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animals that have recovered as fully as possible from the primary mfectron
(usually after a period of at least 4 wk following inoculation), the expertmental
induction of reactivation involving either a systemic or local sttmulus, leading
to vnus shedding with or without clinical disease in the skin or eye. In the first
stage, animals usually are infected at a particular site in the skin or eye with
doses and strains of vn-us that produce as many latently infected survivors as
possible and under circumstances that leave as little residual damage as pos-
sible at the maculation site. It is particularly important to maintain the integrity
From Methods MI Molecular Medrcme, Vol 10 Herpes 8mplex Wrus Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ


273
Hill and Shimeld
274
of the pertpheral nerves, as these transport reactivated vuus from the ganglion
to the periphery and may be involved in the transmission of signals to the neu-
ronal nucleus during reactivation (2). Moreover, recognition of a recurrent
lesion is not possible on the background of a badly damaged tissue. Cornea1
nerve damage with a resultant loss of cornea1 sensitivity that accompames
chronic severe stromal keratitis is the most common outcome of primary mfec-
non of the eye m the mouse (3). A secondary bacterial infection may contrib-
ute to this severity, but even when the infection is prevented by antibiotic
treatment the keratitis remains severe (3). We were the first group to report
recurrent cornea1 disease and shedding of vnus m the tears of mice, but such
signs were only seen in the small proportion of mice that survived primary
mfection with undamaged eyes (4,).To increase the incidence of mice that have
the potential to develop recurrent cornea1 disease now we routmely use passive
immunization prior to cornea1 maculation of virus (5,6). Such treatment pro-
tects from stromal keratitis and death while allowing a high incidence of latent
mfectton to be established in the ophthalmic part of the trigemmal ganglion.
In addition to these models of true recurrent mfection, the zosteriform spread
model is also described since it has been argued that, even though this mvolves
only the primary infection, it provides a useful analog of recurrent disease (7,8).
In this zosteriform model, virus is inoculated in the skm of the neck where
primary infection occurs. From this site virus spreads, probably by retrograde
axonal flow to the cervical sensory ganglia (mainly the second to fourth) and
the central nervous system (CNS). Further spread, probably at various points
within the nervous system, allows virus to reach axons that do not supply the
inoculation site itself but that supply skin within the same dermatome as this
site. Via these axons, the vnus then spreads, probably via orthograde flow, at
approx d 3 after the primary inoculation, to these more “distant” skin sites; m
this case mcludmg the ear pinna on the inoculated side. This m turn produces
clinical herpetic lesions on the pmna (erythema as early as d 4 or 5 and lesions
on d 5 or 6). Hence, like true recurrent lesions following reactivation of latent
infection (described later), zosteriform lesions on the pinna are relatively short-
lived, virus is delivered to the skin via nerves and smce the zosteriform lesions
do not develop until d 5 or 6, like recurrent lesions they develop in the presence
of some degree of immunity. The phenomenon of zosteriform spread is not
restricted to skin; virus can be delivered to the eye via nerves following inocu-
lation of the snout (9). Zosteriform disease also has the advantage of develop-
ing within a well defined time with respect to the primary maculation and after
inoculation in the neck can occur m 100% of animals. The analogy between
zosteriform and recurrent disease, however, is not perfect since the dose of
virus delivered to the skm m zosteriform spread almost certainly is far greater
than that m a true recurrence. Moreover, the nature of the immunity present m
275
Models of HSV in the Mouse
the two sltuatlons will be different since with zosterlform disease the immune
responses to the virus will be in the early stages of development, whereas m
recurrent disease the animal may have had immunity for many months.
In both the ear and the zosterlform model, the pmna offers a number of
advantages for the observation of herpetic disease. These include the follow-
mg: the provlslon of a flat, relatively hairless area that 1seasy to inoculate and,
m albino animals, on which lesions are easily visible (m albinos, even the mild-
est inflammatory response is visible as erythema). Other indicators of disease
are provided by measurement of ear thickness, isolation of virus or hlstologlcal
observations from small punch biopsies, development of ear paralysis result-
ing from demyelinatlon in the seventh cranial nerve root (10, II). Moreover,
the flat relatively halrless pmna allows easy application of drugs.
It is commonly stated that mice do not show spontaneous reactlvatlon of
latent infection, as evidenced by the development of spontaneous recurrent
disease or the presence of vu-us at the periphery m the absence of climcal dis-
ease. In fact, spontaneous shedding of virus in the tear film does occur m mice
(4,22--Z+˜), but at a much lower mcldence (3-0.8%) than in the rabbit In addi-
tion, virus has been isolated from clinically normal mouse skin (15) and, more
rarely, spontaneous recurrent skin lesions are observed (16). It has been argued
that this apparently low incidence of spontaneous reactivation in the mouse 1sa
disadvantage of this animal for experimental studies with HSV. However, the
absence of frequent spontaneous disease gives the advantage of a clean “back-
ground” for experimentally induced reactivation. Moreover, as already men-
tloned, the immunological advantages of the mouse, provide strong arguments
for the continued use of this animal.
Three models will be described: for brevity these will be referred to as the
ear model, eye model (both involving latent and recurrent infection), and the
zosteriform model (the analog of cutaneous recurrent infection but involving
primary disease only).

