Journal
of General Virology (1999), 80, 2149–2155. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Herpes simplex virus type 1 infection has two separate modes
of spread in three-dimensional keratinocyte culture
Veijo Hukkanen,1, 2 Hannamari Mikola,1 Marja Nyka$ nen2, 3 and Stina Syrja$ nen2, 3
Department of Virology1, MediCity Research Laboratory2 and Department of Oral Pathology, Institute of Dentistry3, University of Turku,
Kiinamyllynkatu 13, FIN-20520 Turku, Finland
This study describes the outcome of herpes simplex virus type 1 (HSV-1) infection in an
organotypic raft culture of spontaneously immortalized HaCat keratinocytes and human fibroblasts, as related to the virus load and epithelial stratification and differentiation. In this model, a
confluent monolayer of HaCat keratinocytes was formed 60 h after seeding. Inoculation of HSV-1
before induction of differentiation by lifting of the culture to the air–liquid interface always resulted
in a productive infection, but the virus yield was highest when the inoculation took place 72 h after
seeding. Even at 0n1 p.f.u. per culture, the HaCat cultures became HSV positive. Infection of the fullthickness epithelium at 5 p.f.u. per culture resulted in a productive infection of the whole
epithelium. The HaCat cells were about 10 times more sensitive to HSV-1 infection than the Vero
cells in which the virus stocks were titrated. The raft cultures infected 30 min after lifting were
negative by HSV-1 culture, and no HSV-1 antigen was detected by immunocytochemistry. PCR
showed the presence of HSV-1 DNA and in situ hybridization showed reactivity with a latencyassociated RNA probe, indicating the presence of a non-productive infection. Two different
patterns of virus spread in epithelia were found : (i) lateral spread through the superficial layers of
the epithelium and (ii) a demarcated infection throughout the whole thickness of the epithelium at
the margins of the culture.
Introduction
Organotypic three-dimensional (raft) culture of keratinocytes provides a tissue culture model with characteristics of
differentiated epithelium (Asselineau & Prunieras, 1984).
Previously, raft cultures have been successfully applied to the
culture of human papillomaviruses (Meyers & Laimins, 1994).
We were the first to demonstrate the applicability of this
organotypic tissue culture to the study of herpes simplex virus
type 1 (HSV-1) infection (Syrja$ nen et al., 1996). A similar HSV1 culture system has also been described by Visalli et al. (1997).
Productive HSV infection involves the coordinately regulated and sequentially ordered expression of defined classes of
viral genes, the α, β and γ genes (Honess & Roizman, 1974,
1975). Lytically HSV-infected cells are subject to structural
alterations, accompanied by microscopically visible cytopathic
effects, including reticular degeneration and ballooning of the
cells, as well as formation of multinucleated giant cells
Author for correspondence : Hannamari Mikola.
Fax j358 2 2513 303. e-mail Hanna.Mikola!utu.fi
0001-6127 # 1999 SGM
(Roizman & Sears, 1996). In the organotypic culture of
epithelial cells, lytic HSV-1 infection shows features typical of
an epithelial infection in vivo (Syrja$ nen et al., 1996 ; Visalli et al.,
1997). Lytic infection was always detectable throughout the
whole epithelium when HSV-1 (5 p.f.u.) was inoculated on the
epithelial monolayer (HaCat) at the time of confluence before
lifting the cultures. Visalli et al. (1997) applied HSV-1 to the top
of a cornified full-thickness epithelium (epithelial cells from
foreskin and ectocervix) grown for 8 days. They reported that
HSV-1 could penetrate into the basal cells and initiate
replication there. Some areas were infected, whereas some
fields of the culture remained apparently normal by light
microscopy. The limitation of virus spread appeared to be
dependent on the HSV strain or mutant used (Visalli et al.,
1997).
HSV establishes a latent infection in sensory neurons
(Roizman & Sears, 1996). In the numerous in vitro models
established in different cell types (Levine et al., 1980 ; Rapp,
1984 ; Nilheden et al., 1985 ; Biswal et al., 1988), replication of
HSV is restricted by various treatments of the cell cultures,
having little resemblance to the early events of a latent
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V. Hukkanen and others
infection in the sensory neurons of animals (Roizman & Sears,
1996). In our recent study, we also described a form of HSV1 infection without cytopathic effects in epithelial raft culture.
