To a Model Pathogen, Spring Viremia of Carp Virus
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Comparative Medicine Vol 53, No 5 Copyright 2003 October 2003 by the American Association for Laboratory Animal Science Pages 514-521 Susceptibility of Zebrafish (Danio rerio ) to a Model Pathogen, Spring Viremia of Carp Virus George E. Sanders, DVM,1, 2,* William N. Batts, and James R. Winton, PhD1 To improve our understanding of the genetic basis of fish disease, we developed a pathogen model, using zebrafish (Danio rerio ) and spring virema of carp virus (SVCV). Replicate groups of 10 fish were acclimated to 20 or 24°C, then were exposed to SVCV concentrations of 103 to 105 plaque-forming units per milliliter (PFU/ml) of water and ob- served daily. In a second trial, fish were acclimated to 15°C, and replicate groups of 10 fish were exposed to SVCV at a concentration of 105 PFU/ml; however, the temperature was raised 1°C/wk. Moribund fish were collected for histo- logic examination, and dead fish were assayed for virus by use of cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Mortality exceeded 50% in fish exposed to 105 PFU of SVCV/ml at the lower tem- peratures. Clinical signs of disease became evident seven days after viral exposure and were observed most consis- tently in fish of the 105 PFU/ml groups. Affected zebrafish were anorectic and listless, with epidermal petechial hemorrhages followed by death. Use of plaque assays and RT-PCR analysis confirmed presence of SVCV at titers > 104 PFU/g of tissue. Histologic lesions included multifocal brachial necrosis and melanomacrophage proliferation in gills, liver, and kidneys. These results indicate that zebrafish are susceptible to infection by SVCV under condi- tions that mimic a natural route of exposure. Zebrafish (Danio rerio) are small (3 to 4 cm), freshwater, tropical cyprinids that grow optimally between 25 and 31°C (Fig. 1A) (40). Females exhibit exceptional fecundity and can produce over 200 eggs/wk; resulting embryos develop ex utero, and are transparent, which facilitates visualization and ma- nipulation (4, 8-11, 37). Zebrafish have a short generation time (3 to 4 months), compared with that of other, larger teleosts used in fish disease research (e.g., rainbow trout, Oncorhynchus mykiss), but only a few common diseases affect them naturally (8, 40). These diseases, none of which currently includes a viral etiol- ogy, are the external parasites Piscinoodinium pillulare (velvet dis- ease), Ichthyophthirius multifiliis (ichthyophthiriosis), and Gyrodactylus and Trichodina spp. and the internal freshwater pro- tozoan, Pseudoloma neurophilia (microsporidiosis), gram-negative bacteria (Aeromonas spp. and Flavobacterium columnare), acid- fast bacteria (typically, Mycobacterium marinum, M. fortuitum, or M. chelonae [mycobacteriosis]), and various monogenetic nema- todes (intestinal capillariasis) (3, 8, 20, 24, 34, 40). Due to its use in developmental and genetic research, the zebrafish has become a powerful model organism for study of vertebrate biology, and a large number of characterized strains are maintained at various research facilities and zebrafish stock centers (4, 9-11). Some of the mutant strains of zebrafish have pathologic conditions similar to human diseases, including hematopoietic, cardiovascular, renal, endocrine, and neurologic disorders (4, 9-11). The full genome sequence of the zebrafish is being completed, facilitating development of a full range of ge- Figure 1. Normal female zebrafish (A) and female zebrafish infected with 105 plaque-forming units (PFU) of spring viremia of carp virus Received: 1/27/03. Revision requested: 4/22/03. Accepted: 6/04/03. (SVCV)/ml (B). Notice presence of extensive epidermal hemorrhages. 1Western Fisheries Research Center, 6505 NE 65th Street, Seattle, Washington 98115, and 2Department of Comparative Medicine, University of Washington nomic tools and novel gene expression assays (11, 28, 39). Thus, School of Medicine, T-160 Health Sciences Center Box 357190, Seattle, Wash- ington 98195-7190. a functional disease model for this species would be a great as- *Corresponding author. set in advancing our understanding of the genetic basis of fish 514 Spring viremia of carp virus in zebrafish disease as well as providing a possible model for diseases of buffer) was seeded into each well of a 24-well cell culture plate. other vertebrate hosts. Cells were incubated for 24 to 48 h at 25°C to become confluent The viral pathogen that we selected for this model was spring monolayers. Ten-fold, serial dilutions (10-1 to 10-5) of viral viremia of carp virus (SVCV). Currently listed as a species of the sample were created in sterile 1.5-ml Eppendorf tubes with genus Vesiculovirus within the Rhabdoviridae (2, 13, 38), SVCV is MEM-5-T. A 0.1-ml volume of each serial dilution was added to an