Current Eye Research
2004, Vol. 29, Nos. 2–3, pp. 119–125
Acetylsalicylic acid reduces viral shedding induced by
thermal stress
Bryan M. Gebhardt, Emily D. Varnell and Herbert E. Kaufman
Lions Eye Research Laboratories, LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans,
Louisiana, USA
Curr Eye Res Downloaded from informahealthcare.com by Aston University on 09/08/14
Abstract
Purpose. To investigate the effect of acetylsalicylic acid on
ocular shedding of herpes simplex virus type 1 (HSV-1).
Materials and methods. Mice that were latent for the McKrae
strain of HSV-1 were treated with acetylsalicylic acid, a non-
specific inhibitor of cyclooxygenases, either prophylactically
or at the time of heat stress–induced viral reactivation. The
For personal use only.
effect of the drug on viral shedding in the tear film, infec-
tious virus in the cornea and trigeminal ganglion, and viral
DNA in the cornea and trigeminal ganglion was determined.
Results. Acetylsalicylic acid inhibited heat stress–induced
shedding of virus in the tears and reduced the numbers of
corneal and trigeminal ganglion homogenates containing
virus. Intraperitoneal therapeutic and oral prophylactic plus
therapeutic treatments were similar in their ability to inhibit
reactivation.
Conclusions. The results indicate that a cyclooxygenase
inhibitor such as acetylsalicylic acid can reduce recurrent
viral infection in mice. These findings may implicate
prostaglandins as agents in the viral reactivation process and
suggest that therapy to suppress viral reactivation using non-
toxic inhibitors of prostaglandin synthesis may be effective
in humans.
Keywords: acetylsalicylic acid; cyclooxygenase; heat stress;
herpesvirus; mouse; prostaglandins; reactivation
Introduction
Studies by Blyth et al.,1 Hill et al.,2 and Harbor et al.3 showed
that prostaglandins enhanced the spread of herpes simplex
Received: November 20, 2003
Accepted: January 21, 2004
Correspondence: Bryan M. Gebhardt, Ph.D., LSU Eye Center, 2020 Gravier Street, Suite B, New Orleans, LA 70112-2234, USA.
Fax: 504-412-1315; E-mail: bgebha@lsuhsc.edu
DOI: 10.1080/02713680490504588
virus type 1 (HSV-1) in cell culture and that they are involved
in the induction of viral reactivation. Studies by Newton4–6
also revealed that the addition of prostaglandin inhibitors to
cell culture significantly reduced viral replication.
The mechanisms whereby prostaglandins stimulate viral
reactivation and promote viral replication have not been
extensively investigated. In a study by Trofatter and Daniels,7
the mechanism of action of prostaglandins in enhancing
replication of HSV-1 was investigated by adding phosphodi-
esterase inhibitors and theophylline to virus-infected cell cul-
tures. It was found that these inhibitors delayed the growth
and inhibited cell-to-cell spread of the virus. It was also noted
that the prostaglandin inhibitors suppressed the production
of interferon by human mononuclear leukocytes, and it was
speculated that prostaglandins may have a role in exacerbat-
ing HSV infection by altering the balance of interferon syn-
thesis in vivo.7 Clearly, further investigation of the role of
prostaglandins in promoting viral replication in vivo is
required.
Recently, there has been a resurgence of interest in the role
of prostaglandins in mediating HSV-1 reactivation. The
topical use of latanoprost, a prostaglandin F2 alpha analog,
in the treatment of glaucoma led to reports indicating an
association of the treatment with viral reactivation and
epithelial keratitis. Wand et al.8 reported the appearance of
herpetic keratitis in three patients undergoing treatment with
latanoprost. It was noted that the keratitis resolved once the
topical hypotensive medication was stopped. Kaufman et al.9
confirmed these observations in a study in rabbits. They
reported that topical latanoprost treatment of rabbits infected
with HSV-1 during the acute phase resulted in more severe
© 2004 Taylor & Francis Ltd.
 120 B.M. Gebhardt et al.
epithelial keratitis compared with animals treated with bal-
anced salt solution. Furthermore, treatment of rabbits during
latency twice daily with latanoprost resulted in a statistically
significant increase in the number of corneas exhibiting
corneal epithelial lesions compared with placebo-treated
corneas. These observations suggested a note of caution to
ophthalmologists regarding this possible undesirable side
effect of topical prostaglandin treatment.
These findings also led to speculation on the broader
meaning of this phenomenon relative to the molecular mech-
anisms of HSV-1 reactivation and the possibility of future
development of prostaglandin antagonists, such as cyclooxy-
genase 2 (COX-2) inhibitors, which might achieve therapeu-
tically effective concentrations in target tissues, including the
eye and nervous system, and thereby prevent viral reactiva-
Curr Eye Res Downloaded from informahealthcare.com by Aston University on 09/08/14
tion and disease.
As an initial foray into determining the effect of inhibi-
tion of cyclooxygenases (COX) on HSV-1 reactivation, we
tested acetylsalicylic acid (ASA) in mice. We report here that
prophylaxis and treatment concomitant with the reactivation
stimulus reduces the frequency of viral reactivation follow-
ing heat stress–induced reactivation.
For personal use only.
Materials and methods
Virus and virological analyses
The McKrae strain of HSV-1 was propagated in Vero cells
(American Type Culture Collection, Manassas, VA, USA),
titered by viral plaque assay, and stored at -70°C until it was
used to infect animals.
Ocular swabs were placed in 0.2 ml of complete tissue
culture medium consisting of RPMI-1640, 10% fetal bovine
serum (FBS), and an antibiotic–antimycotic mixture (Life
Technologies, Grand Island, NY, USA). The swabs in culture
medium were stored at 4°C until testing by plaque assay. The
determination of infectious virus in the cornea and in the
trigeminal ganglia of animals was performed in a similar
manner. Pairs of corneas and ganglia from individual mice
were placed in separate tubes in 0.2 ml of complete medium,
homogenized, and clarified by centrifugation at 14 000 ¥ g
for 5 min, after which the supernatant was tested for infec-
tious virus on Vero cells. Swab supernates and tissue
homogenates were observed daily for cytopathic effect for
14 days.
Animals and animal infection
Male BALB/c strain mice (The Jackson Laboratory, Bar
Harbor, ME, USA) were infected at 5 weeks of age by topical
application of 1 ¥ 105 plaque-forming units of the McKrae
HSV-1 strain following superficial scratching of the cornea.
Survival of the mice was ensured by giving each animal
0.1 ml of human immune serum intraperitoneally. Documen-
tation of viral infection was determined by culturing ocular
swabs taken 3 and 5 days after infection. Animals without
infectious virus on both days 3 and 5 in both eyes were
excluded from use in these experiments. Thirty days after
infection, the eyes were swabbed one additional time to
establish that infectious virus was no longer present on the
ocular surface. Groups of uninfected age- and sex-matched
mice were used as controls.
Experimental design
A preliminary experiment was performed to determine
the approximate effective dose of ASA. Groups of six mice
per group latent for HSV-1 were heat stressed and
given intraperitoneal injections of 1.0, 0.5, 0.1, 0.05, or
0.01 mg ASA suspended in saline. The lowest concentrations
had no effect on shedding, and the highest concentrations
appeared to make the mice lethargic; the intermediate
dosage, 0.1 mg, inhibited viral shedding and did not appear
to have a deleterious effect on the appearance or behavior of
the mice.
Two types of experiments were performed. In one, the
mice were treated therapeutically. Mice latent for HSV were
immersed in 43°C water up to their necks for 10 min. After
the stress, the animals were dried and given an intraperitoneal
injection of 0.1 mg of ASA (Sigma Chemical Co., St. Louis,
MO, USA) suspended in 0.1 ml saline. At 8 and 16 hr
after hyperthermia, the animals were again treated with
ASA. Twenty-four hours after hyperthermia, the eyes were
swabbed, the mice were sacrificed, and the corneas and
trigeminal ganglia analyzed for infectious virus and viral
DNA.
In the other, mice were treated prophylactically and
therapeutically. Mice latent for HSV were treated orally with
0.1 mg of ASA in 0.1 ml saline three times at 8-hr intervals
1 day before hyperthermic stress. The animals were heat
stressed, treated with ASA, and treated again at 8 and 16 hr
after stress. As above, the eyes of the prophylactically treated
mice were swabbed at 24 hr after stress and then sacrificed
for analysis of infectious virus and viral DNA in their
corneas and trigeminal ganglia.
Control groups for these studies included latent, stressed
animals treated with saline; latent, not-stressed animals
treated with ASA; uninfected, stressed animals treated with
ASA or saline; and uninfected, not stressed animals treated
with ASA or saline.
Body temperature measurement
Rectal temperatures of untreated and mice treated orally with
ASA were measured before, immediately after, and 5 min
after hyperthermic stress using a rectal probe linked to a
digital thermometer (Physitemp BAT-10 Multipurpose
Thermometer; World Precision Instruments, Inc., Sarasota,
FL, USA). Temperature readings from groups of 10 mice
were used to determine the mean and standard deviation of
the readings obtained from mice before and after hyperther-
mic stress.
 Acetylsalicylic acid and herpesvirus 121

