Download Reactivation of latent infection and induction of recurrent herpetic

Journal o f General Virology (1990), 71, 397-404.
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397
Reactivation of latent infection and induction of recurrent herpetic eye
disease in mice
C. Shimeld, 1. T. J. Hill, 2 W. A. Blyth 2 and D. L. Easty 1
Departments of 1Ophthalmology and 2Microbiology, The Medical School, University of Bristol, University Walk, Bristol
BS8 1TD, U.K.
During primary ocular infection of mice with herpes
simplex virus type 1 (HSV-1) strain McKrae, dendritic
corneal ulcers developed and many eyes became
permanently damaged. When primary infection had
subsided, latent infection was detected in the three
parts of the trigeminal ganglion and in the superior
cervical ganglion. Such latently infected mice were
treated with cyclophosphamide, dexamethasone and
u.v. irradiation, or cyclophosphamide and dexamethasone alone. After treatment with immunosuppressive
drugs and u.v. irradiation infectious virus was isolated
from the ophthalmic part of the trigeminal ganglion,
and in eyelids and eyewashings; recurrent herpetic eye
disease was seen but only in eyes undamaged by
primary infection. After treatment with cyclophosphamide and dexamethasone alone there was a lower
incidence of virus isolated from eyewashings and no
recurrent disease was seen. There was a good correlation between the pattern and distribution of recurrent
lesions and the distribution of cells stained due to the
presence of virus antigens.
Introduction
of virus in the tears. The incidence of such shedding was
further increased by the addition of timolol eye drops to
this arduous and time-consuming schedule of treatment
(Harwick et al., 1987). However none of these workers
commented on recurrent eye disease.
We now report on the development of a model of
reactivation, virus shedding and recurrent herpetic eye
disease in mice.
Detailed studies of animal models for recurrent eye
disease caused by herpes simplex virus (HSV) provide
valuable information on the pathogenesis of this condition and on the events surrounding the reactivation of
latent infection. In rabbits, after inoculation of the
cornea with HSV, latently infected animals show a high
rate of spontaneous shedding of virus in tears and on
some occasions this is associated with recurrent corneal
disease (Nesburn et al., 1967; Se Kwon et al., 1981 ; Hill
et al., 1987a; Haruta et al., 1987). Such frequent
shedding (which is probably preceded by reactivation of
latent virus in the sensory or sympathetic ganglia
supplying the eye) complicates attempts to induce
reactivation by specific stimuli as it is difficult to
dissociate experimentally induced events from those
occurring spontaneously. However in latently infected
mice the incidence of spontaneous shedding of virus in
eye secretions is very low (Tullo et al., 1982; Willey et al.,
1984).
Using iontophoresis of epinephrine into the cornea
Willey et al. (1984) were able to induce reactivation of
virus in the trigeminal ganglion (TG) and shedding of
virus from the eyes of latently infected mice. Gordon et
al. (1986) were unable to repeat this work but after
testing a variety of regimes, they found that iontophoresis of 6-hydroxydopamine, combined with epinephrine
and prednisolone phosphate eye drops, caused shedding
Methods
Mice. N/H/OLA inbred mice were obtained originally from Olac;
they were maintained as a breeding colony in the Department of
Microbiology. All were used at 8 weeks old; any with abnormal eyes
were rejected (Tullo et al., 1983).
Inoculation. Mice were anaesthetized by intraperitoneal injection of
sodium pentobarbitone and inoculated by scarification of the left
cornea with a 26-gauge needle (Tullo et al., 1983) through 5 gl drop of
medium containing 104 p.f.u. HSV type 1 (HSV-I) strain McKrae.
Control mice were inoculated in the same way with a preparation of
uninfected Vero cells made in a similar manner to the virus inoculum
(mock inoculum).
Examination of eyes and isolation o f virus from eyewashings. Mice were
anaesthetized and the cornea, iris and lids were examined for signs of
disease using a slit lamp microscope. Eyewashings were put onto Vero
cells for the isolation of virus (Tullo et al., 1983).
