Download Recurrence of Herpes Simplex Virus Vin Rabbit Eyes

Recurrence of herpes simplex virus
in rabbit eyes: Results of a
three-year study
Peter R. Laibson and Sidney Kibrick
Spontaneous reactivation of herpes simplex virus in rabbit ocular tissue was found on 112
occasions in 50 rabbits for up to 3 years after the primary eye infection. To detect these
spontaneous reappearances of virus, cultures for herpes simplex virus were obtained 5 or 6
clays a week from the cul-de-sac of rabbit eyes for various periods up to 3 years. Keratitis
was also intermittently observed but 72 per cent of the viral reactivations occurred without
coincidental corneal disease. Once the rabbit eye is infected with herpes simplex virus a
chronic inapparent infection ensues which probably persists for the life of the animal.
Key words: chronic ocular herpes simplex infection, herpes simplex virus,
virus reactivation, herpes simplex keratitis, epinephrine, ophthalmic ointment, time factors,
virus isolation, cornea, corneal ulcer, corneal vascularization, fluorescein stain.
D
In 1961 during attempts to induce reactivation of herpes simplex virus in eyes
of rabbits with healed herpetic corneal
lesions, several instances of spontaneous
virus release were observed.1 In these studies eyes were not examined for evidence
of accompanying herpetic disease.
Spontaneous shedding of virus from the
rabbit eye was subsequently also noted to
stimulate reactivations of herpetic keratitis
in this host. It was observed at that time
that spontaneous shedding of virus (that is,
without preceding stimulation) occurred
either with or without accompanying evidence of corneal disease.2'3
The present report provides information
as to the long-term natural history of herpes
simplex ocular infection in the rabbit. It is
based on periodic examinations of the rabbit eye for presence of virus and lesions
over a 3 year period after primary infection.
Results of attempts to induce reactivation
of virus during the last year of this period
isease caused by herpes simplex virus
in man is frequently recurrent in nature.
Although the rabbit is not a natural host
for this virus, it has been employed for
studies with herpes simplex ever since
Griiter demonstrated that this virus could
replicate in rabbit tissue.
From the Cornea Service, Wills Eye Hospital and
Research Institute of Temple University Medical
Center, Philadelphia, Pa., and the Evans Memorial Department of Clinical Research, University
Hospital, and the Departments of Microbiology
and Medicine, Boston University School of Medicine, Boston University Medical Center.
Supported by Public Health Service Grants NB
06355 from the National Institute of Neurological Diseases and Blindness and AI-04305 from
the National Institute of Allergy and Infectious
Diseases.
Presented in part at the meeting of the Association
for Research in Ophthalmology, Tampa, Fla.,
April 28, 1968.
Manuscript submitted Aug. 13, 1968; manuscript
accepted Sept. 4, 1968.
346
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are also described. These data indicate that
once herpetic infection of the rabbit eye
occurs, the virus persists as a chronic infection for up to 3 years (the length of time
the animals were studied).
Materials and methods
The initial herpetic corneal infection was produced by the Rodanus strain of herpes simplex
virus. A 0.1 ml. suspension of virus containing approximately 105-5 TCD50 per 0.1 ml. for human
amnion tissue culture was placed in the inferior
cul-de-sac of albino male and female rabbits and
the lids were gently massaged against the cornea
for 30 seconds. Typical dendritic figures were
noted in 48 to 96 hours, with many eyes proceeding to geographic corneal epithelial and stromal
involvement with iridocyclitis. Viral cultures of
each infected eye were obtained during the course
of the infection to verify the cause of the keratitis.
At least 4 weeks after the initial herpetic infection,
cultures were obtained on the surviving animals,
generally 5 or 6 days weekly. The mortality rate
from the primary infection was approximately 25
per cent and was usually due to herpetic encephalitis.
Cultures were taken from the eyes with a sterile
cotton-tipped applicator which was rotated in the
lower cul-de-sac, then across the cornea and into
the upper cul-de-sac, without prior topical anesthesia. This technique did not denude the epithelium and the rabbits soon accustomed themselves
to the daily routine. The conjunctival and corneal
epithelium was examined with fluorescein for punctate, dendritic, or geographic staining and occasional animals were viewed with the biomicroscope.
Swabs obtained from the eyes were either inoculated immediately into culture tubes or the specimens stored at -65° C , depending on the availability of human amnion or rabbit kidney tissue
culture tubes. Virus isolations were performed according to standard procedures which have been
described in a previous report.2 Identification of
isolates was confirmed by neutralization tests in
cultures of human amnion cells.