The presence of HSV-1 was demonstrated by in situ detection
of latency-associated RNA (LAT RNA) (Stevens et al., 1987 ;
Wagner et al., 1988) and by PCR for HSV-1 DNA (Syrja$ nen et
al., 1996). The appearance of this type of infection was
dependent on the timing of virus application relative to
epithelial differentiation, requiring virus infection early after
induction of stratification (Syrja$ nen et al., 1996). Epithelial
latency of HSV in humans has been suggested previously on
the basis of indirect evidence in an HSV reactivation study
(Spruance et al., 1991). Persistent HSV-1 infection in murine
non-neuronal cells has also been suggested (Maggs et al.,
1998). The non-productive form of HSV-1 infection (Syrja$ nen
et al., 1996) would support the concepts of epithelial latency or
persistence.
In the present report, we describe the spread of HSV-1
infection in an organotypic culture and describe the establishment of HSV-1 infection as a function of the virus load,
growth properties of the epithelium and the level of epithelial
differentiation.
Methods
Virus. HSV-1 strain F, obtained from the ATCC (Manassas, VA,
USA) was used throughout the study. The virus stock was prepared in
human foreskin fibroblasts (HFF) as described previously (Syrja$ nen et al.,
1996) and stored at k70 mC at a concentration of 3n6i10' p.f.u.\ml
(Vero cell infectious units).
Organotypic cell culture. HaCat cells (Boukamp et al., 1988) were
grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 1 % non-essential amino acids, 2 mM -glutamine, 50 µg\ml
streptomycin, 100 U\ml penicillin and 10 % foetal calf serum (Gibco
BRL). Human skin fibroblasts were prepared as described previously
(Syrja$ nen et al., 1996) and grown in supplemented DMEM.
The fibroblast–collagen gel was prepared as described previously
(Syrja$ nen et al., 1996). Briefly, human skin fibroblasts were suspended in
a solution of Vitrogen 100 collagen (Celtrix Pharmaceuticals) in DMEM
at pH 7n4 at a density of 280 000 cells per 0n7 ml and plated on 24-well
tissue culture dishes (Costar). The cells were maintained at 37 mC in
Green’s medium [DMEM–HamF12 (3 : 1) medium containing 10 % foetal
calf serum, 4 mM glutamine, 5 µg\ml insulin (Sigma), 0n18 mM adenine
(Sigma), 0n4 µg\ml hydrocortisone (Sigma), 0n1 nM cholera toxin (Sigma)
and 5 ng\ml epidermal growth factor (Boehringer Mannheim)]. Ascorbic
acid was added to the medium just before use to a final concentration of
50 µg\ml.
For organotypic cultures, HaCat cells were added onto fibroblast–
collagen gels at a density of 200 000 cells per well. The cultures were
grown submerged in Green’s medium for 52–78 h as indicated and lifted
to the air–liquid interface by using a stainless steel grid support. The
cultures were infected with HSV-1 at time-points of 24 h before lifting or
at 0n25–6 h after lifting as described. The amount of infectious virus
varied between 0n1 and 10& p.f.u. per culture, expressed as Vero cell
infectious units. The cultures were harvested 1 week post-infection (p.i.).
The cultures were collected at the times indicated and divided into equal
parts for HSV-1 culture, microscopical analyses and DNA extraction.
For microscopy and immunohistochemistry, the samples were fixed in
buffered 10 % formalin for 24 h.
CBFA
PCR for HSV-1 DNA. The PCR test for HSV-1 DNA was carried
out as described previously (Syrja$ nen et al., 1996) with gD-l primers
described by Aurelius et al. (1991). The specimens were DNA extracts
from frozen cultures. Positive controls were HSV-1 (F) virus, 1 p.f.u. from
a 10* p.f.u.\ml stock per reaction. The working spaces and devices were
designed to minimize contamination.
In situ hybridization. In situ hybridizations were carried out
according to our previous protocol (Syrja$ nen et al., 1996) with
digoxigenin (DIG)-labelled single-stranded RNA probes. The LAT RNA
probe contained sequences from the 0n5 kb HpaI–SalI subfragment of
HSV-1 (F) BamHI fragment B. The DIG RNA probe for αTIF (VP16)
mRNA was transcribed from plasmid pRB3717 (McKnight et al., 1987 ; a
gift from Bernard Roizman, University of Chicago, IL, USA) by using T7
polymerase.
