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J Virol, June 1998, p. 5256-5261, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Isolation and Propagation of Human Papillomavirus
Type 16 in Human Xenografts Implanted in the Severe Combined
Immunodeficiency Mouse
William
Bonnez,1,*
Carrie
DaRin,1
Christine
Borkhuis,1
Karen
de
Mesy Jensen,2
Richard C.
Reichman,1 and
Robert
C.
Rose1
Departments of
Medicine1 and
Pathology,2 University of Rochester
School of Medicine and Dentistry, Rochester, New York 14642
Received 29 October 1997/Accepted 13 February 1998
 |
ABSTRACT |
We report the isolation and propagation of human papillomavirus
type 16, the main agent of cervical cancer, using human foreskin fragments implanted in severe combined immunodeficiency mice. The
infection produced viral particles, and with each passage of the virus
it caused lesions identical to intraepithelial neoplasia, the precursor
to carcinoma.
| |
TEXT |
Human papillomaviruses (HPVs) are
small DNA viruses that infect stratified squamous epithelia. Over 100 different HPV genotypes have been identified or characterized, and an
association between the genotypes and the tissues they infect has been
established (3). For example, HPVs infecting the anogenital
region form a group distinct from the HPVs infecting the nongenital
epithelia. Anogenital HPVs cause lesions ranging from anogenital warts
(condylomata acuminata) to squamous cell cancer of the cervix,
including intraepithelial neoplasias (3). Each anogenital
HPV carries a different oncogenic risk. For example, HPV types 6 and 11 (HPV-6 and -11) are the agents of condyloma acuminatum and low-grade
intraepithelial neoplasias, while HPV-16 and -18 cause intraepithelial
neoplasias and cancers.
The inability to passage HPVs in vitro has been a major obstacle
to working with these viruses. In 1985, Kreider et al. used human
cervix fragments infected with an extract of pooled human condylomas and grafted them under the renal capsule of athymic (nude)
mice to isolate the first HPV and reproduce some of the histologic
features of HPV infection (27). This isolate,
HPV-11Hershey, was subsequently passaged in other human
tissues, primarily human foreskins, using the same animal model
(17, 24-26). We subsequently showed that this viral strain
could be more efficiently propagated and provide a more versatile model
by using the severe combined immunodeficiency (SCID) mouse
(9).
HPV-11Hershey was until recently the sole HPV reported to
have been propagated and passaged experimentally. Kreider et al. described the isolation of HPV-1, the agent of plantar warts, in the
athymic mouse xenograft model using human fetal foot skin (28), but passage of the isolate was not described. In a
chamber grafted on the panniculus carnosus of SCID mice, Sterling et
al. obtained the differentiation of a uterine cervical cell line (W12) stably infected with HPV-16 that produced histologic features of
intraepithelial neoplasia, as well as viral antigen and particles; however, the virus was not passaged through uninfected cells or tissues (41). More recently, Brandsma et al. were able
to introduce with a gene gun naked full genomic HPV-16 DNA in human
foreskin fragments that were subsequently grafted onto the flank of
SCID mice (12). Lesions that had histologic features of HPV
infection and contained HPV antigen and DNA from type 16 developed, but the production of virions and the passage of the infection to uninfected tissue were not demonstrated. Other investigators have successfully grafted human lesions of recurrent respiratory
papillomatosis or epidermodysplasia verruciformis and observed the
maintenance of the full differentiation of the grafts, but they did not
isolate and propagate the HPVs contained in these tumors (29,
39). In 1997, Christensen et al. isolated simultaneously HPV-40
and HPV-LVX82/MM7 from an anal condyloma coinfected with these two viruses (14). These two relatively uncommon viruses, which
are found in anogenital condylomas and intraepithelial neoplasias, were
subsequently copassaged.
Production of HPV particles and associated cytopathic
effects in vitro have been demonstrated in differentiated
epithelia developed on a raft system, either by using naturally
infected cell lines or by transfection of HPV DNA into keratinocytes
(19, 32, 33). However, these promising models have not yet
permitted the propagation of free viral particles in vitro. In the
present study, we report the isolation and passage of HPV-16, and the concomitant production of intraepithelial neoplasia, using the human
xenograft SCID mouse model.
