[CANCER RESEARCh 26 Part 1: 364-369,March 1966]

Summary

Murine leukemia virus, isolated from Ha/ICR Swiss miceduring the course of experiments which originated with assay ofhuman leukemias and tumors for the presence of oncogenicagents, was recovered within 1—2weeks from organs of mice injected at birth with cell-free filtrates from leukemic tissues. Theincidences of leukemia induced by filtrates made from organs ofmice at weekly intervals from 2 to 13 weeks after injection atbirth were comparable to those obtained with filtrate from organsof leukemic mice. There was about 1 log greater infectivity in

filtrates from pooled kidney-liver-spleen than from brain, asmeasured by incidence ofleukemia in injected mice. Dose-responsecurves showed a decrease in incidence and increase in latency ofleukemia development with increasing dilutions of filtrates.Similar growth and dose-response curves were obtained in anumber of experiments. Virus could not be recovered from weanling mice, which are resistant to induction of leukemia by virus,until 8 weeks after infection. It is suggested that weanling micemay develop an early immunity to virus infection which may belost in time. Recovery of virus from Sprague-Dawley rats 2 weeksafter injection at birth was poor; virus could not be recovered at5 and 8 weeks of age. However, virus which was highly infectious,as determined by high incidence of leukemia in mice, could be recovered from rat leukemias induced by the mouse virus.

Introduction

The isolation in our laboratory of a leukemia virus from HalICR Swiss mice was 1st reported in 1963 (1). It was isolatedduring the course of experiments which originated with the assayof human tumors and leukemias for the presence of oncogenic

agents. Some of the properties of this virus were reported in 1964(2). These are : induction of lymphocytic leukemia in approximately 80% of Swiss mice injected at birth with cell-free filtrates;retention of infectivity after storage at room temperature for 24hr and at 4°Cfor 48 hr ; some loss of infectivity following storageat —20°Cand in the CO2 deepfreeze box for varying periods;

inactivation at 56°Cfor 30 mm; resistance to ether; and neutralization by specific rabbit antiserum. RF strain mice and SpragueDawley rats are susceptible to infection when injected at birth;weanling Swiss mice are resistant to infection.

This agent is now in its 18th consecutive passage in Ha/ICRmice, and continues to induce leukemia in 80—90% of mice, infected at birth, at 8—9months of age. The somewhat higherincidence in females has been maintained. The incidence of

1 This work was supported in part by grants from the John A.

Hartford Foundation and the USPHS.

spontaneous leukemia in mice of this strain remains very low,less than 1%; 2 of 916 control mice (1 male, 1 female) observedfor a period of 10—12months during the past 1—2years developedthymic and generalized leukemia at 9 months of age.

Further studies have been concerned with recovery of thisvirus from animals in the preleukemic period and establishmentof growth and dose-response curves based on the incidence ofleukemia in assay mice. The results of these studies are reportedhere.

Materials and Methods

Pregnant Ha/ICR Swiss mice were obtained from Roswell

Park Memorial Institute breeding colonies. Sprague-Dawleyrat litters were obtained by randomly mating rats obtained fromthe Charles River Breeding Laboratories. All animals receivedDerwood-Morris (Teklad) diet and tap water ad libitum. Intercurrent infections were treated with Achromycin (Lederle),50 mg/500 ml of drinking water, for 1—2weeks. All animals wereinspected biweekly, and sacrificed in extremis whenever possible.Histologic sections were taken as necessary to establish thediagnosis of leukemia or other cause of death.

Cell-free filtrates were made routinely from the kidney, liver,spleen and lymphoid tissues of leukemic mice, and from kidney,liver, and spleen (KLS) of nonleukemic mice. Tissues werehomogenized in sterile phosphate-buffered saline to which 1%penicillin-streptomycin had been added. The final dilution was a10% w/v suspension of tissue. The homogenate was spun downfor 1 hr at 5000 rpm in a model L Spinco centrifuge (No. 40rotor) at 4°C.The supernatant fluid was removed and passedthrough 0.45-j.& millipore filters, using E. coli markers. Postfiltration sterility was also checked in Sabouraud's and thioglycollate broth.

