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Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumours of pleuronectes
VIROLOGY 126, 466-479 (1983)
Analysis of the Genome of Fish Lymphocystis Disease Virus Isolated Directly from Epidermal Tumours of Pleuronectes GHOLAMREZA DARAI, *'1 KERSTIN ANDERS, *'2 HANS-GEORG KOCH,* HAJO DELIUS,t HANS GELDERBLOM,$ C. SAMALECOS,~ AND ROLF M. FLUGELw *Institut fiir Medizinische Virologie der Universitdt Heidelberg, Im Neuenheimer Feld 324, Heidelberg, Federal Republic of Germany, tEuropean Molecular Biology Laboratory, Heidelberg, West Germany, $Robert-Koch-Institut des Bundesgesundheitsamtes, West Berlin, and w fiir Virusforschung am Deutschen Krebsforschungszentrum, Heidelberg, Federal Republic of Germany Received May 3, 1982," accepted December 7, 1982 Virions of fish lymphocystis disease virus (FLDV), a m e m b e r of the iridovirus family, were isolated directly f r o m lymphocystis disease lesions of individual flatfishes and purified by sucrose and s u b s e q u e n t cesium chloride g r a d i e n t centrifugation to homogeneity as judged by electron microscopy. The isolated FLDV D N A s a p p e a r to be heterogeneous in size. Contour length m e a s u r e m e n t s of 43 DNA molecules gave an average length of 49 _+ 23 t~m, corresponding to 93 _+ 44 • 106 D. Molecular w e i g h t e s t i m a t i o n s of FLDV DNA by restriction enzyme analysis resulted in only 64.8 • 106 D indicating an excess length of the DNA of about 50%. FLDV DNA w a s sensitive to l a m b d a 5'-exonuclease and to E. coli 3'-exonuclease I I I w i t h o u t preference of any one t e r m i n a l DNA restriction fragment. D e n a t u r a t i o n and reannealing e x p e r i m e n t s of FLDV DNA resulted in the f o r m a t i o n of circular DNA molecules of 34.25 #m contour length (= 65.22 • 106 D). This result suggests t h a t FLDV D N A contains directly repeated sequences at both ends and t h a t it is terminally redundant. FLDV DNA is methylated in cytosine. FLDV DNA did not hybridize with frog virus DNA indicating t h a t the two iridoviruses are not closely related to each other. Restriction enzyme a n a l y s i s and S o u t h e r n blot hybridizations revealed t h a t FLDV isolates can be classified into two different strains: FLDV s t r a i n 1 occurs in flounders and plaice, w h e r e a s s t r a i n 2 is usually found in lesions of dabs. INTRODUCTION
pects. Although FLDV has a DNA genome, it is assembled in the cytoplasma of cells (Templeman, 1965), a feature shared by several other viruses, such as certain frog and insect viruses and African swine fever virus. Thus, FLDV has been classified as belonging to the family of iridoviridae (Matthews, 1979). LD frequently appears in pleuronectidae (flatfish): Pleunectes platessa (plaice), Platichtys flesus (flounder), Limanda limanda (dab), and Trigla gurnardus (gurnard). LD can be induced experimentally in Lepomis macrochirus (bluegill) (Dunbar and Wolf, 1966) and by subdermal injection of plaice and flounder (Russell 1974). Since the discovery of LD in 1874 by Lowe, attempts have been made to isolate (Wolf et al., 1966) and propagate FLDV in vivo and in vitro with limited
Lymphocystis disease (LD), an important fish disease, is characterized by papilloma-like lesions. The tumours are due to enormous hypertrophy of dermal host cells which is induced specifically by fish lymphocystis disease virus (FLDV) (Lopez et al., 1969). The mechanisms of this nonmalignant transformation and tumour induction are unknown. As a first step towards understanding the underlying mechanisms, the structure and properties of the causal virus must be elucidated. FLDV has many interesting biological as1 To w h o m r e p r i n t r e q u e s t s should be addressed. 2 P r e s e n t address: I n s t i t u t fiir H a u s t i e r k u n d e der Christian-Albrechts-Universit~it Kiel, Kiel, West Germany. 0042-6822/83 $3.00 Copyright 9 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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DNA ANALYSIS OF FISH IRIDOVIRUS
success (Wolf, 1966; Wolf and Quimby, 1973; Walker and Hill, 1980). The polypeptide composition of FLDV and a virion-associated ATPase activity was recently described (Flfigel et al., 1982). This is the first report on the FLDV DNA structure and properties of the causal agent of LD. MATERIALS AND METHODS
Virus isolation and purification. Virions of FLDV were isolated from lymphocystis tissues and purified according to published methods (Parr et al., 1977) which were modified as follows: FLDV was isolated from 1 to 5 g of lymphocystis tissues of freshly caught fish by homogenisation of lymphocystis material in TNE buffer (0.05 M Tris-HC1, 0.001 M EDTA, 0.1 M NaC1, pH 7.4) at 4 ~ The suspension was clarified from cell debris by centrifugation at 3000 rpm for 20 min at 4 ~ The s uper na t a nt was centrifuged again under the same procedure. The cell-free s u p e r n a t a n t was centrifuged through a 30% sucrose (w/w) cushion in a Spinco SW27 rotor at 25,000 rpm for 120 min at 4 ~ The virus pellets were resuspended in TNE buffer and layered on to 35-ml gradients of 25 to 60% (w/w) sucrose and recentrifuged for 20 hr in a Spinco SW27 rotor at 25,000 rpm at 4 ~. The virus band was collected and centrifuged in a CsC1 gradient (10-35% w/w) for 24 hr in a Spinco SW41 rotor at 30,000 rpm at 10 ~ The virus band in the middle of the gradient was harvested, dialysed against TNE, and used for negative-staining a n d / o r extraction of viral DNA. Electron microscopy. Lymphocystis tissue specimens from flounder, plaice, and dabs were fixed in glutaraldehyde immediately after excision and processed for thin-section electron microscopy as described earlier (Gelderblom et al., 1967). The purity of the virus preparations was examined by a negative-staining technique (Gelderblom et al., 1974). For electron microscopic length measurements the DNA was spread with cytochrome c using the formamide spreading technique described by Davis et al. (1968). The DNA was spread from a hyperphase containing
467
30% formamide, 0.1 M Tris-HC1, pH 8.5, 1 mM EDTA onto a hypophase containing 10% formamide, 10 mM Tris-HC1, pH 8.7, 1 mM EDTA. Samples were picked up on Parlodion-covered copper grids, stained with uranyl acetate, and rotary-shadowed with platinum. Relaxed, circular PM 2 and T7 DNA were used as internal length references. For the circularization of the single-stranded DNA a 50-#1 aliquot of FLDV DNA (4 #g/1 ml) in 0.01 M Tris-HC1, pH 7.4, 1 m M E D T A , and 60% formamide was denatured by immersing the tube into boiling water for 90 sec. After addition of 2 M CsC1 to a final concentration of 0.2 M, the sample was incubated for 30 min at 37 ~. An aliquot was spread from 30% formamide onto a 10% formamide hypophase as described above. Thermal denaturation. The melting temperature of FLDV DNAs was determined in a Gilford Model 250 spectrophotometer equipped with thermal cuvettes and a t h e r m o p r o g r a m m e r as described previously (Darai et al., 1980). Buoyant density analysis. The buoyant density of FLDV DNAs was determined by ultracentrifugation in CsC1 at 44,000 rpm and 25 ~ for 40 hr as described previously (Darai et al., 1980). Enzymes. Restriction endonucleases were purchased from Biolabs (Beverly, Miss.), BRL (Neu-Isenburg, FRG), or Boehringer (Mannheim, FRG). Incubations were carried out according to standard procedure for each enzyme, and the resulting DNA fragments were separated on 0.5 or 0.8% agarose slab gels (Seakem, Biomedical, Rockland, Miss.). Electrophoresis at constant voltage was performed according to Sharp et al. (1973). E. coli 3'exonuclease III and Lambda 5'-exonuclease were purchased from BRL (Neu-Isenburg, FRG). Nick translation. Aliquots (0.5 #g) of FLDV DNAs of flounder and dab and frog virus 3 DNA (kindly provided by Dr. A. Granoff, Memphis, Tenn.) were labeled in vitro according to Rigby et al. (1977). Each sample (25 #l) contained 40 ttCi or [~/-32P]dCTP (New England Nuclear, Dreieich, FRG; sp act 6000 Ci/mmol). Reactions were started by addition of 0.2 units
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D A R A I ET AL.
of D N A p o l y m e r a s e I and 0.1 m u n i t s of D N A s e I, s t o p p e d a f t e r 30 min a n d separ a t e d f r o m n o n i n c o r p o r a t e d molecules over S e p h a d e x G 150 columns w i t h 10 m M NH4HCO3. Blot hybridization. D N A s w e r e cleaved by different r e s t r i c t i o n endonucleases and 0.5 #g w e r e s e p a r a t e d b y 0.6% or 0.8% a g a rose slab gel electrophoresis. The D N A f r a g m e n t s were t r a n s f e r r e d to nitrocellulose sheets as described by S o u t h e r n (1974). F o r h y b r i d i z a t i o n the nitrocellulose sheets were p r e i n c u b a t e d in 6 X SSC, D e n h a r d ' s buffer (0.2% BSA, 0.2% polyvinyl pyrrolidone 360, 0.2% Ficoll 400), and calf t h y m u s D N A 30 t t g / m l for a t l e a s t 4 h r a t 62 ~ A f t e r w a r d s h y b r i d i z a t i o n w a s c a r r i e d out by i n c u b a t i o n of the nitrocellulose sheet in 50% f o r m a m i d e , 5 • SSC w i t h [X-32P]dCTP-labeled d e n a t u r e d D N A s for 24 h r at 42 ~. T h e n the sheets w e r e w a s h e d extensively in 2 • SSC c o n t a i n i n g 0.1% SDS dried at 25 ~ for a few hours, b a k e d at 80 ~ for 30 m i n and exposed to Xr a y films (Kodak XAR-5).