2. Materials
1. Strain and dose of virus: in the eye model a dose of lo4 PFU of HSV-1 strain
McKrae; in the ear model doses of 105-IO6 PFU of HSV- 1 strain SC I6 have been
used routinely (see Note 1).
2. Mice: 8- and 4-wk-old female NIH mice (inbred strain, H2 q) for the eye and ear
models, respectively (see Note 2) Four- or eight-week ammals are suitable for
the zosteriform model This strain is available from Harlan UK Ltd (Blcester,
Oxfordshire). All animals should be Specific Pathogen Free (see Note 3)
3. AnesthetIcs:
a. 60 mg/mL Sodium pentobarbltone (Sagatal, Rhone Meneux, Harlow, Essex, UK);
b. 10mg/2 mL Mtdazolamhydrochloride (Hypnovel, RocheProducts,Welwyn
Garden City, UK);
Hill and Shimeld
276
c 0 3 15 mg/mL Fentanyl cttrate and 10 mg/mL fluanisone (Hypnorm, Janssen
Pharmaceutical, Oxford, UK),
d. 100 mg/mL Ketamme (Vetalar, Parke-Davts Veterinary, Pontypool, Gwent,
UK); and
e. 20 mg/mL Xylazme (Rompun, Bayer plc, Bury St. Edmunds, Suffolk, UK)
4. Antibiotic cream Chloramphemcol eye ointment BP 1% w/w (Damels Pharma-
ceutical, Derby, UK).
5 Cornea1 stains
a. 2% w/v Fluorescem sodium BP (Fluorescein mmims eye drops); and
b. 1% w/v Rose Bengal (Rose Bengal minims eye drops), both from Smith and
Nephew Pharmaceuticals, Essex, UK)
6. For retinal exammatton.
a Mydriatic* 1% cyclopentolate hydrochloride BP with 0 01% benzalkonmm
chloride (Mydrtlate l%, Boerhmger Ingleheim, Berkshire, UK); and
b. 2% Sodium carboxymethyl cellulose with 0 0 1% benzalkomum chloride eye drops
7. Shredded paper bedding supplied by W M Ltlhco & Son (Betchworth, Surrey, UK).
8. Hanovia UV lamp (Slough, Buckmghamshire, UK) or similar that emits a peak
of 4 02 mj/cm2s at 320 nm

3. Methods
3.1. Setting Up Infected Mice
3.1.1. Anesthesia
1. 0.2 mL of neat Sagatal inJected mtraperitoneally is used for terminal anesthesia
(see Note 4)
2 For surgical anesthesia two methods are used (see Note 5). Either dilute hypnorm
1:8 m sterile water or dilute hypnovel 1.8 in sterile water InJect as two separate
inJections (0.2 mL of each for a 25-30 g mouse) subcutaneously m the scruff
of the neck or add 1 mL Vetalar and 0.5 mL Rompun to 8.5 mL sterile water
and use the mixture at 0.25-0.3 mL for a similar size animal, given by mtra-
peritoneal inJection.
3. For sedation mJect 0 2 mL of the described dtlutton of hypnorm subcutaneously
m the scruff of the neck
3.1.2. Labeling Mice
Mice should be mdlvidually labeled by tattooing the tall with Indian mk.
For this, use a I-mL syringe contammg tnk and fitted with a 26-gage needle.
Marks are made on the tail by injecting a very small amount in a line under
the skin. A mark at the tip of the tail represents a single unit (up to 4 can be
tattooed) and a mark at the base of the tail 5 U. Thus an animal with four
marks at the ttp 1s number 4, one with one mark at the tip and one mark at the
base 1snumber 6 and so on. If done carefully these marks last for the lifetime
of the animal.
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Models of HSV in the Mouse
3.1.3. Methods and Sites of inoculation
In all cases animals must be grven surgical anesthesia before inoculation.
3.1.3.1. THE EYE MODEL

