434 DISCUSSION AND PRELIMINARY REPORTS

parent virus in this respect. Once the HA( -) mutant appeared in virus progeny, how- ever, the virus readily multiplied in any mice carrying tumor cells. The finding that the HA( -) mutant appeared at the initial passage of the parent virus and became dominant at the next passage is not incom- patible with Cassel’s finding that the hemag- glutinin titer of virus preparations gradually diminished with the passage level in tumor cells.

REFERENCES

1. CASSEL, A., Virology, 3, 514-526 (1957). 2. VOGEL, J., and SHELOKOV, A., Science 126,358-

359 (1957). 3. ODA, M., Virology 21, 533-539 (1963).

MASAHIKO ODA Institute for Infectious Diseases University of Tokyo Minato-ku, Tokyo, Japan

Accepted April 27, 1964

Morphology of Shope Papilloma Virus

Associated with Nucleic Acid-Induced

Tumors of Cottontail Rabbitsl,’

Deoxyribonucleic acid (DNA) prepara- tions prepared by phenolic extraction of viral material from Shope papillomas of cottontail rabbits have been shown to be infectious and tumorigenic in domestic rab- bits (I). Tumorigenic DNA-containing ex-

1 The study was supported in part by grants from the National Institutes of Health (USPHS C-2669, C-5698, and C-6173).

2 Some of the results of the studies described in this paper were reported at the First Annual Meeting of the American Society for Cell Biology, November 1961.

tracts have also been obtained from papil- lomatous tissue of domestic rabbits (a).

The present paper deals with DSA-in- duced tumors of the cottontail rabbit, the natural host for Shope papillomatosis. The demonstration of whole virus particles as- sociated with the tumorigenicity and in- fectivity of the resulting tumors is presented.

The nucleic acid extracts were prepared from glycerinated tumor tissue of cottontail rabbits. Our standard extraction procedure (I) was employed, and in some cases the ex- tracts were further processed by et,hanol precipitation. The DNA content of such final aqueous extracts varied from approxi- mately 80 to 500 pg/ml. Two standard ex- tracts, one ethanol-precipitated extract and one extract pretreated with antiviral anti- serum, were inoculated in O.l-ml doses into clipped and shaved skin of cottontail rab- bits.3 The intradermal injection and punc- ture method of inoculation was employed (.2).

Among 24 inoculation sites tested in 4 cottontail rabbits, 22 sites, or 91.6%, gave rise to gross tumors, one of which is shown in the photograph in Fig. 1. The microscopic section of the tumor illustrates vast pro- liferation of epithelial cells and a thick layer of keratin covering the surface (Fig. 2). These gross and microscopic findings were no different from those of papillomas of rabbit skin induced by “intact” virus prepa- rations. From 4 fair-sized tumors induced by viral nucleic acid, 10% aqueous extracts were prepared and titrations for intact virus were carried out in domestic rabbits. The infectivity titer based on 100% end point

3 Cottontail rabbits were obtained from Earl Johnson Farm, Rago, Kansas.

FIG. 1. Panillomatous growth induced in skin of a cottontail rabbit with a DNA-containing extract. Photographed on the 86thday after inoculation.

FIG. 2. Photomicrograph of a section of DNA-induced tumor showing great proliferation of epithelial cells and a large amount of keratin at the surface. The tumor was removed 90 days after inoculation from the same rabbit whose tumor appears in the photograph of Fig. 1. Magnification: X20.

FIGS. 3-8. Electron micrographs of Shope papilloma virus particles negatively stained with sodium phosphotungstate. The bars in Figs. 3 and 4 represent 1000 A; those in Figs. 5-8 represent 506 A.

FIG. 3. Virus particles collected from an aqueous extract of DNA-induced cottontail tumor. FIG. 4. Virus particles from a virus-induced papilloma

FIGS. 5-7. Virus particles from DNA-induced tumors illustrating capsomeres FIG. 8. A damaged virus particle (arrow) and a cluster of “capsomeres” (C) from a DNA-induced tumor.