Immunohistochemistry. The presence of HSV-1 antigen was
demonstrated in paraffin sections of the epithelial cultures by immunohistochemistry with a polyclonal rabbit antibody against HSV-1
(BioGenex Laboratories) diluted 1 : 100. The sections were pretreated in
a microwave oven with 10 mM citric acid, pH 6n0 for 5 min.
Immunohistochemistry was performed with a staining automate (Dako
TechMatem 500) according to the manufacturer’s instructions. Formalinfixed sections from biopsy samples taken from labial herpes were used as
positive controls. In the negative controls, the primary antibody was
omitted from the reaction mixture. Antibodies against ICP0 and
thymidine kinase of HSV-1 were kindly provided by Bernard Roizman
(University of Chicago).
Detection of infectious HSV-1. Infectious HSV-1 was detected in
the culture supernatants and homogenates by a previously described
immunoperoxidase system in Vero cells (Ziegler et al., 1988). Stock virus
was titrated in duplicate wells in HFF, Vero and HaCat cells.
Results
HaCat cells are spontaneously transformed keratinocytes
derived from the skin. In an organotypic raft culture, the HaCat
keratinocytes stratify and form a differentiated epithelium
8–12 cell layers thick. In our model, only slightly parakeratinized epithelium is formed, without any orthokeratosis
normally present in the skin. Thus the cultured epidermis is not
as differentiated as the skin epidermis in vivo. During the first
days of culture, before lifting to the air–liquid interface,
keratinocytes grow horizontally as a monolayer until reaching
confluence. We determined the time needed to reach confluence to understand the effect of differentiation and stratification of the keratinocytes on the outcome of HSV-1
infection.
HaCat cultures on a fibroblast–collagen layer were allowed
to grow for 48–120 h, before being infected with HSV-1 while
still in the culture medium. The morphological appearance of
the keratinocyte layer was followed with respect to the
continuity of the monolayer, because horizontal growth might
affect the replication and spread of the virus within the
epithelium. At 50–54 h post-seeding, the HaCat cell monolayer was 80–90 % complete. At that point, the epithelial cells
resembled basal cells from normal skin. The keratinocyte layer
was confluent 60 h after seeding, after which they became
proliferation-arrested. Morphologically, the epithelial cells
were flattened and maintained the same phenotype for
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HSV infection in raft cultures
Table 1. Detection of HSV-1 in HaCat keratinocyte raft cultures infected with different amounts of the virus 24 h before
induction of differentiation and stratification
IHC, Immunohistochemistry. The number of HSV-1-positive parallel cultures of those tested is shown.
HSV-1 culture/immunoperoxidase detection of HSV-1
Culture supernatants
Input virus (p.f.u.
per culture)
0
0n1
0n5
Culture homogenates
3 days p.i.
(1 : 50)*
6 days p.i.
(1 : 250)
8 days p.i.
(1 : 250)
8 days p.i.
(1 : 10)
8 days p.i.
(1 : 100)
Detection of
HSV-1 by IHC
0\2
1\3
3\3
0\2
3\3
3\3
0\2
3\3
3\3
0\2
3\3
3\3
0\2
3\3
3\3
0\2
3\3
3\3
* The dilution of the original culture supernatant or homogenate tested is shown.
Table 2. Titration of HSV-1 virus stock in HFF, B-Vero and HaCat cells
The mean number of plaques per culture is shown for each cell line and each dilution ; means were determined
from two parallel cultures.
Dilution of virus stock
Cell line
Experiment 1
HFF
B-Vero
HaCat
Experiment 2
HFF
B-Vero
HaCat
10−5
10−6
13
7n5
82
1
0n5
8
1n2i10'
0n6i10'
8n1i10'
21n5
25
133n5
2
2n5
13
2n1i10'
2n5i10'
13n2i10'
96–120 h after inoculation. Application of HSV-1 before
induction at different time-points resulted in a productive
infection but the virus yield was found to be highest when
infection took place at 72 h after seeding (data not shown).
