer. The virus caused epi-zootic mortality among young kokanee salmon,and it was implicated with high summer mortality among adult kokanee salmon in a freshwater lake. METHODS OF CONTROL The only certain method of control is avoidance, and that requires that sources of the virus be known. Since low tem-peratures seem to be required for the virus to produce dis-ease, it may be possible to minimize mortality by holding infected fish at temperatures of 15 C or higher. There is no assurance, however, that the virus will be eliminated by such a practice. ANNOTATED BIBLIOGRAPHY Sano, T. 1975. Viral diseases of cul-tured fishes in Japan. In S. Egusa, ed. Interna- --tional seminar on fish diseases. Tokyo, Japan. (In press). Under the subtitle "Sus-pected viral disease, 1. An 7 epizootic in landlocked Oncorhynchus nerka," the author reports annual epi-zootics beginning in 1970 among fry. Histopatholog-ical findings are described. A syncytium-producing agent was isolated, and its effect on RTG-2 cells described. Syncytium formation was found in interstitial cells of the kidneys of victims of natural epizootics. Elec-tron microscopy showed en-veloped viral particles hav-ing the size, shape, and replication features of a herpesvirus. Wolf, K. 1975. Fish viral diseases in North America and new research findings of the Eastern Fish Disease Laboratory. In S. Egusa, ed. InternatIOnal seminar on fish diseases. Tokyo, Japan. (In press). A review of problem viruses of North American fishes, covers literature from the period 1971-75. Recent find-ings at the Eastern Fish Dis-ease Laboratory concern myxo-sporidian whirling disease, corynebacterial kidney dis-ease, and the new herpesvirus of rainbow trout. Services provided by the laboratory's Section of Biological Stan-dards are described. The purpose, distribution, and plans for FISH HEALTH NEWS are discussed. Wolf, K., and W. G. Taylor. 1975. Salmonid viruses: a syncytium-forming agent from rainbow trout. Fish Health News 4: 3. The report documents the isolation from brood stock rainbow trout of a newly recognized virus which was not neutralized by anti-sera against previously known salmonid viruses. Its cytopathic effects in RTG-2 cells are described, and key features of its characterization are given. Wolf, K., R. L. Herman, R. W. Darlington, and W. G. Taylor. 1975. Salmonid viruses: effects of Herpesvirus salmonis in rainbow 8 trout. Fish Health News 4: 8. Experimental infection was carried out in rainbow trout fry at 10 C. Gross external and internal signs are de-scribed, as are several clinical findings. The report includes results of electron microscopic exam-ination of infected cultures and details of histopatho-logic changes found in exper-imentally infested fry. 

HERPESVIRUS DISEASE OF SALMONIDS

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HERPESVIRUS DISEASE OF SALMONIDS

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