1. The eyes of all mice used for ocular experiments should be exammed using a slit-
lamp microscope (see the following) before use; any with ocular abnormalities
should be discarded (see Note 6).
2. To produce mice suitable for the mduction of recurrent infection, 24 h before
inoculation of virus on the cornea mice are given an ip injection of 0.5 mL of
rabbit serum containing antibodies to HSV-1 (see Note 7). The serum is diluted
to give a dose of 800 ED50 (see Note 8).
3. While viewing through a dissectmg microscope, the mouse is held on its side, the
index finger and thumb are placed either side of the eye and by application of
gentle downward pressure the globe is proptosed and partially immobilized
4. The required dose of vnus, m a volume of 5 pL is placed onto the eye and the
cornea 1s lightly scarified in a grid pattern of five horizontal and five verttcal
lines using the sharp edge of the bevel of a 26-gage needle. The amount of dam-
age done to the cornea by scarification IS critical, since the cornea1 epithehum IS
very heavily innervated by sensory nerves and slight deviattons m the extent of
damage will directly affect the amount of vu-us entering nerve endings and thus
the severity of disease The inoculator must establish the “correct” amount of
damage for his system and then aim to repeat this amount consistently (see Note 9).
5. With the drop of moculum remaining on the cornea, the animal is then left on its
side to recover from anesthesia
6 For all ocular studies it is recommended that saw dust bedding 1s avoided since
the small particles can enter the eye easily and produce cornea1 damage. To avoid
this, mice maculated on the cornea are routinely housed on shredded paper,
3.1.3.2. THE EAR MODEL

Two methods of maculation can be used. In the first, as originally described
(16), virus is inoculated under the epidermis of the upper surface of the pinna
(see Note 10). Because of the thinness of the pinna, this is technically demand-
mg and some practice IS necessary. Use of an illuminated large low power
magnifying glass may be helpful.
1. With the mouse anesthetized and the dorsal surface of the pinna uppermost, the
pinna IS grasped gently between the index finger and thumb.
2. The inoculum, usually 10 pL, IS delivered from an accurately graduated glass
syringe through a short bevel, 26-gage 3/8-in (0 45 x 10 mm) needle. The needle
should enter in the middle area of the dorsal surface of the pinna The most com-
monly encountered problem 1spenetration of the needle through the pmna There-
fore, it is recommended that a rubber guard is worn on the tip of the index finger.
A suitable guard can be made from a cutoff small rubber teat.
HIII and Shimeld

