<<

. 44
( 61 .)



>>

ammals (uninfected) should be Included m each experiment W damage m such
animals IS predommantly ulceration of the cornea1 epithehum that conforms to a
regular pattern, the cornea may be slightly swollen and have a diffuse hght mfiltrate
There will also be a mild iris hyperemla and eyelids may be swollen slightly In such
eyes, healmg 1scomplete by d 3-4 after W treatment In contrast, m eyes with virus
induced disease, eplthehal lesions are irregular m shape, and accompanied by dense
focal mfiltratlon In addition, there 1susually a severe iris hyperemla Recurrent hd
lesions start as vesicles that rapldly progress to ulcers and then scabs
It should be noted that the hght from sht-lamp microscopes 1spowerful and a
lengthy exposure of the eye may produce drying and break up of the tear film that
may be misinterpreted as a sign of disease, especially if vital stains are used Occa-
slonal opening and closing of the hds will restore the mtegrlty of the tear film
15 Assessment of erythema should be done only on unanesthetized ammals smce
anesthetics may alter the picture by causmg changes m the pertpheral blood vessels.
16. Good lllummatlon 1s essential for dlssectlons and so on, under the microscope;
fibre optic lamps that easily can be adjusted to different posltions are the most useful
17. For certam experiments, such as tracing viral spread, it may be necessary to dlvlde
the ganghon mto its three parts ophthalmic, maxillary, and mandibular; this 1s
best done zn srtu (12) For lmmunohistochemical studies, perfuslon with fixatives
under terminal anesthesia gives the best tissue preservation Fixation before dls-
section helps the somatotopic organization of the tissue to be maintained and this
1sof particular value if you wish precisely to locate evidence of infection
18 The carotid vein usually 1sfull of blood, whereas the artery often IS empty and more
difficult to find. Superior cervical ganglia vary in size in different mouse strains
They are bean-shaped and translucent and they may be confused with lymph nodes
that are of a similar size and shape but usually are opaque and yellowish. Standard
histological techniques can be used to check that the correct tissue has been removed
19 There is no danger of accidentally removing cervical ganglia durmg the pre-
paratory dissection since they are held quite firmly m place m small concavities
m the vertebrae
20 The major sites of latency m the ear model are the second, third, and fourth cer-
vical ganglia with the highest mcidence in the second
21. When sensitivity in detecting latency 1s important in particular studies, the addl-
tlon of hypomethylatmg reagents to the explant medium should be considered
since these significantly Increase the incidence of reactivation Concentrations of
these drugs found to be effective in the medium are 8 mA4 azacytldme for mouse
ganglia (37), 50 mA4 for guinea pig ganglla (38), 8 m44 L-ethlonine for mouse
ganglia (3 7), 5 mM bzs-acetamlde for gumea ptg gangha (39).
More recently, m an adaptation of the coculture method, attempts were made
to provide better oxygenation of the explanted gangliomc tissue in the hope that
this would aid its survival and thereby increase the chance of reactivation For
this, ganglia or ganglion fragments are placed m glass tubes, 1 x 10 cm, on the
287
Models of HSV in the Mouse
inside face of which, at one end, a monolayer of Vero cells has been grown. The
tubes contain 1 mL of tissue culture mamtenance medium and are rotated at 10
rev/h on a roller drum at 35°C. The monolayers are examined dally for 3 wk for
evidence of cpe caused by HSV. Medium m the tubes IS changed every 2-3 d and
precautions are taken to avoid crosscontammatron between tubes. This roller tube
method gave a srgmficantly hrgher Incidence of latent mfectron compared wrth
the statrc, 5-d culture/homogenizatton procedure (40).
In the ear model, depending on the studres Involved, the three cervrcal ganglia
can be incubated separately or together
22. Before sampling from the tar1 vem, put the mace m a cage and warm them wrth
one or two angle poise lamps until the tail veins are drlated A close watch must
be kept; hyperthermra, whrch IS preceded by sweating, may result m death. Use-
ful blood letting mstruments can be made by breaking off the plasttc mount from
23-gage x 1-in. needles For bleeding, place the mouse m a restramer, posmoned
so that the tad hangs downward and is freely accessible Insert a needle mto a tall
vem and catch the blood m a sterile Eppendorf tube or A4 cup. Remove the needle
and press on the vein with a pellet of cotton wool. Bleeding pigmented mice from
the tall vein IS difficult Mice of a more nervous drspositron than NIH™s may have
to be anesthetized before this procedure and should not be warmed. Blood flow
will therefore be considerably slower than in warmed mice

