[CANCER RESEARCH 32, 243-246,
February 1972]
Experimental Infection of Human Cervix by Herpesvirus Type 2
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in Organ Culture
Piero C. Balduzzi, Michael A. Nasello, and Marvin S. Amstey
Departments of Microbiology [P. C. B., M. A. N.] and of Obstetrics and Gynecology ¡M.S. A.], The University of Rochester School of Medicine
and Dentistry, Rochester, New York 14642
SUMMARY
A simple experimental
procedure for the study of
herpesvirus infection in organ cultures of human cervical
tissues is discussed. The use of this procedure has allowed us to
conclude that the columnar epithelium and mucous glands of
the endocervix, squamous epithelium of the ectocervix, and
stromal tissues in both endo- and ectocervix all are susceptible
to herpesvirus type 2. Infection spreads more rapidly in
stromal tissue than in the epithelial tissues of endo- and
ectocervix. Cells with intranuclear inclusions and with
"ground-glass" nuclei occur in the same organ culture but in
different proportions,
time after infection.
depending on the type of tissue and the
INTRODUCTION
The relationship between herpesvirus type 2 and human
cervical cancer has been the subject of numerous discussions in
recent years. There seems to be little doubt that an association
between this virus and cervical cancer exists (9, 10); however,
adequate data establishing a causal relationship have not been
published to date. There have been several retrospective
serological and cytological reports suggesting that herpesvirus
type 2 is important in the etiology of human cervical cancer
(6, 7). There also have been reports suggesting that the virus
infection follows the development of epithelial abnormalities
in the cervix (2) and that the outcome of an infection may
depend on the particular strains of herpesvirus type 2(1).
Until now, information about the pathological changes
resulting from infection of human cervical tissues by
herpesvirus derived mainly from 2 sources: the study of
histological sections of cervical tissues obtained as biopsy or
surgical material (3) and the study of cytological specimens
obtained from scraping herpetic lesions or from routine
cervical smears. With both methods, the study of the
development of the infectious process is difficult. Histological
specimens cannot be obtained repeatedly. In the case of
cytopathological studies, information as to the origin of the
infected cells is inadequate most of the time (8). In all cases, it
is not possible to determine with certainty the onset of
infection. An experimental system which would permit the
exact determination of the time of infection and would make
1This work was aided by USPHS Research Grant CA-05206 from the
National Cancer Institute, Grant IN-18L from the American Cancer
Society, and by a grant from the Labor Foundation.
Received August 12, 1971; accepted October 7, 1971.
FEBRUARY
possible a study of the progress of the infection and the
precise identification of the tissues and cells involved in the
process would be desirable and useful. This report describes
such an experimental
model. Human cervical tissues
maintained as organ cultures have been infected with
herpesvirus type 2 and the results of this infection are
described.
MATERIALS AND METHODS
The virus used in these experiments was the Lewis strain of
herpesvirus type 2. This strain was described in a previous
report (1).
Cervical tissues for organ cultures were obtained from
hysterectomy specimens after surgery for benign disease in the
Department of Obstetrics and Gynecology of Strong Memorial
Hospital, Rochester, N. Y. Immediately after the uterus and
cervix were excised, strips of endocwvical and ectocervical
epithelium with some underlying stroma were removed with a
Davol-Simon dermatome. Each strip, approximately 5 mm
wide, 20 mm long, and 1 mm thick, was cut into smaller
fragments, which were washed several times in Tris-buffered
0.9% NaCl solution. Finally, the fragments were transferred to
a test tube containing 1.0 ml of virus suspension (2.7 X IO6
50% tissue culture infectious dose/ml, as assayed in WI 38
cells) in Eagle's minimal essential medium (4) containing 10%
newborn
calf serum, penicillin
(100 units/ml),
and
streptomycin (100 fig/mi).
Control fragments of cervical epithelium were incubated
with minimal essential medium containing serum and
antibiotics, as described above. Both control and infected
fragments were incubated for 2 hr at 37°. Following
incubation, all fragments were placed in 2.5-x6-cm glass,
screw-cap vials previously filled with 10 ml of F-12 medium
(5) containing 10% newborn calf serum, 0.8% Noble's Special
agar (Difco Laboratories, Inc., Detroit, Mich.), and antibiotics,
as described above. Four or 5 fragments could be accommodated
in 1 vial. Finally, each fragment was covered with 1 drop
(approximately 0.05 ml) of the virus suspension or control
medium. Until fixation, the organ cultures on agar were
incubated at 37°in a humidified 5% CO2 atmosphere. No
further feeding or manipulation of the cultures was done. All
tissues were handled aseptically from the operating room to
the last step in the preparation of the organ cultures.
