Biochemical and structural studies of fish lymphocystis disease virions isolated from skin tumours of Pleuronectes

Journalof VirologicalMethods, 13( 1986) 197-205

197

Elsevier JVM 00487

BIOCHEMICAL AND STRUCTURAL STUDIES OF FISH LYMPHOCYSTIS DISEASE VIRIONS ISOLATED FROM SKIN TUMOURS OF PLEURONECTES

C. SAMALECOS Institut fiir Biochemie und Molekularbiologie der FU Berlin, FB Biologic. WE 03, Ehrenbergstr. 26-28, 1000 Berlin 33, F.R. G. (Accepted

23 January

Fish lymphocystis

1986)

disease

viruses

of various

flatfish species and further

disrupted.

In combination

with different

tion and after ultrasonication virus was used for isolation measurements

(FLDV) purified.

of the virus nucleoid

directly

from

could be identified

types of treatment,

and ultracentrifugation,

lymphocystis

disease

Nonidet-P40,

dithiothreitol,

proteases

the inner region of FLDV was studied.

and for further

proteases

digestion

electron

wereshown

microscopy

lesions

only after the purified virus was

study of the viral genome.

diges-

The purified

Contour

of 20 DNA molecules gave an average length of 40.44 f 3.2um. Linesofprecipitation

isolated nucleoid material and FLDV-antibodies

iridovirus

were isolated Subunits

length between

by immunoelectrophoresis.

subunits

immunoelectrophoresis

DNA

analysis

INTRODUCTION

Lymphocystis disease (LD) is a virus-induced infection of freshwater and brackish water fishes, characterised by papilloma-like hypertrophy of the connective tissue (Dunbar and Wolf, 1966). The papillomatous tumours are composed or enlarged ‘lymphocystis’ cells of up to 0.5 nm, containing densely packed isometric virus particles (Walker, 1962; Samalecos et al., 1982). Fish lymphocystis disease virus (FLDV) measures 199-227 nm in diameter (Darai et al., 1983; Tempelman, 1965) and frequently appears in pleuronectidae (flatfish). According to the FLDV morphology, site of assembly and its genetic material, it has been classified into the ‘icosahedral cytoplasmic deoxyribovirus,group’ (Kelly and Robertson, 1973) and more specifically as a separate genus within the iridovirus family (Matthews, 1981). LD can be transmitted experimentally by injection of FLDV (Wolf, 1962). However, the mechanism of induction and tumour formation is unknown. FLDV isolated from PZeuronectesplutessa (plaice), Plarichtys jlesus (flounder), Limanda Iimanda (dab) and Trigla gurnardus (gurnard) were compared using biochemical methods and electron microscopy with frog iridoviruses (FV-3), Tipula iridescent virus (TIV) and African swine fever virus 01660934/86/$03.50

o 1986 Elsevier Science Publishers

B.V. (Biomedical

Division)

198

(ASFV)

(8zel

characterisation

et al.,

1984). The virus

was isolated

directly from the lesions. In view of the wide-spread

disease, at horough MATERIALS

1982; Samalecos,

study oft he basic properties

for further

occurrence

of the

of FLDV was carried out.

AND METHODS

Virus purification Virions of FLDV were isolated from lymphocystis tissues and purified according to published methods (Parr and Burnett, 1977), which were modified by Darai et al. (1983). Isolation of FLD V nucleoid 200 ~1 purified virus suspension was treated for 1 h with 1 ml 25 mM TNE-buffer in 0.5% Nonidet-P40 (N-P40) and 10 mM dithiothreitol (DTT). The virus sample was layered onto a 35-65% saccharose gradient in 10 ml TNE-buffer and centrifuged at 35,000 rpm for 4 h at 4°C in a SW 41 rotor centrifuge. The band obtained from the gradient was characterised morphologically by electron and used for immunoelectrophoretic experiments. Isolation of the DNA of the FLDV DNA isolation was carried out according 1967; Samalecos,

microscopy

to the modified

as virus nucleoid

method

of Hirt (Hirt,

1984).

Spreading of FLD V-DNA For electron published

microscopic

methods

length

measurements,

the DNA was spread according

(Davis et al., 1968). The DNA was spread by pouring

to

a hyperphase

containing 0.5 M ammonium acetate, 1 mM EDTA pH 8.0, 0.1% cytochrome c and 1 pg/ml FLDV-DNA onto a hypophase containing 0.25 M ammonium acetate. The sample was brought into an Edwards-306Gnit andshadowed with platinum-palladium (pt/pd) for 30 s at an angle of 8”. Examinations were carried out with an EM 10A Zeiss electron microscope at 80 kV and a magnification of 4,000. Bacteriophage PM2 DNA (1 pg/ml) was used as a marker molecule. Chemical treatment of the FLD V particles (a) Purified FLDV was fixed for 15 min in 2.5% glutaraldehyde. 1% N-P40 and 50 mM DTT in phosphate-buffered saline (PBS) were added and incubated for 15 min at 20°C. Virions were stained with 1% uranyl acetate for 40 s and washed with 0.01 M Tris-EDTA, pH 7.2. (b) 0.1 ml of the virion samples were fixed for 15 min in 2.5% glutaraldehyde and incubated for 15 min at 25°C in 0.2% pronase in PBS, pH 7.2; and stained and washed as in (a). (c) Particles were incubated for 30 min in 0.5% papain, 1 mM r_-cysteine. Staining and washing were as in (a).