Comparison of the amount of viral DNA in the eye and
trigeminal ganglion
DNA was extracted from pairs of corneas and trigeminal
ganglia using a silica-gel membrane system (DNAeasy
Tissue System; Qiagen, Inc., Valencia, CA, USA). Optical
densities at 260 nm were obtained and 100 ng of DNA was
added to 25 ml of polymerase chain reaction master mix
(Amplitaq Gold PCR Master Mix; Roche Molecular
Systems, Inc., Branchburg, NJ, USA). Two microliters of a
DNA primer mix complementary to the HSV-1 ribonu-
cleotide reductase (RR) gene and water to a volume of 50 ml
completed the reaction mixture. Dilutions of a viral DNA
standard (Advanced Biotechnologies, Inc., Columbia, MD,
USA) were also amplified in each experiment. The viral
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test. In these statistical analyses, p < 0.05 was considered the
minimum significant difference to reject H0: x1 = x2.
Results
Effect of therapeutic intraperitoneal acetylsalicylic acid
on viral reactivation
Fewer HSV-latent mice treated IP with ASA at the time of
and after hyperthermic stress showed infectious virus on the
ocular surface 24 hr after reactivation compared with stressed
mice treated with saline (Table 1). Control, uninfected mice
that were heat stressed, uninfected mice that were not heat
stressed, and latent mice that were not heat stressed did not
have infectious virus on their ocular surface (data not