DISCUSSION AND PRELIMINARY REPORTS 435

FIGS. 1-8

was 1OP for three tumor preparations and lo-” for one preparation.

Partial purification of virus from nucleic acid-induced papillomas of cottontail rab- bits was attempted by two cycles of differ- ential centrifugation applying the classical method of Beard and his co-workers (3). The final pellets were resuspended in a small amount of phosphate-buffered saline (pH 7.5, free of magnesium and calcium) to which an equal volume of 2 %I sodium phos- photungstate was added. The resultant mix ture was mounted on carbon-coated grids and examined in a RCA EMU-2C electron microscope.

The observation of specimens prepared from nucleic acid-induced tumors revealed considerable numbers of spherical particles (Fig. 3) which were identical to the particles observed in specimens prepared from tumors induced by intact virus (Fig. 4). These virus particles are indistinguishable from those previously demonstrated in Shope papilloma virus preparations by other workers (4, 5). The particles measured 56-60 mk in diam- eter. The subunits or capsomeres, with a diameter of approximately 50 A, were clearly illustrated on their surface (Figs. j-7). Within the limited number of fields ex- amined, few defective “hollow” particles were detectable. Occasionally, a damaged virus particle or an accumulation of capso- meres were seen (Fig. 8).

The isolation of an abundance of typical papilloma virus particles from nucleic acid- induced tumors and the demonstration of their infectivity provide substantial evi- dence that these tumors are identical to those produced by intact papilloma virus. It is, therefore, evident that the entire synthetic process for production of intact virus can take place in the natural host when only viral DSA is made available to susceptible cells.

REFERENCES

1. ITO, Y., virobgy 12, 596601 (1960). 2. ITO, Y., and EVANS, C. A., J. Exptl. Med. 114,

485-500 (1961). 3. BEARD, J. W., BRYAN, W. R., and WYCOFF,

R. W. G., J. Inject. Diseases 65, 43-52 (1939). 4. WILLIAMS, R. C., KASS, S. J., and KNIGHT,

C. A., Virology 12, 48-58 (1960).

5. BREEDIS, C., BERWICK, L., and ASDERSOX, T. F., Virology 17, 84-94 (1962).

VELMA C. CHAMBERY Y~HEI ITO

Department of Microbiology School of Medicine, University of Washington, Seattle, Washington and Department of Hygiene, Sara Gakugei (Xational) University, Japan

.Iccepted April 29, 1964

Inhibition of Spreading of HeLa cells after

Infection with Herpes Simplex Virus

The formation of giant cells 8 hours after infection of HeLa cells with herpes simplex virus (I) implies the development of pro- found changes in the cell surface within 8 hours of infection. It was therefore of in- terest to determine how soon after infection detectable alterations in surface properties occur. The spreading of HeLa cells after their adhesion to glass is a biological phe- nomenon that must depend upon the state of the cell membrane, and it seemed likely to be a useful indicator of membrane changes resulting from infection with herpes simplex virus.

HeLa cells were grown in 20% (v/v) calf serum, 10% (w/v) Bacto-tryptose broth in Medium 199 with penicillin 100 units/ml and streptomycin 100 pg/ml; the same me- dium was used in all experiments. The HFE31 strain of herpes simplex virus was stored at -70” as infected chick embryo allantoic and amniotic fluid to which 20% (v/v) rabbit serum had been added. Spread- ing of cells was studied in a simple slide chamber. This was constructed by making a ring of paraffin wax on a chemically clean microscope slide, using a hollow metal cylinder 3’4 inch in diameter. A similar wax ring was made on a 1 inch square coverslip. Cell suspension (0.05 ml) was placed with a dropping pipette inside the ring on the slide and covered with the coverslip so that the wax rings were in apposition. The chamber was sealed by applying the heated metal cylinder to the coverslip so as to melt the wax. The chamber so formed was about 0.2 mm deep and contained an air space about one-quarter of the total volume. -After