Detection o f latent infection. Mice were killed with an overdose of
sodium pentobarbitone and the following tissues were removed from
the left side: the three parts of the TG and the superior cervical
ganglion (SCG). The TG was divided in situ so that part 1 (TG1)
0000-9077 O 1990 SGM
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398
C. Shimeld and others
probably contained all the ophthalmic and some of the maxillary
neurons, part 2 (TG2) contained maxillary neurons and part 3 (TG3),
mandibular neurons (Gregg & Dixon, 1973; Arvidson, 1979; Tullo et
al., 1982). The SCG and the parts of TG were each placed in 0.5 ml of
medium and incubated at 37 °C in 5 % CO2 for 5 days (Tullo et al., 1982).
The tissues were then ground and 50 ~tl of the suspension was put onto
Veto cell monolayers. These were incubated at 37 °C for 2 days before
being fixed and stained so that plaques could be identified.
Reactivation of latent infection. At least 40 days after corneal
inoculation mice were given 5 mg of cyclophosphamide (Koch-Light
Laboratories) intravenously in 0.2 ml of phosphate-buffered saline
(PBS). One day later, 0.2 mg of dexamethasone in 0-2 ml (Dexadresson
diluted in PBS, Intervet Laboratories) was injected via the same route.
In all experiments except one, mice were anaesthetized immediately
after this injection and held for 90 s with the left eye proptosed, below a
Hanovia lamp so that the cornea and lids could be irradiated. The lamp
emitted a peak of 4.03 mJ/cm 2 . s at 320 nm. This irradiation produced
mild erythema in the skin of the pinna of these mice (T. J. Hill & W. A.
Blyth, unpublished results).
Detection of virus reactivated in vivo.
(i) Isolation of infectious virusfrom tissues. The three parts of the left
TG and the left SCG were each ground in 0.5 ml of medium, then
frozen and thawed three times to disrupt all the cells. The resulting cellfree suspensions were put onto a monolayer of Vero cells in 25 cm 2
flasks and incubated at 37 °C in 5 ~ CO2. Cultures were examined daily
for c.p.e, for 5 days. The upper and lower left lids from each mouse were
ground together in 0.5 ml of medium, frozen and thawed three times
and the resulting cell-free suspensions were put onto a monolayer of
Vero cells as above. These cultures were examined for c.p.e, for up to 21
days.
(ii) Detection of virus antigens by peroxidase-antiperoxidase (PAP)
staining. Mice were killed with an overdose of sodium pentobarbitone,
followed by the removal of the left eye and the lids (or part of the lids).
Flat mounts of the corneal epithelium and of the remainder of the eye
(the corneal stroma and endothelium, the uvea and sclera, referred to as
'globe') were prepared and stained by the PAP method for the detection
of HSV-1 antigens, as described by Dyson et al. (1987). Upper and
lower lids were treated separately or selected diseased areas of lids were
fixed in Bouin's fluid for 30 rain to 1 h and then immersed in 70%
ethanol for at least 24 h. They were then embedded in paraffin wax and
serial 5 ~tm sections were cut for PAP staining.
Results
Primary disease and incidence o f latent infection
N i n e t e e n f e m a l e m i c e a n d 20 m a l e m i c e were i n o c u l a t e d
with virus on the cornea. E y e w a s h i n g s were t a k e n for the
isolation o f virus on days 3 to 7 after i n o c u l a t i o n a n d m i c e
were e x a m i n e d for signs o f eye disease on d a y s 3, 5, 7 a n d
10. S e v e n t e e n female m i c e a n d 12 m a l e s survived. O n
d a y 25 the eyes o f female m i c e were e x a m i n e d , the m i c e
were killed a n d tissues were t a k e n to d e t e r m i n e the
i n c i d e n c e o f l a t e n t infection in the three p a r t s o f the T G
a n d in the S C G ; on d a y 43 after i n o c u l a t i o n m a l e m i c e
were t r e a t e d similarly.
Eye disease followed a s i m i l a r p a t t e r n in b o t h sexes.
T h r e e d a y s after i n o c u l a t i o n 50 % o f c o r n e a s h a d m u l t i p l e
d e n d r i t i c ulcers, a c c o m p a n i e d in some m i c e by localized
c o r n e a l haze. L i d disease (swelling, ulcers a n d scabs) was
seen in the m a j o r i t y o f m i c e on d a y 7. By d a y 10, 50% o f
surviving mice had opaque corneas with peripheral
v a s c u l a r i z a t i o n a n d swollen lids d e v o i d o f lashes.