DNA standard consisted of 1 : 2 dilutions of a concentrate

containing 1 ¥ 105 copies/ml. Experimental and control
samples were heated to 94°C for 1 min followed by 35 cycles
at 58°C for 1.5 min, 72°C for 40 s, and 94°C for 1 min. A final
extension cycle at 70°C for 2 min was followed by storage at
4°C. Amplicons from the standards and corneal and gan-
glionic DNA were electrophoresed on 2% agarose gels and
stained with ethidium bromide. The quantity of viral DNA in
the corneal and ganglionic samples was determined by com-
For personal use only.
shown).
Similarly, fewer of the homogenates of corneal tissue and
trigeminal ganglia from the animals treated therapeutically
with ASA contained infectious virus compared with
homogenates from latent, stressed animals treated with saline
(Table 2). Latent mice that were not stressed did not shed
infectious virus.

paring the amplicon band intensities from the viral DNA

standards to the viral amplicon densities of the tissue extracts
using a gel imaging instrument (Eagle Eye II, Eagle Sight
Software; Stratagene, La Jolla, CA, USA). Each amplifica-
tion experiment was repeated three times to confirm the
results. The amount of viral DNA in a tissue extract was
expressed as the number of genome copies based on the
amplicon band density compared to the amplicon band den-
sities of the viral DNA standards.
Statistical methods
Analysis of normally distributed data was performed using
the one-way ANOVA and Tukey’s post hoc t test. The corre-
lation coefficient and its statistical significance were deter-
mined by regression analysis. The statistical significance of
HSV-1 reactivation in mice was evaluated by the Cox–Mantel
Effect of prophylactic and therapeutic oral
acetylsalicylic acid on viral reactivation
Fewer mice treated orally with ASA 1 day before and 1 day
after receiving the reactivation stimulus had infectious virus
in the ocular tear film compared with saline-treated mice that
underwent reactivation (Table 3). Control animals that were
latent for virus but not hyperthermically stressed and animals
that had not been infected and were hyperthermically
stressed did not have infectious virus in their ocular tear film
(data not shown).
Fewer of the corneas and trigeminal ganglia of mice that
had been prophylactically treated with oral ASA contained
infectious virus compared with homogenates from saline-
treated animals that had been hyperthermically stressed
(Table 4). Control animals that were latent but not stressed
and animals that had been stressed but were not latent for

