for cell cultures, were raised in the Animal Production Section of the National Institutes of Health; they included hamsters, Cricetus auratus, and NIH strain white mice. The animals were received and kept in an area.free of the polyoma virus and separate from areas where rhesus monkeys were housed. Injections were made subcutaneously with inocula of 0.2 ml for newborn hamsters and 0.1 ml for newborn mice. The animals were observed for survival and macroscopic tumors every day or every other day. All dead animals were examined for the presence of tumors in the internal organs as were certain tumor-bearing animals that were killed. The tissues were fixed in sodium acetate-buffered formalin or in sodium acetate-buffered mercuric chloride "B5" (Lillie, 1954), embedded, sectioned, and stained with hematoxylin-eosin routinely, or in some instances with Masson's trichrome, periodic acid-Schiff, or phosphotungstic acid-hematoxylin techniques.

Other Methods Used to Characterize the Oncogenic Agent

For thermal stability experiments, the RMKC extract was sealed in glass ampules in 5-ml amounts and immersed in a water bath at 70° for 30 minutes. Another portion of the same extract was mixed with diethyl ether, stored at 4° for 18 hours, then freed of ether by passing nitrogen through the fluid. An additional portion of the extract was filtered through a very fine Corning sintered glass filter.

An aliquot of a similar RMKC extract, in a tube closed with a rubber stopper and tape, was stored in a dry ice chest for 353 days, then thawed and used.

Hemagglutination tests were carried out on the virus strains isolated from the hamster tumors by the same method used for testing the polyoma virus, namely the use of equal parts of a series of twofold virus dilutions and 0.4% washed guinea pig erythrocytes and incubation at 4° for 18 hours Eddy et al., 1958). In addition, duplicate mixtures of one virus strain, A426, and erythrocytes were prepared; one set was incubated at 4° and the other at 25°. Also, duplicate dilutions of virus were mixed individually with goat, cow, sheep, and horse

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erythrocytes and one set of each was incubated at 4° and the other at 25°.

RESULTS

Virus from Hamster Tumors

One hamster, A426, that developed a subcutaneous tumor 131 days after injection of RMKC extract was killed on the 203rd day. Cell cultures were prepared from the tumor tissue in 2-ounce prescription bottles. A sheet of cells grew over the flat surface of one side of the bottle. The medium was changed at weekly intervals. After the cultures had been incubated for 29 days, the cells from ten culture bottles were detached from the glass by freezing and thawing, concentrated to one-tenth volume by centrifugation at 4000 rpm for 10 minutes, and ground to a fine powder over dry ice. The powdered extract was allowed to thaw and then injected into 11 newborn hamsters. Five of these hamsters, observed at 125 days of age, had developed subcutaneous tumors (Table 1).

Some of the fluid from the hamster tumor cell cultures which manifested only minor nonspecific or questionable cytopathic changes were transferred to cultures of C. aethiops kidney. By the seventh day multiple, small vacuoies were seen in the cytoplasm of the cell cultures that were characteristic of SV40. The cytopathic changes progressed so that by the 14th day only masses of brownish cellular debris remained. Transfer of this virus, A426, to mouse embryo cell cultures caused no cytopathic · changes of any kind.

First, second, and third passage of strain A426 virus in C. aethiops kidney cell cultures also elicited subcutaneous tumors when injected into newborn hamsters. The inoculum for obtaining the third C. aethiops kidney passage virus was the ultimate dilution, 10-, of second passage virus. Control C. aethiops kidney cell cultures that had not been incubated with virus failed to produce tumors in hamsters.

Tumors also failed to develop when antiSV40 rabbit serum was combined with the virus before injecting into hamsters. Normal rabbit serum did not exhibi- this inhibitory effect (Table 1).

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covered from a hamster, that had been injected when newborn with a filtered lot of an extract of RMKC that was different from the lot that stimulated the production of a tumor in hamster A426. This hamster developed a subcutaneous tuinor 203 days after

injection and was killed on the 304th day. A portion of the tumor was removed and trypsinized. Some of the packed cells obtained from the trypsinized tumor were added to 2-ounce prescription bottle cultures of C. aethiops kidney and incubated

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for 14 days. Cytopathic changes occurred in these cultures that were typical of SV40. Third passage virus A528 in C. aethiops kidney cell cultures when injected into newborn hamsters, induced tumors in 10 of 13 animals in 118 days. The virus titer as determined in roller tube cultures of C. aethiops kidney was 10- and the titer was reduced to 10-3 when the virus dilutions were combined with anti-SV40 rabbit serum.