References
1 Stanberry, L R (1992) Pathogenesis of herpes simplex vtrus infection and animal
models for Its study Cur-r Topics Micro Immunol 179, 15-30
2 Hill, T J (1985) Herpes simplex virus latency, m The Herpesvzruses, vol 3
(Rorzman, B , ed.), Plenum, New York, pp. 175-240
3. Claoue, C., Hodges, T., Blyth, W A., Hill, T J , and Easty, D L (1988) Neural
spread of herpes stmplex vnus to the eye of the mouse. microbrologrcal aspects
and effect on the blink reflex Eye 2,3 18-323.
4. Shimeld, C., Hill, T J., Blyth, W. A., and Easty, D L. (1990) Reactrvatton of
latent infection and mductron of recurrent herpetrc eye disease m mace J Gen
Vii-01 71, 397-404
5. Shimeld, C., Hill, T J., Blyth, W A., and Easty, D. L. (1989) An Improved model
of recurrent herpetrc eye disease in mice. Curr Eye Res 8, 1193-1205.
6 Shimeld, C., H111,T. J., Blyth, W. A., and Easty, D. L. (1990) Passrve rmmuniza-
tion protects the mouse eye from damage after herpes simplex vu-us infection by
limrting the spread of vnus in the nervous system. J Gen Vzrd 71,681-687.
7. Blyth, W A., Harbour, D, A , and Hill, T. J (1983) Pathogenesrs of zosteriform
spread of herpes simplex vnus in the mouse. J Gen Vzrol 65, 1477-1486.
8. Hill, T J , Blyth, W A., Harbour, D A., Tullo, A B., Easty, D. L , and Shrmeld,
C. (1984) Antiviral agents, antnnflammatory agents and models of herpes srm-
plex m the mouse, m Herpeswas (Rapp, F., ed.), Alan R. Liss, NY, pp. 601-615
9. Shimeld, C., Easty, D. L , Tullo, A. B., Blyth, W. A., and Hill, T. J. (1984) Spread
of herpes stmplex vu-as to the eye following cutaneous moculatron m the skin of
Hill and Shimeld
288
the mouse, m Herpettc Eye Diseases (Maudgal, P C and Mtssotten, L., eds ), Dr.
W. Junk, Dordrecht, Belgium, pp 3945.
10. Hill, T. J , Blyth, W. A , Harbour, D. A , Berrie, E. L., and Tullo, A B. (1983) Latency
and other consequences of mfectton of the nervous system with herpes simplex vuus
m Immunology of Nervous System Infections. Progr Bram Res 59, 173-l 84
11 Hill, T J. (1983) Herpes viruses in the central nervous system, m Y,,es znDemyeh-
natzng Dzsease (Mtms, C , et al , eds.), Academic, London, pp 29-45
12 Tullo, A B , Shimeld, C , Blyth, W A , Hill, T. J , and Easty, D L (1982) Spread
of vu-us and dtstrtbutton of latent mfectton followmg ocular herpes simplex m the
non-immune and immune mouse J Gen Vzrol 63,95-l 0 1
13 Willey, D E , Trousdale, M D., and Nesbum, A. B (1984) Reacttvation of murme
latent HSV infectton by epmephrme iontophoresis. Invest Ophthalmol Vzs Set
25,945-950
14 Gordon, Y J. (1990) Pathogenests and latency of herpes simplex virus type 1
(HSV-1). an ophthalmologist™s view of the eye as a model for the study of the
virus-host relationshtp, m Immunobzology and Prophylaxes of Human Herpesvt-
rus Znfectzon (Lopez, C et al., eds ), Plenum, NY, pp. 205-209
15. Hill, T J , Harbour, D A , and Blyth W. A (1980) Isolation of herpes stmplex vuus from
the skm of clintcally normal nuce dunng latent tnfection J Gen Vzrol 47,205207
16 Hill, T. J., Fteld, H. J , and Blyth, W A (1975) Acute and recurrent mfectton with
herpes simplex vu-us m the mouse. a model for studying latency and recurrent
dtsease J Gen Vtrol 28,341-353
17 Hill, T J., Blyth, W. A , and Harbour, D A (1978) Trauma to the skm causes
recurrent herpes simplex m the mouse J Gen Virol 39,2 l-28
18 Hill, T. J , Blyth, W A , and Harbour, D A. (1982) Recurrent herpes simplex m
mace topical treatment wtth acyclovn cream Anttviral Res. 2, 135-146
19 Shimeld, C., Lewkowicz-Moss, S., Lipworth, K., Hill, T J , Blyth, W. A., and
Easty, D. L. (1986) Anttgens of herpes stmplex vn-us m whole cornea1 epithehal
sheets from mice Arch Ophthalmol 104, 1830-1834
20. Dyson, H , Shtmeld, C , Hill, T J , Blyth, W A., and Easty, D. L. (1987) Spread
of herpes simplex vuus within ocular nerves of the mouse* demonstratton of viral
antigen in whole mounts of eye tissue. J Gen. Vtrol. 68,298!&2995.
2 1 Williams, N., Shtmeld, C , and Hill, T J (1992) The dtstrtbutton of viral antigens