199

(d) 0.1 ml of the virus suspension was dissolved in 9.9 ml PBS at pH 7.2 and centrifuged at 40,000 rpm in a SW 41 rotor for 30 min at 4°C. The pellet containing now the virus particles was resuspended in 0.5 ml buffer (20 mM Tris-HCl, pH 7.0,50 mM NaCl and 0.5% N-P40) and treated with ultrasonication. The lysate was layered onto a 10 ml saccharose gradient (35-60% in PBS, pH 7.2) and centrifuged at 40,000 rpm for 1 h at 4OC. There was a small band in the gradient, which was collected. It was stained with 3% phosphotungsticacid(PTA), pH 7.0, andexaminedundertheelectron microscope. Negative staining

One drop of virus suspension was placed on Pioloform-coated and carbon stabilised 400-mesh copper grids. After 2 min of adsorption, the grids were washed twice with distilled water for 10 s successively. For the negative staining, 3% PTA at pH 7.0 or 1% uranyl acetate, pH 4.5 were used. Staining was carried out for 30 to 60 s at 25°C. Preparation of anti-FLDV-antibodies

Purified FLDV-antigen was injected into a rabbit. After 20 days, antibody production was shown by immunoelectrophoresis. RESULTS

Ultrastructure of the FLD V-nucleoid

After treatment of the purified FLDV with N-P40 and DTT (Fig. 2) the virus membrane was separated to a great extent from the internal structure of the particle, whereas structural changes of the nucleoid were not observed and the staining of the nucleoid itself did not supply further information. More detailed information about the nucleoid was obtained by treatment with pronase or papain-cysteine (see Chemical Treatments b, c) (Fig. 3, 4) or after incubation and high speed centrifugation of the virus particles and then PTA-staining (see Treatment d). Usually aggregates of loose subunits of 3.5-4 nm in diameter were obtained (Fig. 5). Occasionally subunits in the region of the nucleoid were seen in infected fish tumour tissue (Fig. 6a, b). Antigenic properties of FLDV

Determination of the protein concentration of the nucleoid fraction (Fig. 7) was carried out according to Lowry et al. (1951) and was found to be 0.6 mg/ml. Using immunoelectrophoresis, a precipitation of anti-FLDV-antibodies with samples of the virus interior structural antigens was observed, whereas no precipitation occurred using a fish tissue homogenate (FTH) of a healthy animal with anti-FLDV-antibodies (Fig. 9). Measurement of the length of FLDV-DNA

Twenty measurements of contour length of the FLDV-DNA molecules were carried out. Measurements resulted in values of 40.44 f 3.2 urn (Fig. 10). 20 values of

Fig. 1. Outer shelt and nucleoid Fig. 2. Individual

membrane

virus particles

showing

0.5% N-P40 and 50 mM DTT. Negative Fig. 3. Selected virus particles Negative

staining

of untreated various

staining

with evidence

with 1% uranyl acetate.

FLDV after PTA staining.

disrupted

forms of the icosahedral

of virus after using 1% uranyl

of regular

X 156,000.

repeating

acetate.

units after treatment

shell. Treatment

with

X78,000. with 0.2% pronase.

X93,500.

Fig.4. ParticlesofFLDV,onwhichtheregulararrangementofthecapsomeresformingthecapsidcanbeseen. Treatment X156.000.

with 0.5% papain,

5 mM t_-cysteine and

1mM EDTA. Negative staining with 1% uranyl acetate.

AFLDV Fig. 5. Aggregates Fig. 6a,b. Nucleoid Xl 10,000 (b).

of subunits subunits

after centrifugation. of the embedded

Staining

AFTH

with 3% PTA. X176,000.

virus particles

in fish tissue. Magnification:

X176,000

(a);

202

Bacteriophage (Fig.

PM2 DNA 3.38 f 0.15 urn molecules

were used as internal

standard

10).