The virus did not cause hemagglutination of guinea pig erythrocytes, nor were cytopathic changes seen when the virus was incubated with mouse embryo cell cultures.

The appearance of the tumors that were induced by both strains of virus, A426 and A528, recovered from the hamster tumors were identical with those that developed following the injection of newborn hamsters with RMKC extracts (Eddy et al., 1961).

tissue surrounded the tumor at the periphery and invaginated to lobulate the tumor parenchyma. The tumor cells contained pleomorphic, anaplastic nuclei within a sparse amount of cytoplasm, as shown in Fig. 1. The nuclear shapes ranged from ovoid to rhomboid (Fig. 2). The nuclear chromatin was marginated and irregularly arranged within the nucleus; most cell nuclei were vesiculated, and a few were hyperchromatic. There were large numbers of multinucleated giant cells in the tumors (Figs. 1 and 2). The tumors appeared, therefore, to be undifferentiated sarcomas.

In 2 of the 6 animals there were also tumors in the lungs. One of these tumors was approximately 2 x 2 x 2 mm, the other a diffuse reddish purple mass, which replaced most of the normal lung. The lung with the massive tumor had a more highly differentiated tumor cell of a fibrosarcomatous form (Fig. 3). Adjacent muscle fibers of the dia

Strain 776

Thirteen hamsters at 1 day of age were injected with SV40, strain 776. Tumors developed in 11 of these animals after 156 to 380 days, and the tumors were macroscopically indistinguishable from the tumors induced by RMKC extracts (Eddy et al., 1961). One animal was missing on the 249th day and one animal has survived without an apparent tumor for 391 days. This animal cannot yet be considered negative since tumors have been observed in hamsters injected with RMKC extracts after long periods of time, one as late as 589 days after injection.

The tissues of 6 of the 11 tumor-bearing hamsters that had been injected with SV40, strain 776, were examined histologically. The tumors at the site of inoculation were elevated, rounded, and firm, and varied in size from 13 x 5 x 5 cm to 3 x 2.5 x 2 cm. On cut surface the tumors appeared as rounded masses of tissue surrounded by a firmly adhering dense capsule.

The cells from the subcutaneous tumors did not invade the body cavity directly. The tumors contained varying amounts of necrotic cells and hemorrhage. In those areas of tumors with intact cells, there were many small, congested capillaries. Dense fibrous

(Fig. 4). The parietal pericardium and the pleural surface of the diaphragm at other sites was infiltrated to a small extent with tumor cells. The other lung tumor cell type was a sarcoma unlike that observed in the other hamsters.

The tumors in the subcutaneous tissue were similar to those observed in hamsters inoculated with RMKC extracts and to those induced by the polyoma virus in hamsters and guinea pigs. On the basis of histological difference, it would be difficult to separate the subcutaneous tumors induced in the hamster either by RMKC extract, SV40, or the polyoma virus.

Mice

Newborn mice were injected subcutaneously with an RMKC extract and with SV40, strain 776. Seventeen mice were injected with a preparation of RMKC extract that induced tumors in 12 of 14 hamsters 149 to 217 days later. One mouse was missing on the 282nd day and one died on the 293rd day; no tumors were seen. The remaining 15 mice have been under observation for 344 days and no tumors have developed.

Ten mice received SV40, strain 776. One was missing after 245 days, two after 278

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days, and one after 309 days. The remaining six nice have survived 343 days and no tumors are evident.

Biological and Physical Properties of RMKC Extracts

The properties of the RMKC extracts that were investigated were concordent with those described by Sweet and Hilleman (1960) for SV40. Heating an extract to 70° for 30 minutes increased either the la tent period or the incidence of tumor forma tion, so that after 361 days only 3 of 12 animals developed tumors. The oncogenic property of the extract was unaffected by treatment with diethyl ether, and only a slight delay in the development of tumors occurred with an extract which was filtered through a very fine sintered glass filter or

stored at -70° for as long as 353 days (Table 1).

SV40 in RMKC Extracts

Since the observation that tumors were induced in hamsters by RMKC extracts was made prior to the report by Sweet and Hilleman (1960), the extracts that induced these tumors had not been checked for the presence of SV40. Portions of four extracts had been stored at -70° for periods of time ranging from 9 to 20 months. These four extracts were thawed and titrated for cytopathic changes in C. aethiops kidney cell cultures. The results are shown in Table 3. An extract that failed to induce tumors in hamsters caused no changes in C. aethiops kidney cell cultures that were typical of SV40, whereas characteristic cytopathie