Labeling the 5'-terminus of FLD V DNA. The e x p e r i m e n t s w e r e p e r f o r m e d u s i n g T4 polynucleotide k i n a s e and 20 p m o l [~,-32P]ATP (sp act 6000 C i / m m o l ) according to described p r o c e d u r e s (Chaconis and Van de Sande, 1980).
RESULTS
Electron Microscopy A t o t a l of 30 fish w i t h LD lesions c a u g h t n e a r the D o g g e r b a n k a r e a s w e r e a n a l y s e d individually, including 20 flounders (F1 to F20), six dabs (D1 to D6), and four plaice (P1 to P4). A section of l y m p h o c y s t i s tissue of each species of fish w a s e x a m i n e d by electron microscopy. As shown in Fig. 1, the large n u m b e r of h o m o g e n e o u s v i r u s particles per unit a r e a and the high cont e n t of a p p a r e n t l y i n t a c t virions as s h o w n in Fig. l a is r e m a r k a b l e , since F L D V w a s not p r e p a r e d in cell cultures, b u t f r o m p a p i l l o m a s g r o w n in vivo. Such h o m o g e neous m a s s e s of v i r i o n s were r o u t i n e l y observed in m a n y p r e p a r a t i o n s f r o m different fish species. The following d i a m e t e r s were found for the particles: F L D V - F 227.5
+_ 12.5 rim, F L D V - P 198.8 _+ 12.9 nm, a n d F L D V - D 200.5 _+ 12 n m (Fig. l a and c).
D N A Analysis and Properties The D N A was e x t r a c t e d f r o m purified F L D V particles as described p r e v i o u s l y ( D a r a i et al., 1982). Use of h o m o g e n e o u s virions as the s t a r t i n g m a t e r i a l was essential for a final F L D V D N A p r e p a r a t i o n of high purity. The yield of viral D N A w a s a b o u t several h u n d r e d m i c r o g r a m s f r o m each t u m o r . The p r e p a r a t i o n s h a d a r a t i o of a b s o r b a n c e at 260 a n d 280 n m of 1.9 to 2.0. Isopycnic CsC1 g r a d i e n t c e n t r i f u g a t i o n resulted in one h o m o g e n e o u s p e a k of v i r a l DNA. No D N A p e a k w a s detectable at the b u o y a n t density of cellular DNA. T h e b u o y a n t density of F L D V D N A was f o u n d to be p = 1.690 g • m1-1. The c o r r e s p o n d i n g values of a G + C c o n t e n t which was calculated according to S c h i l d k r a u t et al. (1962) w a s in a g r e e m e n t with the value o b t a i n e d f r o m the Tm m e a s u r e m e n t . Det e r m i n a t i o n of the u v - a b s o r b a n c e - t e m p e r a t u r e profile of the D N A in 0.1 • SSC resulted in a Tm value of 65.5 _+ 0.8 ~ This c o r r e s p o n d s to a (G + C) content of the D N A of 30.7 + 1.7%.
Restriction Endonuclease Analysis In o r d e r to identify a n d c h a r a c t e r i s e the F L D V g e n o m e s D N A s of different F L D V isolates w e r e cleaved w i t h r e s t r i c t i o n endonucleases and the r e s u l t i n g D N A f r a g ments were separated electrophoretically on a g a r o s e slab gels. R e p r e s e n t a t i v e results of this a n a l y s i s a r e given in Fig. 2A to F. F i g u r e 2A shows the BstEII cleavage p a t t e r n s of F L D V D N A isolated f r o m individual flounders (lane 1 and 2), plaice (lane 3) and dabs (lane 4 to 6). The f r a g ment patterns demonstrate that FLDV D N A of flounders (F7 a n d F9) and of plaice (P1) are indistinguishable, b u t clearly diff e r e n t f r o m those of dab (D1 to D3). The s a m e r e s u l t was o b t a i n e d for the r e m a i n der of all o t h e r flounders tested, t h r e e plaice, and t h r e e dabs, which were separ a t e l y and individually a n a l y s e d ( d a t a not shown). The cleavage p a t t e r n s in Fig. 2B and E, o b t a i n e d a f t e r digestion of the s a m e
D N A A N A L Y S I S OF F I S H I R I D O V I R U S
FIG. 1. Electron m i c r o g r a p h s of fish l y m p h o c y s t i s disease virus; (a) T h e lower m i c r o g r a p h of the u l t r a t h i n section exemplifies t h e h u g e a m o u n t of v i r u s p a r t i c l e s p r e s e n t in an e p i d e r m a l t u m o r cell of a lesion of l y m p h o c y s t i s disease (magnification: X9000). (b) T h e fine s t r u c t u r e of t h e intact complex virion is best revealed in u l t r a t h i n section. A fringe of p r o t r u s i o n s a b o u t 2.5 n m in l e n g t h s u r r o u n d s t h e hexagonal, m e m b r a n e - b o u n d particle. The envelope s t r u c t u r e c o n s i s t s of two layers, an o u t e r u n i t m e m b r a n e s t r u c t u r e a n d an i n n e r m o r e electro-dense layer closely a t t a c h e d to t h e f o r m e r . T h e concentric core inside t h e virions is s e p a r a t e d f r o m t h e composite m e m b r a n e by a broad e l e c t r o n - t r a n s l u c e n t region (magnification: X130,000). (c) N e g a t i v e s t a i n i n g of F L D V virions; in f a i n t l y s t a i n e d r e g i o n s a f r i n g e on t h e virion s u r f a c e is visible. D u e to osmotic shock t h e o u t e r m e m b r a n e of t h e virion is broken a n d also p a r t l y dissociated into two layers, while t h e core s e e m s to r e m a i n c o m p a c t a n d stable (magnification: X130,000).
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FIG. 2. Agarose slab gel electrophoresis of fish lymphocystis disease virus DNAs; FLDV DNAs (1 ttg) of each species were digested with restriction endonucleases BstEII (A), EcoRI (B), PstI (C), BglII (D), BamHI (E), and HpaII, and MspI (F). The resulting DNA fragments were separated in 0.5% (A, B, C, and E) and in 0.8% (D and F) agarose slab gels (35 X 20 X 0.3 cm) and run at 75 V for 18 hr (A, B, D, E, and F) and/or 24 hr (C) in electrophoresis buffer at 4 ~ Lanes of A, B, and E: 1, FDLV-F7 DNA; 2, FLDV-F9 DNA; 3, FLDV-P1 DNA; 4, FLDV-D1 DNA; 5, FLDV-D2 DNA; 6, FLDV-D3 DNA. Lane 1 in B, 10 #g FLDV-F7 DNA were used for demonstration of the fragment. (C) Lanes: 1, FLDV-F7 DNA; 2, FLDV-F9 DNA; 3, FLDV-P1 DNA; 4, FLDV-D1 DNA; 5, FLDVD2 DNA; and 6, FLDV-D3 DNA. (D) Lanes: 1, FLDV-F12 DNA; 2, FLDV-F4 DNA; 3, FLDV-F2 DNA; 4, FLDV-F1 DNA; 5, FLDV-F7 DNA; 6, FLDV-F9 DNA; 7, FLDV-P DNA; 8, FLDV-D1 DNA; 9, FLDV-D1 DNA; and 10, FLDV-D3 DNA. M represents positions of EcoRI DNA fragments of lambda DNA which served as internal marker. (F) Cleavage pattern of different FLDV DNAs after digestion (1 ttg) with HpaII enzyme (lane 1 to 4) and MspI enzyme (lane 5 to 8). Lanes: 1 and 5, FLDV-D1 DNA; 2 and 6, FLDV-P1 DNA; 3 and 7, FLDV-F12 DNA; 4 and 8, lane M: lambda DNA digested with BglII as internal marker.
DNA ANALYSIS OF FISH IRIDOVIRUS
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F L D V D N A s as in Fig. 2A w i t h E c o R I (2B) and B a m H I (2E) respectively, confirm t h e results described above for the B s t E I I enzyme. A v a r i a t i o n in the r e s t r i c t i o n p a t t e r n of individual fish of one species (e.g., flounder) w a s not found w h e n these t h r e e e n z y m e s w e r e used. The s a m e holds t r u e
w h e n flounder F L D V D N A and plaice F L D V D N A w e r e cleaved w i t h the PstI enzyme (Fig. 2C). The dab F L D V D N A p a t t e r n s of individual dabs w e r e indistinguishable f r o m each o t h e r (Fig. 2C, lanes 4, 5, and 6), but different f r o m those of flounders and plaice (lane 1 to 3). Accord-
472
DARAI ET AL. flounder and dab viral D N A s hybridize to each o t h e r and thus seem to be closely related. The molecular w e i g h t of F L D V of different isolates calculated f r o m the sizes of individual D N A f r a g m e n t s obtained after electrophoretic s e p a r a t i o n of digested F L D V D N A s with a v a r i e t y of r e s t r i c t i o n e n z y m e s was found to be 64.8 • 106 D as shown in Table 1.
