[CANCER
RESEARCH
34, 712-715, April
19741
Nonspecific Antiviral Substances in Human Milk Active against
Arbovirus and Murine Leukemia Virus1
A. Howard
Fieldsteel
Life Sciences Division, Stanford Research institute, Menlo Park, California 94025
SUMMARY
tumor virus, prompted this report of work carried out in
1965 and 1966 (3). Their work reconfirms our original
Forty-three individual human milk samples obtained 2 observations on viruses infecting human beings (8). The
weeks to 17 months postpartum were divided into five large experiments
described
here further demonstrate
that
pools. Theseplus three large individual human milk samples human milk apparently has broader antiviral activity
obtained 2 and 5 months postpartum
were tested for that encompasses murine leukemia viruses.
antiviral activity against Japanese B encephalitis virus,
Friend leukemia virus, and Rauscher leukemia virus. All
MATERIALS
AND METHODS
milk samples had a high degree of antiviral activity against
the three viruses. The bulk, ifnot all, ofthe antiviral activity
Through the courtesy of Mrs. James Duggan, Northern
appeared to be in the cream fraction and was remarkably
heat stable. Heating the milk to temperatures up to 100°for California Director of the La Leche League, 43 individual
30 mm had no effect upon its ability to destroy the milk samples were obtained from women 2 weeks to 17
infectivity of Japanese B encephalitis virus or inhibit the months postpartum. A sample of each was frozen individ
ually, and the remainder was divided into 5 large pools, each
ability of Friend leukemia virus to induce splenomegaly.
consisting of equal quantities of milk from 5 to 9 different
INTRODUCTION
It has been known for many years that human milk
contains potent antiviral substances unrelated to specific
antibody. In 1962, Sabin and Fieldsteel (8) published the
results of a series of experiments carried out during 1949 to
1952 in which they demonstrated that neutralizing activity
was present in human milk against all viruses tested,
including JBE,2 St. Louis encephalitis, West Nile, dengue,
yellow fever, Western equine encephalitis, and Herpesvirus
hominis. The substance(s) present in milk differed from
antibody in that it resisted heating at 100°for 30 mm, was
largely associated with the cream fraction, and was absent
from the protein fractions known to be associated with
poliomyelitis antibody in human and bovine milks.
These studies are particularly pertinent at this time in
view of the search for a possible virus etiology of human
mammary cancer. This effort has been spurred by reports of
the presence of both RNA-directed DNA polymerase
activity and particles resembling both type B (mouse
mammary tumor) and type C (murine and feline leukemia)
viruses in human milk (2, 7, 11- 14). The recent rediscovery
by Sarkar
el al. (9) that milk does indeed have antiviral
activity, which could adversely affect the murine mammary
I This
work
was
supported
in
part
by
a research
and
development
grant
from Stanford Research Institute and by USPHS Grant CA-07868 from
the National Cancer Institute.
2 The
Friend
abbreviations
leukemia
phate-buffered
used
virus;
are:
RLV,
JBE,
Japanese
B
encephalitis
Rauscher leukemia
virus;
virus;
saline.
Received November 28, 1973; accepted December 26, 1973.
712
FLV,
PBS, phos
women. Each pool contained 300 to 900 ml of milk. These
pools plus 3 individual milk samples that were obtained in
large quantities were tested for antiviral activity.
The Nakayama strain ofJBE, FLy, and RLV were used
to determine antiviral activity in a standard neutralization
test. A pool of the Nakayama strain of JBE was prepared
from the brains of mice showing signs of encephalitis
following intracerebral inoculation when newborn. A 20%
suspension was prepared in 50% normal rabbit serum
0.9% NaC1 solution, distributed in sealed ampules, and
stored at —70°.
A pool of FLY was prepared from infected mouse spleens
as previously described (4). The RLV was kindly supplied
by Dr. Frank J. Rauscher, Jr., of the National Cancer
Institute as a 10% suspension of infected spleen.
For initial testing, the milk was sonically disrupted for 30
sec to emulsify the fat and prevent separation of the cream.
Serial decimal dilutions of virus were mixed with undiluted
milk and incubated before inoculation. With JBE, incuba
tion was carried out for 2 hr at 37°,followed by intracere
bral inoculation of 0.03 ml of each virus-milk mixture into
groups of five 3-week-old Swiss Webster mice (Simonsen
Laboratory,
Gilroy, Calif.) U nprotected mice developed
encephalitis and died. The tests were terminated in 14 days.
With FLV and RLV, incubation with milk was carried out
for I hr at 37°,and the mixtures were inoculated i.p. in 0.2
ml amounts into groups of ten 4- to 5-week-old BALB/c
mice from our own colony. If no protection occurred, the
mice developed typical splenomegaly. In some instances
mice died from a ruptured spleen, but all tests were
terminated 35 days after inoculation and all spleens weigh
ing 0.4 g or greater were considered to be positive for Friend
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A ntiviral A ctivity of Human
disease. As a practical matter, spleens in these tests were
either clearly negative, with a mean weight for 564 spleens
of 121 mg ±43, or clearly positive, with a mean weight for
148 spleens of 1480 mg ±748. The reason for the large
standard
deviation for the latter spleens is that they
represented mice inoculated with doses of FLY ranging
from < 1 to l0@@50% infectious dose.