First isolation of HPV.
Fragments of single biopsies of
clinical condyloma acuminatum from 11 patients were collected and
snap-frozen in liquid nitrogen. (Protocols were approved by the
University of Rochester Committee on Animal Research, Highland Hospital
[Rochester, N.Y.] Human Investigation Committee, and the
Investigational Review Board of the University of Rochester.) Each was
separately ground in phosphate-buffered saline with sterile sand, using
mortar and pestle, as previously described (9, 10). The
material was clarified by centrifugation at 1,000 × g
for 10 min, and the supernatant was stored at 
80°C. Individual
aliquots of these 11 sample suspensions were pooled and pelleted by
centrifugation at 100,000 × g for 1 h at 4°C.
The single pellet was resuspended in 760 µl of phosphate-buffered saline (identified as original inoculum) before being aliquoted and
stored at 80°C. A neonatal human foreskin from routine circumcision was prepared as previously reported (9, 10) and cut into fragments of 1 by 1 mm. These were incubated in 250 µl of the original inoculum for 1 h at 37°C. One graft was implanted under the skin of the external ear, and another was implanted under the renal
capsule on both sides of three 5- to 8-week-old female SCID (C.B-17/Icr
Tac-scidfDF) mice (Taconic Farms). The mice were sacrificed
12 weeks later. None of the grafts implanted at the ear site grew. Of
the six renal grafts, only one enlarged slightly; the cubic root of its
three perpendicular dimensions, or geometric mean diameter (GMD), was
1.82 mm, compared to approximately 1 mm for the original implant.
The grafts were split; one part was fixed in buffered formalin, and the
other part was snap-frozen in liquid nitrogen. Histologic (hematoxylin-eosin stain) criteria of HPV infection were preestablished and were the presence of any two of the following elements: acanthosis, koilocytosis, and parakeratosis (8-10, 22). All of the
grafts had a normal histology except the enlarged graft, which was
positive for HPV. It had the cytoarchitecture of an epidermal
cyst, with evidence of acanthosis and parakeratosis. Koilocytes
were essentially absent, but a basaloid hyperproliferation with
several unusual mitotic figures in the upper third of the stratum
acanthosum was noted. All of these features were consistent with
intraepithelial neoplasia (Fig. 1).
This graft was also strongly positive for the presence of HPV capsid
antigen (data not shown), as determined by a previously described
streptavidin-peroxidase method (10, 43). The primary
antibodies were from pre- and postimmune sera from a rabbit immunized
with a 
-galactose fusion protein encoded by a 480-bp fragment
derived from the L1 open reading frame of HPV-6b and bearing the common
papillomavirus antigen (42). In the whole study, positive
controls were included and the corresponding uninfected foreskins were
used as negative controls. Viral DNA was extracted from the frozen
sample and submitted to PCR amplification using the degenerate L1
primers MY09 and MY11 (30). An approximately 450-bp fragment
consistent with the presence of HPV was amplified.
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FIG. 1.
First isolation; histology (hematoxylin-eosin stain) of
the single renal graft positive for the presence of HPV after the first
passage. In addition to the acanthosis and parakeratosis, there is a
mild basaloid proliferation and several mitotic figures are present in
the stratum spinosum (bar, 600 µm).
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Second passage and typing of the HPV isolate.
The HPV-positive
graft was processed as for the preparation of the original inoculum by
low- and high-speed centrifugation; 60 µl of the resuspended lysate
was allocated for HPV DNA PCR, and 450 µl was used for further
passage. A neonatal human foreskin prepared as before was incubated in
225 µl of the lysate for 1 h at 37°C. Single infected grafts
were implanted under the renal capsule on both sides of six 5- to
8-week-old female SCID mice. The experiment was repeated, using a
different foreskin on six additional SCID mice. These mice were
sacrificed 19 weeks later. Of the 24 implants placed under the renal
capsule of the 12 mice, 20 were still visible at euthanasia 19 weeks
later, and all had the appearance of solid cysts. Their mean (standard
deviation [SD]) GMD was 1.98 (0.87) mm. Fifteen grafts were
ultimately available for histology, and five had evidence of HPV
infection, including acanthosis and parakeratosis, as well as
basaloid proliferation, dyskeratosis, and mitoses high in the
stratum spinosum. None had koilocytosis. One of the five grafts
was positive for capsid antigen by immunocytochemistry (data not
shown). Transmission electron microscopy of a paraffin-embedded section
(16) of that sample revealed the presence of intranuclear
clusters of 30-nm particles in cells of the stratum granulosum (data
not shown). The inoculum prepared for the subsequent passage from 11 of
the collected grafts contained approximately 55-nm viral particles
(Fig. 2) whose morphology was consistent
with papillomavirus, as shown by electron microscopy after negative
staining with 2% phosphotungstic acid.
