Viral Structural Proteins and Genome Analyses of the Rhabdovirus of Penaeid Shrimp (RPS)
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DISEASES OF AQUATIC ORGANISMS Vol. 19: 187-192, 1994 Published August 25 Dis. aquat. Org. I Viral structural proteins and genome analyses of the rhabdovirus of penaeid shrimp (RPS) Yuanan Lu, Philip C. Loh Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822, USA ABSTRACT: The viral structural proteins of the recently isolated rhabdovirus of penaeid shrimp (RPS), and that of the mammalian vesicular stomatitis virus (VSV) and 3 other fish rhabdoviruses, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV) and rhabdovirus carpio (RC), were comparatively analyzed by sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) and Western blot procedures. The number of major bands identified and the pro- file of the viral polypeptides of RPS resembled that of VSV and RC but were different from that of IHNV and VHSV. Western blot analysis using polyclonal antiserum against RPS identified the viral origin of the 4 malor proteins and an extra protein of RPS. Also, the analysis indicated that although RPS is closely related to RC, it is only partially related to IHNV, VHSV, and VSV The genoinic RNA of RPS extracted by the pronase-SDS-urea method and analyzed by polyacrylamide-agarose gel electro- phoresis was determined to be a single component havlng a molecular weight of 3.6 X 10' daltons. KEY WORDS: SDS-PAGE . Rhabdoviruses . RPS . Viral proteins . Viral RNA INTRODUCTION These viral proteins were named: L (large protein), G (glycoprotein), N (nucleoprotein), NS (nonnucleopro- The Rhabdoviridae is a well-defined family of viruses tein) and M (matrix protein) (Wagner et al. 1972). and its members are found in plants, vertebrates Based on the electrophoretic patterns of the viral struc- (including fish), invertebrates, and arthropods. Four tural proteins, viruses belonging to the Rhabdoviridae rhabdoviruses, Y-organ virus, Rhabdo-like virus A are classified into 2 major genera, Lyssavirus and (RhVA), Rhabdo-like virus B (RhVB) and Enveloped Vesiculovirus (Matthews 1982, Wagner 1987). Among helical virus (EHV),are known from Crustacea, exclu- the fish rhabdoviruses, members of the lyssavirus sively in crabs. Except for the first crab rhabdovirus to group are conlposed of IHNV (infectious hematopoi- be found, Y-organ virus, which was from European etic necrosis virus), VHSV (viral hemorrhagic sep- shore crab Carcinus maenas (Chassard-Bouchaud ticemia virus), HRV (hirame rhabdovirus), and SHRV & Hubert 1975, Chassard-Bouchaud et al. 1976), these (snakehead rhabdovirus). Members of the vesi- crab viruses were found in blue crab Callinectes culovirus group include RC (rhabdovirus carpio), PFV sapidus (Jahromi 1977, Yudin & Clark 1978, Johnson (pike fry rhabdovirus), EVEX (eel virus European X), 1984). Studies on these crustacean rhabdoviruses are and EVA (eel virus of America). The RPS represents limited and little is known about their biochemical and the first rhabdovirus isolate from cultured penaeid biological properties. Recently a new crustacean rhab- shrimp; it would be of interest to characterize its viral dovirus named rhabdovirus of penaeid shrimp (RPS) structural proteins and viral RNA, and to assign its was isolated from cultured penaeid shrimp (Lu et al. genotype. 1991) and some biological characteristics of this shrimp rhabdovirus have been described (Lu & Loh 1992, Lu et al. 1994). MATERIALS AND METHODS Rhabdoviruses isolated from vertebrates are mainly composed of 4 to 5 structural proteins (Kang & Prevec Cell cultures and viruses. In this study 2 cell lines, 1970, Mudd & Summers 1970, Wagner et al. 1970). epithelioma papulosum cyprini (EPC) and buffalo green O Inter-Research 1994 188 Dis. aquat. Org. 19: 187-192, 1994 monkey kidney (BGMK),were employed. Monolayers lose membranes (Bio-Rad) using a modified method of of EPC cells were grown in Eagle's minimum essential Towbin et al. (1979) at 150 mA for 1.5 h in a home- medium (MEM) supplemented with 10 % fetal bovine made electrophoretic transfer apparatus. The nitrocell- serum (FBS), 100 IU ml-' penicillin and 100 pg ml-' ulose membranes were blocked with 5 % (w/v) nonfat streptomycin in tissue culture grade plastic container dried milk (Carnation, Los Angeles, CA) in phosphate- at 20°C. The BGMK cells were grown at 37 "C in a mix- buffered saline (PBS), pH 7.4, for 1 h or overnight at ture of 2 media, 2x Leibovitz medium (L-15) and lx 4°C and then rinsed twice in 0.05% Tween-20/PBS MEM, plus the same supplements as described above. and once with PBS only, at 5 min per wash. After incu- RC and IHNV provided by Professors J. L. Fryer and bation with rabbit anti-RPS IgG diluted in 1 % (w/v) J. C. Leong, Oregon State University, Corvallis, Oregon, nonfat milk/PBS for 1 h, the membranes were washed USA, and VHSV received from Dr J. R. Winton, National 3 times as above and then treated with secondary anti- Fisheries Research Center, Seattle, Washington, USA, body, goat anti-rabbit IgG (H + L) conjugated with were grown in EPC cells. VSV (vesicular stomatitis peroxidase (Kirkegaard & Perry Laboratories, Inc., virus), Indiana strain, received from Dr Sandra Ho- Gaithersburg, MD, USA) diluted in 1 % nonfat rnilk/PBS rakami, University of Florida, Gainesville, Florida, USA, for another 1 h. The membranes were washed again -..