Fifty rabbits constituted the study group, 38
infected bilaterally and 12 in one eye. All rabbits
were individually caged and cross infection was
not noted during these studies. Bilateral viral cultures were performed intermittently on the 12 rabbits infected in only one eye and viral isolation
was never obtained from the uninfected eye. Rabbits were observed for periods ranging from 115
to 1,131 days after their initial infection, with
variable interspersed periods of rest. These rest
periods were employed to reduce the total number
of daily cultures and observations to a level which
could conveniently be handled.
Due to the intermittent shedding of virus from
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Recurrence of herpes simplex virus 347
the rabbit eye, the following criteria were established to characterize any single episodes of viral
reactivation. Positive cultures separated by 7 days
or more of negative cultures were considered as
representing distinct episodes of virus reactivation.
When cultures for virus were positive in both eyes
simultaneously (that is, separated by less than 7
days of negative cultures), this event was considered a single episode of virus reactivat.on for that
rabbit. Although corneal lesions demonstrable by
fluorescein staining were intermittently observed
in most rabbits they were not considered as evidence of virus reactivation in the absence of positive cultures.
Results
In 50 rabbits, followed in some instances
up to 3 years, there were 112 separate episodes of one or more days of spontaneous
virus release. Spontaneous reappearance of
virus occurred in all rabbits under study
at least once, and one rabbit had seven
separate episodes of virus release (Table
I).
Half of the spontaneous viral reappearances (57 of 112) were noted for only one
day, and at the other extreme, during fourteen episodes of reactivation virus was
Table I. Recurrence of herpes simplex
virus in rabbit eyes (112 episodes of
spontaneous reactivation in 50 rabbits)
Episodes
per rabbit
1
2
3
4
5
6
7
No. of
rabbits
22
12
6
6
1
2
1
No. of
episodes
22
24
18
24
5
12
7
Table II. Recurrence of herpes simplex
virus in rabbit eyes (112 episodes of
spontaneous reactivation in 50 rabbits)
Duration of episodes
(days)
1
2- 4
5- 7
8-11
>H
No. of episodes
57
24
17
7
7
348 Laibson and Kibrick
cultured for at least 8 days (Table II).
Many of the reactivations occurring for just
1 or 2 days would have been missed had
cultures not been obtained at least 5 or 6
days a week. As cultures were obtained
only once a day, we still may have missed
reactivation episodes occurring at other
times on that day; therefore, the figures for
virus isolation must be considered minimal.
The number of rabbits observed for 3
years was limited but from Table III it is
apparent that virus may be found in the
eyes of rabbits for as long as 3 years after
primary infection. Spontaneous reactivations were noted in 5 of 13 rabbits studied
between 601 and 700 days after initial infection, in 3 of 8 rabbits between 701 and
800, and in 1 of 4 from 901 to 1,000 days.
These "late" episodes of spontaneous reactivation varied in duration as did the
earlier ones. Thus of 8 such episodes between Days 601 and 800, five persisted for
one day or less and the remainder for 3, 8,
and 10 days respectively.
From 70 to 77 per cent of the rabbits
developed spontaneous reactivations during the first 200 days after primary infection. By 400 days this had dropped to 35
per cent, and it remained at that level over
the next year (Table III). After 2 years
only a small number of rabbits was available for observation, and in this group
spontaneous virus release and corneal disease were infrequently observed.
Since previous experiments in our laboratories had shown that epinephrine in ointment form could induce reactivation of
herpes simplex virus in the rabbit eye,4 this
technique was employed to demonstrate
persistence of virus in those rabbits which
had survived more than 701 days after
primary infection. Such treatment was associated with 7 additional episodes of virus
release from these animals. The total of
both induced and spontaneous reactivations
during various intervals from 701 to 1,131
days is given in Table IV.