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FIG. 2.
First isolation. The electron micrograph of a negatively
stained preparation of the inoculum prepared from grafts collected from
the second-passage experiment shows a 55-nm viral particle of
morphology similar to that of papillomavirus capsids among cellular
debris (bar, 55 nm).
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The DNA in the same preparation was amplified by PCR with the MY09 and
MY11 primers, and the amplicon was cloned into the
pAMP cloning system
(Gibco-BRL, Gaithersburg, Md.). It was also
amplified by PCR using
HPV-16 L1-specific primers, by methods
previously described
(
37). The resulting amplification product
was then cloned
into a baculovirus transfer vector, pVL-1392,
for expression in insect
cells. Both the MY09-MY11 and full L1
amplicons were independently
sequenced in both directions, using
standard methods on a model 373 series Dye-Deoxy DNA sequencer
(Applied Biosystem, Inc. Foster City,
Calif.). The MY09-MY11 amplicon
sequence was identical to that of the
reference strain (
34,
38) except at seven nucleotide
positions (A6693C, G6719A, A6801T,
C6852T, C6863T, C6968T, and G6992A).
Five of the base changes
were silent, one caused a conservative change
from threonine to
serine (A6801T), and one yielded a nonconservative
substitution
from threonine to proline (A6693C). DNA sequence of the
full L1
clone from that preparation contained the same changes in
the
overlapping region, as well as 11 additional nucleotide
substitutions
outside the region amplified by MY09 and MY11
(G5696A, C5826T,
T5909C, C6163A, A6178C, C6240G, T6245C, G6252A,
A6432G, C6557T,
and G7058T). These mutations resulted in seven
amino acid changes:
histidine to tyrosine (C5862T), threonine to
asparagine (C6163A),
asparagine to threonine (A6178C), histidine
to aspartic acid (C6240G),
glycine to serine (G6252A), threonine
to alanine (A6432G), and
leucine to phenylalanine (G7058T). All
of these nucleotide substitutions
fall in positions where
variation has also been observed in HPV-16
variants, except for G6252A
(
35). In keeping with a naming practice
established with the
strain HPV-11
Hershey isolated in the athymic
mouse model
(
17), we refer to this isolate as
HPV-16
Rochester-1k.
Third passage of HPV-16Rochester-1k.
Eleven grafts
recovered from the second passage were pooled. As before, they were
ground and subjected to low- and high-speed centrifugation to prepare
an infectious inoculum. A neonatal human foreskin was prepared and cut
into squares of 1 by 1 mm for implantation under the renal capsule and
3 by 3 mm for subcutaneous grafting. Each group of fragments was
incubated separately for 1 h at 37°C in 125 µl of the
inoculum. The renal grafts were grafted in the usual manner, one per
kidney, in six 6-week-old male SCID mice. Individual subcutaneous
grafts were implanted under each flank in replacement of two stacked
1-cm-diameter round glass coverslips that had been placed 2 weeks
earlier to elicit the formation of a vascular bed (41). The
experiment was replicated a total of four times with different foreskin
donors. The mice were sacrificed 27 weeks postgrafting. From 35 surviving mice, we retrieved 30 renal and 28 subcutaneous grafts with
mean (SD) GMDs of 2.65 (0.77) and 2.75 (0.83) mm, respectively. The
renal implants (GMD of 
1 mm), which were twice as small as the
subcutaneous implants (GMD of 2 mm), eventually reached the same
size. Only four of the grafts were examined by histology. All
demonstrated the features of HPV infection and of moderate to severe
intraepithelial neoplasia already observed after the first passage
(Fig. 3). The DNA from a viral inoculum
prepared with available portions of 48 of the collected grafts was
amplified by PCR with the MY09 and MY11 primers. The amplicon was
probed with an extensive series of type-specific oligonucleotide probes
corresponding to the anogenital HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 66, 68, and 73 and the novel types MM4, LVX-82/MM7, and MM8 (2, 21, 31). It
contained only HPV-16.