Virus was isolated f r o m e y e w a s h i n g s o f n e a r l y all m i c e
on d a y s 3 to 6; on d a y 7 the i n c i d e n c e o f isolation was
80% in m a l e s a n d 65% in females.
L a t e n t infection was d e t e c t e d in T G 1 f r o m 82% o f
animals. It was also d e t e c t e d , in T G 2 (58 % in males, 71
in females), in T G 3 (33% in males, 4 1 % in females) a n d
in S C G (50% in males, 35% in females). I n two m a l e a n d
t h r e e female m i c e l a t e n t i n f e c t i o n was n o t d e t e c t e d in
a n y o f the tissues tested.
Reactivation o f latent infection
F i f t y - s e v e n d a y s after virus i n o c u l a t i o n on the cornea, 47
m a l e m i c e (the s u r v i v o r s f r o m a group o f 75) were t r e a t e d
w i t h c y c l o p h o s p h a m i d e , d e x a m e t h a s o n e a n d u.v. i r r a d i a t i o n ; 31 f e m a l e m i c e (the survivors f r o m a g r o u p o f 75)
were t r e a t e d s i m i l a r l y on d a y 54 after inoculation. T h e d a y
o f t r e a t m e n t w i t h d e x a m e t h a s o n e was defined as d a y 0.
Eyes were e x a m i n e d i m m e d i a t e l y before the m i c e were
t r e a t e d w i t h c y c l o p h o s p h a m i d e a n d e y e w a s h i n g s were
t a k e n for virus isolation at this t i m e a n d d a i l y for 5 days.
T h e eyes o f g r o u p s o f 10 to 14 m i c e were e x a m i n e d for signs
o f r e c u r r e n t disease on d a y s 2, 3 a n d 4. T h e y were t h e n
killed a n d t h e i r tissues r e m o v e d for the isolation o f virus or
for P A P staining to d e t e c t HSV-1 antigens.
Infectious virus was first isolated f r o m one o f 14 T G 1
s a m p l e s f r o m m a l e m i c e on d a y 2 a n d four o f 14 on d a y 3;
it could not be isolated f r o m this tissue in f e m a l e mice.
O n l y a small a m o u t o f virus w a s isolated, a n a v e r a g e o f
3 p.f.u, a n d a m a x i m u m o f 6 p.f.u, p e r sample. I n f e c t i o u s
virus was isolated f r o m a small p r o p o r t i o n o f lids f r o m
m a l e a n d female m i c e at all t i m e s t e s t e d ; the m a x i m u m
was from five o f 10 f e m a l e m i c e on d a y 4. W h e r e
infectious virus was isolated c.p.e, a p p e a r e d w i t h i n 5
d a y s o f culture, e x c e p t on two o c c a s i o n s w h e n c.p.e, was
first seen after 16 days. I n f e c t i o u s virus was not isolated
f r o m S C G at a n y t i m e ( T a b l e 1 a).
R e c u r r e n t c o r n e a l disease ( d e n d r i t i c u l c e r a t i o n w i t h or
w i t h o u t a s s o c i a t e d c o r n e a l haze) was seen in one m a l e
m o u s e on d a y 3 a n d one f e m a l e m o u s e on d a y 4. Virus
a n t i g e n was seen in cells b o r d e r i n g b r a n c h e d ulcers in the
c o r n e a l e p i t h e l i a l sheets f r o m these m i c e (Fig. 1). It was
also seen in this tissue on d a y 3 in small groups o f cells
a l t h o u g h these two c o r n e a s were n o t ulcerated. Virus
a n t i g e n was n e v e r seen in globes ( T a b l e 1 a). I n a d d i t i o n
to those m i c e w i t h r e c u r r e n t c o r n e a l disease, six m i c e h a d
r e c u r r e n t lid disease (ulcers or scabs). Such disease was
seen in two m a l e m i c e on d a y 2 a n d four f e m a l e mice, one
on d a y 2, one on d a y 3 a n d two on d a y 4 ( T a b l e l b).