For the growth and dose-response curves study, organs werecollected from at least 5 animals and pooled at weekly intervalsfor a period of from 1 to 13 weeks after injection of newborn mice.From previous data, this time interval covered the period todevelopment of leukemia. Brain, and kidney, liver and spleen

(KLS) were collected by 2 operators, one taking brain, the otherthe abdominal viscera, with separate instruments, to avoidcontamination of one set of specimens with the other. The amountof blood contaminating the brain tissue was nil. The tissueswere stored in the CO2 deepfreeze box until time to make thefiltrates. Filtrates were made from each group of organs, anddilutions were made out to 10@ for injection. Thirty newbornmice were injected with each preparation, making a total of4680, in order to assure an adequate number for evaluation ofresults.

None of the animals from which organs were collected for the

364 CANCER RESEARCH VOL. 26

Recovery of Murine Leukemia Virus Prior to Development ofLeukemia: Growth Curve and Dose Response1

R. F. BUFFETT,J. 1. GRACE,JR., L. A. DIBERARDINO,AND E. A. MIRANDRoswell Park Memorial Institute, Buffalo, New York

on July 6, 2020. © 1966 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Leukemia Virus Growth and Dose-Response Curves

growth and dose-response curves study were grossly leukemic;histologic sections were not taken. However, the incidence ofleukemia in the passage from which these tissues were collected,after all animals had been sacrificed for collection of tissues,was 67 % over a period of from 12 to 40 weeks.

Results

The results of the growth and dose-response curves study areshown in Tables 1 and 2. Virus was recovered from kidney, liver,and spleen (KLS) as early as 1 week after infection at birth.Recovery of virus from these organs at 2 weeks after infectionwas comparable to that at weekly intervals up to 13 weeks (Table 1) . Similarly, virus was recovered from brain, in good titer,2 weeks after infection at birth, and at regular intervals up to 13weeks (Table 2). The incidence of leukemia in females was moreconsistent because of variable mortality as a result of fighting inmales ; the incidence of leukemia in females is therefore indicatedin parentheses. The experiment was terminated at 36 weeks(9 months).

Numerous attempts have been made to isolate a leukemiavirus from normal Ha/ICR mice, at all ages, without success.

Dose-response curves, comparing infectivity of filtrates fromKLS and brain, as measured by incidence of leukemia in femalemice, are shown in Chart 1. There is about 1 log greater infectivity in filtrates from KLS. These curves were obtained byaveraging the % leukemia.s in females for the 2- to 13-week assayintervals (1st week excluded). The ID50 titer of infectivity foreach filtrate is shown in Table 3.

Latency of leukemia development in female mice at the 10-folddilutions of filtrates is shown in Table 4. Since the latencies fromthe 2- to 13-week assays were very similar, the cumulative mcidence for each dilution was obtained by averaging the % leukemias for the 2—13weeks at each dilution. It can be seen readilythat not only does the incidence of leukemia fall off with each10-fold dilution of filtrate, but also the latency period is comparably extended in both groups, and to a somewhat greaterdegree in the animals receiving filtrates from brain and tissue.

Several dose-response experiments have been carried out, on asmaller scale, since the larger growth and dose-response curvesstudy was undertaken, and the results are comparable to thoseobtained in the earlier study (Table 5). As is to be expected,there are variations among filtrates. The material used in Experiment 5 was stored as homogenate for 3—6weeks in the CO2 deepfreeze box before filtrates were prepared. From l)ast experience,we would expect this to reduce infectivity somewhat (2). As inthe earlier study, latency of leukemia development was extendedby increasing dilutions of virus (Table 6).

Recovery of virus from infected animals in the preleukemicperiod has been confirmed a number of times (Table 7). Cell-freefiltrates from pooled KLS from mice at intervals from 2 to 8weeks after injection at birth have produced results similar tothose obtained in the larger growth and dose-response curvesstudy. In each instance, the mice from which virus was recoveredhad been injected with filtrates from leukemic tissues fromdifferent donor mice.