Structure o f F L D V D N A
FIG. 3. Reciprocal blot hybridization of 82P-labeled DNAs of FLDV-F16and FLDV-D2to PstI fragments of the same DNAs. (A) [32P]DNAof FLDV-F16 was hybridized to a Southern blot of PstI digest of 0.5 ttg DNA of FLDV-D2(lane 1) and 0.5 ttg DNA of FLDVF16 (lane 2). (B) [s2P]DNAof FLDV-D2 was hybridized to a Southern blot of PstI digests of 0.5 t~g DNA of FLDV-D2 (lane 1) and 0.5 ttg DNA of FLDV-F16 (lane 2). Time of exposure was 24 hr for A and B, and C is the blot shown in B exposed for 6 days.
ing to these results two different s t r a i n s of F L D V exist; F L D V s t r a i n 1 is found in flounders and plaice, w h e r e a s F L D V s t r a i n 2 is usually associated with dabs inflicted with LD. This result is in a g r e e m e n t with the F L D V polypeptide p a t t e r n s which distinguish flounder and plaice f r o m the pattern of dab (Fliigel et al., 1982). To ascertain these results, D N A s of F L D V s t r a i n 1 and 2 were s e p a r a t e l y 32p_ labeled by nick t r a n s l a t i o n a c c o r d i n g to Ribgy et al., (1977), hybridized by the S o u t h e r n blot m e t h o d to nonradioactive, PstI-digested D N A of F L D V of F16 and D2, reciprocally. The resulting a u t o r a d i o g r a p h s of Fig. 3A, B, and C show t h a t
In o r d e r to d e t e r m i n e the t e r m i n a l f r a g m e n t s of F L D V DNA, purified D N A was first digested with either the },-5'-exonuclease or the 3'-exonuclease I I I of E. coli followed by digestion with the restriction e n z y m e BstEII. The D N A cleavage patterns shown in Fig. 4A and B do not show a selective digestion of a p a r t i c u l a r f r a g m e n t instead a g r a d u a l d i s a p p e a r a n c e of all viral D N A f r a g m e n t s is observed. Exp e r i m e n t s in which B a m HI, Pst I or BgIII enzymes were used, for either of the two exonucleases gave the same results. Exp e r i m e n t s were p e r f o r m e d using the guanidinium h y d r o c h l o r i d e m e t h o d as described previously by Robinson et aL (1973). No evidence for a t e r m i n a l l y bound D N A protein was found. A n o t h e r m e t h o d to search for t e r m i n a l f r a g m e n t s was employed, n a m e l y labeling the 5'-termini of F L D V D N A by T4 polynucleotide kinase with [~/-32P]ATP followed by digestion with different restriction endonucleases and s u b s e q u e n t electrophoretic s e p a r a t i o n on agarose gels. As shown in Fig. 5 it was found t h a t radioactive p h o s p h a t e was inc o r p o r a t e d into most D N A f r a g m e n t s ind e p e n d e n t of the restriction enzyme used.
Electron Microscopy o f F L D V D N A Purified viral D N A molecules of different F L D V isolates were examined in the electron microscope using c y t o c h r o m e c s p r e a d i n g (Davis et al., 1971). An average length of 49 • 23 # c o r r e s p o n d i n g to a b o u t 93 • 44 • 106 D was found by m e a s u r i n g 43 molecules. The c o m p a r i s o n between the molecular weight values obtained by restrict• enzyme analysis and c o n t o u r length m e a s u r e m e n t s indicates t h a t the
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FIG. 4. Lambda 5'-exonuclease and E. coli 3'-exonuclease-restriction endonuclease digest of fish lymphocystis disease virus DNA. Agarose slab gel electrophoresis of FLDV DNAs were treated with lambda exonuclease a n d / o r exonuclease III for 2 and 30 min and thereafter cleaved with restriction endonuclease B s t E I I and run under the same conditions and in the same gel in comparison to untreated DNA: (B) Lane 2, 1 ttg of FLDV-F12 DNA digested with BstEII; lane 1, BstEII cleavage pattern of 1 #g of the same DNA which was pretreated with lambda exonuclease. Lane 3 and 4, control experiment using three shrew herpesvirus (THV-2) DNA (Darai et al., 1981) under the same conditions. THV-2 DNA was digested with EcoRI. The white arrows in lane 4 label the two terminal factors (H and J) which disappeared after a 5 min t r e a t m e n t with lambda exonuclease (lane 3). (A) Lane 2, FLDV-F16 DNA (1 ttg) digested with BstEII. Lane 1 and 3, B s t E I I digests of aliquots of the same DNA after t r e a t m e n t with lambda exonuclease for 2 min (lane 1) and for 30 min (lane 3). Lane 5, FLDV-F16 DNA (0.5 #g) digested with BstEII. Lane 4 and 6, B s t E I I digests of aliquots of the same DNA after t r e a t m e n t with exonuclease III for 2 min (lane 4) and for 30 min (lane 6). Phage lambda DNA (2 t~g) digested with H i n d I I I served as marker (M).
DNA molecules are on the average longer than expected from the restriction enzyme analysis. While in most preparations FLDV DNA contained so many nicks that only small
single-stranded fragments were obtained, two separate preparations from two different isolates (FLDV-F16, F17) yielded DNA single strands approaching in length the sizes measured for the double-stranded
DNA ANALYSIS OF FISH IRIDOVIRUS DNA. In these p r e p a r a t i o n s it was possible to observe circular D N A molecules a f t e r d e n a t u r a t i o n / r e n a t u r a t i o n . These circles were p a r t i a l l y single s t r a n d e d and m i g h t show, e.g., as in the molecule s h o w n in Fig. 6, s i n g l e - s t r a n d e d D N A tails w i t h i n the double-stranded region. Three circles could be m e a s u r e d and had an a v e r a g e molecular w e i g h t of 65.22 • 106 D. F i g u r e 6 represents a single s t r a n d of m o r e t h a n unit size being circularized by a f r a g m e n t of D N A c o m p l e m e n t a r y to sequences on both sides of the a t t a c h m e n t point of the extra D N A tails. These tails are s e p a r a t e d by one unit length of the F L D V genome. The small n u m b e r of i n t a c t D N A molecules obtained is p r o b a b l y due to the fact t h a t the F L D V D N A was isolated f r o m virions g r o w n in vivo for an u n k n o w n period of time. In addition, the p r o b a b i l i t y of circle f o r m a t i o n could be considerably reduced by the relatively large e x t e n t of the overall redundancy.
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F L D V D N A is Methylated and N o t Closely Related to Frog Virus 3 D N A F L D V D N A s were digested with the H p a I I (C~CGG) and MspI (c~MeCGG) enzymes; the l a t t e r cleaves D N A in the presence of a 5-methyl g r o u p at the internal cytosine residue in the recognition sequence of the H p a I I enzyme (Waalwijk and Flavell, 1978). As shown in Fig. 2F, lanes 5, 6, and 7, F L D V D N A f r o m dab, plaice, and flounder were cut m a n y times with MspI indicating t h a t the i n t e r n a l C in the recognition sequence of this enzyme is heavily m e t h y l a t e d , since the H p a I I enzyme leaves the F L D V D N A s i n t a c t (Fig. 2F, lanes 1, 2, and 3). A similar result was obtained by Willis and G r a n o f f (1980) for frog virus DNA. I t was of i n t e r e s t to s t u d y the relatedness of both these viral D N A s to each other. FV 3 D N A was labeled with 32p as described in Materials a n d Methods and hybridized a g a i n s t unlabeled FLDV D N A f r o m different fish species by the S o u t h e r n blot m e t h o d (1975). As a control FV 3 D N A was included in these experiments. As shown in Fig. 7, no h o m o l o g y
FIG. 5. Autoradiograph of 82p-5'-labeled FLDV-F12 DNA digested with different restriction endonucleases: lane 1 - PstI, lane 2 = BstEII, lane 3 = Bali, lane 4 - BamHI. Lane 1 to 4, 1 ttg unlabeled FLDVF12 DNA containing 0.5 #g 32P-5'-labeled FLDV-F12 DNA (5 • 108 cpm) (lane 1 to 4). Phage lambda DNA digested with BglII (fragment A, B, C, and D) served as marker (5). Time of exposure was 7 days.