I n f e c t i o u s virus was isolated f r o m lids o f all eight m i c e
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Recurrent herpetic eye disease in mice
399
Eye disease, isolation of virus and staining of virus antigens following treatment of latently infected mice*
with cyclophosphamide, dexamethasone and ~.v.~ irradiation
T a b l e 1.
(a) Incidence
HSV-1 antigen
stained by PAP
Isolation of infectious virus
Day after
treatment:~
TS 1§
- 1
0
1
ND
ND
ND
Male mice
Female mice
-
II
SCG
Lids
ND
ND
ND
ND
ND
ND
0/47 ¶
0/42
0/41 * *
2
3
4
1/14 (7)
4/14 (29)
0/14
0/14
0/14
0/14
3/14 (21)
1/14 (7)
1/14 (7)
I
ND
ND
ND
ND
ND
ND
ND
0/10
O/ll
0/10
ND
3/10 (30)
1/ll (9)
5/10 (50)
0
ND
0/10
O/ll
0/10
1
2
3
4
Corneal
epithelium
Eyewashings
Globe
ND
ND
ND
2/41 (5)
2/27 (7)
0/14
ND
ND
ND
0/14
2/14 (14)
0/14
0/31
0/31
0/14
0/14
0/14
ND
ND
1/31 (3)
1/31 (3)
4/21 (20)
3/10 (30)
biD
ND
ND
0/10
1/ll (9)
1/10 (10)
ND
0/10
O/ll
0/10
(b) Details of mice with evidence of recurrent ocular infection
Male mice
Female mice
Mouse
number
State of cornea'~t
immediately
before
treatment
Virus from eyewashings
on day
Day
killed
-0
I
V
2
-
2
3
4
5
6
V
V
N
N
N
2
2
3
3
4
-
1
V
2
-
V
V
V
N
N
N
N
V
V
2
2
3
3
4
4
4
4
4
.
.
2
3
4
5
6
7
8
9
10
0
1
2
3
4
Infectious
virus
from lids
-
11111
> 100
-
> 100
> 100
-
> 100
-
11
.
.
.
.
-
Cornea~:
<50
-
-
+
> 100
<50
> 100
1
+
+
-
+
-
+
-
>
100
-
-
+
3
2
100
100
100
100
3
100
+
+
-
-
+
+
+
>
1
41
> 100
84
4
76
9
>
>
>
.
.
Recurrent
disease
Antigen in
corneal
epithelium
>
Lids§§
-
+
-
* Inoculated 57 days (male) or 54 days (female) previously with 1 x 10~ p.f.u, on left cornea.
t Treatment: 5 mg of cyclophosphamide intravenously (i.v.) followed 24 h later by 0-2 mg of dexamethasone i.v. and 90 s of u.v. irradiation to the
left eye.
Dexamethasone was given on day 0.
§ TG1, ophthalmic part of trigeminal ganglion.
IIND, Not determined.
¶ Number with virus or antigen/number tested. Percentage value is given in parentheses.
** Six mice died from anaesthetic.
"~i"V, Vascularization of the cornea; N, normal. All lids were normal.
:~:~ Dendritic ulceration with or without associated corneal haze or infiltrate.
§§ Ulcers or scabs.
IIII Number of p.f.u.
with recurrent disease and from the lids of a further six
from
mice without such disease. Virus was also isolated from
noteworthy
the eyewashings
of three
of the mice
lesions and three other mice. However,
with
recurrent
virus antigens
were seen in corneal epithelia and/or virus was isolated
the
the
lids
these
latter
three
animals.
It
is
in
corneal
e p i t h e l i a l s h e e t s w e r e all n o r m a l w h e n e x a m i n e d
on day
-1
corneas
of
that the lids of mice which yielded virus and
(Table
of mice
1 b).
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with
virus
antigens
400
C. Shimeld and others
Fig. 1. (a) Corneal epithelial sheet on day 3; (b) and (c) sections of lids on day 5 after treatment with cyclophosphamide, dexamethasone
and u.v. irradiation. Mice were inoculated on the cornea 57 days (a) or 92 days (b and c) previously with 1 x 10~ p.f.u. (a) Virus antigen
(dark staining) in cells around a branched ulcer, around two small punctate ulcers and in a plaque; bar represents 500 ~tm. (b) Cells
containing antigen in conjunctival epithelium, bar represents 100 ~tm. (c) Cells containing antigen in glandular tissue; bar represents
100 p.m.
