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11. VAN REGENMORTEL, M. H. T’., Virology 23, 495 (1964).

12. CLELAND, W. W., Biochemistry 3,480 (1964). 13. FRANCKI, R. I. I<., Virology 34,694 (1968). 14. KAPER, J. M., and JENIFER, F. G., Bio-

chemislry 6, 440 (1967). 15. KAPER, J. ?\2., and JENIFER, F. G., Virology

35, 71 (1968). 16. LEBERMAN, It., Symp. Sot. Gen. Microbial. 18,

183 (1968). J. RI. KAPER

The George Washington University Washington, D. C. 60006

rlccepled Oclober 18, 1968

Virus-like Particles in Galls on Sugar-

cane Plants Affected by Fiji Disease

The causal agent of Fiji disease has not been determined. Early workers (e.g., 1, .2) noticed the presence of vacuolate intracellu- lar bodies in the leaf galls which typify the disease. They suggested that parasitic amoebae might be responsible. More re- cently, however, there has been agreement that, the vacuolate bodies are X-bodies and that, a virus is responsible (3). Circumst,antial evidence consistent with this hypothesis is: (a) no known bacterial or fungal pathogen has been consistently isolated from, or de- tected in, infected plants; and (b) the disease is transmit’ted by leafhoppers of the genus Perliinsiella after a latent period of many days. ?Jone of this evidence shows conclu- sively that a virus is involved, especially since Mycoplasnaa-like organisms have been associated with certain leafhopper-borne dis- eases formerly attributed to viruses (C-7).

To determine whether electron micros- copy would provide more convincing evi- dence of the nature of the causal agent of Fiji disease, galls from infected plants were sectioned and viewed in an electron micro- scope. Other galls u-ere ground in a mortar and the sap examined. This paper reports that numerous viruslike particles were regu- larly observed.

Diseased and healthy plant material of t,he suscept’ible sugar-cane variety, Q.70, was collected at t’he Queensland Bureau of Sugar Experiment Stations Pathology Farm near Brisbane. Calls were cut off t,he leaves with

a scalpel; those taken from expanded leaves were hard and tough (“old galls”), whereas those from folded spindle leaves were soft and cheesy (“young galls”). Small pieces of gall tissue were fixed for 20 hours in 4.1% glutaraldehyde buffered to pH 7.2 with sodium cacodylate, washed with 0.2 M sucrose also buffered to pH 7.2, and post fixed in 1% osmium tetroxide for 20 hours. They were then dehydrat’ed in ethyl alcohol and embedded in Epon 812 with the aid of vacuum infiltration. Sections were cut \vit’h glass knives on a Porter-Blum ultramicro- tome, and stained with uranyl acetate and lead citrate. Pieces of leaf from healthy plants of Q.70 mere treated similarly.

Crude gall extracts were made by grinding single galls in a mortar with one or two drops of 2-3 % potassium phosphotungstate, pH 5 or 6. A drop of this extract was placed on a carbon-coated electron microscope grid, and excess fluid was removed with filt’er paper before viewing in a Siemens Elmiskop IA electron microscope. Xormal midrib and lamina tissues from both diseased and healthy leaves were treat’ed similarly as controls.

Viruslike particles were regularly de- tected in scatt’ered groups of phloem cells in sections of galls (Fig. 1). The particles typically had a darkly staining core of about 40 rnp diameter surrounded by a light’ly staining shell of 65-70 rnp diameter (Fig. 2). The core and t,he shell occasionally showed a hexagonal out’line.

The particles were found in the cytoplasm, either more or less evenly dispersed (Fig. I), or in groups (Fig. 3). Occasionally the groups of particles had a somewhat regular arrange- ment, but this fell short of complete crystal- lization. Some part’icles were embedded in or were near darkly staining cytoplasmic mate- rial (Figs. 3-5). This material was often rather diffuse in cells of young galls but was more commonly clearly defined and vacuo- late in cells of old galls. The clearly defined, vacuolate regions were usually 1-12 P in diameter.

In addition to these particles, others re- sembling the shells without the cores were someGmes found. These shells were always associated with the part’icles which con-

FIG. 1. Section of an old gall, showing a group of cells with numerous viruslike particles (V) and one cell with darkly staining cytoplasmic material resembling viroplasm (VP). X 5500.

140

FIG. 2. Section of a young gall, showing viruslike particles (V) which have a dense core surrounded by a more lightly staining shell, viroplasm (VP), and cell wall (Cl+‘). X 60,000.

FIG. 3. Section of an old gall, showing two vacuolate inclusion bodies composed ol viroplnsm (VP), and numerous viruslike particles (V) which are either free in the cytoplasm or associated with viroplasm (VP). x 14,000.

141

FIG. 4. Viroslike particles associated with viroplasm (VP). Arrows indicate shells withollr. darkly staining cores. X 60,000.