At various intervals after infection, infected and control
tissue fragments were fixed, embedded in paraffin, serially
sectioned (6 to 8 sections/fragment),
and stained with
1972
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
243
P. C. Balduzzi, M. A. Nasello, and M. S. Amstey
hematoxylin and eosin. Each section was then examined by
light microscopy at low and high magnification.
RESULTS
Seven different surgical specimens were used in attempts to
establish organ cultures from strips of cervical epithelium.
Successful cultures, as judged by the morphology of the cells
and by the presence of occasional mitosis in the fragments,
were established in 5 instances; the 2 unsuccessful attempts
resulted from drying of the tissue fragments.
Organ cultures prepared as described above showed
excellent histológica! details of glands, surface epithelium,
stromal elements, and vascular endothelium for as long as 23
days in culture (the longest time any culture was held).
Occasionally, there was slight stromal degeneration with focal
areas of necrosis in the middle of the culture fragments,
beginning at II to 12 days after the culture was established.
At the same time, there was little or no change in the
epithelium (Fig. 1). Fig. 2 illustrates a portion of control
ectocervix after 8 days in organ culture.
When organ cultures were exposed to herpesvirus type 2,
evidence of infection appeared in both the stromal and
epithelial cells at the surface of endocervical fragments as early
as 48 hr postinfection.
Obvious intranuclear inclusions
characteristic of herpesvirus infection were apparent at this
time in endocervical tissues and, after 72 hr, in ectocervical
organ cultures (Figs. 3 and 4). These intranuclear inclusions
appeared as large, homogeneous masses surrounded by a clear
halo. There were very few multinucleated cells or giant cells.
Cells with ground-glass nuclei and chromatin margination or
vacuolated nuclei did not appear until 5 to 6 days after
infection. These cells were observed more frequently in the
stroma and squamous epithelium of the ectocervix than in the
columnar cells of the endocervix. In the squamous epithelium,
infection affected the basal or parabasal cell layers, while the
superficial layers appeared normal (Fig. 5). After 5 or 6 days
in culture, the herpes infection extensively involved stromal
elements of the endocervical cultures (Fig. 6).
After 8 days in culture, all cellular types were affected by
herpesvirus infection. From this time through the following
week, the infection progressed rapidly and involved most
cellular elements. Intranuclear inclusions in some cells and
ground-glass nuclei with chromatin margination in others
could readily be identified in squamous and columnar
epithelium, stromal fibroblasts, and vascular endothelial cells.
At this time, the number of cells with intranuclear inclusions
was approximately
equal to the number of cells with
ground-glass nuclei. Only a few cells in any low-power
microscopic field contained vacuolated nuclei.
Infected squamous cells at the basal and parabasal areas in
the epithelium seemed to separate from each other, and a very
loose cellular arrangement resulted after 8 to 10 days.
However, the normal architecture of the tissue was preserved
in all fragments. This was true through 19 days of infection,
the longest time infected cultures were held. No evidence for
cytological atypia, other than obvious herpetic changes, was
present up to 19 days postinfection.
244
DISCUSSION
The results of this investigation show that the experimental
model used here, i.e., organ culture of human cervical tissues,
is a useful tool for the study of herpesvirus infections. This
procedure is simple, economical, rapid, and easily adapted to a
number of investigations with different viral agents. The agar
medium used as a support of the tissue fragments is commonly
used in our laboratory for the overlay of cells in tissue
cultures. No attempts were made here to identify a better
medium or to extend the period of cultivation of the
fragments by addition of fluid medium or transfer of tissues to
new containers. Presumably, experimentation with conditions
of growth could prolong the survival of the tissues beyond the
3 weeks used in this study. In its simplicity, this procedure is
remarkably reliable. Out of 7 attempts to obtain survival of
the specimens up to 3 weeks, only 2 were unsuccessful; the
failure in these 2 cases was probably due to the drying of the
organ cultures. Infection of the fragments was consistently
obtained with exposure to a virus suspension at 37°for 2 hr;
however, it was not possible to infect the tissues simply by
covering the fragments in the vial with a drop of virus
suspension.
This experimental procedure has allowed us to determine
the cell types affected by the virus, the relative rate of spread
of the infectious process in different tissues of the cervix, and
the morphological alteration affecting cells of different types.
In this study, the 1st sign of infection appeared after 48 hr in
fragments of endocervix and after 72 hr in ectocervical tissue.
In fragments fixed at a later time, the progression of infection
could be accurately followed. The herpetic infection under
these experimental conditions spreads more rapidly in the
stromal cells than in the columnar epithelial cells. This
observation is interesting in light of the findings of a recently
reported case in which herpesvirus infection and microinvasive
carcinoma of the uterine cervix occurred simultaneously. In
that case, the only cells apparently affected by the virus were
epithelial cells (3).