I n a f u r t h e r e x p e r i m e n t , 104 m a l e m i c e t h a t h a d
s u r v i v e d p r i m a r y i n o c u l a t i o n were e x a m i n e d . T w e n t y four o f t h e m were f r o m a g r o u p o f 75 i n o c u l a t e d 40 d a y s
previously, 80 f r o m a g r o u p o f 124 i n o c u l a t e d 92 d a y s
previously. T h e 52 w i t h n o r m a l c o r n e a s a n d lids were
selected; 29 were t r e a t e d w i t h c y c l o p h o s p h a m i d e , d e x a m e t h a s o n e a n d u.v. i r r a d i a t i o n , a n d 23 w i t h cyclophosp h a m i d e a n d d e x a m e t h a s o n e only. E y e w a s h i n g s were
t a k e n before t r e a t m e n t a n d d a i l y for 7 days. F r o m d a y 3,
m i c e were e x a m i n e d e a c h d a y for signs o f r e c u r r e n t eye
disease. I f virus was isolated f r o m e y e w a s h i n g s a n d / o r
r e c u r r e n t disease was seen, the mouse was killed, a n d the
c o r n e a l e p i t h e l i u m , globe a n d selected lid tissue were
t a k e n for P A P staining.
I n the g r o u p o f m i c e t r e a t e d w i t h only c y c l o p h o s p h a m i d e a n d d e x a m e t h a s o n e , n o n e s h o w e d signs o f recur-
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Recurrent herpetic eye disease & mice
401
Table 2. Recurrent eye disease, isolation of virus and staining of virus antigens in male mice*
HSV-I antigen stained by PAP
Virus from eyewashings on day
Mouse
number
- 1
1
2
3
4
5
6
.
.
.
7
8
9
10
11
0
-
2
.
.
.
.
-
.
.
-
.
.
69
52
>
4
5
> 1005
-
- K§
- K
> 100
> 100
> 100
3
3
53 K
> 100 K
54
-
46
-
-
.
> 100
> 100
.
.
-
.
3
-
.
-
-
.
2
1
100
19
> 100
80
> 100
-K
6
Corneal
epithelium
> 100 K
D ¶
> 100
12
-
Globe
Upper
Lower
Cornea
Lids
-
NDII
+
+
ND
ND
+
+
+
ND
3
4
5
-
4
4
4
4
5
-
N D
N D
-
-
ND
.
ND
ND
--
--
5
-
-
-
+
+
+
-
-
ND
-K
19
> 100 K
-
Day recurrent
disease first seen
Lids~"
-
23
1K
K
+
.
-
N D
,
-
.
-
.
ND
* Inoculated on the cornea at least 40 days previously with 1 x 104 p.f.u. All had normal eyes immediately before treatment. Mice were treated as
previously except numbers 10 and 11 which received no u.v. irradiation.
t From mice 1 and 3, the suspected recurrent lid lesions were taken for staining and from mice 4, 5 and 9, whole lids (upper and lower) were taken.
:~Number of p.f.u.
§ K, Killed.
IIND, Not determined.
¶ D, Died from anaesthetic.
rent disease a n d only two of 23 shed virus in the tear film
(Table 2). O n e mouse had virus i n eyewashings o n day
- 1 (before s t i m u l a t i o n ) a n d for the next 5 consecutive
days; virus was present in high titres. Tissues from these
mice showed n o evidence of the H S V a n t i g e n w h e n
e x a m i n e d by P A P s t a i n i n g (Table 2).
I n the group treated with c y c l o p h o s p h a m i d e , dexam e t h a s o n e a n d u.v. i r r a d i a t i o n , five mice had r e c u r r e n t
lid lesions, three of these also had corneal lesions a n d
a n o t h e r mouse had only corneal lesions; thus six of 27
mice (two mice died from anaesthetic, one o n day 1, one
o n day 2) showed some sign of r e c u r r e n t disease. T h e r e
was good correlation b e t w e e n clinical disease a n d the
presence of virus a n t i g e n in tissue a n d all b u t one mouse
with lid disease had virus a n t i g e n s i n its diseased tissue.