between the D N A s of FV 3 and FLDV from various isolates (flounder, dab, or plaice) was detectable u n d e r the conditions used. DISCUSSION The analysis of the genome of F L D V as reported here shows t h a t the F L D V genome is a double s t r a n d e d and linear D N A molecule of 4 9 _ 12 #m in length corresponding to a molecular weight of 93 + 44 • 106 D. The exceptionally wide r a n g e in molecular weight could reflect the fact t h a t
FIG. 6. Electron m i c r o g r a p h of a circular F L D V - F 1 6 D N A molecule g e n e r a t e d by d e n a t u r a t i o n a n d r e n a t u r a t i o n as described in M a t e r i a l s a n d M e t h o d s . The a r r o w s p o i n t to t h e excess single s t r a n d s . The t r i a n g l e s m a r k t h e t r a n s i t i o n points b e t w e e n single- a n d d o u b l e - s t r a n d e d regions. 476
D N A A N A L Y S I S OF F I S H I R I D O V I R U S
viral DNA was directly isolated from FLDV grown in vivo for an unknown, but probably long period of time which could lead to degradation of DNA. Besides, single- and double-stranded DNA degrading enzymatic activities were detectable in purified virions of FLDV (R.M.F., unpublished observations), The molecular weight calculated from restriction enzyme data resulted in 64.8 • 10GD which when compared to the value obtained by electron microscopy indicate t h a t FLDV DNA has an average redundancy of approximately 50 %. Denaturation and renaturation of FLDV DNA revealed circular DNA molecules of 34.25 ~m length with two protruding single-stranded ends. This contour length corresponds to a molecular weight of 65.22 • 106 D, in agreement with the value obtained by restriction enzyme analysis. Although circular DNA molecules were formed upon renaturation between two single DNA strands only, the low number of DNA circles is explained by the long concatemer-like structure of the FLDV genome which decreases the probability of circle formation due to its relatively high average redundancy. Taken together, these data indicate that the DNA isolated is highly redundant but to a variable extent. The results of the experiments performed with either the 5'specific exonuclease or 3'-exonuclease and with 5'-polynucleotide kinase are in support of a variable redundancy a n d / o r a possible circularly permuted genome (Thomas, 1970; Tye et aL, 1974). Thus, the DNA structure of FLDV might be similar to t h a t of the genome of frog virus 3, another iridovirus similar to FLDV, which has been reported to be circularly permuted and terminally redundant (Goorha and Murti, 1982). A structural feature common to both FLDV and FV 3 DNA is the methylation in cytosine residues (Willis and Granoff, 1980). As revealed by hybridization experiments no homology was detectable between the DNAs of FLDV and frog virus under the conditions used. Similar hybridization experiments were performed in Dr. Granoff's laboratory in Memphis,
1 23[M]4
477
56
FIG. 7. A u t o r a d i o g r a p h of blot h y b r i d i z a t i o n between f r o g virus 3 (FV 3) D N A a n d fish l y m p h o c y s t i s disease v i r u s (FLDV) D N A of different isolates. [h-82P]dCTP n i c k - t r a n s l a t e d FV 3 D N A w a s h y b r i d ized to S o u t h e r n blots of EcoRI digests of D N A s of FV 3 a n d FLDV DNAs. L a n e s 1, 2, a n d 3, FV 3 DNA; lane 1, 0.06 #g = 10 8 pmol; lane 2, 10 -4 pmol; a n d lane 3, 10 -5 pmol. L a n e 4, F L D V - F 1 2 DNA; lane 5, F L D V D2 DNA; a n d lane 6, F L D V - P 1 DNA. L a n e 4 to 6 c o n t a i n 1 ~g each of D N A (10 -2 pmol). X-s2P-labeled phage l a m b d a D N A (5 • 10a cpm) digested with EcoRI served as i n t e r n a l m a r k e r (M).
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DARAI ET AL.
Tennessee, with the same r e s u l t (personal communication). To establish a gene library, FLDV DNA f r a g m e n t s were inserted into the pAT153 plasmid vector of E. coli. Bacterial colonies h a r b o r i n g r e c o m b i n a n t plasmids were selected. The analysis of the isolated amplified clones is now in progress, and will be of g r e a t help for f u r t h e r molecular and biological studies of fish lymphocystis disease virus, p a r t i c u l a r in view of the fact t h a t FLDV c a n n o t yet be efficiently groWn in tissue culture.
ACKNOWLEDGMENTS We are grateful to Dr. Allan Granoff, Division of Virology, St. Jude Children's Research Hospital, Memphis, Tennessee, for critical comments and to Dr. Rakesh Goorha of t h a t institution for performing hybridization experiments of frog virus 3 and fish lymphocystis disease virus DNA. The authors t h a n k Mrs. B. Holder for preparation of the manuscript.
REFERENCES
CHACONIS,G., and VAN DE SANDE,J. H. (1980). 5'-s2P labeling of RNA and DNA restriction fragments. I n "Methods in Enzymology" (L. Grossmann and K. Moldave, eds.), Vol. 65, pp. 75-85. Academic Press, New York. DARAI, G., MATZ, B., FLUGEL, R. M., GRAFE, A., GELDERBLOM, H., and DELIUS, H. (1980). An adenovirus from tupaia (tree shrew): Growth of the virus, characterization of viral DNA, and transforming ability. Virology 104, 122-138. DARAI, G., FL~GEL, R. M., MATZ, B., and DELIUS, H. (1981). DNA of tupaia herpesviruses. In "Herpesvirus DNA" (Y. Becker, ed.), pp. 345-361. Martinus Nijhoff Publishers, The Hague, Boston, London. DARAI, C., Z(3LLER, L., MATZ, B., DELIUS, H., SPECK, P. T., and FLI~GEL, R. M. (1982). Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy. J. BacterioL 150, 788-794. DAVIS, R. W., SIMON, M., and DAVIDSON, N. (1971). Electron microscopy heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In "Methods in Enzymology" (L. Grossmann and K. Moldave, eds.), Vol. 21, pp. 413-428. Academic Press, New York. DUNBAR, C. E., and WOLF, K. (1966). The cytological course of experimental lymphocystis in bluegill. J. Infect. Dis. 116, 466-472.
FLUGEL, R. M., DARAI, G., and GELDERBLOM, H. (1982). Viral proteins and adenosine triphosphate phosphohydrolase activity of fish lymphocystis disease virus. Virology 122, 48-55. GELDERBLOM, H., BAUER, H., FRANK, H., and WIGAND, R. (1967). The structure of group II adenoviruses. J. Gen. ViroL l, 553-560. GELDERBLOM, H., OGURA, H., and BAUER, H. (1974). On the occurrence of oncornavirus-like particles in HeLa cells. Cytobiology 8, 339-344. GOORHA, a., and MURTI, K. G. (1982). The genome of frog virus 3, and animal DNA virus, is circularly permuted and terminally redundant. Proc. Nat. Acad. Sci. USA 79, 248-252. LOPEZ, D. M., SIGEL, M. M., BEASHLEY, A. R., and DIETRICH, L. S. (1969). Biochemical and morphological studies of lymphocystis disease. Nat. Cancer Inst. Monogr. 31,223-236. LOWE, K. (1874). Fish disease. IV. Trans. Norfolk and Narwich Nat. Soc. Fishes, 21-56. MATTHEWS, R. E. F. (1979). Classification and Nomenclature of Viruses. "Third Annual Report of the International Committee on Taxonomy and Viruses" pp. 174-176. Karger, Basel. MURTI, K. G., GOORHA, R., and GRANOFF, A. (1982). Structure of frog virus 3 genome: Size and arrangement of nucleotide sequences as determined by electron microscopy. Virology 116, 275-283. PARR, R. P., BURNETT, J. W., and GARON, C. F. (1977). Structural characterization of the Molluscum contangiosum virus genome. Virology 81, 247-256. RIGBY, P. W. J., DIECKMANN,M., RHODES, C., and BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. MoL Biol. 114, 237-251. ROBINSON, A. J., YOUNGHUSBAND, H. B., and BELLET, A. J. D. (1973). A circular DNA protein complex from adenoviruses. Virology 56, 54-69. RUSSELL, P. H. (1974). Lymphocystis in wild plaice Pleuronectes platessa (L.) and flounder, Platichthys flesus (L.), in British coastal waters: A histopathological and serological study. J. Fish Biol. 6, 771-778. SCHILDKRAUT, C. L., MARMUR, J., and DOTY, P. (1962). Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsC1. J. Mol. Biol. 4, 430-443. SHARP, P. A., SUGDEN, B., and SAMBROOK, J. (1973). Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055-3061. SOUTHERN, E. U. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioL 98, 503-517. TEMPLEMAN, W. (1965). Lymphocystis disease in American plaice of the Eastern Grand Bank. J. Fish. Res. Board CanacL 22, 1345-1355.
DNA ANALYSIS OF FISH IRIDOVIRUS THOMAS, C. A., JR. (1970). The rule of the ring. J. Cell PhysioL Suppl. 1, 13-34. TYE, B. K., HUBERMAN, J. A., and BOTSTEIN, D. (1974). Nonrandom circular permutation of phage P22 DNA. J. Mol. Biol. 85, 501-532. WAALWIJK, C., and FLAVELL, R. A. (1978). Msp I, an isoschizomer of Hpa II which cleaves both unmethylated and methylated Hpa II sites. Nucleic Acids Res. 5, 3231-3236. WALKER, D. P., and HILL, B. Z. (1980). Studies on the culture, assay of infectivity and some in vitro properties of lymphocystis virus. J. GeE. Virol. 51,385395.
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WILLIS, D. B., and GRANOFF, A. (1980). Frog virus 3 DNA is heavily methylated at CpG sequences. Virology 107, 250-257. WOLF, K. (1966). The fish viruses. Adv. Virus Res. 12, 36-98. WOLF, K., GRAVELL, M., and MALSBERGER, R. G. (1966). Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science 151, 1004-1005. WOLF, K., and QUIMBY, M. C. (1973). Fish viruses: buffers and methods for plaquing eight agents under normal atmosphere. Appl. Microbiol. 25, 659664.