FIG. 5. Section of a young gall, showing a deeply indented nucleus (AV), ratherdiffuseviroplasm (VP), and viruslike particles (V). X 10,000.

112

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FIG. 6. Section of a young gall, showing :- -aLiered viruslike particles, endoplasmic reticulum (ER), golgi bodies (G), mitochondria (AT), and cell wall (CW). X 24,000.

tained cores, and they were typically in the areas of darkly staining cytoplasmic material (Fig. 4).

Nuclei were usually present in the par- ticle-containing cells of young galls, but were often absent from such cells of old galls. If present, the nuclei sometimes appeared to be distorted and deeply indented (Fig. 5). Mitochondria, endoplasmic reticulum, and Golgi bodies were often clearly defined in particle-containing cells of young galls (Fig. 6) but were often degenerate or lacking in such cells of old galls. Viruslike particles were never observed in the nucleus or mito- chondria and did not appear to be associated with endoplasmic reticulum or Golgi bodies (Fig. 6). Chloroplasts were never observed in cells which contained the particles.

No such particles were observed in the sections of healthy sugar-cane leaves.

Viruslike particles were regularly ob- served in crude gall extracts (Fig. 7). The particles appeared to consist of an inner

body about 60 rnp diameter, surrounded by an outer ring of outside diameter about 75 rnp. Capsomeres could sometimes be resolved on the outer ring and also on the inner body, although less clearly.

Extracts made from normal (non-gall) leaf t’issues of diseased plants or from leaf t’issues of healthy plants failed to yield similar par- ticles.

The particles found in sections and ex- tracts of galls from sugar-cane plants af- fected by Fiji disease are believed to be those of the causal virus. They resemble those of maize rough dwarf virus (8), clover wound tumor virus (9), rice dwarf virus (IO), and reo virus (11) . All the viruses have a core of about 40-50 rnp diameter surrounded by a shell of 60-75 rnp diameter. The high electron density of the core after staining with uranyl acetate suggests that it contains nucleic acid, whereas the low electron density of the shell is consistent, with its being protein. Clover wound tumor, rice dwarf and reo

FIG. 7. Extract of a gall, negatively stained with potassium phosphotungstate, showing several viruslike particles. X 100,000.

viruses are known to contaiu double- stranded RNA and to have 92 capsomeres. Further work with purified preparations of sugar-cane Fiji disease virus will be re- quired to determine whether it is similar in these respects.

The darkly staining regions in the cells containing particles are believed to be equiv- alent to the intracellular inclusions seen in the light microscope (e.g., 1,2, 12). Both are vacuolate and of similar size, i.e.,usually 1-12 p in diameter. The presence of both com- plet,e particles and empty “protein” shells in and around the inclusion bodies may in- dicate that they are equivalent to the sites of particle assembly or “viroplasm” re- ported from cells infected by maize rough dwarf, clover wound t,umor, and reo viruses (8, 9, 1s).

On the basis of biological characters, _ sugar-cane Fiji disease virus would appear

144 SHORT COM~MUNICATIONS

to resemble maize rough dwarf virus more than rice dwarf or clover wound tumor viruses. Sugar-cane Fiji disease and maize rough dwarf viruses are both transmitted by leafhoppers of the family Delphacidae, whereas rice dwarf and clover wound tumor viruses are both transmitted by leafhoppers of the family Jassidae. Further, clover wound tumor virus is not known to infect grasses. Whet’her any serological relation- ship exist’s between these four viruses has not been reported.

ACKNOWLEDGMENTS

We wish to thank Mr. D. H. Gowanlock and Mr. J. J’. Hardy, Electron Microscope Center, University of Queensland, for their expert tech- nical assistance in cutting the sections and taking the electron micrographs. The senior author was supported by Grant 5G of the Australian Research Grants Committee.

REFERENCES

1. ILICWHORTER, F. P., Philippine Agriculturist 11, 103-111 (1922).

2. KUNKEL, L. O., Hawaiian Sugar Planters’ Assoc. Expt. Sta. Bull. 3, 99-107 (1924).

8. HUGHES, C. G., and R~DINSON, P. E., in “Sugar-cane Diseases of the World” (J. P. RIart,in, E. V. Abbot.t, and C. G. Hughes, eds.), Vol, 1, pp. 389-400. Elsevier, Amster- dam, 1961.

4. 1101, Y., TERANAKA, M., YORA, K., and ASUYAMA, H., Ann. Phytopathol. Sac. Japan 33, 259-266 (1967).

5. LIN, S., and LEE, C., Taiwan Sugar Expt. &a. Ann. Rept. 1967-1968, pp. 17-22 (1968).

6. ~IARAMOROSCH, K., SHIKATA, E., and GRANA-

DOS, R. R., Trans. N.Y. Acad. Sci. 30,

841-855 (1968). 7. BO\VYER, J. W., ATHERTON, J. G., TEAKLE, D.