Moreover the p a t t e r n of s t a i n e d cells in epithelial sheets
corresponded to the p a t t e r n of u l c e r a t i o n a n d o e d e m a in
corneas.
All mice with r e c u r r e n t disease shed virus into the tear
film. I n mice with corneal lesions virus could be isolated
from eyewashings for at least 3 consecutive days. T h e
titre of virus in these samples was far higher t h a n those
from mice that shed virus o n one day only.
I n mice 1 a n d 3 (Table 2) only diseased areas of eyelids
were investigated. Virus a n t i g e n was p r e s e n t i n two
separate foci in eyelid e p i d e r m i s from mouse 3 b u t it was
n o t seen in mouse 1. Cells stained for virus a n t i g e n were
seen in three separate foci in the lower lid from m o u s e 4;
two were in c o n j u n c t i v a l e p i t h e l i u m a n d the other was at
the j u n c t i o n of this tissue with eyelid epidermis. Both lids
from mouse 5 showed staining. I n the lower lid it was
p r e s e n t i n a single focus in the c o n j u n c t i v a l e p i t h e l i u m ;
in the other lid it was more extensive with foci i n the
e p i d e r m i s o f the eyelid, i n hair follicles a n d in g l a n d u l a r
tissue b e n e a t h the c o n j u n c t i v a (Fig. 1).
Drug treatment and u.v. irradiation of uninfected mice
T e n mice were i n o c u l a t e d o n the cornea with the m o c k
i n o c u l u m a n d t h e n treated 77 days later with cyclophosp h a m i d e , d e x a m e t h a s o n e a n d u.v. irradiation. Eyewashings were t a k e n b u t discarded a n d eyes were e x a m i n e d
i m m e d i a t e l y before injection of c y c l o p h o s p h a m i d e a n d
t h e n daily for 5 days. O n days - 1 a n d 0 all eyes were
n o r m a l . O n day 1, four mice showed haze, suggesting
oedema, in the central c o r n e a ; five had corneal epithelial
deficits. These were ovoid, e x t e n d e d from the m e d i a l to
lateral c a n t h u s a n d were u n d e r l a i d by haze. N i n e mice
h a d such deficits by day 2 a n d i n a d d i t i o n two h a d iris
h y p e r a e m i a . O n day 3, five corneas were n o r m a l ; the
others had very small patches of slight haze in the centre
a n d all corneas were n o r m a l by day 4. Lid disease was n o t
seen at any time.
Over the period of o b s e r v a t i o n n o signs of systemic
toxicity were seen. However, i n other e x p e r i m e n t s seven
of 16 ( 4 4 ~ ) died w i t h i n 22 days of similar t r e a t m e n t .
D i s c u s s i o n
As reported by others (Willey et al., 1984) d e n d r i t i c
lesions were seen d u r i n g p r i m a r y infection following
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402
C. Shimeld and others
inoculation of the cornea with HSV-1 strain McKrae. In
contrast this type of ulcer was rarely seen after a similar
inoculation with strain SC16 (Tullo et al., 1983).
However, with both strains the incidence and timing of
lid disease, and the proportions of surviving mice that
had severely and irreversibly damaged corneas, were
similar (Tullo et al., 1983). In addition the distribution of
latent infection was similar to that found when SC 16 was
inoculated at this site (Tullo et al., 1982). The incidence
of spontaneous shedding was low; in the present work
only one of 130 latently infected mice was found to have
virus in eyewashings before treatment to induce reactivation. This confirms previous reports for the McKrae
strain (Willey et al., 1984) and SC16 (Tullo et al., 1982).
However a disadvantage of the McKrae strain is
its greater neurovirulence when compared with
strain SC16.
The most commonly recognized, diagnosed and
reported sign of recurrent herpetic eye disease in humans
is the corneal dendritic ulcer (Duke Elder, 1965). After
treatment with immunosuppressant drugs and u.v.
irradiation this characteristic lesion was seen on the
corneas of latently infected mice. Confirmation that
these lesions were caused by virus was provided by the
close similarity between the pattern of the lesion and that
of cells containing the virus antigen. Recurrent lid
disease, also confirmed by the presence of virus antigen,
was seen more frequently than recurrent corneal disease.