Analysis of the Genome of Fish Lymphocystis Disease Virus Isolated Directly from Epidermal Tumours of Pleuronectes GHOLAMREZA DARAI, *'1 KERSTIN ANDERS, *'2 HANS-GEORG KOCH,* HAJO DELIUS,t HANS GELDERBLOM,$ C. SAMALECOS,~ AND ROLF M. FLUGELw *Institut fiir Medizinische Virologie der Universitdt Heidelberg, Im Neuenheimer Feld 324, Heidelberg, Federal Republic of Germany, tEuropean Molecular Biology Laboratory, Heidelberg, West Germany, $Robert-Koch-Institut des Bundesgesundheitsamtes, West Berlin, and w fiir Virusforschung am Deutschen Krebsforschungszentrum, Heidelberg, Federal Republic of Germany Received May 3, 1982," accepted December 7, 1982 Virions of fish lymphocystis disease virus (FLDV), a m e m b e r of the iridovirus family, were isolated directly f r o m lymphocystis disease lesions of individual flatfishes and purified by sucrose and s u b s e q u e n t cesium chloride g r a d i e n t centrifugation to homogeneity as judged by electron microscopy. The isolated FLDV D N A s a p p e a r to be heterogeneous in size. Contour length m e a s u r e m e n t s of 43 DNA molecules gave an average length of 49 _+ 23 t~m, corresponding to 93 _+ 44 • 106 D. Molecular w e i g h t e s t i m a t i o n s of FLDV DNA by restriction enzyme analysis resulted in only 64.8 • 106 D indicating an excess length of the DNA of about 50%. FLDV DNA w a s sensitive to l a m b d a 5'-exonuclease and to E. coli 3'-exonuclease I I I w i t h o u t preference of any one t e r m i n a l DNA restriction fragment. D e n a t u r a t i o n and reannealing e x p e r i m e n t s of FLDV DNA resulted in the f o r m a t i o n of circular DNA molecules of 34.25 #m contour length (= 65.22 • 106 D). This result suggests t h a t FLDV D N A contains directly repeated sequences at both ends and t h a t it is terminally redundant. FLDV DNA is methylated in cytosine. FLDV DNA did not hybridize with frog virus DNA indicating t h a t the two iridoviruses are not closely related to each other. Restriction enzyme a n a l y s i s and S o u t h e r n blot hybridizations revealed t h a t FLDV isolates can be classified into two different strains: FLDV s t r a i n 1 occurs in flounders and plaice, w h e r e a s s t r a i n 2 is usually found in lesions of dabs. INTRODUCTION
pects. Although FLDV has a DNA genome, it is assembled in the cytoplasma of cells (Templeman, 1965), a feature shared by several other viruses, such as certain frog and insect viruses and African swine fever virus. Thus, FLDV has been classified as belonging to the family of iridoviridae (Matthews, 1979). LD frequently appears in pleuronectidae (flatfish): Pleunectes platessa (plaice), Platichtys flesus (flounder), Limanda limanda (dab), and Trigla gurnardus (gurnard). LD can be induced experimentally in Lepomis macrochirus (bluegill) (Dunbar and Wolf, 1966) and by subdermal injection of plaice and flounder (Russell 1974). Since the discovery of LD in 1874 by Lowe, attempts have been made to isolate (Wolf et al., 1966) and propagate FLDV in vivo and in vitro with limited
Lymphocystis disease (LD), an important fish disease, is characterized by papilloma-like lesions. The tumours are due to enormous hypertrophy of dermal host cells which is induced specifically by fish lymphocystis disease virus (FLDV) (Lopez et al., 1969). The mechanisms of this nonmalignant transformation and tumour induction are unknown. As a first step towards understanding the underlying mechanisms, the structure and properties of the causal virus must be elucidated. FLDV has many interesting biological as1 To w h o m r e p r i n t r e q u e s t s should be addressed. 2 P r e s e n t address: I n s t i t u t fiir H a u s t i e r k u n d e der Christian-Albrechts-Universit~it Kiel, Kiel, West Germany. 0042-6822/83 $3.00 Copyright 9 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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success (Wolf, 1966; Wolf and Quimby, 1973; Walker and Hill, 1980). The polypeptide composition of FLDV and a virion-associated ATPase activity was recently described (Flfigel et al., 1982). This is the first report on the FLDV DNA structure and properties of the causal agent of LD. MATERIALS AND METHODS
Virus isolation and purification. Virions of FLDV were isolated from lymphocystis tissues and purified according to published methods (Parr et al., 1977) which were modified as follows: FLDV was isolated from 1 to 5 g of lymphocystis tissues of freshly caught fish by homogenisation of lymphocystis material in TNE buffer (0.05 M Tris-HC1, 0.001 M EDTA, 0.1 M NaC1, pH 7.4) at 4 ~ The suspension was clarified from cell debris by centrifugation at 3000 rpm for 20 min at 4 ~ The s uper na t a nt was centrifuged again under the same procedure. The cell-free s u p e r n a t a n t was centrifuged through a 30% sucrose (w/w) cushion in a Spinco SW27 rotor at 25,000 rpm for 120 min at 4 ~ The virus pellets were resuspended in TNE buffer and layered on to 35-ml gradients of 25 to 60% (w/w) sucrose and recentrifuged for 20 hr in a Spinco SW27 rotor at 25,000 rpm at 4 ~. The virus band was collected and centrifuged in a CsC1 gradient (10-35% w/w) for 24 hr in a Spinco SW41 rotor at 30,000 rpm at 10 ~ The virus band in the middle of the gradient was harvested, dialysed against TNE, and used for negative-staining a n d / o r extraction of viral DNA. Electron microscopy. Lymphocystis tissue specimens from flounder, plaice, and dabs were fixed in glutaraldehyde immediately after excision and processed for thin-section electron microscopy as described earlier (Gelderblom et al., 1967). The purity of the virus preparations was examined by a negative-staining technique (Gelderblom et al., 1974). For electron microscopic length measurements the DNA was spread with cytochrome c using the formamide spreading technique described by Davis et al. (1968). The DNA was spread from a hyperphase containing
467
30% formamide, 0.1 M Tris-HC1, pH 8.5, 1 mM EDTA onto a hypophase containing 10% formamide, 10 mM Tris-HC1, pH 8.7, 1 mM EDTA. Samples were picked up on Parlodion-covered copper grids, stained with uranyl acetate, and rotary-shadowed with platinum. Relaxed, circular PM 2 and T7 DNA were used as internal length references. For the circularization of the single-stranded DNA a 50-#1 aliquot of FLDV DNA (4 #g/1 ml) in 0.01 M Tris-HC1, pH 7.4, 1 m M E D T A , and 60% formamide was denatured by immersing the tube into boiling water for 90 sec. After addition of 2 M CsC1 to a final concentration of 0.2 M, the sample was incubated for 30 min at 37 ~. An aliquot was spread from 30% formamide onto a 10% formamide hypophase as described above. Thermal denaturation. The melting temperature of FLDV DNAs was determined in a Gilford Model 250 spectrophotometer equipped with thermal cuvettes and a t h e r m o p r o g r a m m e r as described previously (Darai et al., 1980). Buoyant density analysis. The buoyant density of FLDV DNAs was determined by ultracentrifugation in CsC1 at 44,000 rpm and 25 ~ for 40 hr as described previously (Darai et al., 1980). Enzymes. Restriction endonucleases were purchased from Biolabs (Beverly, Miss.), BRL (Neu-Isenburg, FRG), or Boehringer (Mannheim, FRG). Incubations were carried out according to standard procedure for each enzyme, and the resulting DNA fragments were separated on 0.5 or 0.8% agarose slab gels (Seakem, Biomedical, Rockland, Miss.). Electrophoresis at constant voltage was performed according to Sharp et al. (1973). E. coli 3'exonuclease III and Lambda 5'-exonuclease were purchased from BRL (Neu-Isenburg, FRG). Nick translation. Aliquots (0.5 #g) of FLDV DNAs of flounder and dab and frog virus 3 DNA (kindly provided by Dr. A. Granoff, Memphis, Tenn.) were labeled in vitro according to Rigby et al. (1977). Each sample (25 #l) contained 40 ttCi or [~/-32P]dCTP (New England Nuclear, Dreieich, FRG; sp act 6000 Ci/mmol). Reactions were started by addition of 0.2 units
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of D N A p o l y m e r a s e I and 0.1 m u n i t s of D N A s e I, s t o p p e d a f t e r 30 min a n d separ a t e d f r o m n o n i n c o r p o r a t e d molecules over S e p h a d e x G 150 columns w i t h 10 m M NH4HCO3. Blot hybridization. D N A s w e r e cleaved by different r e s t r i c t i o n endonucleases and 0.5 #g w e r e s e p a r a t e d b y 0.6% or 0.8% a g a rose slab gel electrophoresis. The D N A f r a g m e n t s were t r a n s f e r r e d to nitrocellulose sheets as described by S o u t h e r n (1974). F o r h y b r i d i z a t i o n the nitrocellulose sheets were p r e i n c u b a t e d in 6 X SSC, D e n h a r d ' s buffer (0.2% BSA, 0.2% polyvinyl pyrrolidone 360, 0.2% Ficoll 400), and calf t h y m u s D N A 30 t t g / m l for a t l e a s t 4 h r a t 62 ~ A f t e r w a r d s h y b r i d i z a t i o n w a s c a r r i e d out by i n c u b a t i o n of the nitrocellulose sheet in 50% f o r m a m i d e , 5 • SSC w i t h [X-32P]dCTP-labeled d e n a t u r e d D N A s for 24 h r at 42 ~. T h e n the sheets w e r e w a s h e d extensively in 2 • SSC c o n t a i n i n g 0.1% SDS dried at 25 ~ for a few hours, b a k e d at 80 ~ for 30 m i n and exposed to Xr a y films (Kodak XAR-5).