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FIG. 3.
First isolation. Histology (hematoxylin-eosin stain) of
one of the renal grafts from the third passage experiment shows
prominent signs of intraepithelial neoplasia with basaloid
proliferation, nuclear pleomorphism, dyskeratosis, and multiple
aberrant mitoses throughout the stratum spinosum (bar, 100 µm).
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Second isolation and propagation of HPV-16.
Using the original
inoculum prepared from clinical lesions, we attempted to reisolate the
virus. A human neonatal foreskin was prepared as before and cut into 3- by 3-mm squares that were incubated for 1 h at 37°C in 50 µl
of the original viral inoculum. Each infected foreskin fragment was
implanted subcutaneously in three 6- to 7-week-old male SCID mice to
replace a cephalad and a caudad stack of two 1-cm-diameter round glass
coverslips that had been placed under the left flank 2 weeks earlier.
Four weeks later, the skin overlaying the caudad grafts was incised and
the grafts were left exposed externally. The mice were sacrificed 24 weeks after grafting. The grafts were collected and split in two parts,
one for formalin fixation and the other for freezing. The experiment
was duplicated with another foreskin.
One of the six grafted mice died. In the five surviving mice, 9 of 10 implanted grafts were recovered. Their mean (SD) GMD
was 2.50 (0.71)
mm. Three of the five grafts that had been externalized
remained so,
forming small papillomas (Fig.
4). The
internal grafts
formed solid cysts. Eight of the grafts were
interpretable by
histology and immunocytochemistry. Five, including one
of the
external grafts, had histologic evidence for the presence of HPV
(acanthosis and parakeratosis) (data not shown); one of them was
also
positive by immunocytochemistry, and three others had features
of
intraepithelial neoplasia (basaloid proliferation, dyskeratosis,
and
dyskaryosis).
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FIG. 4.
Second isolation. A mouse from the first-passage
experiment demonstrates an externalized, cutaneous graft. Magnified
view of the lesion (insert) reveals a papillomatous appearance.
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Four of the five grafts were used to prepare, in the manner already
described, an inoculum for a second passage. Three sets
of glass
coverslips were implanted on each side under the flank
skin of 12 6-week-old male SCID mice. Twenty days later, the coverslip
stacks were
removed and each was replaced with 3- by 3-mm square
foreskin fragments
that had been incubated in 100 µl of the viral
inoculum. The foreskin
fragments implanted on the left side of
the animals came from a
different donor than the fragments implanted
on the right side. Twelve
weeks after grafting, the cephalad grafts
on each mouse were
externalized through an incision to allow the
graft to become
cutaneous. The mice were sacrificed 16 weeks after
grafting. Two of the
12 mice died before the end of the experiment.
In the 10 surviving
mice, 56 of the 60 implants were present at
euthanasia; none were
external. Their mean (SD) GMD was 2.05 (0.50)
mm. Thirteen of the 53 available for histology were not interpretable
because of incomplete or
altered histologic architecture. Of the
40 interpretable grafts, 34 were positive for the presence of
HPV (acanthosis and parakeratosis),
and 21 of those exhibited
the features of intraepithelial neoplasia,
grades 1 to 2 (Fig.
5). Only 1 of the 40 samples was positive for HPV capsid by immunocytochemistry.
From the
viral inoculum prepared from 42 of the retrieved grafts,
PCR with the
MY09 and MY11 primers amplified a DNA fragment that
was cloned into
pAMP and whose sequence was found to be identical
to that of the
reference HPV-16 strain (
34,
38). This strain
is referred as
HPV-16
Rochester-1sc.