Effect of virus load on the outcome of HSV-1 infection
HaCat keratinocyte cultures were infected at different m.o.i.
of HSV-1 (F) in order to see whether there was a limit of input
virus below which the cultures did not support productive
infection. All cultures were infected similarly with HSV-1 1 day
before lifting with different amounts of virus from 0n1 to 5
p.f.u. (Vero cell infectivity) per culture (Table 1). Even at 0n1
p.f.u. per culture, the HaCat culture supernatants were HSV-1positive at 6 days p.i. and at 0n5 p.f.u. or more per culture, a
strong productive infection always ensued. We determined the
sensitivity of HaCat cells for HSV-1 and found that HaCat cells
are about 10 times more sensitive than Vero cells (Table 2).
Calculated titre (p.f.u./ml)
Infection of the full-thickness epithelium (cultured for 7
days after lifting) at 5 p.f.u. per culture resulted in a productive
infection in 2\3 of the cultures, with morphological alterations
in almost the entire epithelium (Table 3). When inoculation was
done with 10$ p.f.u., large, multinucleated giant cells were seen.
Infection with 10% p.f.u. caused almost total lytic destruction of
the epithelium. Some fibroblasts also became infected. These
features were even more prominent when inoculation was
done with 10& p.f.u.
Effect of time of virus application on differentiationinduced cultures
Organotypic cultures of HaCat keratinocytes were infected
at 0n25–6 h after lifting to the air–liquid interface, i.e. after
induction of stratification and differentiation. Virus infection
was studied by cultivation of the infectious virus from epithelia
and supernatants, by immunohistochemical analysis, by PCR
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V. Hukkanen and others
Table 3. Detection of HSV-1 in HaCat keratinocyte raft cultures infected as full-thickness
epithelium 7 days after lifting
The number of positive parallel cultures of those tested is shown.
Supernatant
Input virus (p.f.u.
per culture)
0
5
10$
Homogenate
3 days p.i.
(1 : 50)*
5 days p.i.
(1 : 50)
7 days p.i.
(1 : 10)
0\3
1\3
3\3
0\3
2\3
3\3
0\3
2\3
3\3
* The dilution used for testing is shown.
Table 4. Detection of HSV-1 at 7 days p.i. in HaCat keratinocyte cultures infected with 5 p.f.u. HSV-1 per culture at
various times after induction of differentiation
Supernatants and homogenates were diluted 1 : 50 and 1 : 10, respectively. The number of positive parallel cultures of those tested is shown. IHC,
Immunohistochemistry ; , not done.
HSV-1 culture from raft cultures
Time of virus
application (h)
Uninfected
0n25
0n5
1
2
4
6
Supernatant
Homogenate
HSV-1 DNA PCR
LAT in situ
hybridization
HSV-1 antigen
detected by IHC
0\1
3\3
0\3
3\3
2\3
2\3
2\3
0\1
3\3
0\3
3\3
3\3
3\3
2\3
0\1
3\3
2\3
3\3



0\1
3\3
3\3
3\3



0\1
3\3*
0\3
2\3*
2\3*
1\3
1\3*
* Positive IHC staining for HSV-1 antigen was observed at the margins of the cultures.
for HSV-1 DNA and by in situ hybridization for LAT and αTIF
RNA. The cultures were analysed at 7 days p.i.
By culture\immunoperoxidase detection, HSV-1 infection
was detected in HaCat raft cultures infected 15 min and
60 min after lifting (Table 4). The cultures infected 30 min
after lifting were negative by culture and no HSV-1 antigen
was detected by immunohistochemical staining (Fig. 1 a, b). An
interesting staining pattern was detected in the specimens
infected 60 min or more after lifting. We observed a productive
infection at the margins of the cultures, where the epithelium
had grown laterally beyond the fibroblast–collagen layer and
reached the surface of the medium (Fig. 2). In these cultures,
PCR showed the presence of HSV-1 DNA and in situ
hybridization showed reactivity with the LAT RNA probe, but
rarely with the probe for αTIF RNA, outside the productively
infected margin area (Fig. 2 b–d). The DIG-based detection
system resulted in an intensive background staining of the
collagen matrix. The productive infection of the margins was
CBFC
observed in many different experimental settings and contributed to the positive virus culture detection in specimens
infected after lifting.
The spread of HSV-1 infection in epithelial cultures
In cases where HSV-1 was applied onto the epithelium
60 min or more after lifting, we observed two distinct patterns
of virus spread : lateral spreading on superficial layers of the
epithelium and a sharply demarcated infection throughout the
thickness of the epithelium at the margins of the cultures (Fig.