The incidence of leukemia in mice injected as weanlings withcell-free filtrates from leukemic tissues was previously reportedat 8% in 26 to 47 weeks (2). A more definitive age susceptibilitystudy was undertaken (Table 8). Although there was a progres

TABLE 1

GROWTH AND 1)osE-REsP0NsE CURVES OF MURINE LEUKEMIA

VIRUS RECOVERED FROM KIDNEY, LIvEII, AND SPLEEN

(PooLED) OF Ha/ICR SWISS MICE

%LEUKEMIASINDUCEDBY10-FOLDDILUTIONSOFCELL-FREEFILTRATESb

a Ha/ICR Swiss mice, injected at birth with cell-free filtratefrom leukemic t issues from 2 donor mice (pooled).

b Total number of mice surviving at 56 days of age = 2107. All

mice were injected s.c. in the back with 0.1 ml of filtrate, at birth.100 10% w/v preparation.

C Incidence in: A, all mice; F, female mice.

d Averages of % leukemia.s induced by filtrates from 2- to 13-

week-old donors (1-week-old donors excluded).

sive fall in incidence of leukemia in mice infected up to 10 daysafter birth, the incidence fell off sharply at 14 days and thereafter. Also, as the incidence of leukemia fell, the latency periodwas extended (Table 9).

It was of interest, then, to determine whether the virus reproduces in, and can be recovered from, weanling mice even thoughthey do not develop leukemia; i.e., whether there might be infection, which is recoverable, without overt manifestation ofdisease.

When filtrates from pooled KLS from mice, injected as weanlings (4—6weeks of age) with a filtrate from leukemic tissues,were assayed in newborn mice, leukemia virus could not be recovered until 8 weeks after infection (Table 7). The incidenceof leukemia (48 % of all mice, 50% of females) was somewhatlower than in assays from mice infected at birth, and the latencyperiod was extended to from 5 to 11 months as compared with3—9months (see Tables 1 and 4, KLS 10°).The potency of thefiltrate which the weanling mice received was checked in newbornHa/ICR mice; 77% of all mice (93% of females) developed leukemia in 3—6months.

It has been previously reported (2) that a small number ofSprague-Dawley rats (17 %) developed leukemia following injection at birth with cell-free filtrate from leukemic mice ; a filtrate

from rat leukemic tissues, in turn, induced leukemia in 77 % ofHa/ICR mice in 16-44 weeks.

Since the incidence and latency of leukemia in Ha/ICR mice

AGE OFDONOR

ANIMALSa (wk)

23456

89

10111213

Average―

10—S

A F

3 14

100 10—1 10-'

A FA' F A F

10—2

A F

3757 70 10 20

725359715360634731574473

807073857573828050786792

796757606750635648585060

868480778560826967736073

503356524441373336374335

576792506350584663626350

4043

725311728141150

3

50671340601838181856

7

10—S

AF

7747

13 17

3146

30773

8

1615411558

11

1112438

73

1034

6

2712117

18

1714141010

1258 76 60 76 41 60 22 29

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1234567

8910111213

U)4

Li

D 60Li.-J

@4O

20

DILUTIONSCUMIJLATWE

INCIDENCE (%) OF LEUKEMIA IN FEMALES―

g_12b 12-16 16—20 20—24 24—28 28—3232—36KLSC1000.8351425417610—10131123367610_I003712206010@0000492910—i00000.741210—b00000011Brain100024917276110—I00.7141227611020000.7352310—s0000.611810-i000000810-b0000009

R. F. Buffelt, J. T. Grace, Jr., L. A. DiBerardino, and E. A. Mirand

TABLE 3ID50 OF LEUKEMIA \IRUS PER GM EQUIVALENT OF TISSUE IN

ORGAN FILTRATESC

TABLE 2

GROWTH AND DOSE-RESPONSE CURVES OF MURINE LEUKEMIA

VIRus RECOVERED FROM BRAIN OF Ha/ICR SWISS MICE

% LEUKEMIASINDUCEDBY10-FOLDDILUTIONSOPCELL-FREEFILTRATESb LOGDILUTIONOFVIRUSFROM

AGEOFDONOR ANIMALSb @c Brain

(wk) _____________ _____________ ____________________________

AGEOFDONOR 10° 10-' 10—2

ANIMAl-Ba (wk)

AC F A F A F

1 7 8 38

2 53 67 43 82 18 363 55 60 43 60 14 254 62 75 33 605 12 7 73 77 10 76 38 58 37 607 46 92 53 58 16 30

8 52 50 14 38 32 429 63 63 77 85 20 5210 40 57 23 43 14 1911 50 77 42 56 13 2412 77 63 54 62 15 2313 54 67 52 46 30 46

