As discussed in a previous section, certain murine leukemia viruses are specific with respect to age, strain, and species of the host. It was considered of interest, therefore, to study the influence of these factors on infection by the leukemia virus recovered from Sarcoma 37.

Age

The susceptibility of BALB/c mice in various age groups to virus-induced leukemia was examined in a series of 4 experiments. In experiment 1 (table 2) the virus source material was Sarcoma 37; in experiments 2 to 4 (table 2) it was leukemic tissue induced by whole-cell inoculation. The agent was extracted from these tissues according to procedure B and in the case of Sarcoma 37, concentrated to 0.59 or 0.75 gm. equivalents per ml.4 The concentration of virus material derived from the leukemic tissues was either 0.25 or 0.5 gm. equivalents per ml.

Table 2. Susceptibility Of Balb/C Mice, At Various Ages, To Virus-Induced Leukemia

Experiment No.

Virus source

Age at inoculation (days)

Number positive

Number inoculated*

Incidence (percent)

Average latent period** (months)

1

Sarcoma 37

1-16

88/88

100

8. 4

2

Leukemic tissue

31

5/5

100

4. 5

3

Leukemic tissue

42-67

65/69++

94. 2

4. 4

4

Leukemic tissue

121-246

114/120++

95.0

6. 2

*Number Inoculated and surviving to weaning age where applicable. **Average time-to-death with leukemia after virus inoculation.

++Remaining mice died without tumor during observation period; cause of death unknown.

Mice 5 less than 30 days old received 0.1 ml. of extract subcutaneously, whereas the older mice were inoculated intraperitoneally with 0.2 ml. of virus concentrate.

As shown in column 3 (table 2), the mice in the combined experiments ranged in age from less than 1 through 246 days at the time of inoculation. In each experiment the mice were approximately equally distributed at 1- or 2-day intervals throughout the designated age bracket.

The percentage incidence data (column 5) indicate that death with leukemia was essentially 100 percent in experiments 1 through 4. It is evident, therefore, that the virus is capable of eliciting leukemia in mice inoculated as late as 8 months of age.

Strain

Although inbred BALB/c mice are used as test animals in most studies carried out in this laboratory, 8 additional strains and 5 F1 hybrid groups of mice were tested for susceptibility to the leukemia virus. Mice were inoculated at less than 1 day, at 4, or 6 days of age. A 0.1 ml. inoculum containing 0.26 to 0.5 gm. equivalent per ml. virus concentrate was employed. The inbred strains tested were: BALB/c (reference strain), C3H, C3Hf, I, A/LN, DBA/2, RIII, C57BL, and general purpose Swiss mice. The following hybrid groups of mice were also inoculated:

4 AI gm. equivalent per ml. concentrate is equal to 1.0 ml. of final suspension for every gm. of tissue processed. 4 All mice used in these studies and the strain specificity studies were from the inbred colonies of Dr. h. B. Andervont, National Cancer Institute.

(BALB/c X C3H)F1, (BALB/c X RIIDFx, (BALB/c X I)F1, (BALB/c X C57BL)F1, and (BALB/c X DBA/2)F1, The incidence of the viral-induced leukemia among the hybrid animals was 100 percent, with an average latent period of 3.3 months. With the exception of the C57BL mice, death with leukemia was also 100 percent in all inbred strains inoculated.6

The mean time-to-death with leukemia, when considered on the basis of age of the host within a given strain, did not vary significantly from the mean time-to-death of the reference (BALB/c) strain. The leukemia virus is therefore not highly strain specific. However, a low incidence of leukemia obtained for the C57BL mice (2 of 7 mice positive) might indicate a degree of resistance of this mouse strain. This possibility is now being investigated.

Species

In view of the apparent lack of strain specificity of this leukemia virus, it was considered of interest to extend the host studies to include animals of other species (45).

Sprague-Dawley rats 7 less than 36 hours of age were inoculated with the agent derived from a pool of virus-induced mouse leukemic thymus glands and lymph nodes. The extract was prepared by differential centrifugation alone, i.e., according to procedure A, and the final product, concentrated to 0.5 gm. equivalents per ml., was suspended in either 0.05 M pH 6.8 sodium citrate buffer or 0.12 M sodium chloride solution. The rats received the virus intracranially, intraperitoneally, or sub-cutaneously. The sodium chloride suspensions were used for the intracranial inoculations, and both citrate and sodium chloride suspensions were employed for the intraperitoneal and subcutaneous routes of administration.

The results of the experiments 16 weeks after virus inoculation are summarized in table 3. From the incidence data (column 4) and the mean time-to-death figures (column 5) it is apparent that the virus, originally derived from mouse leukemic tissues, is effective in producing the same disease in rats at a high incidence level after a short period of latency.8

The recovery of virus from the induced rat leukemic tissues has been demonstrated by the passage of cell-free extracts of this material in newborn rats and also in mice. Although the first rat "selective virus passage" experiment is still in progress, both rats and mice are dying with leukemia 7 weeks after inoculation of the rat-derived virus preparation. The leukemia agent, therefore, has not acquired host specificity after a single passage in the rat.

It is of interest to note that the virus-induced rat leukemia is also freely transplantable within the strain of rat. The neoplastic cells, when inoculated subcutaneously in adult Sprague-Dawley rats, produce a localized leukemia or lymphosarcoma at the site of implantation.

6 This incidence value excludes mice which died of unknown causes during the observation period, 7Furnished by the Animal Production Unit of the National Institutes of Health.

8 comparative assay of a 10^-1 dilution of the same virus preparation in newborn BALB/c mice produced leukemia In 100 percent of the recipients, with a mean time-to-death of 15.9 weeks.

Table 3. Virus-Induced Lymphocytic Leukemia In Sprague-Dawley Rats*

Virus dilution (log)

Number positive

Average latent period++ (months)

Route of inoculation

number inoculated**

Incidence (percent)

Intracerebral

0 -1 (0.05 ml.)

8/13 16/26

61. 0 61. 5

3. 1 3. 5

Intraperitoneal

-1 (0.2 ml.)

21/32

65. 6

3. 5

Subcutaneous

-1 (0.2 ml.)

17/24

70.8

3. 1

*Results at 16 weeks of experiments still in progress. **Age of rats at inoculation: <36 hours ++Average time-to-death with leukemia.