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FIG. 5.
Second isolation. Histology (hematoxylin-eosin) of a
subcutaneous graft from the second-passage experiment exhibits features
of acanthosis, parakeratosis, and dyskeratosis (bar, 100 µm).
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We were able to repeatedly propagate HPV-16 in human foreskin fragments
grafted in the SCID mouse and to obtain intraepithelial
neoplasia in
the implants. We obtained two distinct lineages for
the propagation of
HPV-16, one through renal implants (HPV-16
Rochester-1k)
and
one through subcutaneous implants (HPV-16
Rochester-1sc). By
extrusion, we were also able to convert some of the subcutaneous
grafts
into cutaneous lesions. These lesions had the appearance
of small
papillomas without any clinical attributes of malignancy,
which is
consistent with the HPV-16-associated papules found on
penile skin
(
1).
The histopathology of the lesions that we observed varied from benign
to premalignant. Because some of the lesions were cystic,
some of the
features of HPV infection such as papillomatosis and
hyperkeratosis
could not be assessed. None of the samples we reviewed
had frank
koilocytosis. Most HPV-16-associated lesions of the
penis reveal
intraepithelial neoplasia on histology, and koilocytosis
is an
inconsistent component of anogenital intraepithelial neoplasia
(
1,
22). However, Brandsma et al. reported that koilocytosis
was a
prominent element in the histology of lesions induced by
the
transfection of full-genomic HPV-16 DNA in human foreskins
subsequently
grafted onto SCID mice (
12). The histology of the
majority
of our lesions containing HPV also revealed mild to severe
intraepithelial neoplasia, as seen clinically in penile lesions
associated with HPV-16 (
1). Keratinocytes transfected with
HPV-16 DNA and implanted subcutaneously in the athymic mouse can
differentiate and exhibit histologic features similar but not
strictly
identical to those of intraepithelial neoplasia, and
they do not
produce viral particles (
18,
44). W12 cells, which
are
derived from a low-grade cervical neoplasia lesion, have retained
episomal HPV-16 DNA (
40,
41). When grafted on the flank
panniculus
carnosus of SCID mice, the cells differentiate, display some
of
the histologic features of the original lesion, and produce viral
particles. This and other data established a link between HPV-16
and
intraepithelial neoplasia. However, our study demonstrates
for the
first time that an intraepithelial neoplasia lesion whose
histology is
identical to the natural lesion can be created by
infecting normal skin
with an inoculum containing HPV-16 viral
particles, the presumed
natural agent of infection. It thus fulfills
one of the classic Koch's
postulates of being able to reproduce
the disease after inoculation of
the healthy host (
20). Of note,
in the human xenograft
athymic mouse model, neither HPV-40 nor
HPV-LVX82/MM7 reproduced the
intraepithelial neoplasia lesions
with which they can be associated in
the natural host (
14).
However, in contrast to HPV-16,
neither of these two viruses appears
to be associated with anogenital
cancer (
11,
31). In our study,
depending somewhat on the
experiment, variation existed in the
grades, from mild to severe, of
the intraepithelial neoplasias
and in the nature of the histologic
features, such as differentiation,
nuclear abnormalities, and mitotic
activity. This variation may
possibly be a reflection of the titer of
the inoculum and the
susceptibility of the foreskin donor. No squamous
carcinoma was
noted, which is not surprising considering that it takes
about
a decade for high-grade lesions to evolve into cervical cancer
(
36).
The presence of HPV in the lesions was confirmed by the detection of
viral capsid antigen in some of the samples. These samples
were few, an
observation in agreement with the finding that only
5 to 8% of
clinical bowenoid papillomatosis lesions (a variant
of intraepithelial
neoplasia of the external genitalia) exhibit
this antigen (
13,
23). The demonstration of approximately
55-nm viral particles
resembling HPV in the inoculum prepared
from the lesions of a second
passage of the isolate excludes any
carryover from the original
inoculum. Furthermore, since it is
reasonable to assume that these
capsids were of HPV-16, it is
remarkable that the xenograft SCID mouse
model allowed the production
of extractable virions, something that has
not been reported previously
for HPV-16 or intraepithelial neoplasias.