Corneal staining with fluorescein was observed during 31 of the 112 episodes of
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In vestigative Ophthalmology
June 1969
Table III. Recurrence of herpes simplex
virus in rabbit eyes (spontaneous
reactivations after primary herpetic
infection)
No. of No. of rab- Per cent
with
Days after
rabbits bits with
positive
positive
primary herpetic under
nfection
culture
culture
culture
Day
40
31- 100
77
31
70
26
37
Day 101- 200
50
18
36
Day 201- 300
35
Day 301- 400
11
31
38
Day 401- 500
9
24
38
Day 501- 600
8
21
38
Day 601- 700
5
13
38
Day 701- 800
3
8
0
Day 801- 900
0
4
25
Day 901-1 000
1
4
0
Day 1.001-1 131
0
2
Table IV. Recurrence of herpes simplex
virus in rabbit eyes (induced and
spontaneous reactivations after primary
herpetic infection)
No. of rabbits
with
Days after primary No. of rabbits
herpetic infection under culture reactivations
Day 701- 800
Day 801- 900
Day 901-1,000
Day 1,001-1,131
spontaneous virus reactivation. This staining varied from minimal punctate dots with
no conjunctival injection to marked geographic comeal ulcers accompanied by
severe limbal injection and corneal vascularization. Dendritic figures were also observed in the transition of punctate changes
to obvious ulceration. The appearance of
punctate corneal staining in the absence of
positive virus cultures was often noted, but
it was unusual to find corneal ulceration
without recovery of herpes simplex virus.
Virus recovery was more common in the
absence of visible ocular disease, and even
on multiple consecutive days of virus isolation, demonstrable corneal disease was not
always present.
Twenty-three of the 50 rabbits developed
at least one corneal ulcer during a spon-
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taneous virus reactivation. The longest interval over which virus was recovered in
any single reactivation period was 21 days,
with a total of 9 positive cultures noted
during this time, although 11 positive cultures were recorded during a shorter spontaneous reactivation episode.
Episodes of spontaneous reactivation involving both eyes occurred 8 times. During
7 of these 8 episodes virus was isolated on
2 days or more from each eye. In these
rabbits corneal ulcers were more common
than in animals with unilateral involvement. Thus, 6 of the 8 developed such
ulcers, and of these, 4 also developed corneal vascularization during the course of
the reactivation.
Discussion
Although herpes simplex virus has been
recovered intermittently from the eye in
man and experimental animals, there have
been no long-term animal studies to determine the possibility of spontaneous reappearance of this virus years after the
initial herpetic infection. The rabbit, the
laboratory animal of choice for ocular experimentation with herpes simplex virus,
was long thought to recover from the initial
ocular infection without developing recurrent herpetic keratitis. Recent evidence,
however, indicates that herpetic keratitis in
this animal is not a self-limited disease,
and corneas which heal without scarring
(by slit lamp examination) are subject to
spontaneous reinfection with this agent.'10
Herpes simplex virus can persist for long
periods at other sites in the rabbit. This
agent was recovered from the saliva of 2
rabbits, 24 and 33 months after intraperitoneal inoculation.7 In addition, several investigators have reported reactivation of
herpetic encephalitis well after the primary
infection.8'9
The frequent and spontaneous reappearances of herpes simplex virus in the rabbit
eye following primary infection indicate
that this is the natural course of the experimental disease in this host. These recurrences of herpetic ocular infection in
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Recurrence of herpes simplex virus 349
the rabbit are similar to those naturally
occurring with this agent in man. The
physical characteristics of the corneal disease, the response to antimetabolite therapy, and viral persistence with or without
keratitis also appear similar in man and
rabbit.
This study documents 112 episodes of
spontaneous viral reactivation in 50 rabbits, over a period of almost 3 years. In
addition to 4 spontaneous reactivations
with onset between 701 and 1,131 days
after primary infection, 7 episodes of reactivation were induced during the same
period with topical epinephiine ointment
as the incitant. The longer the interval after
primary infection the less frequently were
spontaneous reactivations observed. The
fact that virus reactivation could be induced with epinephiine ointment following
long periods during which virus was not
demonstrable indicated that virus (or viral
precursor) remained in these hosts. These
findings suggest that once this agent is
placed on the rabbit eye it produces a
chronic inapparent infection which probably persists for life.
During the 112 periods of virus recovery,
coincidental pathological changes in the
cornea were noted 31 times (28 per cent).
Therefore, herpes simplex virus, isolated
from the eyes of these rabbits, did not
cause detectable disease 72 per cent of the
time. The mechanism whereby this virus
may persist in the eye in the absence of
ocular disease is unknown. A similar phenomenon has also been demonstrated for
herpes simplex virus in the upper respiratory tract10 and the human female genital
tract.11 The relative role of host susceptibility and such local factors as trauma,
anoxia, and fever in inducing reappearance
of virus with or without disease still remain to be determined.
The reisolation of virus from rabbit ocular tissue as long as 3 years after primary
infection indicates persistence of virus in
this host. Neither the state of the virus nor
the site where it persists during quiescent
periods, however, is presently known.
Investigative Ophthalmology
June 1969
350 Laibson and Kibrick
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