DISCUSSION

The fine structure of the complex shell of FLDV was described using negative staining, and ultrathin sectioning of the virus particles in previous publications (Darai et al., 1983; Samalecos, 1984). It seems to consist of 2 adjacent unitary membranes and therefore has morphologically the characteristics of the unit cytoplasmic membrane. An attempt has been made in this work to describe the inner structure of FLDV. As can be seen in Fig. 1, a membrane lies below the capsid, which encloses the electron dense inner bodies. The untreated virus particle in Fig. 1 showed that the bodies enclosed in the virions were separated from the complex shell by a broad electrontranslucent region. We have called them nucleoid. Fig. 7 shows isolated nucleoids, which are sometimes surrounded by membrane structures. The nucleoid region of FLDV was estimated after lysis of the external structure of the virus particles with N-P40 and DTT, with proteases and through high speed centrifugation (see Treatments a-d). The arrangements and number of the FLDV nucleoid subunits could not be defined, as was reported from other iridoviruses: Tipula iridiscent virus (TIV) (Wingley, 1970) and African swine fever virus (ASFV) (Carrascova et al., 1984). Staining with 2.5% glutaraldehyde before lysis stabilises partly the internal structure of FLDV and so possible structural degradations can be avoided. Some electron microscopic pictures show large amounts of empty viruses (Fig. 8), since empty capsids are a frequent by-product of the virus reproduction cycle. In March, we found through DNA extraction a maximum of intact virus DNA as compared to the amounts of DNA found in the tumour samples in December, where its level was at a minimum (Darai, pers. commun.). Electron microscopic investigation carried out here showed an unusually large amount of empty virus capsids compared to those in the December samples, Walker and Hill (1980) suggest that increased temperatures in in vitro experiments

lead to increased

amounts

of lymphocystis

cells. Berthiaume

et al. (1984)

report on ‘empty’ FLDV particles, but the virus was grown on bluegill fry (BF-2) cells. We cannot definitively say that temperature differences are the sole factor influencing the amount mental

of lymphocystis

pollution,

Fig. 7. Intact

virus nucleoids.

Fig. 8. Depleted

antibody

Staining

virus particles

Fig. 9. Precipitation anti-FLDV

antibodies.

cells, since also other external

may have an unknown

parameters,

on this subject.

e.g. environ-

We would not like

with 3% PTA. X80,000.

(without

nucleoid).

of 10 pl, 15 ul antigen Between fish-tissue

no lines of precipitation

influence

are seen.

X50,000.

from isolated homogenate

virus nucleoid

of FLDV

20 ul (FTH) of the healthy

with 15 $/cm*

rabbit

animal and anti-FLDV

203

Fig. 10. Electron Methods,

circular

micrograph

of the FLDV genome. DNA (1 pg/ml) was spread as described

Bacteriophage

PM2 was used as an internal length reference.

X20,900.

in Materialsand

204

to try to suggest

interpretations

of the amount

of the empty

virus particles

seen in

March and December, respectively, since temperature and eventually the other parameters mentioned above are not conclusive enough in comparison with the results of experiments between

done in vitro. However,

the empty particles

we had the impression

and the reciprocal

amount

that a correlation

of the isolated

exists

DNA.

The genome of FLDV consists of ds-DNA, as seen from the smooth contour of the spreaded DNA. The length of the DNA, observed and measured in electron microscopic studies is 40.44 + 3.2 urn. Analysis

of the genome by treatment

with the restriction

endonuclease Bst EII results in different patterns of the fragments of the DNA from viruses from flounder and dab (Darai et al., 1983). This might explain the differences in the structural size of the viruses of the two different fish species of FLDV. The determination of differences in the size of FLDV was reported in Samalecos (1984, 1986). The determination of antigenic properties of the FLDV nucleoid shows that there are determinants of at least two virus nucleoproteins, as shown by immunoelectrophoresis with rabbit anti-FLDV-antibodies. The homologous immunoreactions shown in Fig. 9 are virus specific because there were no reactions against uninfected fish tissue with the respective hyperimmunesera. One of the many morphologically (Samalecos et al., 1982; ijzel et al., 1982; Darai et al., 1983; Almeida et al., 1967; Stolz, 1973; Els and Pini, 1977; Aubertin et al., 1971; Carracosa et al., 1984; Willis et al., 1977) and biochemically related features of FLDV-iridoviruses as opposed to other iridoviruses like ASFV, FV-3 and TIV is the degradation of the virus membrane, necessary for electron microscopic examination of the subunits in the internal capsid area. ACKNOWLEDGMENTS

1 would like to thank Demitris Lopez

Pila and Professor

results.

I am also grateful

Hadjiyiannis,

Dr. Uwe Hollihn,

Dr. E.-R. Lochmann

for the intensive

to Dr. Darai for providing

work was supported by the Robert-Koch-Institut eral Health Agency) West Berlin, FRG.

Professor

Dr. Juan

discussion

of the

me with the fish specimens.

des Bundesgesundheitsamtes

This (Fed-

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