3), presumably at the loci exposed to the culture medium
during incubations. This infection was not limited to the
basal\suprabasal cell layers ; instead, the infection appeared to
spread along and from the most superficial cell layers. The
basal and parabasal cells remained HSV-1 antigen-negative by
immunohistochemistry. Occasional infected fibroblasts were
found in cultures infected with large amounts of HSV-1.
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HSV infection in raft cultures
(a)
(b)
Fig. 1. Absence of morphological signs of
HSV-1 infection and of HSV-1 α and β
class protein expression in raft cultures
infected 30 min after lifting. Detection by
immunohistochemistry using anti-ICP0
(a) and anti-thymidine kinase (b)
antibodies, diluted 1 : 200. Haematoxylin
counter-staining. Magnification i100.
(a)
(a)
(b)
(c)
(d)
Fig. 2. Pattern of HSV-1 infection in
cultures infected after lifting. (a) Infection
of keratinocytes at 5 p.f.u. 60 min after
lifting. Productive infection at the margin
of the culture can be seen, as shown by
immunohistochemistry. More centrally, a
restricted area was also HSV-1 antigenpositive, while all other areas of the
epithelium remained negative.
Magnification i40. (b) In situ
hybridization with LAT RNA probe. A
fraction of the LAT-positive cells are αTIF
RNA negative [shown in (d)]. The
collagen matrix shows strong background
staining because of the DIG-based
detection. Magnification i100. (c) The
sample hybridized for αTIF RNA, showing
positive signals at the margin of the
sample and in the adjacent area.
Magnification i40. (d) High-power view
of the in situ αTIF-positive area at the
margin of the epithelium. Magnification
i100.
(b)
Fig. 3. The spread of HSV-1 infection in
the epithelial cultures. Inoculation with 5
p.f.u. HSV-1 was done 6 h after lifting.
(a) Two distinct patterns of HSV-1
infection were found : HSV-1 antigen at
the margin of the epithelium and focal
antigen expression in the superficial cells.
Magnification i40. (b) Focal staining
pattern in the surface layers of the
epithelium. Magnification i250.
Discussion
We have previously used organotypic keratinocyte cultures
to study the replication and spread of HSV-1 in epithelium
(Syrja$ nen et al., 1996). Visalli et al. (1997) utilized a slightly
different organotypic model. In both studies, a productive
HSV-1 infection was established with morphological characteristics of herpes lesions in vivo.
The models presented by us (Syrja$ nen et al., 1996) and by
Visalli et al. (1997) are not similar in all respects, which may
account for the observed differences in virus spread and the
outcome of the infection. We obtained HSV-1 infection
throughout the whole epithelium (Syrja$ nen et al., 1996),
whereas Visalli et al. (1997) found a productive infection in the
basal and parabasal cells of their epithelium. One explanation
for the contradictory results might be the inoculation time of
the virus. We applied HSV-1 before lifting, when epithelial
cells were confluent, while Visalli et al. (1997) inoculated HSV1 on the surface of the full-thickness epithelium grown for 7
days. In our current model, a productive infection of the whole
epithelium and even fibroblasts was produced by high input
virus on the fully developed epithelium. When low titres were
used to infect the epithelium 4–6 h after lifting, the infection
appeared to spread along and from the most superficial cell
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V. Hukkanen and others
layers. The basal and parabasal cells remained HSV-1 antigennegative by immunohistochemistry (Fig. 3). In our model, it is
unlikely that the virus penetrates the epithelium to initiate
replication only in the basal cells.
According to earlier raft culture studies, fully keratinized
epidermis in vitro is 10 times more permeable than human
epidermis ex vivo (Regnier et al., 1990). In raft cultures,
keratinocytes move from the basal layers to the cornified layer
within a maximum period of 7 days, with virtually complete
replacement of all viable keratinocytes in 14 days (MacCallum
& Lillie, 1990). This movement might also prevent virus
penetration to the basal cells, suggested by Visalli et al. (1997).
The epithelium produced in our model is more immature and
proliferative than the epithelium produced by Visalli et al.
(1997), which might also affect the outcome of the infection.
There are essential differences in the origin of keratinocytes
and fibroblasts in the two culture models. We use a spontaneously transformed HaCat keratinocyte cell line and human
fibroblasts derived from the skin, whereas Visalli et al. (1997)
used human foreskin and ectocervical keratinocytes as well as
mouse 3T3 fibroblasts. We have recently shown that fibroblasts
can modulate the phenotype of the epithelium in vitro (Atula et
al., 1997). The use of human cells allows the complex
interactions between keratinocytes and fibroblasts, including
the actions of cytokines and growth factors, to take place.