Table 1. Effect of therapeutic intraperitoneal acetylsalicylic acid (ASA) treatment on the

numbers of ocular surface swabs positive for virus in HSV-latent, heat-stressed mice.

Treatment group Infectious virus (no. positive/total)

Experiment 1 Experiment 2 Experiment 3

+ASA 2/12 (17%)* 3/12 (25%)* 3/12 (25%)*

-ASA 8/12 (67%) 9/12 (75%) 7/12 (58%)

Ocular surface swabs were obtained 24 hr after heat stress and at the same time from latent and
non-latent, stressed and unstressed controls. None of the ocular surface swabs from the eyes
of any of the control mice contained infectious virus (data not shown).
* Significantly different from saline-treated mice (-ASA), p < 0.05.
 122 B.M. Gebhardt et al.
Table 2. Effect of therapeutic intraperitoneal acetylsalicylic acid
(ASA) treatment on the number of corneas and trigeminal ganglia
containing infectious virus in HSV-latent, heat-stressed mice.
Treatment group Infectious virus (no. positive/total)
Cornea
+ASA 5/36 (14%)*
-ASA 11/36 (31%)
Trigeminal ganglia
+ASA 8/36 (22%)*
-ASA 15/36 (42%)
Twenty-four hours after hyperthermic stress, mice were sacrificed
and their corneas and ganglia homogenized and tested for infectious
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virus. None of the homogenates from any of the control mice con-
tained infectious virus (data not shown).
* Significantly different from saline-treated tissues (-ASA),
p < 0.05.
Table 3. Effect of oral prophylactic and therapeutic acetylsalicylic
acid (ASA) treatment on the number of ocular swabs positive for
herpes simplex virus in HSV-latent, heat-stressed mice.
Treatment groups Infectious virus (no. positive/total)
For personal use only.
Experiment 1 Experiment 2
+ASA 4/10 (40%)* 3/14 (21%)*
-ASA 8/12 (67%) 8/12 (67%)
Ocular surface swabs were obtained 24 hours after heat stress and
at the same time from latent and nonlatent, stressed and unstressed
controls. None of the ocular surface swabs from the eyes of any of
the control mice contained infectious virus (data not shown).
* Significantly different from saline-treated mice (-ASA), p < 0.05.
virus did not have infectious virus in their corneas or trigem-
inal ganglia.
Viral DNA concentration in the cornea and
trigeminal ganglion
The quantity of viral DNA in the corneal extracts was con-
sistently near or below the limit of detection (40 genome
copies), and thus, the amount of viral DNA in the cornea was
too low to be accurately compared among the various groups
of animals. The levels of viral DNA found in the trigeminal
ganglia of intraperitoneal ASA-treated, stressed animals
treated therapeutically was consistently lower than the
amounts found in the saline-treated, stressed mice (Table 5).
This experiment was repeated two additional times with
similar outcomes.
Prophylaxis of latent mice with oral ASA prior to hyper-
thermically induced reactivation was associated with
significantly less viral DNA in the trigeminal ganglia of
the heat-stressed, ASA-treated animals compared with the
saline-treated mice that underwent hyperthermically induced
Table 4. Effect of oral prophylactic and therapeutic acetylsalicylic
acid (ASA) treatment on the number of corneas and trigeminal
ganglia containing infectious virus in HSV-latent, heat-stressed
mice.
Treatment groups Infectious virus (no. positive/total)
Cornea
+ASA 2/14 (14%)*
-ASA 7/14 (50%)
Trigeminal ganglia
+ASA 4/14 (29%)*
-ASA 9/14 (64%)
Homogenates of corneas and trigeminal ganglia obtained 24 hr after
stress were cultured for infectious virus. None of the homogenates
from any of the control mice contained infectious virus (data not
shown).
* Significantly different from saline-treated tissues (-ASA),
p < 0.05.
reactivation (Table 6). Latent mice treated with ASA and
latent mice not treated with ASA that were not stressed had
similar levels of DNA in their ganglia (Table 6). Viral DNA
was not found in the trigeminal ganglia of uninfected mice
regardless of whether or not they were hyperthermically
stressed.
Body temperature changes following
hyperthermic stress
The mean body temperatures of mice with latent HSV-1
prophylactically treated with ASA or saline were recorded
before and after stress. Both groups of heat-stressed mice had
transiently elevated body temperature immediately after
hyperthermia (Table 7); body temperature returned to normal
5 min later in both groups (Table 7). There was no significant
difference in the mean temperatures of the ASA and saline-
treated mice before or after heat stress (Table 7).
Discussion
A case has been made for a role for prostaglandins in herpes
viral reactivation. The results reported in this study using a
nonspecific inhibitor of cyclooxygenases support this idea.
Latanoprost, a prostaglandin analog that is widely used as a
glaucoma medication, has been reported to induce herpes
viral reactivation in patients; Wand et al.8 recorded several
instances of recurrent herpetic corneal disease subsequent to
prolonged latanoprost treatment in patients. Kaufman et al.9
confirmed that latanoprost treatment of the ocular surface
was associated with an increased risk of viral recurrences as
evidenced by the appearance of lesions in rabbits treated with
latanoprost. The results reported here are the first to show
that suppression of prostaglandin synthesis by a cyclooxy-
genase inhibitor inhibits the reappearance of infectious virus
on the ocular surface.
 Acetylsalicylic acid and herpesvirus 123