Average― 50 61 45 61 16 23

10-I 10-'

A F A F

9

All mice

0—1.7—1.7—1.4—1.7—1.2—1.5—1.5—2.3—1.8—1.8—2.1—2.0

All mice

—1.2—3.1—2.6—2.4—2.8—2.6—2.2—2.6—2.3—2.1—2.7—2.0—2.3

Females

—1.5—3.6—3.6—3.3—3.3—3.4—2.4—3.4—3.1—3.3—3.5—3.0—2.8

Females

0—2.4—2.1—2.0—1.6—2.1—2.@—1.8—2.4—2.4—2.5—2.2—2.4

6

715

13187

1768

8

3

88

107

4834

5

5

43347

10

5

358729

8

10-'

A F

4 107 13

87

317 3110 19387 17

a Cell-free filtrates prepared from tissues obtained at weekly

5 9 intervals following inoculation of newborn Ha/ICR mice. ID50— — = titer at which 50% of assay animals died of leukemia (Reed

a Ha/ICR Swiss mice, injected at birth with cell-free filtrate Muench).

from leukemic tissues from 2 donor mice (pooled). bHa/ICR Swiss mice, injected at birth with cell-free filtrateb Total number of mice surviving at 56 days of age = 2075. All from leukemic tissues of 2 donor mice (pooled).

mice were injected s.c. in the back with 0.1 ml of filtrate, at birth. CPool of kidney, liver, and spleen.100 = 10% w/v preparation.

C Incidence in: A, all mice; F, female mice. TABLE 4

d Averages of % leukemias induced by filtrates from 2- to 13- LATENCY OF LEUKEMIAS INDUCED BY 10-FOLD DILUTIONS OF

week-old donors (1-week-old donors excluded). CELLFREE FILTRATE: GROWTH AND DOSE-RESPONSECURVES STUDY

FROM KLS —

FROM BRAIN-@-@-

100 10.-I ,o@2@@@

DILUTIONS OF CFF

CHART 1. Dose-response curves, comparing infectivity of cellfree filtrates (CFF) from pooled kidney, liver, and spleen (KLS)and brain; incidence of leukemia in female Ha/ICR Swiss mice.Each point = average of % leukemias induced by filtrates from2- to 13-week-old donors (from Tables 1 and 2).

which received filtrate from rat leukemic tissues were comparableto those obtained when mice receive filtrates from mice, eventhough the incidence in rats is low, an attempt was made torecover virus from rats in the 1st 8 weeks after injection at birthwith a cell-free filtrate from leukemic mouse tissue. Virus could

a Averages of % leukemias induced in female Ha/ICR mice by

filtrates from 2- to 13-week-old donor mice (1-week-old donorsexcluded).

b Weeks of age.

C Pool of kidney, liver, and spleen.

be recovered 2 weeks after injection, but not at 5 and 8 weeks.The incidence was very low (27 %) as compared with that inmice which receive filtrates from fully leukemic rats. The ratswhich were not sacrificed for recovery assays were kept to deter

366 CANCER RESEARCH VOL. 26

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% LEUKEMIASINDUCEDBY10-FoLDDILUTIONSOFCELL-FREEFILTRATESbEXPERIMINT―10010'10'10-'10-'10'1633872833416373110448@60621700570632

TIs@ FROMINTECTION

(wk)ANIMALS

INFECTED AS

-______________b !Newborn Vseanl,ng Newborn

Ha/ICR Ha/ICR Sprague-DawleySwiss SwissRats225/40e

(62.5)1/19 (5)8/30(27)318/27(66.67)419/36(53)420/25(80)413/19(68)516/24

(66.67)0/230/20623/41(56)0/27812/25

(48)0/27

AGE ATINJECTION―

(days)INCIDENCE

OFLEUKEMIAbTotal

No. of micecNo. of female micecNo. of malemiceC027/29

(93)17/17 (100)10/12(83)329/42(69)20/24 (83)9/18(50)723/41(56)12/21 (71)8/20(40)1014/40(35)8/13 (62)6/27(22)142/40(5)2/17(12)0/23170/380/130/25212/28

(7)2/15(13)0/13244/30

(13)4/20(20)0/10280/300/200/10

Leukemia Virus Growth and Dose-Response Curves

TABLE 5

DOSE RESPONSE TO INFECTION WITH MURINE LEUKEMIA VIRUS

TABLE 7

RECOVERY OF VIRUS FROM INFECTED ANIMALS IN THE

PRELEUKEMIC PERIOD―

a Ha/ICR Swiss mice were injected at birth with cell-free fil

trates from organs from leukemic mice.b The numbers of mice surviving at 56 days of age ranged from

30 to 240/experimental group.C Mortality from intercurrent disease was unusually high,

affecting the total incidence of leukemia.