-.P m- ;m RCh4U 0c.11.z YVUJ ylO~llllll uul-14%Lbrrd. and pr~teinbads on th~membranes were visualized Virus purification. Monolayer cultures of EPC cells by incubating in a freshly prepared DAB substrate grown in 150 cm2 tissue culture grade plastic flasks [0.5 mg ml-' 3,3'-diaminobenzidine tetrahydrochloride (Corning) were infected with the different viruses at a with 0.02 % (v/v) H202in PBS] for approximately 90 S. multiplicity of infection of 0.01 to 0.1 pfu cell-'. Preparation of viral RNA. Viral RNA was extracted Rhabdovirus of penaeid shrimp and RC-infected cul- by the method of Kelly (1972). Briefly, purified virus tures were incubated at 20 "C while IHNV- and VHSV- was mixed with pronase E (Sigma) at a final concentra- infected cultures were incubated at 18"C, and VSV- tion of 50 pg ml-' in a 1.5 m1 microcentrifuge tube infected cuitures were held at 37°C. With the total (Fisher Srand, Fisher Scientific Allied Co.). The mix- destruction of the entire cell monolayer (usually 2 to ture was incubated at 37 "C for 1 h, and then SDS and 3 d postinfection), the infected cultures were frozen reagent grade urea crystals were added to a final con- and thawed once; the virus was concentrated and puri- centration of 1 % (v/v) and 4 M, respectively. The mix- fied using the protocol described previously (Lu et al. ture was then incubated for 10 min at 37°C. The SDS 1991).A minor modification was further purification of was removed by precipitation with 2 % (v/v) potassium the banded virus by sucrose gradients: 1 discontinuous acetate for 20 min in the cold, followed by slow speed (20 to 50%, w/v) and 2 continuous (10 to 50%, w/v) centrifugation at 1000 X g for 10 min. The recovered sucrose gradients. Purity of the finally banded viruses aqueous phase containing RNA was then transferred was evaluated by transmission electron microscopy to a fresh tube, mixed with 3 volumes of cold 100% and the virus stored at -70°C. Concentrations of viral ethanol, and precipitated overnight at -20°C. The protein and RNA were determined by the methods of RNA was recovered by sedimentation at 10 000 X g for Ehresmann et al. (1973). 10 min at 4 "C and was resuspended in a small volume Polyacrylamide gel electrophoresis. Analysis of viral of lx Tris-acetate-EDTA buffer (0.04 M Tris, 0.02 M structural proteins on SDS-PAGE was conducted using Na-acetate, and 0.002 M di-Na EDTA, pH 7.8). a 4 % stacking gel and 10 % resolving slab gel (Laemrnli Electrophoresis of RNA. A vertical slab unit SE400 1970) prepared as described in a manual by Hoefer (Hoefer Scientific Instruments), an apparatus routinely Scientific Instruments (San Francisco, CA, USA). Viral used for protein analysis, was adopted for the purpose proteins were dissociated by mixing an equal volume of analyzing the viral genomic RNA. Electrophoresis of of the purified virus and 2x denaturing buffer (0.125 M RNA was carried out in slab gels consisting of 2 % poly- Tris, pH 6.8, 4% SDS, 20% glycerol, and 10% mer- acrylamide (0.1% bisacrylamide) and 0.6% agarose. captoethanol), heated in boiling water for 1.5 min, and The gels were prepared by mixing 12.5 m1 8% acryl- then chilled in ice. Following the separation of con- amide, 25 m1 of 2x Tris-acetate-EDTA buffer (0.08 M stituent proteins by electrophoresis at 30 mA/200 V for Tris, 0.04 M Na-acetate, and 0.004 M di-Na EDTA), 3.5 to 4 h, gels were stained with silver nitrate and the and 12.5 m1 2.4 % agarose (Ultrapure Agarose, electro- molecular weights of the viral proteins were deter- phoresis grade, Bethesda Research Laboratories, mined by comparing their relative mobility with inter- Gaithersburg, MD, USA). The slab gels were allowed nal SDS-PAGE molecular weight standards (Bio-Rad to polymerize overnight. The extracted viral RNA Laboratories, Richmond, CA, USA). samples were mixed with equal volume of gel-dye Western blot analysis. Following the separation of solution (0.1% Bromophenol blue, 50% sucrose, and viral constituent proteins by SDS-PAGE, the banded 0.1 M EDTA, pH 6.8).RNA molecular-weight marker I1 proteins were immediately transferred to nitrocellu- (Boehringer, Mannheim, Germany) was used to esti- Lu & Loh: Shnmp rhabdovlrus mate the relative mobility of the viral RNAs. Electro- Table 1. Estimated molecular weights of viral structural pro- phoresis was carried out in lx Tris-acetate-EDTA teins of the viruses RPS, VSV, RC, IHNV, and VHSV based on buffer for 2.5 to 3 h at 150 V/68 + 1 mA.