Table 5. Effect of intraperitoneal therapeutic acetylsalicylic acid (ASA) treatment on the

amount of viral DNA in the trigeminal ganglion.

Treatment Tissue Viral DNA (genome equivalents)

Heat stressed
+ASA Trigeminal ganglia 2.1 ¥ 104 ± 0.1*
-ASA Trigeminal ganglia 3.5 ¥ 106 ± 0.3
Not stressed
+ASA Trigeminal ganglia 1.5 ¥ 102 ± 0.09
-ASA Trigeminal ganglia 1.7 ¥ 102 ± 0.06

Ganglia from six mice were pooled for quantitative analysis of viral DNA.
* Significantly different from heat-stressed, saline-treated trigeminal ganglia (-ASA), p < 0.05.
Curr Eye Res Downloaded from informahealthcare.com by Aston University on 09/08/14

Table 6. Effect of oral acetylsalicylic acid (ASA) prophylaxis and treatment on viral DNA
concentration in the trigeminal ganglion.

Treatment Tissue Viral DNA (genome equivalents)

Heat stressed
+ASA Trigeminal ganglia 4.9 ¥ 104 ± 0.6*
-ASA Trigeminal ganglia 3.6 ¥ 106 ± 0.7
For personal use only.

Not stressed
+ASA Trigeminal ganglia 2.3 ¥ 103 ± 0.04
-ASA Trigeminal ganglia 3.1 ¥ 103 ± 0.07

Ganglia from six mice were pooled for comparative analysis of viral DNA.
* Significantly different from heat-stressed, saline-treated trigeminal ganglia (-ASA), p < 0.05.

Table 7. Effect of acetylsalicylic acid (ASA) on body temperature of hyperthermically

stressed mice.

Treatment groups Mean body temperature (°C)

Before Immediately after 5 Minutes after

hyperthermia hyperthermia hyperthermia

Latently infected, 37.7 ± 0.4 40.3 ± 0.3 36.8 ± 0.2

saline-treated
Latently infected, 37.6 ± 0.4 40.1 ± 0.2 37.1 ± 0.4
ASA-treated

Groups of 10 mice were prophylactically treated with oral ASA as described in “Materials and
methods.” Immediately before and after heat stress and 5 min after heat stress, the rectal
temperature of each animal was determined. There was no statistically significant difference
in the body temperature of the two groups of mice at the three time points measured.