In humans, recurrent lid disease due to HSV (Jacobiec et
al., 1979; Egerer & Stry, 1980) appears to occur less
frequently than corneal disease although the difference
may be artificial because corneal disease is more likely to
be recognized and reported than localized lid disease.
Recurrent herpetic conjunctival disease has also been
reported in man (Brown et al., 1968; Nauheim, 1969;
Colin et al., 1980). In this present study no such disease
was seen, but only the bulbar conjunctiva of mice can be
examined clearly. However subclinical disease was
present in this tissue as virus antigens were seen in
epithelial cells at this site.
Isolation of virus from eyewashings provides further
evidence of infection although this does not help in
identifying the precise site of infected cells. When
corneal disease was present (usually with lid disease)
large amounts of virus were isolated for at least 2
consecutive days. However when clinical disease was not
seen virus was isolated only sporadically and in small
amounts. Four mice shed only small amounts of virus in
tears on one occasion only; in three of these no recurrent
disease was seen. Shedding of virus in the absence of
clinical disease has been reported also in humans and
rabbits (Kaufman et al., 1967; Haruta et al., 1987). By
chance the likely source of such virus was identified in
one mouse when virus antigens were demonstrated in
corneal epithelium.
This study and others (Metcalf & Michaelis, 1984;
Stulting et al., 1985) show that primary infection with
HSV in the mouse cornea is often followed by severe and
irreversible eye damage. It is noteworthy that recurrent
eye disease and/or shedding of virus was seen only in
those mice with undamaged eyes. In mice with damaged
eyes recognition of recurrent lesions might be difficult
but the absence of such lesions did not appear to be due to
a failure of reactivation in the ganglia since, on some
occasions, virus was isolated from TG1 of such mice
following treatment with cyclophosphamide, dexamethasone and u.v. irradiation. In eyes damaged from the
primary disease the sub-epithelial plexus of the cornea is
severely depleted whereas in undamaged corneas the
plexus appears normal (C. Shimeld, unpublished results).
Similar observations have been reported in rabbits after
inoculation of the cornea with HSV-1 (Asbell &
Beuerman, 1985). Since an intact nerve supply to the
cornea or to the skin is necessary for induced shedding of
virus in tears (Rootman et al., 1988) or the production of
recurrent herpetic skin lesions (Hill et al., 1983)
respectively, the absence of the nerve plexus in damaged
eyes may provide a further explanation for the absence of
recurrent disease in such eyes. Other factors may
influence the production of recurrent eye disease. For
example in rabbits, the strain of virus is known to affect
its incidence (Hill et al., 1987b) and the strain of mouse
was shown to play a role in influencing the incidence of
recurrent skin disease in mice (Harbour et al., 1981).
Following treatment to induce reactivation, only small
amounts of virus were isolated from TG 1. This observation, the times at which virus was isolated and the number of animals yielding virus were similar to previous
results involving different reactivation stimuli in latently
infected mice (Openshaw et al., 1979; Harbour et al., 1983).
A nerve supply to the cornea is required to produce
shedding of virus in the tear film (Rootman et al., 1988).
Therefore the reactivated virus in TG 1 was the most likely
source of virus to produce recurrent ocular disease and
shedding in the tears. However in humans (Shimeld et al.,
1981 ; Tullo et al., 1985 ; Cooket al., 1986), in mice (Abghari
& Stulting, 1988) and in rabbits (O'Brien & Taylor, 1989)
there is evidence that the cornea may be persistently or
latently infected with virus. Therefore reactivation may
also occur in this tissue.
The small amounts of reactivated virus isolated from
TG1 may account for the absence of virus antigens
within ocular nerves. During primary infection, when
much larger amounts of virus are present in ganglia
(Blyth et al., 1984) such antigens are frequently seen in
Schwann cells of ocular nerves on days 2 to 6 after
corneal inoculation of mice with HSV-1 strain SC16
(Dyson et al., 1987).
