[CANCER RESEARCH 32, 1729—1733, August 1972]
Early Replication of Herpesviruses in Naturally Occurring Frog
Tumors'
Robert Gilmore McKinnell, Virginia L. Ellis, David C. Dapkus, and Lyle M. Steven, Jr.
Department of Zoology, University ofMinnesota, Minneapolis, Minnesota 55455
SUMMARY
MATERIALS AND METHODS
Renal adenocarcinomas of the leopard frog, Rana pipiens,
collected in Kandiyohi County, Minn., were fixed for electron
microscopy prior to and 4, 7, 13, and 16 days after the entry
of host frogs into lakes for overwintering. Fifteen tumors were
studied. No herpesviruses were detected in 4 tumors of
prehibernating frogs. Two tumors of frogs autopsied on Day 4
of overwintering did not show viruses. Mature herpesviruses
were observed in 2 tumors of frogs that had been in lake water
for 7 days. The temperature of the water was 9°on Day 4 and
5°on Day 7. Three of 7 tumors from frogs that had been in
the lake for more than 7 days did not contain viruses detected
with the electron microscope. All tumors examined from frogs
that had been in lake water for more than I month contained
viruses.
Tumor Frogs. Frogs that are hibernating in lakes are
exceedingly difficult to obtain. Accordingly, frogs were
collected between October 14 and October 26, 1971 , in the
immediate vicinity of Diamond Lake, located north of
Atwater, Kandiyohi County, Minn. Frogs were congregating
near the margins of lakes prior to entering the water for
hibernation. The collected frogs were put in 4 cages, measuring
1 x 2 x 3 feet, made of wooden frames covered with hardware
cloth. The cages were placed in the lake on October 28, 1971.
At this time, the bulk of their uncollected compatriots were
also entering the water. Although the frogs of this study were
collected a short time prior to their entry into the lake and
were stored in cages, it is likely that they were exposed to
temperatures similar to those they would have encountered if
they had entered the lake in a normal manner. We dropped the
cages into the lake about SO feet from the shore in water 3 to
4 feet deep. We took frogs from the cages for autopsy in the
field on November 1, 4, 10, and 13, i97i . Tumors were fixed
INTRODUCTION
The chronology of virus replication in spontaneous renal
adenocarcinomas of northern leopard frogs, Rana pipiens, is
important to at least 3 aspects of viral oncology. First, an
understanding of natural transmission will not be possible until
we know when in the life cycle of the host frog the presumed
oncogenic agent is replicated and when it is released. Secondly,
laboratory studies suggest that the virus remains latent until
there is a drop in temperature. Does cold lake water similarly
stimulate virus replication? Finally, if viral assembly proceeds
stepwise, it should be possible to observe ontogenetic stages of
the virus during or early in replication.
Eighteen tumors of frogs that were obtained from the
overwintering lakes from December 1, 1970, through May 4,
197 1, were found to contain viruses (9). Previously, 11 renal
tumors of frogs emerging from overwintering lakes were
observed to contain viruses (1 1). Six frog tumors obtained
after spawning also contained herpesviruses (10). Thus, it
seems reasonable to conclude that spontaneous tumors of
frogs obtained during or shortly after the cold season in
Minnesota
contain viruses. In contrast,
it was previously
shown
that tumors of prehibernating frogs are devoid of viruses
detectable by electron microscopic examination (1 8). It was
the purpose of the present study to ascertain how soon after
the onset of hibernation virus replication begins.
‘This study
was
supported
by Grant
990-CT
from
the Damon
Runyon Memorial Fund for Cancer Research, Inc., New York, N. Y.,
and Grant GB-29482 from the National Science Foundation.
ReceivedMarch 17, 1972; accepted April 28, 1972.
in
the
field
for
electron
microscopy.
Four
tumors
of
prehibernating frogs were dissected within 24 hr of capture on
October 19, 1971 (Table 1).
Electron Microscopy.
Our procedure for electron
microscopy has been described elsewhere (9).
RESULTS
Tumors of Prehibernating Frogs. No virus particles were
detected after extensive examination by electron microscopy
of 4 tumors (Table 1). Typical tumor cells (Fig. 1) contained
small mitochondria
and poorly developed endoplasmic
reticulum.