Labeling the 5'-terminus of FLD V DNA. The e x p e r i m e n t s w e r e p e r f o r m e d u s i n g T4 polynucleotide k i n a s e and 20 p m o l [~,-32P]ATP (sp act 6000 C i / m m o l ) according to described p r o c e d u r e s (Chaconis and Van de Sande, 1980).
RESULTS
Electron Microscopy A t o t a l of 30 fish w i t h LD lesions c a u g h t n e a r the D o g g e r b a n k a r e a s w e r e a n a l y s e d individually, including 20 flounders (F1 to F20), six dabs (D1 to D6), and four plaice (P1 to P4). A section of l y m p h o c y s t i s tissue of each species of fish w a s e x a m i n e d by electron microscopy. As shown in Fig. 1, the large n u m b e r of h o m o g e n e o u s v i r u s particles per unit a r e a and the high cont e n t of a p p a r e n t l y i n t a c t virions as s h o w n in Fig. l a is r e m a r k a b l e , since F L D V w a s not p r e p a r e d in cell cultures, b u t f r o m p a p i l l o m a s g r o w n in vivo. Such h o m o g e neous m a s s e s of v i r i o n s were r o u t i n e l y observed in m a n y p r e p a r a t i o n s f r o m different fish species. The following d i a m e t e r s were found for the particles: F L D V - F 227.5
+_ 12.5 rim, F L D V - P 198.8 _+ 12.9 nm, a n d F L D V - D 200.5 _+ 12 n m (Fig. l a and c).
D N A Analysis and Properties The D N A was e x t r a c t e d f r o m purified F L D V particles as described p r e v i o u s l y ( D a r a i et al., 1982). Use of h o m o g e n e o u s virions as the s t a r t i n g m a t e r i a l was essential for a final F L D V D N A p r e p a r a t i o n of high purity. The yield of viral D N A w a s a b o u t several h u n d r e d m i c r o g r a m s f r o m each t u m o r . The p r e p a r a t i o n s h a d a r a t i o of a b s o r b a n c e at 260 a n d 280 n m of 1.9 to 2.0. Isopycnic CsC1 g r a d i e n t c e n t r i f u g a t i o n resulted in one h o m o g e n e o u s p e a k of v i r a l DNA. No D N A p e a k w a s detectable at the b u o y a n t density of cellular DNA. T h e b u o y a n t density of F L D V D N A was f o u n d to be p = 1.690 g • m1-1. The c o r r e s p o n d i n g values of a G + C c o n t e n t which was calculated according to S c h i l d k r a u t et al. (1962) w a s in a g r e e m e n t with the value o b t a i n e d f r o m the Tm m e a s u r e m e n t . Det e r m i n a t i o n of the u v - a b s o r b a n c e - t e m p e r a t u r e profile of the D N A in 0.1 • SSC resulted in a Tm value of 65.5 _+ 0.8 ~ This c o r r e s p o n d s to a (G + C) content of the D N A of 30.7 + 1.7%.
Restriction Endonuclease Analysis In o r d e r to identify a n d c h a r a c t e r i s e the F L D V g e n o m e s D N A s of different F L D V isolates w e r e cleaved w i t h r e s t r i c t i o n endonucleases and the r e s u l t i n g D N A f r a g ments were separated electrophoretically on a g a r o s e slab gels. R e p r e s e n t a t i v e results of this a n a l y s i s a r e given in Fig. 2A to F. F i g u r e 2A shows the BstEII cleavage p a t t e r n s of F L D V D N A isolated f r o m individual flounders (lane 1 and 2), plaice (lane 3) and dabs (lane 4 to 6). The f r a g ment patterns demonstrate that FLDV D N A of flounders (F7 a n d F9) and of plaice (P1) are indistinguishable, b u t clearly diff e r e n t f r o m those of dab (D1 to D3). The s a m e r e s u l t was o b t a i n e d for the r e m a i n der of all o t h e r flounders tested, t h r e e plaice, and t h r e e dabs, which were separ a t e l y and individually a n a l y s e d ( d a t a not shown). The cleavage p a t t e r n s in Fig. 2B and E, o b t a i n e d a f t e r digestion of the s a m e
D N A A N A L Y S I S OF F I S H I R I D O V I R U S
FIG. 1. Electron m i c r o g r a p h s of fish l y m p h o c y s t i s disease virus; (a) T h e lower m i c r o g r a p h of the u l t r a t h i n section exemplifies t h e h u g e a m o u n t of v i r u s p a r t i c l e s p r e s e n t in an e p i d e r m a l t u m o r cell of a lesion of l y m p h o c y s t i s disease (magnification: X9000). (b) T h e fine s t r u c t u r e of t h e intact complex virion is best revealed in u l t r a t h i n section. A fringe of p r o t r u s i o n s a b o u t 2.5 n m in l e n g t h s u r r o u n d s t h e hexagonal, m e m b r a n e - b o u n d particle. The envelope s t r u c t u r e c o n s i s t s of two layers, an o u t e r u n i t m e m b r a n e s t r u c t u r e a n d an i n n e r m o r e electro-dense layer closely a t t a c h e d to t h e f o r m e r . T h e concentric core inside t h e virions is s e p a r a t e d f r o m t h e composite m e m b r a n e by a broad e l e c t r o n - t r a n s l u c e n t region (magnification: X130,000). (c) N e g a t i v e s t a i n i n g of F L D V virions; in f a i n t l y s t a i n e d r e g i o n s a f r i n g e on t h e virion s u r f a c e is visible. D u e to osmotic shock t h e o u t e r m e m b r a n e of t h e virion is broken a n d also p a r t l y dissociated into two layers, while t h e core s e e m s to r e m a i n c o m p a c t a n d stable (magnification: X130,000).
469
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FIG. 2. Agarose slab gel electrophoresis of fish lymphocystis disease virus DNAs; FLDV DNAs (1 ttg) of each species were digested with restriction endonucleases BstEII (A), EcoRI (B), PstI (C), BglII (D), BamHI (E), and HpaII, and MspI (F). The resulting DNA fragments were separated in 0.5% (A, B, C, and E) and in 0.8% (D and F) agarose slab gels (35 X 20 X 0.3 cm) and run at 75 V for 18 hr (A, B, D, E, and F) and/or 24 hr (C) in electrophoresis buffer at 4 ~ Lanes of A, B, and E: 1, FDLV-F7 DNA; 2, FLDV-F9 DNA; 3, FLDV-P1 DNA; 4, FLDV-D1 DNA; 5, FLDV-D2 DNA; 6, FLDV-D3 DNA. Lane 1 in B, 10 #g FLDV-F7 DNA were used for demonstration of the fragment. (C) Lanes: 1, FLDV-F7 DNA; 2, FLDV-F9 DNA; 3, FLDV-P1 DNA; 4, FLDV-D1 DNA; 5, FLDVD2 DNA; and 6, FLDV-D3 DNA. (D) Lanes: 1, FLDV-F12 DNA; 2, FLDV-F4 DNA; 3, FLDV-F2 DNA; 4, FLDV-F1 DNA; 5, FLDV-F7 DNA; 6, FLDV-F9 DNA; 7, FLDV-P DNA; 8, FLDV-D1 DNA; 9, FLDV-D1 DNA; and 10, FLDV-D3 DNA. M represents positions of EcoRI DNA fragments of lambda DNA which served as internal marker. (F) Cleavage pattern of different FLDV DNAs after digestion (1 ttg) with HpaII enzyme (lane 1 to 4) and MspI enzyme (lane 5 to 8). Lanes: 1 and 5, FLDV-D1 DNA; 2 and 6, FLDV-P1 DNA; 3 and 7, FLDV-F12 DNA; 4 and 8, lane M: lambda DNA digested with BglII as internal marker.
DNA ANALYSIS OF FISH IRIDOVIRUS
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F L D V D N A s as in Fig. 2A w i t h E c o R I (2B) and B a m H I (2E) respectively, confirm t h e results described above for the B s t E I I enzyme. A v a r i a t i o n in the r e s t r i c t i o n p a t t e r n of individual fish of one species (e.g., flounder) w a s not found w h e n these t h r e e e n z y m e s w e r e used. The s a m e holds t r u e
w h e n flounder F L D V D N A and plaice F L D V D N A w e r e cleaved w i t h the PstI enzyme (Fig. 2C). The dab F L D V D N A p a t t e r n s of individual dabs w e r e indistinguishable f r o m each o t h e r (Fig. 2C, lanes 4, 5, and 6), but different f r o m those of flounders and plaice (lane 1 to 3). Accord-
472
DARAI ET AL. flounder and dab viral D N A s hybridize to each o t h e r and thus seem to be closely related. The molecular w e i g h t of F L D V of different isolates calculated f r o m the sizes of individual D N A f r a g m e n t s obtained after electrophoretic s e p a r a t i o n of digested F L D V D N A s with a v a r i e t y of r e s t r i c t i o n e n z y m e s was found to be 64.8 • 106 D as shown in Table 1.