Interferon secreted by human fibroblasts is supposedly
effective on human keratinocytes in our model.
As in our previous study, we obtained a form of HSV-1
infection where there was no production of infectious virus,
whereas HSV-1 was detectable by PCR and RNA in situ
hybridization (Syrja$ nen et al., 1996). In agreement with the
observations of Visalli et al. (1997), there were areas of the
culture that remained apparently normal, without any evidence
of HSV-1 infection, as viewed by immunohistochemistry. On
the other hand, we found that the margins of the epithelial
cultures were heavily infected with HSV-1 in many different
experimental settings (Fig. 2 a ; Fig. 3 a, b). This could be
explained by direct contact of the keratinocytes with the
culture medium as a result of an overgrowth exceeding the
limits of the collagen–fibroblast bed. Horizontally growing
epithelial cells are actively proliferating cells that might support
virus replication. Visalli et al. (1997) reported that the infection
does not proceed if the virus is distributed to the culture from
below, diluted in the medium. However, in those experiments,
the collagen–fibroblast layer was present between the virus
and the keratinocytes. When a basal cell divides, one of the
daughter cells migrates upward and starts differentiation. Thus,
infected basal cells spread the infection upwards during
differentiation and migration from the basal layers to the
surface of the epithelium. This might result in a sharp boundary
between the infected and non-infected epithelium, especially if
the virus yield is high, as shown in Figs 2 (a) and 3 (a).
We found that the HaCat cells are about 10 times more
sensitive to HSV-1 than the Vero cells used for initial virus
CBFE
titration (Table 2). Thus the p.f.u. values used for infection
probably represent 10 times greater virus input. The nonproductive infection observed therefore cannot be merely the
result of an insufficient number of HSV-1 particles in the
inoculum. The non-productive form of HSV-1 infection is
strictly dependent on the early timing of virus application on
keratinocytes after induction of differentiation. This raises the
need for further analysis of the early expression of transcription
factors, growth factors and other signalling molecules in the
epithelium. The stratified layers of keratinocytes may express
a different profile of transcription factors and growth factors
than the cells shortly after induction of differentiation. The
effect of HSV-1 infection at the margins of the culture blurs the
opportunities to observe the non-productive infection by virus
culture, since it would be positive for infectious virus even
when there was a mixed productive and non-productive
infection in different parts of the culture. The possibility that
the LAT probe reacts just with random productively infected
cells is, however, minimal, since the immunohistochemical and
αTIF hybridizations for the same regions remained negative.
There is some indirect support for the concept of epithelial
HSV-1 latency in humans (Spruance et al., 1991) and in mice
(Maggs et al., 1998). We cannot yet define our non-productive
HSV-1 infection as latency, since there is no reactivation model
for it at present.
We acknowledge Professor Bernard Roizman, University of Chicago,
for the HSV clone, the ICP0 and thymidine kinase antibodies and the
protocols of producing the virus stock. Financial support was from the
Paulo Foundation, the Turku University Foundation and from the
Medical Research Council, the Academy of Finland.
References
Asselineau, D. & Prunieras, M. (1984). Reconstruction of ‘ simplified ’
skin : control of fabrication. British Journal of Dermatology 111 (Suppl. 27),
219–222.
Atula, S., Grenman, R. & Syrja$ nen, S. (1997). Fibroblasts can modulate
the phenotype of malignant epithelial cells in vitro. Experimental Cell
Research 235, 180–187.
Aurelius, E., Johansson, B., Sko$ ldenberg, B., Staland, A/ . & Forsgren,
M. (1991). Rapid diagnosis of herpes simplex encephalitis by nested
polymerase chain reaction assay of cerebrospinal fluid. Lancet 337,
189–192.
Biswal, N., Patel, A. G. & Max, S. R. (1988). Regulation of viral and
cellular genes in a human neuroblastoma cell line latently infected with
herpes simplex virus type 2. Molecular Brain Research 3, 95–106.
Boukamp, P., Petrussevska, R. T., Breitkreu$ tz, D., Hornung, J.,
Markham, A. & Fusenig, N. E. (1988). Normal keratinization in a
spontaneously immortalized aneuploid human keratinocyte cell line.