enveloped, negative-sense, single-stranded RNA virus having a each corresponding set of three wells of the 24-well plate, and bullet-shaped virion approximately 60 to 90 nm wide and 90 to 180 the plates were incubated for 15 to 20 min to allow viral adsorp- nm long. The virus is ether, heat, and acid labile, stable at pH 7 to tion to cells. Negative-control wells received 0.1 ml of MEM-5-T, 10, and replicates optimally between 20 and 22°C (13, 14, 35, 38, and positive-control wells received 0.1 ml of a serial dilution of 41). This virus is the causative agent of acute infectious dropsy of frozen stock SVCV. After incubation, wells were subsequently the common carp (Cyprinus carpio) or spring viremia of carp (14, covered by one milliliter of methylcellulose overlay (MEM-5-T 17, 38, 41). In its normal hosts, SVCV is transmitted horizontally containing 0.75% [wt./vol.] methyl cellulose, and100 IU of peni- (including via mechanical and biological vectors), the incubation cillin, 100 mg of streptomycin [Gibco BRL], 2.5 mg of amphotericin period is temperature dependent (average 10 to 17 days), and the B [Fungizone, Gibco BRL, Grand Island, N.Y.], and 100 mg of gen- portal of entry is the gills, with subsequent viremia and dissemi- tamicin sulfate [Gibco BRL]/ml, buffered to pH 7.8 with 1M Tris nation to the kidneys, liver, spleen, heart, and gastrointestinal and sodium bicarbonate) and incubated at 20°C for 72 to 96 h. tract (1, 2, 14, 38, 41). We selected SVCV for the viral component Viral titer was expressed as plaque-forming units per milliliter. of this model because this pathogen replicates well near the nor- Animal selection, housing, and husbandry. The AB mal temperature of our host (25 to 31°C) and is extremely patho- strain of zebrafish was selected because it is a wild-type strain genic to cyprinid species indigenous to Europe (2, 25, 41). that is commonly used in research. Fish were bred in-house Because SVCV causes a disease that is notifiable to the Office In- from breeders maintained in a closed colony for multiple gen- ternational des Epizooties (O.I.E.), the in vivo component of this erations. This strain is also similar genetically to the Tübingen research was carried out within the confines of an aquatic ani- strain from Germany (12, 40). Zebrafish were reared under the mal biohazard level-three laboratory (ABL-3) located at the guidelines provided by the Guide for the Care and Use of Labo- Western Fisheries Research Center (WFRC) to prevent possible ratory Animals (27) and the United States Public Health Ser- introduction to the local environment (14, 25, 31, 32). Experimen- vice Policy on the Humane Care and use of Laboratory Animals. tal work with this pathogen should be limited to laboratories ad- The protocol for experimental use of these animals was ap- equately equipped to handle this agent safely to prevent its proved by the Institutional Animal Care and Use Committee of introduction into naïve aquatic environments. the University of Washington (Seattle, Wash.). Zebrafish were spawned using standard conditions (40), and Materials and Methods approximately 200 juveniles were transferred at two months of Cell culture. Mycoplasma-free cultures of epithelioma age to the wetlab of the WFRC from a zebrafish colony at the papulosum cyprini (EPC) cells were obtained from Dr. G. University of Washington (Seattle, Wash.). Fish were maintained Kurath (WFRC, Seattle, Wash.). Cells were grown without anti- in 5-L opaque containers, which received sand-filtered, ultravio- biotics in minimal essential medium (MEM) supplemented with let-irradiated, fresh water in flow-through manner (2.4 L/min) 0.3% tryptose phosphate broth (TPB; Difco Laboratories De- with one air-stone per container. Fish were maintained at 26°C troit, Mich.), 10% fetal bovine serum (FBS; Hyclone, Logan, and a 14/10-h cycle of light and dark. Utah), and 2 mM L-glutamine (Gibco BRL, Grand Island, N.Y.), Fish were fed No. 2 salmon starter diet (Bioproducts, Warrenton, then buffered to a pH of 7.8 by addition of sodium bicarbonate Oreg.) and were monitored twice daily. Food was withheld 24 h be- (MEM-10-SB). Cells were removed from the flasks after addi- fore and after movement. On reaching an average size of three to tion of a trypsin-versene solution and were split at a ratio of 1:3, four cm at eight months of age, fish were transferred to the ABL-3 using standard aseptic techniques (21, 30, 42). Cell cultures facility and were maintained as described. were incubated at 25°C for initial growth and were maintained Prior to the start of and during this project, zebrafish from at 20°C when the monolayers became confluent. the re-circulating system where the fish for this experiment Virus propagation. The reference strain of SVCV (ATCC were spawned, and other systems within this colony were VR-1390, Manassas, Va.) was used for in vitro and in vivo work.