A Retrovirus in Chinook Salmon (Oncorhynchus tshawytscha) with Plasmacytoid Leukemia and Evidence for the Etiology of the Disease 1

A plasmacytoid leukemia (PL) has caused mortalities in chinook salmon (Oncorhynchus tshawytscha) reared in seawater netpens in western British Columbia, Canada, since 1988. Kidney or eye tissues from 11 of 13 fish from netpens with clinical PL had reverse transcriptase (RT) activity. This RT activity was associated with virus particles of retrovirus morphology and buoyant density. In a transmission experiment, PL-positive donor fish tissues also had RT activity and virus particles of retrovirus morphology and buoyant density, as did recipient fish tissues following development of the disease 6 weeks postinjection with a tissue homogenate from the donor fish. Kidney and spleen tissues from fish that developed PL following injection with an inoculum that was passed through a 0.22-~m filter, in a separate experiment (M. L. Kent and S. C. Dawe. Further evidence for a viral etiology in the plasmacytoid leukemia of chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Org., in press, 1992), also exhibited RT activity. The virus particles observed by electron-microscopic examination of tissues or sucrose fractions from PL-positive fish were enveloped and were about l l0-nm diameter with a central electron-dense core. Polypeptides of about Mr 120,000, 80,000, 42,000, 27,000, 25,000, 22,000, and 19,000 were observed when purified virus particles were examined by polyacrylamide gel electrophoresis analysis. Many infectious neoplasms of animals, including fishes, are caused by retroviruses. The evidence in this study shows the presence of a retrovirus in chinook salmon with PL and further suggests a retroviral etiology of the disease. We are tentatively calling this virus salmon leukemia virus. I N T R O D U C T I O N A PL 3 of chinook salmon (Oncorhynchus tshawytscha), referred to as marine anemia by fish farmers, has caused extensive mortal i ty at numerous seawater netpen facilities in western British Columbia, Canada, since 1988 (1, 2). The disease has been characterized as a proliferation and infi l trat ion of plasmablasts into the visceral organs and retrobulbar tissue of the fish (1). Al though no etiological agent of PL was identified, the disease has been shown to be caused by an infectious agent, inasmuch as intraspecific t ransmission was repeatedly achieved by injection of thoroughly homogenized tissue from affected chinook salmon (2, 3). The disease can be t ransmit ted from chinook salmon to sockeye (Oncorhynchus nerka) and Atlantic sa lmon (Salmo salar) (2, 3). Transmiss ion between different species (chinook and sockeye salmon) and different genera (chinook and Atlantic salmon) support the assumption that the disease is caused by an infectious agent, as opposed to t ransplanta t ion of neoplastic Received 4/17/92; accepted 9/23/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported by funds from the Department of Fisheries and Oceans/Natural Sciences and Engineering Research Council of Canada Science Subvention Program, and the British Columbia Ministry of Fisheries and Aquaculture. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: PL, plasmacytoid leukemia; RT, reverse transcriptase; TNE, 0.01 M Tris-HCl, pH 7.5, 1.0 mM EDTA, 0.l M NaCI; PI, postinjection; TCA, trichloroacetic acid; DNAP, DNA polymerase; SLV, salmon leukemia virus. cells. Furthermore, PL has been recently t ransmit ted with an inoculum passed through a 0.22-~m filter. 4 Infections by the intranuclear microsporidium, Enterocytozoon salmonis, and bacterial kidney disease, caused by Renibacterium salmoninarum, are frequently observed in fish with PL. However, t ransmission studies demonstrated that these 2 conditions were not the pr imary cause of PL, as the disease could be t ransmit ted in the absence of ei ther of these pathogens (2, 3). Essentially all infectious neoplasms are caused by oncogenic viruses, and these t ransmission studies led us to hypothesize that PL is caused by an oncogenic virus, perhaps a retrovirus. Retroviruses are known etiological agents of many leukemias and leukemia-like diseases in higher vertebrates (4), and have also been associated with hemic (5-9) and nonhemic neoplasms (10-17) in several fish species. In this paper, we describe the presence of a retrovirus in chinook salmon with PL and the evidence suggesting a retroviral etiology of the the disease. M A T E R I A L S A N D M E T H O D S Source of Material: Field Fish. Thirteen different chinook salmon with clinical PL from several private aquaculture facilities were brought to the Department of Fisheries and Oceans Pacific Biological Station in Nanaimo, British Columbia, Canada. All had been reared in seawater netpens that contained fish experiencing a PL-associated epizootic. The fish showed typical signs of PL, such as swollen kidney and spleen, pale gills, bloody ascites, and bilateral exophthalmia. Samples were collected and processed for histological and electron-microscopic examination. Approximately 10 g of kidney tissue from 11 of these fish and 10 g of tumor tissue from the orbit of the eye from 2 of these fish were removed and processed for RT analysis. Ten g of a dermal sarcoma from walleye pike (Stizistedion vitreum) were used as a positive control, as RT activity and retrovirus particles have been observed in this neoplasm (15, 17). In addition, 20 ml of supernatant from a BF-2 cell culture 5 days postinoculation with the snakehead retrovirus (18) were centrifuged at 100,000 • g for 1 h and resuspended in 0.5 ml of TNE and then used as an additional positive control for the RT assay. Ten g of kidney tissue from normal chinook salmon from an unaffected population were used as a negative control. Source of Material: Transmission Study Fish. A transmission study was conducted using a tissue homogenate prepared from kidney and spleen of PL-affected fish. Kidney tissues from 3 PL-positive chinook salmon from one of the netpen facilities previously mentioned were diluted 1:4 (v:v) in minimum essential medium and thoroughly homogenized with a Polytron (Brinkmann Instruments) at 4~ The homogenate was examined by wet mount preparation (5 fields of view) to ensure that no intact cells remained. This homogenate was used as the donor inoculum for the transmission study. The donor inoculum was examined for RT activity and by electron microscopy. Thirty apparently healthy chinook salmon (average weight, 60 g) were used as recipient fish and were given i.p. injections of the donor inoculum (0.5 ml/fish). The fish were held in tanks with flow-through fresh water at about 13~ Twenty control fish were given injections of 0.5 ml TNE and similarly maintained. At 6 weeks PI, kidney and spleen from 3 recipient fish with clinical and histological signs of PL and 3 control fish were collected for RT analysis. The tissues from these 4 M. L. Kent and S. C. Dawe. Further evidence for a viral etiology in the plasmacytoid leukemia of chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Org., in press, 1992.