Structure o f F L D V D N A
FIG. 3. Reciprocal blot hybridization of 82P-labeled DNAs of FLDV-F16and FLDV-D2to PstI fragments of the same DNAs. (A) [32P]DNAof FLDV-F16 was hybridized to a Southern blot of PstI digest of 0.5 ttg DNA of FLDV-D2(lane 1) and 0.5 ttg DNA of FLDVF16 (lane 2). (B) [s2P]DNAof FLDV-D2 was hybridized to a Southern blot of PstI digests of 0.5 t~g DNA of FLDV-D2 (lane 1) and 0.5 ttg DNA of FLDV-F16 (lane 2). Time of exposure was 24 hr for A and B, and C is the blot shown in B exposed for 6 days.
ing to these results two different s t r a i n s of F L D V exist; F L D V s t r a i n 1 is found in flounders and plaice, w h e r e a s F L D V s t r a i n 2 is usually associated with dabs inflicted with LD. This result is in a g r e e m e n t with the F L D V polypeptide p a t t e r n s which distinguish flounder and plaice f r o m the pattern of dab (Fliigel et al., 1982). To ascertain these results, D N A s of F L D V s t r a i n 1 and 2 were s e p a r a t e l y 32p_ labeled by nick t r a n s l a t i o n a c c o r d i n g to Ribgy et al., (1977), hybridized by the S o u t h e r n blot m e t h o d to nonradioactive, PstI-digested D N A of F L D V of F16 and D2, reciprocally. The resulting a u t o r a d i o g r a p h s of Fig. 3A, B, and C show t h a t
In o r d e r to d e t e r m i n e the t e r m i n a l f r a g m e n t s of F L D V DNA, purified D N A was first digested with either the },-5'-exonuclease or the 3'-exonuclease I I I of E. coli followed by digestion with the restriction e n z y m e BstEII. The D N A cleavage patterns shown in Fig. 4A and B do not show a selective digestion of a p a r t i c u l a r f r a g m e n t instead a g r a d u a l d i s a p p e a r a n c e of all viral D N A f r a g m e n t s is observed. Exp e r i m e n t s in which B a m HI, Pst I or BgIII enzymes were used, for either of the two exonucleases gave the same results. Exp e r i m e n t s were p e r f o r m e d using the guanidinium h y d r o c h l o r i d e m e t h o d as described previously by Robinson et aL (1973). No evidence for a t e r m i n a l l y bound D N A protein was found. A n o t h e r m e t h o d to search for t e r m i n a l f r a g m e n t s was employed, n a m e l y labeling the 5'-termini of F L D V D N A by T4 polynucleotide kinase with [~/-32P]ATP followed by digestion with different restriction endonucleases and s u b s e q u e n t electrophoretic s e p a r a t i o n on agarose gels. As shown in Fig. 5 it was found t h a t radioactive p h o s p h a t e was inc o r p o r a t e d into most D N A f r a g m e n t s ind e p e n d e n t of the restriction enzyme used.
Electron Microscopy o f F L D V D N A Purified viral D N A molecules of different F L D V isolates were examined in the electron microscope using c y t o c h r o m e c s p r e a d i n g (Davis et al., 1971). An average length of 49 • 23 # c o r r e s p o n d i n g to a b o u t 93 • 44 • 106 D was found by m e a s u r i n g 43 molecules. The c o m p a r i s o n between the molecular weight values obtained by restrict• enzyme analysis and c o n t o u r length m e a s u r e m e n t s indicates t h a t the
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FIG. 4. Lambda 5'-exonuclease and E. coli 3'-exonuclease-restriction endonuclease digest of fish lymphocystis disease virus DNA. Agarose slab gel electrophoresis of FLDV DNAs were treated with lambda exonuclease a n d / o r exonuclease III for 2 and 30 min and thereafter cleaved with restriction endonuclease B s t E I I and run under the same conditions and in the same gel in comparison to untreated DNA: (B) Lane 2, 1 ttg of FLDV-F12 DNA digested with BstEII; lane 1, BstEII cleavage pattern of 1 #g of the same DNA which was pretreated with lambda exonuclease. Lane 3 and 4, control experiment using three shrew herpesvirus (THV-2) DNA (Darai et al., 1981) under the same conditions. THV-2 DNA was digested with EcoRI. The white arrows in lane 4 label the two terminal factors (H and J) which disappeared after a 5 min t r e a t m e n t with lambda exonuclease (lane 3). (A) Lane 2, FLDV-F16 DNA (1 ttg) digested with BstEII. Lane 1 and 3, B s t E I I digests of aliquots of the same DNA after t r e a t m e n t with lambda exonuclease for 2 min (lane 1) and for 30 min (lane 3). Lane 5, FLDV-F16 DNA (0.5 #g) digested with BstEII. Lane 4 and 6, B s t E I I digests of aliquots of the same DNA after t r e a t m e n t with exonuclease III for 2 min (lane 4) and for 30 min (lane 6). Phage lambda DNA (2 t~g) digested with H i n d I I I served as marker (M).
DNA molecules are on the average longer than expected from the restriction enzyme analysis. While in most preparations FLDV DNA contained so many nicks that only small
single-stranded fragments were obtained, two separate preparations from two different isolates (FLDV-F16, F17) yielded DNA single strands approaching in length the sizes measured for the double-stranded
DNA ANALYSIS OF FISH IRIDOVIRUS DNA. In these p r e p a r a t i o n s it was possible to observe circular D N A molecules a f t e r d e n a t u r a t i o n / r e n a t u r a t i o n . These circles were p a r t i a l l y single s t r a n d e d and m i g h t show, e.g., as in the molecule s h o w n in Fig. 6, s i n g l e - s t r a n d e d D N A tails w i t h i n the double-stranded region. Three circles could be m e a s u r e d and had an a v e r a g e molecular w e i g h t of 65.22 • 106 D. F i g u r e 6 represents a single s t r a n d of m o r e t h a n unit size being circularized by a f r a g m e n t of D N A c o m p l e m e n t a r y to sequences on both sides of the a t t a c h m e n t point of the extra D N A tails. These tails are s e p a r a t e d by one unit length of the F L D V genome. The small n u m b e r of i n t a c t D N A molecules obtained is p r o b a b l y due to the fact t h a t the F L D V D N A was isolated f r o m virions g r o w n in vivo for an u n k n o w n period of time. In addition, the p r o b a b i l i t y of circle f o r m a t i o n could be considerably reduced by the relatively large e x t e n t of the overall redundancy.
475
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1 2 345
F L D V D N A is Methylated and N o t Closely Related to Frog Virus 3 D N A F L D V D N A s were digested with the H p a I I (C~CGG) and MspI (c~MeCGG) enzymes; the l a t t e r cleaves D N A in the presence of a 5-methyl g r o u p at the internal cytosine residue in the recognition sequence of the H p a I I enzyme (Waalwijk and Flavell, 1978). As shown in Fig. 2F, lanes 5, 6, and 7, F L D V D N A f r o m dab, plaice, and flounder were cut m a n y times with MspI indicating t h a t the i n t e r n a l C in the recognition sequence of this enzyme is heavily m e t h y l a t e d , since the H p a I I enzyme leaves the F L D V D N A s i n t a c t (Fig. 2F, lanes 1, 2, and 3). A similar result was obtained by Willis and G r a n o f f (1980) for frog virus DNA. I t was of i n t e r e s t to s t u d y the relatedness of both these viral D N A s to each other. FV 3 D N A was labeled with 32p as described in Materials a n d Methods and hybridized a g a i n s t unlabeled FLDV D N A f r o m different fish species by the S o u t h e r n blot m e t h o d (1975). As a control FV 3 D N A was included in these experiments. As shown in Fig. 7, no h o m o l o g y
FIG. 5. Autoradiograph of 82p-5'-labeled FLDV-F12 DNA digested with different restriction endonucleases: lane 1 - PstI, lane 2 = BstEII, lane 3 = Bali, lane 4 - BamHI. Lane 1 to 4, 1 ttg unlabeled FLDVF12 DNA containing 0.5 #g 32P-5'-labeled FLDV-F12 DNA (5 • 108 cpm) (lane 1 to 4). Phage lambda DNA digested with BglII (fragment A, B, C, and D) served as marker (5). Time of exposure was 7 days.