and Langerhans cells during the zostertform spread of herpes simplex vu-us to the
skin of mice. Arch Vtrol 122, 349-358
22. Lynas, C , Laycock, K , Cook, S., Hill, T J , Blyth, W A., and Maitland, N J
(1989) Detection of herpes simplex vnus type 1 gene expression m latently and
productively infected mouse ganglia using the polymerase chain reaction. J Gen
Vu-01 70,2345-2355
23. Wtlliams, L. E., Nesburn, A B., and Kaufman, H. E (1965) Experimental induc-
tion of disciform keratms Arch Ophthalmol. 73, 112-l 14
24 Stroop, W G. and Banks, M. C (1994) Herpes simplex vtrus type 1 strain KOS-
63 does not cause acute or recurrent ocular disease and does not reactivate gangh-
omc latency in VEVO. Acta Neuropath 87, 14-22.
Models of HSV in the Mouse 289
25. Hill, J. M., Rayfield, M A., and Haruta, Y. (1987) Strain specificity of spontane-
ous and adrenergically induced HSV- 1 ocular reactivation. Curr Eye Res 6,9 l-97
26. Harbour, D. A., Hill, T. J., and Blyth, W A (1981) Acute and recurrent herpes
simplex m several strains of mice. J. Gen Vzrol. 55,3 l-40.
27. Laycock, K., Lee., S. F., Brady, R. H., and Pepose, J. S. (1991) Charactertzatlon
of a murine model of recurrent herpes simplex viral keratitis induced by ultravio-
let B radiation Invest Ophthalmol Vis Scz 32,2741-2746
28. Blatt, A. N., Laycock, K. A., Brady, R. H., Traynor, P., Krogstad, D J., and
Pepose, J. S (1993) Prophylactic acyclovn- effectively reduces herpes simplex
type 1 reactivation after exposure of latently infected mice to ultraviolet B Invest
Ophthalmol Vzs.Scl 34,3459-3465.
29 Sulhvan, D A. and Allansmith, M R. (1985) Hormonal influences on the secre-
tory nnmune system of the eye* androgen modulatton of IgA levels m tears of
rats. J lmmunol 134,2978-2982
30. Kaye, S. B , Shtmeld, C , Grmfeld, E., Maltland, N. J., Hill, T. J , and Easty, D. L.
(1992) Non-traumatic acquismon of herpes simplex virus infection through the
eye Br J Ophthalmol 76,412-418
31 Blyth, W A , Hill, T J , Field, H J , and Harbour, D A (1976) Reactivation of
herpes simplex vnus mfectton by ultraviolet light and possible mvolvement of
prostaglandms J Gen Vzrol 33, 547-550.
32. Harbour, D. A , Blyth, W. A , and Hill, T. J (1983) Recurrent herpes simplex m
the mouse. inflammation m the skin and activation of virus m the ganglia follow-
mg peripheral sttmuh J Gen Vzrol 64, 1491-1498.
33 Hill, T. J , Blyth, W A , and Harbour, D A (1980) Effect of immunosuppression
on recurrent herpes simplex in mice. Znjixt Immunol. 29,902-907.
34 Hennmgs, H. and Elgjo, K (1970) Eptdermal regeneration after cellophane tape
stripping of hairless mouse skin. Cell Tissue Klnet. 3, 243-252.
35 Roat, M I, Romanowski, E., Araulla-Cruz, T , and Gordon, Y. J (1987) The
antiviral effects of rose bengal. Arch. Ophthalmol. 105, 1415-1417.
36. Chodosh, J,, Banks, M. C., and Stroop, W. G. (1992) Rose Bengal mhtbtts herpes
simplex virus rephcatron m Vero and human eptthehal cells m vitro Invest.
Ophthalmol Vis Scz 33,252U2527.
37 Whttby, A, Blyth, W A., and Hill, T. J. (1987) The effect of DNA hypo-
methylating agents on the reactivation of herpes simplex virus from latently
infected mouse ganglia in vitro Arch Vzrol. 97, 137-144.
38. Stephanopoulos, D. E., Kappes, J. C., and Bernstein, D I. (1988) Enhanced in
vitro reactivation of herpes simplex virus type 2 from latently infected guinea ptg
neural tissues by 5-azacytidme J Gen. Vwol. 69, 1079-1083.
39. Bernstein, D I. and Kappes, J. C. (1988) Enhanced zn vztro reactivation of latent
herpes simplex vnus from neural and peripheral tissues with hexamethylene-
bisacetamide. Arch Virol 99,57-65
40. Lynas, C., Hill, T. J., Maltland, N. J., and Love, S. (1993) Latent infection with
herpes simplex vtrus type 2 following mtracerebral moculatton. J. Neurol. Scz
120,107-l 14.
Pathogenesis and Molecular Biology
of HSV Latency and Ocular Reactivation in the Rabbit
James M. Hill, Renjie Wen, and William P. Halford