HPV-16 was identified by PCR and DNA sequencing of the MY09-MY11
amplicon. The sequence of HPV-16
Rochester-1k differed from
the prototype sequence at seven nucleotide positions, only one
resulting in a significant amino acid change. This DNA sequence
was
confirmed in a separate clone of the full L1 open reading
frame. At
least two reasons may account for why the
HPV-16
Rochester-1k sequence differs from that of
HPV-16
Rochester-1sc. The DNA sequences
were each derived
from single clones that may represent different
variants in
the original inoculum. Alternatively, nucleotide errors
may
have been introduced in the HPV-16
Rochester-1k amplicon by
the
Taq polymerase during PCR. This is less likely given the
concordance
of two independent DNA sequences of the
HPV-16
Rochester-1k amplicon.
Conversely, the
sequencing error might have been in the
HPV-16
Rochester-1sc.
Further passage and sequencing of the
isolate should clarify this
issue. The inoculum prepared from the third
passage in the first
set of experiments contained only HPV-16 when
tested, after PCR
amplification, with type-specific
oligonucleotide probes corresponding
to the vast majority of anogenital
HPV types. The isolation of
HPV-16 from clinical condylomata
acuminata was surprising since
this genotype is rarely, if ever, found
in penile condylomata
acuminata (
1). However, when we looked
back at the histologic
diagnosis of the lesions biopsied, we found that
2 of the 11 patients
had mild to moderate penile intraepithelial
neoplasia, while the
remainder had condyloma acuminatum. This finding
is significant
because in a previous study we had found HPV-16 in 17 (36%) of
47 penile intraepithelial neoplasias (
15). It is
therefore conceivable
that different HPV-16 strains from different
patients were present
in the original inoculum. Since this isolation of
HPV-16, we have
attempted five other isolations and propagations of HPV
types,
using the human xenograft SCID mouse model. All were successful
and yielded three HPV-6 and two new HPV-11 strains (
4,
5,
7). The percentages of grafts positive for HPV by histology
after
a first passage were, respectively, 79, 43, and 21% for
our three
HPV-6 isolates and 62 and 50% for our two HPV-11 isolates,
compared
with 17% for HPV-16
Rochester-1k and 63% for
HPV-16
Rochester-1sc.
These variable rates do not permit
conclusions regarding the growth
advantage of a particular genotype
over another, and we cannot
explain why other HPV types did not grow
along with HPV-16. As
with HPV-11
Hershey, we noted that
HPV-16-infected renal grafts
grew larger than subcutaneous grafts
(
6). The ease of isolating
new genital HPV strains in the
human xenograft SCID mouse model
contrasts with several personal failed
attempts and the sporadic
success of others using the athymic mouse
(
14,
27). It supports
our view of the advantages of the SCID
mouse over the athymic
mouse (
9). Another potential factor
in our success was the
addition of a high-speed centrifugation step in
the preparation
of the viral inoculum (
27), since a high
viral titer may be
essential for infectivity (
14,
24).
Our observations are further confirmation that HPV-16 directly causes
intraepithelial neoplasia. The availability of this
HPV-16 human
xenograft model offers novel possibilities. One can
now produce
infectious particles of an oncogenic HPV and study
the full replicative
cycle of the virus. It provides a model that
seems to faithfully
reproduce the natural lesion and thus permits
the investigation of an
authentic pathogenesis of HPV-induced
premalignant lesions. Finally, it
allows for the evaluation of
antiviral strategies.
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ACKNOWLEDGMENTS |
This work was supported by contract NIH-NO1-AI-35159 from the
National Institutes of Health.
We are indebted to Mark H. Stoler for interpreting the pathology of the
patient biopsies. We thank Debbie Pilc, University of Rochester
Xenograft Facility, for excellent assistance, and we thank Elizabeth
Woodward and her staff for providing access to foreskins.
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FOOTNOTES |
*
Corresponding author. Mailing address: Infectious
Diseases Unit, Box 689, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Phone: (716) 275-5871. Fax: (716)
442-9328. E-mail:
william_bonnez{at}medicine.rochester.edu.
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J Virol, June 1998, p. 5256-5261, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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