It was apparent that solid intranuclear inclusions appeared
in the columnar cells of the endocervix earlier than
ground-glass nuclei and that the 2 types of lesions are present
in the same fragment, although in different proportions,
depending on the time after infection. Also, it appears that
ground-glass nuclei are more frequent in cells of the squamous
epithelium
of the ectocervix, while solid intranuclear
inclusions are more frequently observed in cells of the
columnar epithelium of the endocervix and mucous glands.
The ground-glass nuclei with chromatin margination observed
in these experiments appear to be the same as those thought to
be related to herpetic infection by Ng et al. (8). These authors
have also suggested that the ground-glass appearances of nuclei
and solid intranuclear inclusions are the expressions of primary
and recurrent infections, respectively. Although no attempt
was made here to determine antibody levels against herpesvirus
type 2 in the serum of the patients in this study, the presence
of the 2 types of lesions in the same fragment of endocervix or
ectocervix suggests that either type of nuclear alteration may
be associated with primary or secondary herpesvirus infection.
The predominance of 1 type of lesion over the other seems
CANCER RESEARCH VOL. 32
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Herpesvirus Infection of Human Cervix
rather to be related to the type of tissue affected (squamous
epithelium versus columnar epithelium) and perhaps also to
the duration of the infectious process.
No evidence of cytological atypia could be observed as a
result of exposure to herpesvirus up to 19 days (the longest
period during which infected fragments were maintained).
However, the limited duration of the organ culture used in
these experiments does not allow one to draw any conclusions
as to the possibility of inducing these changes in the tissues.
Prolonged organ cultures under optimum conditions must be
obtained in order to determine whether cellular atypia will
occur.
It is realized that the organ culture method represents an
artificial situation in which susceptible cells come in contact
with the virus simultaneously and in which the absence of
inflammatory processes alters the course of infections, as
compared with the in vivo situations; however, it seems to be
adequate for the experimental study of viral infections at the
cellular and tissue level.
Studies are in progress to investigate the effect of antiviral
substances on the initiation and/or progression of herpetic
infection of human cervical tissues.
ACKNOWLEDGMENTS
We thank Dr. Stanley Patten and Dr. Frank Young for reviewing this
manuscript.
FEBRUARY
REFERENCES
1. Amstey, M. S., and Balduzzi, P. C., Genital Herpesvirus (Type II)
Strain Differences. Am. J. Obstet. Gynecol., 106: 924-927, 1970.
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Diagnosis and Significance. Am. J. Obstet. Gynecol., 108:
188-193, 1970.
3. Amstey, M. S., and Patten, S. F. Genital Herpesvirus Infection
Associated with Carcinoma of the Cervix. Obstet. Gynecol., in
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4. Eagle, H. Aminoacid Metabolism in Mammalian Cell Cultures.
Science, 130: 432-433, 1959.
5. Ham, R. G. Clonal Growth of Mammalian Cells in a Chemically
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6. Nahmias, A. J., Josey, W. E., Naib, Z. M., Luce, C. F., and Guest,
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Humans. Am. J. Epidemiol., 91: 547-552, 1970.
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Histopathology of Cervical Herpes Simplex Infection. Cancer, 19:
1026-1031, 1966.
8. Ng, A. B. P., Reagen, J. W., and Linder, E. The Cellular
Manifestations of Primary and Recurrent Herpes Genitalis. Acta
Cytol., 14: 124-129, 1970.
9. Rawls, W. E., Tompkins, W. A. F., Figueroa, M. E., and Melnick, J.
L. Herpesvirus Type 2: Association with Carcinoma of the Cervix.
Science, 161: 1255-1256, 1968.
10. Royston, I., Aurelian, L., and Davis, H. J. Genital Herpesvirus
Findings in Relation to Cervical Neoplasia. J. Reproductive Med.,
4: 109-113,1970.
1972
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
245
P. C. Balduzzì,M. A. Nasello, and M. S. Amstey
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Fig. 1. Control endocervix after 17 days in organ culture. X 280.
Fig. 2. Control ectocervix after 8 days in organ culture. X 280.
Fig. 3. Organ culture of endocervix 2 days after infection with herpesvirus type 2. X 450.
Fig. 4. Organ culture of ectocervix 3 days after infection with herpesvirus, type 2. X 450.
Fig. 5. Basal and parabasal cell effect of herpesvirus type 2 infection of organ cultures of ectocervical fragments 6 days postinfection. X 450.
Fig. 6. Extensive involvement of stromal cells with herpesvirus type 2 six days after infection of endocervical organ cultures. X 450.
246
CANCER RESEARCH VOL. 32
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
Experimental Infection of Human Cervix by Herpesvirus Type 2
in Organ Culture
Piero C. Balduzzi, Michael A. Nasello and Marvin S. Amstey
Cancer Res 1972;32:243-246.
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