Journal of Cell Biology 106, 761–771.
Honess, R. W. & Roizman, B. (1974). Regulation of herpesvirus
macromolecular synthesis. I. Cascade regulation of the synthesis of three
groups of viral proteins. Journal of Virology 14, 8–19.
Honess, R. W. & Roizman, B. (1975). Regulation of herpesvirus
macromolecular synthesis : sequential transition of polypeptide synthesis
requires functional viral polypeptides. Proceedings of the National Academy
of Sciences, USA 72, 1276–1280.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 18:00:05
HSV infection in raft cultures
Levine, M., Goldin, A. L. & Glorioso, J. C. (1980). Persistence of herpes
simplex virus genes in cells of neuronal origin. Journal of Virology 35,
203–210.
MacCallum, D. K. & Lillie, J. H. (1990). Evidence for autoregulation of
cell division and cell transit in keratinocytes grown on collagen at an
air–liquid interface. Skin Pharmacology 3, 86–96.
McKnight, J. L. C., Kristie, T. M. & Roizman, B. (1987). Binding of the
virion protein mediating α gene induction in herpes simplex virus 1infected cells to its cis site requires cellular proteins. Proceedings of the
National Academy of Sciences, USA 84, 7061–7065.
Maggs, D. L., Chang, E., Nasisse, M. P. & Mitchell, W. J. (1998).
Persistence of herpes simplex virus type 1 DNA in chronic conjunctival
and eyelid lesions of mice. Journal of Virology 72, 9166–9172.
Meyers, C. & Laimins, L. A. (1994). In vitro systems for the study and
propagation of human papillomaviruses. Current Topics in Microbiology
and Immunology 186, 199–215.
Nilheden, E., Jeansson, S. & Vahlne, A. (1985). Herpes simplex virus
latency in a hyperresistant clone of mouse neuroblastoma (C1300) cells.
Archives of Virology 83, 319–325.
Rapp, F. (1984). Experimental studies of latency in vitro by herpes
simplex viruses. Progress in Medical Virology 30, 29–43.
Regnier, M., Asselineau, D. & Lenoir, M. C. (1990). Human epidermis
on dermal substrates in vitro : an alternative to animals in skin
pharmacology. Skin Pharmacology 3, 70–85.
Roizman, B. & Sears, A. (1996). Herpes simplex viruses and their
replication. In Fields Virology, 3rd edn, pp. 2231–2295. Edited by B. N.
Fields, D. M. Knipe & P. M. Howley. Philadelphia : Lippincott–Raven.
Spruance, S. L., Freeman, D. J., Stewart, J. C., McKeough, M. B.,
Wenerstrom, L. G., Krueger, G. G., Piepkorn, M. W., Stroop, W. G. &
Rowe, N. H. (1991). The natural history of ultraviolet radiation-induced
herpes simplex labialis and response to therapy with peroral and topical
formulations of acyclovir. Journal of Infectious Diseases 163, 728–734.
Stevens, J. G., Wagner, E. K., Devi-Rao, G. B., Cook, M. L. & Feldman,
L. T. (1987). RNA complementary to a herpesvirus alpha gene mRNA
is prominent in latently infected neurons. Science 235, 1056–1059.
Syrja$ nen, S., Mikola, H., Nyka$ nen, M. & Hukkanen, V. (1996). In vitro
establishment of lytic and nonproductive infection by herpes simplex
virus type 1 in three-dimensional keratinocyte culture. Journal of Virology
70, 6524–6528.
Visalli, R. J., Courtney, R. J. & Meyers, C. (1997). Infection and
replication of herpes simplex virus type 1 in an organotypic epithelial
culture system. Virology 230, 236–243.
Wagner, E. K., Devi-Rao, G. B., Feldman, L. T., Dobson, A. T., Zhang,
Y. F., Flanagan, W. M. & Stevens, J. G. (1988). Physical characterization
of the herpes simplex virus latency-associated transcript in neurons.
Journal of Virology 62, 1194–1202.
Ziegler, T., Waris, M., Rautiainen, M. & Arstila, P. (1988). Herpes
simplex virus detection by macroscopic reading after overnight incubation and immunoperoxidase staining. Journal of Clinical Microbiology
26, 2013–2017.
Received 14 December 1998 ; Accepted 16 April 1999
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