End points were calculated by the Reed-Muench method,
and a neutralization
index (the difference in titer between
the virus controls and the virus-milk mixtures) was deter
mined. A difference between control and test mixture of
1017 or greater was considered indicative of antiviral
activity.
To separate the cream, the milk was centrifuged at 17,300
x g for 20 mm. By tilting the tube, the skim was removed
from the lower layer with a syringe fitted with a 10-cm
cannula leaving the fatty cream layer behind. The cream
was brought back to the original volume of the whole milk
with PBS and then sonically disrupted before use to create a
homogeneous
mixture. Since the cream represented an
insignificant percentage of the whole ( <2%), the skim was
used without adding PBS.
Milk
the antiviral properties of human milk were not due to some
property inherent in milk derived from that area. All the
milk in our present study was obtained from women
residing in the San Francisco Bay area. Table I summarizes
the results of testing these milk samples against both JBE
and FLY. It is apparent that all the poois and individual
milk samples tested were active against both viruses. In a
separate experiment milk Pool 1 was also tested against
RLV. It had a neutralization
index of > 1029, thus
neutralizing all dilutions of virus tested, the most concen
trated being l0'@.
Because milk Pool I contained the largest volume, it was
selected for further study. Since the experiments of Sabin
and Fieldsteel (8) indicated that the substances active
against JBE had a high heat stability, Pool I was heated at
56°,
80°,
or 100°for30 mm and testedagainstboth JBE
and FLY. The results shown in Table 2 confirm not only
that the antiviral substance active against JBE was stable
after heating to 100°for 30 mm but also that identical
results could be obtained with FLY. In the case ofthe latter
virus, there was complete neutralization of all dilutions of
virus starting with 10 ‘,
which was the lowest dilution used.
From the data given in Table 3 for JBE virus,
appear that the antiviral activity is equally divided
the cream and the skim fraction since the difference
these 2 fractions in their ability to neutralize the
RESULTS
it might
between
between
virus is
insignificant. When the cream and the skim fractions were
tested against FLV, the difference betweentheir ability to
Our original experiments (8) were carried out using milk
from women in the Cincinnati, Ohio, area. Therefore, the
present experiments were conducted initially to confirm that
neutralize
the virus was > 1.0 log10 . All dilutions
of the
Table I
Neutralizing activity of individual milk samples and human milk pools on JBE and FL V
Each milk sample was sonically disrupted for 30 sec to emulsify the fat and prevent separation
of the cream. Serial decimal dilutions of virus were mixed with undiluted milk and incubated before
inoculation.
JBE-milk
mixtures
were incubated
at 37° for 2 hr followed
by intracerebral
inocula
tion of 0.03 ml into groups of five 3-week-old Swiss Webster mice. Unprotected mice died of en
cephalitis.
FLV-milk
mixtures
were incubated
groups of ten 4- to 5-week-old BALB/c
for
1 hr at 37° followed
by i.p. inoculation
into
mice. Unprotected mice developed typical splenomegaly.
Antiviral
activity against
virusJBE0FLVCNeutrali
indicated
Neutrali
50% lethalzation50%
zationMonthsdoseindexdtious
doseindex―Milk―postpartum(—log10)(log10)(—log10)(log10)Pool
infec
I1
.5—6.55.23.21
.52.2Pool20.5-3.54.73.11.42.5Pool34.0-5.55.02.81.02.9PooI46.5-124.43.41.42.5PoolS13—175.22.60.83.1Mrs.B.5.04.23.61.42.5Mrs.
P.2.04.63.21.32.6Mrs.A.2.04.83.01.82.1
a Each
0 The
pool
JBE
consisted
virus
of
control
milk
in
tested
equal
in
proportions
parallel
with
from
milk
5 to
Pool
I
9 women.
had
a titer
of
1084;
when
tested
against the remaining milks, the titer was l0_78.
C The
FLV
control
tested
in
parallel
with
milk
Pool
the
amount
I
had
a
titer
of
l0'@;
when
tested
against
the remaining milks, the titer was l0@'.
d The
neutralization
index
is the
log10
of
of
virus
neutralized
when
mixed
with
un
diluted milk. A neutralization index of 1017 or greater was considered to be significant.
APRIL 1974
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713
A . Howard Fieldsteel
Table 2
Effect of heating on antiviral activity of whole human milk
Milk washeatedin a water bath, followed
theAntiviral
by sonication to emulsify the fat and
procedure
was carried
out as described
in Table
rest of
I.the
virusJBE activity against indicated
FLVNeutrali
Milk0 held for 30 mm at:
(log10)Room
50% lethal
dose
(—log10)
zation
index@'
(log10)
5.0
3.4
<0.5
5.4
4.2
4.6
3.0
4.2
3.8
<0.5
<0.5
<0.5>3.2
temperature
56°
80°
>3.2Viruscontrol
100°
50% infec
zation
index0
tious dose
(—log10)Neutraliz
8.4
a A
pool
of
milk
(Pool
I)
in
equal
>3.2
>3.2
3.7
proportions
from
9
women,
obtained
1.5
to
6.5
months
post
partum.