TABLE 6

LATENCY OF LEUKEMIAS INDUCED BY 10-FOLD DILUTIONS OFCELL-FREE FILTRATES

a All assays were done in newborn Ha/ICR Swiss mice. Assay

materials were cell-free filtrates from pooled kidney, liver, andspleen from 3—5donor animals.

5 Experiments terminated at 10 months. Mice assayed at each

time interval received filtrates from different leukemic Swiss mice.C Experiment terminated at 12 months. All weanling mice re

ceived the same filtrate, prepared from pooled organs of 3 leukemic S@vissmice.

d Experiment terminated at 12 months. All rats received the

@48 same filtrate, prepared from pooled organs of 2 leukemic Swiss

mice.e No. with leukemia/no. infected and surviving at 56 days of

age; % in parentheses.

TABLE 8

AGE SUSCEPTIBILITY OF Ha/ICR SWISS MICE TO INDUCTION OFLEUKEMIA BY MURINE LEUKEMIA VIRUS

CUMULATIVE INCIDENCE (%) OP LEURZMIAb

8@l2c12—1616—2020—2424—2828—32

0 3 17 20 30 400 4 4 30 52 650 14 14 25 50 552.510 20 25 32 455 15 37 47 57 59

0 7 27 27 47 530 5 10 23 41 46

0 0 10 14 24 280 3 3 3 9 110 0 0 11 11 110 3 13 31 38 62

0 0 3 3 3 100 0 0.4 1 1.3 1.@

0 0 0 0 0 70 0 0 0 0 00 0 0 0 0 00 0 0 0 0 0

32—36 36—4040—44

EXPERIMENT―

100 dilution12345

10—1dilution45

102 dilution1234

10—sdilution45

10' dilution12

34

47

70

68

48―69

60

59

31141162

17

1.3

7

1100

83

73

34

11

140

83

34

16

63

70

68

70

63

383111

2

7110

a Ha/ICR Swiss mice were injected at birth with cell-free flu

trates from organs from leukemic mice.S The numbers of mice surviving at 56 days of age ranged from

30 to 240/experimental group.C Weeks of age.

d Mortality from intercurrent disease was unusually high,

affecting the total incidence of leukemia.

mine the incidence and latency of leukemia development. Threeof 29 rats (10%) developed leukemia at 4.5—8months of age,while the incidence in Ha/ICR mice was 71 % (96% in females)at 2.5—7months of age. A filtrate from leukemic tissues from

a All animals were injected Ofl the same day with the same cell

free filtrate; 0- to 7-day-old mice received 0.1 ml, 10- to 17-day-oldmice 0.15 ml, and 21- to 28-day-old mice 0.2 ml, s.c. in the back;10% (w/v) preparation.

b Experiment terminated at end of 1 year.

C No. with leukemia/no. of mice injected and survivillg at 56

days of age; % in parentheses.

1 of these rats (the 1st one to develop leukemia) was returnedto both rats and mice ; the incidence of leukemia in rats is only9% (3 of 33) in 3—7months, whereas the incidence in mice is77 % (90% in females).

367MARCH 1966

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AGE ATINJECTIONb@ No. LEUKEMIAS/

@ No. INJECTEDC(days) @No.

OFLEUKE@ASO@CUR@INGATMEEKLYINTERVALS-__________________________8—12—

12—1616—2020—2424—2828—3232—3636—4040—4444—4848-320

@ 27/2910456113@ 29/42274921317

23/4118311114211014/40213215142/4011170/38212/282244/30112280/30

R. F. Buffett, J. T. Grace, Jr., L. A . DiBerardino, and E. A . Mirand

TABLE 9AGE SUSCEPTIBILITY OF Ha/ICR SwISS MICE TO INDUCTIONOF LEUKEMIA BY MURINE LEUKEMIA

VIRuS: LATENCY OF LEUKEMIA I)EVELOPMENT―

a Experiment terminated at end of 1 year.