In in vitro studies, O’Brien et al.10 reported that pro-
staglandin alpha 2 inhibited HSV-1 replication in human and
rabbit cells. However, these investigators also noted that
prostaglandin alpha 2 was not effective in vivo in rabbits and
that, in fact, acute viral infection of the cornea was more
severe in animals treated with the prostaglandin compared
with control placebo-treated animals.
Inglot and Woyton11 treated patients with the nonsteroidal
anti-inflammatory drugs mefenamic acid and indomethacin
and observed an inhibition of virally induced inflammation
in herpetic epithelial lesions and an inhibition of local spread
of lesions. Both of these drugs are thought to act through the
prostaglandin pathway. Overall, the authors noted shortening
of the duration of the disease process.
 124 B.M. Gebhardt et al.
We are investigating the effect of heat stress on the
prostaglandin levels and the increased expression of
cyclooxygenases in heat-stressed mice. In measurements of
body temperature following heat stress, we found that the
effect is transient and not like the febrile illness in patients
that may be followed by a viral lesion such as a cold sore.
Our preliminary studies indicate that there is an increase in
COX-2 gene expression and COX-2 protein in ganglia sub-
jected to heat stress in vitro (unpublished data). Studies in
mice are in progress.
Our observation that the treatment of mice with a non-
specific cyclooxygenase inhibitor reduces the frequency of
infectious virus at the ocular surface and reduces the amount
of viral DNA synthesis in the trigeminal ganglion of heat-
stressed mice is supported by the in vitro studies of Kurane
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et al.12 These investigators incubated the trigeminal ganglia
of mice in various concentrations of indomethacin, tetra-
caine, mepacrine, and mefenamic acid and noted that all of
these inhibitors of prostaglandin synthesis inhibited the reac-
tivation of virus in culture. Given these observations, it is not
surprising that prophylactic treatment with ASA suppresses
viral reactivation and viral DNA replication.
Other models of induced reactivation in mice that involve
the prostaglandin system have been studied. Blyth et al.1
For personal use only.
noted that exposure of the skin of mice to ultraviolet light
resulted in viral reactivation and speculated that this reacti-
vation might involve prostaglandins produced as a result of
inflammation subsequent to the irritation of the skin. Several
investigators have used ultraviolet light as a means of induc-
ing viral reactivation in mice,13–16 although the mechanism
involved has not been elucidated. It is not unreasonable to
speculate that ultraviolet irradiation of the ocular surface
induces a transient inflammatory response, elevated cyclo-
oxygenase expression, and subsequent prostaglandin pro-
duction leading to viral reactivation. On this point, a recent
report by Athar et al.17 showing that ultraviolet treatment of
mouse skin elicits elevated cyclooxygenase 2 (COX-2) pro-
duction in the skin provides direction for further study. We
are currently investigating changes in COX-2 gene expres-
sion in the corneas and trigeminal ganglia of hyperthermi-
cally stressed mice. In a preliminary investigation,18 we
found that hyperthermic stress resulted in a greater than
threefold increase in COX-2 gene expression in the mouse
trigeminal ganglion. In addition, studies are in progress to
investigate selective cyclooxygenase inhibitors to determine
if there is a difference between the COX-1 and COX-2
inhibitors in suppressing herpes viral reactivation in vivo.
In conclusion, our recent studies with latanoprost showing
that this drug increases both the severity and recurrence of
herpetic keratitis in rabbits9 and the current study establish-
ing that a nonspecific cyclooxygenase inhibitor can suppress
viral reactivation in mice provide the impetus for expanded
studies of other prostaglandin inhibitors, particularly those
that target cyclooxygenase intermediates. In the near future,
it may be possible to recommend prophylaxis to patients who
are prone to herpes viral reactivation of genital, orolabial,
and ocular disease to prevent recurrent disease without sig-
nificant drug toxicity. Furthermore, investigations into the
role of prostaglandins in herpes viral reactivation may
provide further clues about the pathways that lead from exter-
nal stimuli such as stress, trauma, chemical injury, and fever
to viral reactivation in the nervous system.
Acknowledgments
This work was supported in part by U.S. Public Health
Service grants R01EY002672 (H.E.K.) and P30EY002377
(departmental Core grant) from the National Eye Institute,
National Institutes of Health, Bethesda, MD, and an unre-
stricted departmental grant from Research to Prevent Blind-
ness, Inc., New York, NY.
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