The nuclei contained
randomly
dispersed
chromatin. Tumor cytology was thus comparable to calid
tumors previously described (9, 18).
Tumors of Hibernating Frogs. Two tumors examined after 4
days of hibernation
were devoid of virus particles.
Herpesviruses were observed in 4 tumors of frogs that had
been in hibernation for 7 and 13 days (Table 1). Empty
capsids,
capsids
containing
double-walled
cores, and
nucleocapsids with dense cores were present in nuclei of the
tumor cells (Fig. 2). Virus particles in the cytoplasm were
typically coated with dense material or were completely
enveloped. Mature, enveloped herpesviruses were also seen free
in the
lumen.
The
marginated chromatin
nuclei.
tumor
cytology
was also typified
by
and dense granular aggregations in the
AUGUST 1972
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1729
McKinnell, Ellis, Dapkus, and Steven
Curiously, 3 of 5 tumors of frogs that had been in
hibernation for 16 days seemed to be devoid ofvirus particles
(Table I). However, 2 of the 16-day tumors showed cytology
typical of algid tumors (9, 1 1). For all 6 algid tumors in this
study, examination by electron microscopy of about 50
randomly chosen thin sections revealed cells containing viruses
temperatures are for a period of about 2 months prior to the
time at which the frogs were placed in Diamond Lake for
overwintering. During this time, there were 27 days when the
temperature fell below 7.5°, a temperature which causes the
appearance of viruses in virus-free tumors in the laboratory
in approximately
prehibernating
50% of the sections.
Examination
was made
of at least 500 randomly chosen thin sections for each of
those tumors lacking viruses in order to verify that the tumors
are indeed “virus
free.―
Chart 1 shows the daily maximum and minimum air
temperatures
for
Willmar
State
Hospital,
located
approximately
10 miles west of Diamond Lake. The
( 12, 15). Despite
Chart
1.
Daily
3 7 21 25 29 3
September
maximum
and
minimum
II 5
October
temperatures
from
the 4 tumors from
1, 1971 , after 4 days in lake water all showed no viruses. The
temperature exceeded 210 on 23 days during this time.
DISCUSSION
Concerning
the Chronology
of Initiation
of Virus
Replication and Its Significance to Epidemiology of the
Tumor. Lucké(7) reported that intranuclear inclusion bodies
were more common in tumors of winter and spring frogs than
in tumors
5 9
these low temperatures,
frogs and the 2 tumors collected on November
of summer
and autumn
frogs. The inclusion
bodies
are now known to be associated with virus particles that can
be detected with the electron microscope (2). Accordingly, it
has been known for some time that the onset of virus
replication probably occurs in the autumn. It was reported
that all tumors (of a sample of 18) collected between
December 1970, and May 197 1, contained virus particles (9).
Since tumors of prehibernating frogs are known to be devoid
of viruses (18), it could be concluded that replication occurs
after the onset of hibernation but prior to December. The
present study has shown that mature viruses are produced in
some tumors as soon as 7 days after the onset of hibernation.
We plan to examine tumors of hibernating frogs daily in the
autumn of 1972 to study more closely the time of initial virus
replication.
What is the significance of virus replication within a week of
the onset of hibernation? This information, combined with
our previous knowledge of the presence of the virus in tumors
from winter through early June, tells us that the host frogs
carry tumors that contain mature, infective viruses for at least
7 months of the year. Oocytes and sperms are fully formed at
the time when the mature, tumor-bearing frogs enter lakes for
overwintering. The gametes are thus potentially exposed to
September 1, 1971, through October 28, 1971, for Willmar State
mature
Hospital,
Kandiyohi
County,
Minn.
Data
abstracted
Climatological
Data,
Environmental
Data Service, United
Department of Commerce, Vol. 77.
simultaneous presence of gametes and viruses for a prolonged
period suggests the possibility of vertical transmission of this
oncogenic agent. Vertical transmission of other oncogenic
from
States
viruses
throughout
the period
of overwintering.
The
Table 1
Chronology of virus replication and environmental data ofspontaneous
natural populations in Minnesota
tumors in
The temperature
of lake water varies with the time of day, wind conditions, locality, depth,
etc. The temperatures
reported were those of the water in the immediate vicinity of the frogs
during collection.
waterOct.