between the D N A s of FV 3 and FLDV from various isolates (flounder, dab, or plaice) was detectable u n d e r the conditions used. DISCUSSION The analysis of the genome of F L D V as reported here shows t h a t the F L D V genome is a double s t r a n d e d and linear D N A molecule of 4 9 _ 12 #m in length corresponding to a molecular weight of 93 + 44 • 106 D. The exceptionally wide r a n g e in molecular weight could reflect the fact t h a t
FIG. 6. Electron m i c r o g r a p h of a circular F L D V - F 1 6 D N A molecule g e n e r a t e d by d e n a t u r a t i o n a n d r e n a t u r a t i o n as described in M a t e r i a l s a n d M e t h o d s . The a r r o w s p o i n t to t h e excess single s t r a n d s . The t r i a n g l e s m a r k t h e t r a n s i t i o n points b e t w e e n single- a n d d o u b l e - s t r a n d e d regions. 476
D N A A N A L Y S I S OF F I S H I R I D O V I R U S
viral DNA was directly isolated from FLDV grown in vivo for an unknown, but probably long period of time which could lead to degradation of DNA. Besides, single- and double-stranded DNA degrading enzymatic activities were detectable in purified virions of FLDV (R.M.F., unpublished observations), The molecular weight calculated from restriction enzyme data resulted in 64.8 • 10GD which when compared to the value obtained by electron microscopy indicate t h a t FLDV DNA has an average redundancy of approximately 50 %. Denaturation and renaturation of FLDV DNA revealed circular DNA molecules of 34.25 ~m length with two protruding single-stranded ends. This contour length corresponds to a molecular weight of 65.22 • 106 D, in agreement with the value obtained by restriction enzyme analysis. Although circular DNA molecules were formed upon renaturation between two single DNA strands only, the low number of DNA circles is explained by the long concatemer-like structure of the FLDV genome which decreases the probability of circle formation due to its relatively high average redundancy. Taken together, these data indicate that the DNA isolated is highly redundant but to a variable extent. The results of the experiments performed with either the 5'specific exonuclease or 3'-exonuclease and with 5'-polynucleotide kinase are in support of a variable redundancy a n d / o r a possible circularly permuted genome (Thomas, 1970; Tye et aL, 1974). Thus, the DNA structure of FLDV might be similar to t h a t of the genome of frog virus 3, another iridovirus similar to FLDV, which has been reported to be circularly permuted and terminally redundant (Goorha and Murti, 1982). A structural feature common to both FLDV and FV 3 DNA is the methylation in cytosine residues (Willis and Granoff, 1980). As revealed by hybridization experiments no homology was detectable between the DNAs of FLDV and frog virus under the conditions used. Similar hybridization experiments were performed in Dr. Granoff's laboratory in Memphis,
1 23[M]4
477
56
FIG. 7. A u t o r a d i o g r a p h of blot h y b r i d i z a t i o n between f r o g virus 3 (FV 3) D N A a n d fish l y m p h o c y s t i s disease v i r u s (FLDV) D N A of different isolates. [h-82P]dCTP n i c k - t r a n s l a t e d FV 3 D N A w a s h y b r i d ized to S o u t h e r n blots of EcoRI digests of D N A s of FV 3 a n d FLDV DNAs. L a n e s 1, 2, a n d 3, FV 3 DNA; lane 1, 0.06 #g = 10 8 pmol; lane 2, 10 -4 pmol; a n d lane 3, 10 -5 pmol. L a n e 4, F L D V - F 1 2 DNA; lane 5, F L D V D2 DNA; a n d lane 6, F L D V - P 1 DNA. L a n e 4 to 6 c o n t a i n 1 ~g each of D N A (10 -2 pmol). X-s2P-labeled phage l a m b d a D N A (5 • 10a cpm) digested with EcoRI served as i n t e r n a l m a r k e r (M).
478
DARAI ET AL.
Tennessee, with the same r e s u l t (personal communication). To establish a gene library, FLDV DNA f r a g m e n t s were inserted into the pAT153 plasmid vector of E. coli. Bacterial colonies h a r b o r i n g r e c o m b i n a n t plasmids were selected. The analysis of the isolated amplified clones is now in progress, and will be of g r e a t help for f u r t h e r molecular and biological studies of fish lymphocystis disease virus, p a r t i c u l a r in view of the fact t h a t FLDV c a n n o t yet be efficiently groWn in tissue culture.
ACKNOWLEDGMENTS We are grateful to Dr. Allan Granoff, Division of Virology, St. Jude Children's Research Hospital, Memphis, Tennessee, for critical comments and to Dr. Rakesh Goorha of t h a t institution for performing hybridization experiments of frog virus 3 and fish lymphocystis disease virus DNA. The authors t h a n k Mrs. B. Holder for preparation of the manuscript.
REFERENCES
CHACONIS,G., and VAN DE SANDE,J. H. (1980). 5'-s2P labeling of RNA and DNA restriction fragments. I n "Methods in Enzymology" (L. Grossmann and K. Moldave, eds.), Vol. 65, pp. 75-85. Academic Press, New York. DARAI, G., MATZ, B., FLUGEL, R. M., GRAFE, A., GELDERBLOM, H., and DELIUS, H. (1980). An adenovirus from tupaia (tree shrew): Growth of the virus, characterization of viral DNA, and transforming ability. Virology 104, 122-138. DARAI, G., FL~GEL, R. M., MATZ, B., and DELIUS, H. (1981). DNA of tupaia herpesviruses. In "Herpesvirus DNA" (Y. Becker, ed.), pp. 345-361. Martinus Nijhoff Publishers, The Hague, Boston, London. DARAI, C., Z(3LLER, L., MATZ, B., DELIUS, H., SPECK, P. T., and FLI~GEL, R. M. (1982). Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy. J. BacterioL 150, 788-794. DAVIS, R. W., SIMON, M., and DAVIDSON, N. (1971). Electron microscopy heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In "Methods in Enzymology" (L. Grossmann and K. Moldave, eds.), Vol. 21, pp. 413-428. Academic Press, New York. DUNBAR, C. E., and WOLF, K. (1966). The cytological course of experimental lymphocystis in bluegill. J. Infect. Dis. 116, 466-472.
FLUGEL, R. M., DARAI, G., and GELDERBLOM, H. (1982). Viral proteins and adenosine triphosphate phosphohydrolase activity of fish lymphocystis disease virus. Virology 122, 48-55. GELDERBLOM, H., BAUER, H., FRANK, H., and WIGAND, R. (1967). The structure of group II adenoviruses. J. Gen. ViroL l, 553-560. GELDERBLOM, H., OGURA, H., and BAUER, H. (1974). On the occurrence of oncornavirus-like particles in HeLa cells. Cytobiology 8, 339-344. GOORHA, a., and MURTI, K. G. (1982). The genome of frog virus 3, and animal DNA virus, is circularly permuted and terminally redundant. Proc. Nat. Acad. Sci. USA 79, 248-252. LOPEZ, D. M., SIGEL, M. M., BEASHLEY, A. R., and DIETRICH, L. S. (1969). Biochemical and morphological studies of lymphocystis disease. Nat. Cancer Inst. Monogr. 31,223-236. LOWE, K. (1874). Fish disease. IV. Trans. Norfolk and Narwich Nat. Soc. Fishes, 21-56. MATTHEWS, R. E. F. (1979). Classification and Nomenclature of Viruses. "Third Annual Report of the International Committee on Taxonomy and Viruses" pp. 174-176. Karger, Basel. MURTI, K. G., GOORHA, R., and GRANOFF, A. (1982). Structure of frog virus 3 genome: Size and arrangement of nucleotide sequences as determined by electron microscopy. Virology 116, 275-283. PARR, R. P., BURNETT, J. W., and GARON, C. F. (1977). Structural characterization of the Molluscum contangiosum virus genome. Virology 81, 247-256. RIGBY, P. W. J., DIECKMANN,M., RHODES, C., and BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. MoL Biol. 114, 237-251. ROBINSON, A. J., YOUNGHUSBAND, H. B., and BELLET, A. J. D. (1973). A circular DNA protein complex from adenoviruses. Virology 56, 54-69. RUSSELL, P. H. (1974). Lymphocystis in wild plaice Pleuronectes platessa (L.) and flounder, Platichthys flesus (L.), in British coastal waters: A histopathological and serological study. J. Fish Biol. 6, 771-778. SCHILDKRAUT, C. L., MARMUR, J., and DOTY, P. (1962). Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsC1. J. Mol. Biol. 4, 430-443. SHARP, P. A., SUGDEN, B., and SAMBROOK, J. (1973). Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055-3061. SOUTHERN, E. U. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioL 98, 503-517. TEMPLEMAN, W. (1965). Lymphocystis disease in American plaice of the Eastern Grand Bank. J. Fish. Res. Board CanacL 22, 1345-1355.
DNA ANALYSIS OF FISH IRIDOVIRUS THOMAS, C. A., JR. (1970). The rule of the ring. J. Cell PhysioL Suppl. 1, 13-34. TYE, B. K., HUBERMAN, J. A., and BOTSTEIN, D. (1974). Nonrandom circular permutation of phage P22 DNA. J. Mol. Biol. 85, 501-532. WAALWIJK, C., and FLAVELL, R. A. (1978). Msp I, an isoschizomer of Hpa II which cleaves both unmethylated and methylated Hpa II sites. Nucleic Acids Res. 5, 3231-3236. WALKER, D. P., and HILL, B. Z. (1980). Studies on the culture, assay of infectivity and some in vitro properties of lymphocystis virus. J. GeE. Virol. 51,385395.
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WILLIS, D. B., and GRANOFF, A. (1980). Frog virus 3 DNA is heavily methylated at CpG sequences. Virology 107, 250-257. WOLF, K. (1966). The fish viruses. Adv. Virus Res. 12, 36-98. WOLF, K., GRAVELL, M., and MALSBERGER, R. G. (1966). Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science 151, 1004-1005. WOLF, K., and QUIMBY, M. C. (1973). Fish viruses: buffers and methods for plaquing eight agents under normal atmosphere. Appl. Microbiol. 25, 659664.