1. Introduction
Research on herpesvnus mfecttons has commanded the attention of a diverse
group of scientists for over a century. Until the advent of the human immuno-
deficiency vnus, the herpes simplex viruses (HSV) were the most mtensively
studied of all vu-uses. During the early part of the nineteenth century, long
before the mfectious agent responsible for cold sores (fever blisters) was iden-
tified, studies suggested that damage to the trigeminal nerve could induce
peripheral herpetic vesicles (I). Gruter demonstrated that a particle of material
from a herpetic blister inoculated into a rabbit eye could cause herpes and that,
in this way, the disease could be transmitted in series from one rabbit to another
(2). Following Gruter™s basic discovery, research conducted m many parts of
the world showed a variety of clinical forms of herpes to be similarly transmis-
sible by inoculation (3). At roughly the same time, Goodpasture proposed that
“the vu-us remains in the ganglia m a latent state after the local lesion has
healed” and discussed in detail the pathology of herpetic infection m humans
and animals (4,.5).
Forty years later, Stevens and colleagues (68) were the first to isolate the
virus from the peripheral sensory ganglia of latently infected rabbits and mice.
Over the past 20 yr, there has been substantial progress in our understanding of
the pathogenesis and natural history of ocular herpetic infection. In recent
years, intensive efforts have been focused on unveiling the molecular mecha-
nisms and the virus-host interactions associated with latent HSV mfection,
reactivation, and recurrence.
The herpesviruses, which are enveloped, double-stranded DNA viruses, are
grouped into three classes:
From Methods m Molecular Medrcme, Vol 10 Herpes Smplex Vfrus Protocols
Edlted by S M Brown and A R MacLean Humana Press lnc , Totowa, NJ