Date fixedNo.
of days of
host hibernationTumors
with
virusesTumors
without
virusesTemperature lake
of
1971Prehibernation04Nov.l,l9714029°Nov.4,i9717205Nov.
19,
197113200.5Nov.13,197l16234
10,
1730
CANCER RESEARCH VOL. 32
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Early Replication ofFrog Herpesviruses
viruses (3, 4) has been known for years, and the susceptibility
of frog eggs to infection by the virus with resulting
tumorigenesis has been demonstrated in tl@elaboratory (17).
Despite the possibility of vertical transmission suggested here,
we do not desire to exclude the alternate possibility of
horizontal transmission of the Luckévirus in breeding ponds,
which is discussed elsewhere (10).
We failed to detect virus particles in some tumors after the
time at which other tumors were replicating viruses. However,
it may be possible that additional
sections would reveal viruses
detected in tumors of frogs taken from their natural habitat.
We failed to detect a sequence of events in the synthesis of the
virus. It may be that assembly
occurs stepwise,
but if it does,
the events all occurred between Day 4 and Day 7 of
hibernation in the tumors that we studied. We observed no
viruses on Day 4, and we observed fully formed viruses on Day
7. If there is a sequential pattern to the assembly of viruses, we
should be able to detect this pattern in the autumn of 1972,
when we plan to autopsy at more frequent intervals after the
onset of hibernation.
in the virus-free tumors. We consider this an unlikely
possibility because of the extensive examination made of those
tumors.
The Stimulation from Latency of Virus Replication by an
Environmental Factor. Winter temperatures in Minnesota are
markedly colder than summer temperatures. Luckd's study (7)
suggested that temperature may affect virus replication. The
suggestion was strengthened
by the light microscopic
observations by Roberts (14) of the effect of temperature on
mitosis and nuclear inclusions. More recently, it has been
shown that virus replication is stimulated in tumors of frogs
maintained at low temperature in the laboratory (I , 12, 13,
15, 16). The first natural habitat in which a frog is confronted
with sustained low temperature after foraging in the warmth
of the summer sun is cold lake water. Prior to the frogs'
entering the lake, the night air may drop to temperatures
colder than the water, although the daytime temperature
frequently may be quite mild even in late October. Hence, it
seems to us that activation of virus replication is mediated
through a low and sustained temperature. If temperature is
indeed the environmental factor that activates Luckétumor
virus replication, then temperature may be added to other
physical factors such as X-irradiation (5, 6) that elicit a change
from latency to virus replication.
Not all tumors of the present study were replicating viruses.
We suggest that, while the genetically similar cells in tissue
culture may respond to viruses simultaneously, it may be
possible that tumors of genetically heterogeneous wild frogs
that have encountered varied environments will respond to
viral challenge asynchronously. Additionally, we do not know
the genome of the oncogenic agent. It, too, may be genetically
variable, thus contributing
to the possible nonuniform
response to temperature as the activating agent of latent
viruses.
Ontogeny of an Oncogenic Virus. Fawcett (2) was not only
the first to describe virus-like particles in thin sections of frog
renal tumors, but he was also the first to propose an
ontological scheme of virus replication. Subsequently, Lunger
et al., (8) suggested an elaborate developmental sequence,
although they were cautious because successive stages in viral
maturation
are difficult
to ascertain
unless a time sequence
system is used. Still more recently, Stackpole (15) described
virus replication
in tumors
implanted
into
the anterior
eye
chamber of host frogs. These ontogenetic studies are cited to
suggest that assembly of viral particles is of substantial interest
and to emphasize that the ontogenetic sequence, if indeed
there is a sequence, is still in an unresolved state. One of the
reasons for undertaking the present environmental study was
to discover if developmental steps of viral synthesis could be
AUGUST 1972
ACKNOWLEDGMENTS
We acknowledge the helpful criticisms of the manuscript by Dr.
Kenyon S. Tweedell, University of Notre Dame, Notre Dame, Ind., and
Dr. Joseph Zanibernard, Colorado Medical Center, Denver, Cob.
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CANCER RESEARCH VOL. 32
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AUGUST
1972
1733
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1972 American Association for Cancer Research.
Early Replication of Herpesviruses in Naturally Occurring Frog
Tumors
Robert Gilmore McKinnell, Virginia L. Ellis, David C. Dapkus, et al.
Cancer Res 1972;32:1729-1733.
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