0 A
neutralization
index
of
l0'@
or
greater
was
considered
Table 3
VWhole
Efftct of whole cream and skim fractions ofhuman
to
be
significant.
milk on infectivity ofJBE
and FL
milk was centrifuged at 17,300 x g for 20 mm. By tilting the tube it was possible to re
movethe skim from the lower layer with a syringefitted with a 10-cmcannula.The creamwasleft
behind and was brought back to the original volume with PBS and then sonically disrupted before
use to create a homogeneous mixture. Since the cream represented <2%, the skim was used without
adding PBS. The rest of the procedure I.Antiviral
was carried out as described in Table
virusJBE
activity against indicated
FLV50%
NeutralizationFraction
lethal
(log10)Whole
dose (—log,0)
>3.2Cream
milkb
>3.2Skim
2.2Virus
4.7
5.4
5.7
control
8.3
a A
0 This
@
neutralization
is the
index
same
pool
of
of
Neutralization
index0 (log10)
50% infectious
dose (—log10)
3.6
2.9
2.6
<0.5
<0.5
1.5
3.7
l0'@
milk
or
used
greater
for
the
was
virus. In addition, on physical separation ofthe cream from
to remain
Fruther
to impart
antiviral
activity
would have
to the skim.
evidence that the cream fraction probably
con
tains all the antiviral activity was obtained in preliminary
experiments
conducted in collaboration
with Dr. J. I.
DeGraw of Stanford Research Institute. Whole milk (Pool
1) was extracted with ethyl ether; the ether-soluble fraction
was evaporated to dryness and reconstituted with PBS. This
fraction when tested against JBE had a neutralization index
714
considered
experiments
FLV ranging from l0
to l0@ were neutralized when they
were tested against the cream. The skim failed to neutralize
the 10 ‘
dilution of FLV but did neutralize dilutions ranging
from 102 to l0@. It is likely that most, if not all, the
antiviral activity is probably in the cream since that fraction
represented less than 2% of the total milk volume. Further
more, the cream as used in these experiments was reconsti
tuted with PBS to the original volume, thus making the final
dilution of the cream fraction I : 100 after mixture with the
the whole milk, only a minute amount ofcream
index0
to
shown
be
significant.
in
Table
2.
of
whereas the ether-insoluble fraction (skim) of the
milk showed no antiviral activity against JBE.
DISCUSSION
The role of viruses in the etiology of human mammary
carcinomas, as in other human cancers, is not well defined.
However, Moore et al. (7), Sarkar and Moore (I 1), and
Schlom et al. ( 13) have not only described the presence of
type B and type C particles resembling oncogenic RNA
tumor viruses in human milk but also have found RNA
dependent DNA polymerase activity in human milk con
taming these particles. There appeared to be complete
correlation between the presence of type B particles and
RNA-dependent
DNA polymerase activity. Furthermore,
by density gradient studies, this activity was found to be in
the same density region (1.16 to 1.19 g/ml) as would be
expected for RNA tumor viruses. Thus, they assumed that
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A ntiviral Activity
the reverse polymerase activity was associated with the virus
particles.
These data are not supported by Jensen and Schidlovsky
(6) and Calafat and Hageman (I ), who suggested that the
virus-like particles might be cellular debris or secreted
products from the mammary gland rather than types B and
C virus particles. Calafat and Hageman were unable to
confirm the report of Sarkar and Moore (10) that some
human milks have a destructive effect on the morphology of
type B particles representing murine mammary tumor virus
from mouse milk.
More recently, Gerwin et al. (5) partially purified DNA
polymerase from human milk and found that, although
some similarities existed between these enzymes and those
associated with RNA tumor viruses, there were also some
significant differences. The enzyme in human milk was
extremely labile compared with that found in the RNA
tumor viruses and differed in its chromatographic
behavior
from the viral enzyme.
In view of these conflicting reports and the known
presence of antiviral substances in many human milks the
possibility should be considered that the virus-like particles
seen in human milk as well as the reverse transcriptase
activity detected in this milk may not be related to any
oncogenic RNA viruses, human or otherwise.
In the present study, we have shown that human milk
contains a potent, heat-stable, lipid-like substance that
inhibits the primary manifestation
of infectivity (spleno
megaly) of both FLV and RLV. The nature of this
substance has not been further delineated, but it appears
certain that it is the same substance that inhibits the
infectivity of JBE and probably is the same substance
described by Sarkar et al. (9). The fact that milk is a natural
substance with a wide spectrum of antiviral activity merits
further study of the nature and mode of action of this
material that appears to be present in a high percentage of
human milk samples.
of Human
Milk
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Human Breast Cancer. Nature, 242: 260-262, 1973.
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Cancer, 28: 1425-1430, 1971.
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APRIL 1974
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
715
Nonspecific Antiviral Substances in Human Milk Active against
Arbovirus and Murine Leukemia Virus
A. Howard Fieldsteel
Cancer Res 1974;34:712-715.
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