SAll animals were injected on the same day with the same cell-free filtrate; 0- to 7-day-old mice received 0.1 ml, 10- to 17-day-old mice 0.15 ml, and 21- to 28-day-old mice 0.2 ml, s.c. in the back; 10%(w/v) preparation.

C Surviving at 56 days of age.

Discussion

The data accumulated here, for a murine leukemia virus fromyet another source, are compatible with those reported for anumber of other murine leukemia viruses which have beenisolated during the past 15 years in other laboratories (4, 5, 7,11—13, 15).

Virus may be recovered from organs of mice within 1—2weeksafter infection (5, 7, 12). Recovery of a leukemogenic agent frombrain tissue was 1st reported by Schoolman et at. (14). Gross (4)and Friend (3) found that the potencies of extracts from brainsfrom leukemic animals were relatively low. Rauscher (12) founda high titer of infectivity in brain tissue from leukemic mice.Spencer (16) recovered i@Joloney virus in 7 days from brains ofmice which were injected intracerebrally ; he also recoveredvirus from brain tissue of leukemic mice which had been injected

i.p. with virus. In all cases, the morphologic findings were thoseof the typical virus-induced disease, and in sharp contrast to themorphologic picture described by Schoolman et al. (14).

It was gratifying, therefore, to find that we could recover virusfrom brain tissue from mice infected with our agent ; the leukemias induced were typical of those induced by virus from

leukemic tissues. As seen in Chart 1 and Table 3, the brainfiltrates were about 1 log less rrntent than the KLS filtrates. Thedata in Table 2 suggest that there may have been an increase inthe amount of virus that was recoverable from brain as theanimals grew older, since responses to the higher dilutions arequite erratic prior to the 9th week. However, the ID50 titersreported in Table 3 would seem not to support this. Except forWeeks 5 and 8, the ID50 titers in females are quite consistent.The explanation of the peculiar results for the 5th week at 10°and 10' dilutions is not apparent; these results lowered theID50 titer for the group. We have not attempted to recovervirus from brains of leukemic mice.

What the pattern of recovery of virus will be in the timeinterval between injection at birth and 1—2weeks of age remainsto be seen. Since induction of high incidence of leukemia with

our virus necessitates the use of newborn mice, adequate exploration of this time interval by assays in mice is almost prohibitive,and awaits the adaptation of an in vitro technic for determinationof virus titer in preparations from leukemic tissues (V. C. Dunkel,personal communication).

The incidence of leukemia may vary considerably, both fromone experiment to another and within a single experiment (Tables1, 2, 5, and 7). This variability is not unexpected and is difficultto overcome because of the number of opportunities in a systemof this type for (a) sampling errors which may occur in obtainingthe original material for virus preparation, in preparation of thematerial, and in administering the exact dosages to newbornmice (and also in retention of the full amount administered),and (b) variability in host response as the result of intercurrentinfections, stress (fighting), etc.

Since the responses of female mice to the various preparationsfrom KLS and brain from 2- to 13-week-old mice were fairlyconsistent, both in time and dose, it seemed permissible to cornbine the results of all groups to establish dose-response curves.These curves (Chart 1), then, represent the approximate doseresponse to the virus that may be anticipated, and not theactual dose response which may be obtained in any given experiment.

The recovery of virus from weanling mice 8 weeks after infection, but not at 2, 5, and 6 weeks, is puzzling. That the viruspreparation used to infect the weanlings was potent is indicatedby the very high incidence and short latency of leukemia development in the control mice injected at birth. One possible explanation is that weanling mice develop an active immunity to thevirus during the 1st 6—8weeks after infection, powerful enoughto neutralize virus and prevent its recovery. But somewherebetween 6 and 8 weeks, immunity wanes, permitting the reproduction of residual virus which can then be recovered in fairlygood titer.