291
Hill, Wen, and Ha/ford
292

1 The a-herpesvnuses are neurotropq tend to have a broad cell specificity, and
can grow to high toters wtth a rapid productive cycle m cell cultures These Include
herpes simplex vnus types 1 and 2 (HSV- 1 and -2) and varrcella zoster vuus.
2 P-herpesvuuses have restricted host ranges and a lengthy rephcatron cycle with
low yields of mfecttous vuus. These include human cytomegalovnus and human
herpesvtrus type 6.
3 y-herpesviruses are lymphotrophm, have a highly restricted range of cells m
which they can replicate, and have a highly regulated latent state of mfectton (9).
This group includes Epstein-Barr vu-us and the newly characterized human herp-
esvirus type 7

HSV infections are common m humans (IO). More than 90% of the popula-
tion has antrbodres to HSV, which generally infects mucocutaneous surfaces,
especially epithelral cells, as in the lip (herpes lablabs) or genitals. Ocular HSV
infection IS a leading cause of blinding keratitrs m industrialized countrtes.
Both HSV- 1 and -2 can infect the eye and produce recurrent ocular infection,
similarly to the recurrences of oral and genital HSV infection. Whereas most
genital infections are caused by HSV-2, approx 85% of ocular isolates are
HSV- 1 (II, 22). HSV-2 ocular infections often are associated with infants born
to mothers with acttve herpetic genital lesions.
Primary ocular infection is an acute keratoconJunctivitis with or without
mvolvement of the perrorbrtal skin. After primary infection, HSV travels along
a neuronal pathway to regional ganglia where it remains dormant (latent).
Under an ill-defined set of circumstances, the virus reactivates and travels to
the site of primary infection to cause recurrent disease. Recurrences m the eye
can produce cornea1 disease and stromal scarring as the result of an mcom-
pletely defined immunologrcal response to the reactivated vnus (13-1.5).
HSV establishes latency in gangliomc neurons following primary infection
at a peripheral site. Viral latency, as operationally defined, IS a state m which
cell-free mfectrous virus particles cannot be demonstrated m tissues at the time
of sacrifice, but mfectrous vu-us can be isolated from the same tissues after
prolonged m vitro cultivatton. Under certam circumstances the latent virus can
be reactivated, resumes rephcatron, and causesrecurrent disease m vivo.
The rabbit has proven to be an excellent model of herpetic eye drsease (16).
Inoculation of the cornea results in herpetic keratrtrs that may be quantified
both clmrcally and vnologically. Rabbit eye models have been useful m exam-
ining the role of viral genes in latency and reactivation. For example, recent
studies using HSV- 1 latency-associated transcript (LAT) mutants have shown
that LAT is not required for establishment of latent infection in rabbit trigemi-
nal ganglia, but is important for efficient reactivation of latent HSV-1 m vrvo
(Z 7,181. The goal of this work IS to describe a reliable rabbit model of ocular
HSV infection, latency, and reactivation.
293
HS V Latency
2. Materials
1. Growth of virus and cells* HSV is propagated in primary rabbit kidney (PRK)
cell monolayers and titered by plaque assay on African green monkey cell (CV- 1)
monolayers. The virus is frozen m small alilquots at -70°C so that the same batch
can be used for multiple experiments.
2. Rabbtts. New Zealand white rabbits (1 S-2 5 kg) can be used for the expertments.
3 3H-dTTP,3H-dCTP,3H-dATP (Amersham, Arlington Heights, IL)
4. Thymidine-5™-Triphosphate (a- 32P) (ICN Biomedicals, Irvine, CA).
5. TRIzol Reagent (Life Technologies, Gaithersburg, MD)
6. Kodak NTB-2 Emulsion (Eastman, New Haven, CT)

<<

. 44
( 61 .)



>>