S., and AHERN, G. A., Australian J. Biol. Sci. 22 (in press) (1969).

8. GEROLA, F.M., and BASSI,~~~., Caryologia 19, 13-40 (1966).

9. SHIKATA, E., and nlARAMOROSCH, K., virology 32, 303-377 (1967).

10. FUKUHI, T., and SHIKATA, E., Tiirology 21, 503-505 (1963).

11. LOH, P. C., HOHL, H. R., and SOERGEL, M.,

J. Bacterial. 89, 1140-1144 (1965). 12. FERGUSON WOOD, E. J., Proc. Roy. Sot. Vie-

toria 49, [N.S.], 308-313 (1937).

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18. DALES, S., GOMATOS, P. J., and Hsu, K. C., viro’irology 25, 193-211 (1965).

D. S. TEAKLE Department of Microbiology University of Queensland Medical School Newton, Queensland, Australia 4006

D. R. L. STEINDL Bureau of Sugar Experiment Stations Brisbane, Queensland, Australia 4000

Accepted October 62, 1968

Reproduction of the Lactate

Dehydrogenase-Elevating (Riley) Virus

in Mouse Embryonic Liver Cell Cultures

Y&fee (1) noted, and Anderson et al. (2) quantitatively confirmed, that the Riley virus could be propagated and maintained by serial passages on primary mouse embryo cell cultures but not in secondary cultures. Rlultiplication of the virus has also been re- ported in primary cultures of adult mouse lung, spleen, liver (3) and macrophsges (+J-@. In each report of successful in vitro cultivation, virus reproduction was inferred from the maintenance of infectivity in the culture medium for a prolonged t’ime or dur- ing serial passage t’hrough cell cultures but increases in titers were not’ noted. In each case the lack of detectable morphological alterations in the host cells was specifically noted. Where tested, infection of t’he cul- tures caused no increase in lact’ate dehydro- genase (LDH) in the medium although t#his enzyme is characteristically elevated 5 to lo- fold in t)he plasma of infect’ed mice. C. W. Kg (personal communication) noted a cyto- pat,hogenic effect (CPE) of Riley virus in cell cultures prepared following mild tryp- sinization of embryonic mouse liver. We have confirmed that a CPE is usually detect- able in such cultures, if the condit,ions are stringently cont,rolled, and have found that bot.h the virus titer and LDH activity in- crease in the culture medium.

A &rain of Riley virus isolated in our lab- oratory from a mouse carrying the Ehrlich nscites carcinoma was used in most experi- ments. Five additional strains, originating from the laboratories of A. 1,. Kotkins (Be- thesda, 1Iaryland), V. Riley (Seattle, Wash-

ington), I’. G. Stansly (Detroit, Michigan) and M. H. Salaman (England), were also tested for their ability to induce CPE. All strains were subjected to at least two se- quential reisolations from mice receiving limiting dilutions (lOeg to lO+l) of plasma from mice infected for 24 hours. Plasma from noninfected mice was used as a control in all experiments.

Cell cultures suitable for demonstrating in- creases in virus titer were prepared from mouse embryonic liver cells disaggregated with 0.025 % trypsin (Baltimore Biological Laboratories, 1: 300) in Hanks’ balanced salt solution for 15 minutes at room tem- perature. Leighton tubes were seeded with about 300,000 cells in lactalbumin hydroly- sate medium with Earle’s salts (Grand Island Biological Co.) supplemented with 20 % fetal calf serum, 20 % bovine amniotic fluid, 0.3 mg/ml L-glutamine, 200 units/ml penicillin and 20 wg/ml streptomycin in an atmosphere of 5% CO, in air. About 10% of the cells attached to the glass. The medium was re- placed in 34 days and virus was added at 6-7 days.

Virus titers were determined by inoculat- ing lo-fold dilutions, prepared in Hanks’ balanced salt solution, into groups of 5 mice per dilution and measuring the plasma LDH levels 4 days later (7). Titers were expressed as the number of doses of virus per ml ca- pable of causing t,he characteristic enzyme elevat’ion in 50% of the test animals (ID,,/ ml) as calculated by t’he met’hod of Reed and J luench (8).

LDH in mouse plasma and in cell culture supernatant’s was determined spectrophoto- met’rically as described by Wrbblewski and LaDue (9).

The concentrations of infectious Rile) virus in the medium bat’hing infected em- bryonic liver cell cultures are shown in Fig. 1. In cultures prepared by a relatively severe trypsinization procedure (0.25 % trypsin for 40 min) a low but approximately consta.nt titer persisted throughout the test period, in agreement with the observationofYlagemann and Swim (3). Cultures prepared by the mild trypsinization procedure showed a drop in titer at 30 minutes postinoculation, followed by a significant rise in titer to a peak by 4