Ultraviolet irradiation appeared to be the major factor
in inducing recurrent lesions since treatment with
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Recurrent herpetic eye disease in mice
cyclophosphamide and dexamethasone alone produced a
very low incidence of shedding of virus in tears, and no
recurrent disease. In contrast treatment with these
immunosuppressive drugs plus u.v. irradiation produced
a much higher incidence of both of these signs of
recurrent infection, u.v. irradiation induces recurrent
skin lesions in man (Wheeler, 1975; Spruance, 1985),
reactivation of latent virus in mice (Blyth et al., 1976)
and, in combination with systemic and topical cortiscosteroids, recurrent corneal lesions in rabbits (Spurney &
Rosenthal, 1972). The dose of irradiation used in the
present study produced temporary and slight damage to
the corneal epithelium and no detectable damage to the
lids and bulbar conjunctiva. U.v. irradiation is known to
produce many changes in the skin including damage to
epithelial cells and their replication (Epstein et al., 1971),
the release of prostaglandins (Harbour et al., 1983) and
the reduction of ATPase staining on Langerhans' cells
(Bergstresser et al., 1980). Its effect on the mouse
cornea has not been well documented but in the rabbit
cornea, swelling (Riley et al., 1987), epithelial loss and
killing of keratocytes (Ringvold & Davanger, 1985) have
been recorded. It is not clear how such observations
relate to reactivation of latent HSV. However, reactivation may arise from the effects of u.v. irradiation on
corneal nerves because in humans, exposure to a low dose
of u.v. irradiation produced a loss of corneal sensitivity
lasting for 4 h (Millodot & Earlam, 1984). In addition,
u.v. irradiation of the corneal epithelium may result in
the release of mediators of inflammation that could also
affect the neuronal cell body.
In latently infected rabbits the combination of
cyclophosphamide and dexamethasone used here was
successful in inducing the shedding of virus in tears
(Stroop & Schaefer, 1986; Rootman et al., 1988) and
recurrent ocular disease (Stroop & Schaefer, 1987). The
disease varied from very severe conjunctivitis and
keratitis to mild conjunctivitis; some rabbits died from
the toxic effects of the drugs. Despite the potent actions
of these drugs on the immune system and the higher dose
of cyclophosphamide than that used previously in
rabbits, drug treatment without u.v. irradiation produced little shedding of virus and no recurrent disease in
mice. However once reactivation has occurred, either
spontaneously or induced by u.v. irradiation, these
immunosuppressive drugs might increase the duration of
shedding of virus from the eye. Evidence for such
activity was provided by one mouse which shed virus
spontaneously before treatment. Previously in spontaneous shedding we found virus only in small amounts on 1
or 2 days (Tullo et al., 1982). In the present study one
mouse was found to be shedding virus spontaneously
before treatment with the drugs. After treatment, the
virus was shed for 6 consecutive days and for 2 of these
days, large amounts were isolated. Even so, and despite
403
the generalized immunosuppression, no disease was
seen. This supports the concept that conditions in the
peripheral ocular tissue have an important role to play in
the development of recurrent disease (Hill & Blyth,
1976).
Besides closely mimicking the clinical features of
recurrent herpetic ocular disease in humans this model
has the following advantages. Firstly, the method of
inducing such disease is far less demanding than
iontophoresis and results in less damage to the cornea.
Gordon et al. (1986) commented on the damage caused
to the mouse cornea after 3 consecutive days of
iontophoresis; indeed such damage may decrease the
chance of inducing or recognizing recurrent disease.
Secondly the staining of HSV-1 antigens in corneal
epithelial sheets allows the specific identification of
virus-infected cells. This can confirm clinical observations and identify subclinical foci of infection. Moreover
lesions caused by the virus can be distinguished from
damage caused by the reactivation stimulus, a distinction which might be difficult to make on clinical
examination alone. The excellent correlation between
recurrent corneal disease, the isolation of virus from
eyewashings and the presence of virus antigens in
corneal epithelial sheets provides complete definition of
recurrent disease. In other systems such definition has
not been achieved. For example in latently infected
rabbits, after iontophoresis, corneal lesions have been
seen in the absence of virus in tears (Haruta et al., 1987;
Hill et al., 1987a). Lastly, compared with models of
recurrent herpetic eye disease in the rabbit (Hilt et al.,
1987a; Haruta et al., 1987) use of mice has the
advantages of economy (thereby allowing larger experimental groups) and the availability of well defined
inbred strains for immunological studies.
This work was funded by the Medical Research Council.
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