Supporting this idea are the results of a pilot study in ourlaboratory (unpublished data). Adult female mice were given

368 CANCER RESEARCH VOL. 26

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Leukemia Virus Growth and Dose-Response Curves

3 injectionsofcell-freefiltrateovera 4-weekperiod; anothergroup of females was similarly treated with cell-free filtratefrom normal mouse KLS. These females were then mated, andtheir litters were challenged with virus at birth. The littersfrom mothers that received the virus preparation were completely protected against induction of leukemia (0/23), whilethe litters from mothers that received the normal tissue preparations, or none at all, were not protected (19/32 and 27/39 de

veloped leukemia in 3—11 months). This suggests that a passiveimmunity is conferred on the offspring from the mother. Thelitters were born to the immunized females at a time intervalafter the last injection that was comparable to the 5- and 6-weekintervals when recovery from weanlings was attempted.

Similarly, Mirand and Grace (9) reported that offspring frommothers injected during pregnancy with Friend virus werepassively immunized. Litters were challenged with Friendvirus at various intervals after birth. Offspring from mothersthat were immunized early in pregnancy (2nd day) and challenged with Friend virus between 1 and 15 days of age wereprotected against development of Friend disease ; after that,

passive immunity was lost. When mothers were injected on the

12th day of pregnancy, there was a delay in the development ofpassive immunity as indicated by the susceptibility of offspring

to infection with Friend virus; as the antibody rose in the

mother, the offspring were protected via lactation. Thus thereappears to be a common denominator, the explanation for whichawaits results of future experiments.

Manaker (7) could recover virus after 1 week from 5 of 7 miceinjected at 1 month of age with his C-60 leukemia virus. At 2weeks, none of 7 test mice developed leukemia, but at 3 and 4weeks, 10 of 12 mice in each group died of leukemia. Averagesurvival times were 14—15weeks. Manaker felt that the virusrecovered from organs at the end of the 1st week may have been

residual virus from the original inoculum of 0.1 ml, and thatvirus produced by infected cells of the new host appeared at theend of the 3rd week. It seems unlikely that the virus recovered atthe end of the 1st week was residual in view of the very small inoculum u@ed and the apparently high potency of the virus thatwas recovered, as indicated by the high incidence and short Ia

tency of leukemia.The susceptibility of rats to induction of leukemia by murine

viruses is well known (6, 8, 10, 12). The leukemias induced in ratsby the Gross and Moloney viruses are lymphocytic and the same asthose induced in mice ; those induced by the Friend and Rauscherviruses are also lymphocytic but differ from the disease in mice.

In all instances, however, the incidence is high and the latencyshort. Also, virus recovered from the leukemic rat induces theoriginal mouse disease, with high incidence and short latency,when returned to mice.

In contrast, only a small percentage of Sprague-Dawley ratsinjected at birth with our agent develop leukemia, and the

latency is greatly extended as compared with that in mice(the disease is the same as in mice). This may be due to thepresence of an inhibitor or antibody in the rat, controlling virusproduction and preventing induction of leukemia in most of theanimals. In some cases, however, this control mechanism may belacking, or not efficient enough to suppress virus production, andsome animals develop leukemias which provide a rich source of

virus, as indicated by the high incidences of leukemia whichresult when filtrates from rat leukemc tissues are returned to

Ha/ICR mice. This could explain our difficulty in recoveringvirus from rats 5 and 8 weeks after injection at birth. The lowincidence of leukemias induced in mice by filtrate from organsfrom 2-week-old rath may be due to a low titer of virus in theseanimals, because of some inhibitory action which increases with

age. Or, in all instances, the l)roblem may have been one ofrandom sampling; we were fortunate to have selected an animal(or 2) that was producing virus at 2 weeks, but, unfortunately,we missed the occasional animal with good virus titer at 5 and8 weeks of age. Further experiments are in @)rogress.

Acknowledgments

The authors wish to acknoWledge the participation of Anton J.Bueschen and Bert A. Lies, Jr., of our NSF Summer ResearchProgram, in the growth and dose-response curves study, and thevaluable technical assistance of Mrs. Veronica R. Alexander andLeslie Adams, Jr.

References

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369MARCH 1966

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1966;26:364-369. Cancer Res   R. F. Buffett, J. T. Grace, Jr., L. A. DiBerardino, et al.   Leukemia: Growth Curve and Dose ResponseRecovery of Murine Leukemia Virus Prior to Development of

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