Attempts to increase the potency of the leukemia virus by repeated passage in newborn mice were undertaken in a group of experiments termed "selective virus-passage series." In principle the techniques were similar to those used to increase the concentration of virus in source tissues in the virus-induced avian neoplasms (46, 47). The series was initiated with leukemia virus derived from Sarcoma 37 and processed according to procedure B. Subsequent passages were effected by inoculation of virus derived from the induced leukemic spleens and lymph nodes. Donors of the source tissues were the first animals in each of the respective passages to exhibit macroscopic evidence of leukemia.

The results to date, based on latent period data, indicate that the potency of the virus has been increased with successive passages. The first passage material induced lymphocytic leukemia in 100 percent of the test animals, with an average latent period of 6.4 months. After eight virus passages in newborn mice, the agent now induces the generalized neoplasm in a high percentage of animals as early as 5.5 weeks after inoculation. One hundred percent of the inoculated mice have leukemia within a latent period of 2.5 months.

In this study, latent period is defined as time from virus inoculation to palpable inguinal lymph nodes and/or spleen. Time-to-death after detection of infiltrated organs varies from 11 to 28 days, with a mean of 19 days.

Pathology Of The Induced Disease. In Mice

Microscopic examination 9 of diseased tissue sections revealed that the lymphocytic neoplasm was widely dispersed and involved many organs and tissues. No neoplasms other than those of the lymphocytic type have been observed either grossly or microscopically in any test animals. In general, a similar histologic picture was recognized in all the mice examined, whether the disease developed after virus inoculation or after the mice received neoplastic cells. No significant variation was detected in mice of different strains or age groups.

The nucleus of the neoplastic cell was round or slightly indented and it was surrounded by a narrow rim of basophilic cytoplasm. Mitotic figures were frequent. In addition to infiltration of the spleen, liver, and lymph nodes by neoplastic cells, mice with the far-advanced disease also showed infiltration of neoplastic cells in the salivary glands, the kidney, the lungs, and the meninges. It should be emphasized that the histologic material was limited to mice with the fully developed disease, and early stages have not been studied. A more comprehensive morphologic study is now in progress.

Mean cell counts of the cardiac blood from leukemic male BALB/c mice are presented in table 4. These values are compared with those reported by Russell et al. (48) obtained from animals of the same subline. From the figures given in the table it is evident that the total leukocyte count was elevated. The erythrocyte count was low, and, as a further indication of anemia, the hemoglobin content, determined by the method of Drabkin and Austin (49), was also reduced.

Table 4. Cardiac Blood Cell Counts Of Male Balb/C Mice

White blood count(mm.3)

Red blood count (mm.3)

Hematocrit (percent)

Hemoglobin (gm/100 ml. blood)

Normal*

8.7 X 103

10.1 X 10^6

46.5

14.5

Leukemic

29.1 X 10»

7.0 X 10^6

34.6

10.3

*From the data of Russell et al. (48).

Macroscopic examination of mice with the virus-induced disease presented the usual picture of a generalized lymphocytic neoplasm (fig. 1). Invariably the mice exhibited marked hepatosplenomegaly. The peripheral and mesenteric lymph nodes showed massive involvement. In 90 to 95 percent of the leukemic animals, the thymus gland was greatly enlarged and sometimes obscured the heart on the anterior surface. Mice that developed leukemia after intraperitoneal implantation of neoplastic cells presented the same picture as that described, with the exception that the thymus gland showed relatively little, if any, gross involvement.

In Rats

The pathology of the virus-induced leukemia in the rat is in general similar to that described for mice.

Macroscopic

Although some rats with the induced disease exhibited the characteristics of a generalized lymphocytic neoplasm that involved the spleen, liver, lymph nodes, and thymus (fig. 2), a high percentage of the inoculated rats showed only marked enlargement of the thymus gland. The average weight of these infiltrated thymus glands was 7.0 gm. (normal thymus, 0.5 gm.).

Microscopic

Histologic studies on rats with the more generalized disease showed the neoplasm involved many organs and tissues.

The neoplastic cell was generally uniform and of large size in comparison with the normal lymphocyte. It contained a single highly basophilic nucleus and a small rim of nongranulated basophilic cytoplasm (fig. 3). Infiltration by these cells destroyed the normal architecture of the thymus and involved lymph nodes, and extended beyond the boundaries to involve adjacent organs and tissues (fig. 4).

Immunological Studies

The antigenic nature of the leukemia virus recovered from Sarcoma 37 is being investigated. Antiserums produced in rabbits by cell-free virus concentrates have been found to neutralize effectively the leukemia virus. Although the specificity and the absolute titer of the antiserums remain to be determined, preliminary findings indicate that a 1:2 dilution of one antiserum completely inactivated an equal volume of 10^-1.3 dilution of virus. Normal serums had no effect on the leukemogenic activity of similar virus preparations.

Biochemical Properties

The results of preliminary investigation into the biochemical nature of an active virus preparation are shown in table 5. The extract was prepared from a pool of virus-induced leukemic lymph nodes and spleens. The final concentration in terms of gm. equivalents per ml. was 0.5. The total nitrogen figure, 0.462 mg. per ml., when related to biological activity, serves as a measure of the degree of separation of nonactive nitrogenous material from the active agent.

Table 5. Results Of Chemical Analyses Of Virus Extract Prepared From Leukemic Lymph Nodes And Spleens

Analysis

Mg./ml.

Total nitrogen

0.

462

Total phosphorus

0.

076

Pentose nucleic acid

0.

274

Deoxypentosenucleic

0.

000

The value (0.076 mg./ml.) given for total phosphorus reflects the lipide-, nucleic acid-, and protein-phosphorus content of the particulate suspension.

With regard to the nucleic acid content, no deoxypentosenucleic acid (DNA) was found as measured by the diphenylamine reaction. The absorption of the nitrogenous bases at 2600 A in perchloric acid extracts of the particulate material was entirely accounted for by the amount of pentose nucleic acid in the acid extract, as measured by the orcinol reaction. These observations strongly indicate that the virus preparation was free of nuclear material and that the agent is an RNA-containing particle.