An alternative sate of maculation for the ear model IS to make use of the
phenomenon of zosteriform spread by inoculating the skm of the neck. It has
been shown that this site of inoculation also leads to latent infection of gangh-
omc neurons that supply the pmna. Hence, recurrent disease can be induced on
the pmna, even though this was not the original site of moculatton (7).
I Before maculation of the neck it is necessary to remove the mr from the site Do not use
chermcal preparation or soaps m this process, since these agents may cause mactivation
of the vnus m the moculum With the mouse anesthettzed, the fur to be removed is
liberally wet with clean, warm water. With the mouse lymg under slight tenston m one
hand the fur IS carefully shaved from the ventral surface of the neck and the skm and
surroundmg fur is then dried, a size 10 round-end scalpel IS best for the shavmg
11 With the mouse lying on tts back, a lo-pL drop of vu-us suspension 1s placed onto
the dry, shaved skm on the ventral surface of the neck, Just caudal to the angle of
the Jaw and ventral to the bottom edge of the pmna
111 The drop is spread out slightly on the skm wtth the shaft of a 26-gage needle and with
the sharp edge of the bevel, 10 parallel scarifications of the neck skm are made m the
cramocaudal dn-ectton, through the moculum fluid During the scartticatton it helps
to keep the skm under slight tension m a cramocaudal dnection. Care should be taken
to avoid scarifymg over the mtdlme otherwtse herpetic lesions ˜111occur on both
ears As m the eye model, the amount of scanficatton IS crmcal and more control can
be gained by observing the procedure under a low power dtssecting microscope
iv The mice are left to recover on their back, with the moculum left m place.
In setting up the zosteriform model itself, the procedure for inoculation of
the neck with virus 1sthe same as described.
3.2. Reactivation of Latent Infection
Mice usually are left for at least 4 wk after the primary infection before
reactivation 1s attempted. For reacttvatton in the eye model mice must have
been passively immuntzed before tnoculation of virus. In all cases, animals
must be given surgical anesthesia for the reactivation procedure.
3.2.7. In the Eye Model by UV Irradiation
1 Turn on a UV lamp and leave to warm up according to the manufacturer™s mstructions
2 Collect eyewashmgs, from the ortgmally inoculated eye, for the tsolatton of vuus
(see the following) to check for spontaneous shedding of virus.
3 Put the mice under the UV lamp, usmg the thumb and index finger exert a slight
downward pressure on the skin around the appropriate eye to proptose the eye
slightly (see Note 11) and hold the mace m position 22 cm from the UV bulb for
90 s (see Note 12) The operator should wear protective glasses that screen out
UV and protect hands with a high factor sun screen and gloves.
4 Apply antibiotic cream to the eye immediately after UV irradiation and daily for
the next 7 d
Models of HSV in the Mouse 279

3.2.2. In the Ear Model by Mild Skin Trauma
For this, cellophane tape is pressed gently onto the upper surface of the pinna
of the orlginally inoculated ear (or the pinna on the same side as the original
neck inoculation). This 1srepeated with a fresh area of tape a further five times
(see Note 13).
3.3. Methods Applicable to the Observation of Recurrent Infection,
Zosteriform Disease, and the Detection of Latent Infection
3.3.1. Clinical Disease
3.3.1 .l. IN THE EYE
1. Surgically anesthetize the mice.
2 Arrange the slit lamp microscope so that you can rest your elbows on the bench
and your hands on the chin rest (to reduce shake) and the hands are more or less
in focus. Hold and support the mouse m a cupped left hand, use the thumbs to
open its eyehds and slightly proptose the eye Focus on the eye by moving the
mouse (rather than movmg the microscope, which IS the normal procedure when
examining human patients) (see Note 14)
3.3.1.2 ON THE PINNA OF THE EAR
In the ear and zosteriform models in albino mice, erythema provides an eas-
11y observed (see Note 15), early, and sensitive indicator of disease in the pinna
(Z6). With respect to recurrent disease obviously it is important to distinguish
between erythema owing to the trauma of the reacttvating stimulus itself, e.g.,
cellophane tape stripping, and erythema due to recurrent disease. With cello-
phane tape stripping it is possible to define criteria to make this distmctlon
(17). For example, if erythema persists after stripping It IS considered to be
significant only if it lasts longer than 3 d, since by this time 98% of stripped
uninfected control mice are normal. If erythema develops after a period of nor-
mality it should remam for at least two consecutive days. By these criteria, the
incidence of recurrent disease following stripping is found to be between 7 and
32% m different groups of animals.
Observation of lesions such as vesicles, pustules, and small scabs,however,
requires the mouse to be anesthetized or at least sedated to allow careful obser-
vation of the skin of the pinna under a dissecting microscope. In the ear model,
vesicles are first visible on d 3 after cellophane tape stripping. Vesicles last
only a few hours and rapldly develop into pustules which then scab, ulcerate,
and heal (28™. The angle of illumination is critical in observing lesions such as
early vesicles (see Note 16).
In the zosteriform model, again the earliest sign of clinical disease m the
pinna is erythema. However, presumably becauseof the larger amounts of virus
Hill and Shlmeld

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