types. Infect Immunol 35, 1125-l 132.
15. Cook, S. D and Hill, J M. (1991) Herpes simplex vnus: Molecular biology and
the posstbihty of cornea1 latency. SW-V.Ophthalmol 36, 140-148.
16 Stanberry, L R. (1994) Ammal models and HSV latency Sem Vu-01 5,2 13-2 19
17 Hill, J. M., Sedaratt, F., Javter, R T., Wagner, E. K., and Stevens, J G (1990)
Herpes simplex virus latent phase transcription facilitates m vtvo reacttvatlon.
Vzrology 174, 117-125.
18 Bloom, D. C., Devt-Rao, G B., Hill, J. M., Stevens, J G., and Wagner, E. K.
(1994) Molecular analysis of herpes simplex virus type 1 durmg epmephrme-
induced reactivation of latently infected rabbits in VIVO. J Vzrol. 68, 1283-1292.
19. Yamamoto, Y. and Hill, J M (1986) HSV-1 recovery from ocular tissues after
viral inoculatton mto the superior cervical ganglion Invest. Ophthalmol Vzs Scz
27, 1447-l 452
20. McNeill, J. I. and Kaufman, H. E. (1979) Local antivlrals m a herpes simplex
stromal keratms model. Arch. Ophthalmol 97,727-729
21. Kwon, B S., Gangarosa, L. P., Park, N. H., Hull, D S., Hineberg, E., Wiggms, C ,
and Hill, J M. (1979) Effect of rontophoretic and topical application of antiviral
agents m treatment of experimental HSV- 1 keratms m rabbits Invest Ophthalmol
Vis. Sci 18, 984-988
22. Rootman, D. S , Haruta, Y., Hill, J. M , and Kaufman, H E. (1989) Trifluridine
decreases ocular HSV- 1 recovery, not recurrent HSV- 1 lesions followmg tlmolol
tontophoresls in the rabbit. Invest Ophthalmol. Vis. SCL 30,678-683.
23. Berman, E J. and Hill, J M. (1985) Spontaneous ocular shedding of HSV-1 in
latently infected rabbits. Invest Ophthalmol Vzs SCL 26, 587-590
24 Hill, J. M., Dudley, J. B., Shlmomura, Y , and Kaufman, H E (1986) Quantlta-
tlon and kinetics of induced HSV- 1 ocular shedding. Curr Eye Res 5,24 l-246
25. Kwon, B. S , Gangarosa, L P , Green, K., and Hill, J. M. (1982) Kinetics of ocu-
lar herpes simplex vu-us shedding induced by epmephrme of iontophoresls. Invest
Ophthalmol. Vu Scz 22,818-823.
26 Lynas, C., Laycock, K A., Cook, S. D., Hill, T. J., Blyth, W. A., and Maitland, N. J. (1989)
Detection of herpes simplex vuus type 1 gene expression in latently and productrvely
infected mouse ganglia using the polymerase chain reaction J, Gen. Viral 70,2345-2355
27. Cantin, E. M., Lange, W , and Openshaw, H. (1991) Application of poly-
merase chain reaction assays to studies of herpes simplex virus latency.
Intervzrology 32, 93-l 00
28. Katz, J. P , Bodin, B T., and Coen, D. M. (1990) Quantitative polymerase chain
reaction analysis of herpes simplex vu-us DNA in ganglia of mice infected with
replication-incompetent mutants J Vwol. 64,4288-4295
Hill, Wen, and Halford
29. Hill, J. M , Halford, W. P., Wen, R , Engel, L S., Green, L. C , and Gebhardt, B
M (1996) Quantitattve analysts of polymerase cham reaction products by dot
blot. Anal Btochem 235,44-48
30 Ramakrtshnan, R., Fmk, D J., Jtang, G., Desat, P , Glortoso, J. C , and Levme, M.
(1994) Competttive quantitative PCR analysis of herpes stmplex vuus type I DNA
and latency-associated transcript RNA m latently infected cells of the rat brain J.
Vwol 68, 1864-1873
3 1 Ogretmen, B., RataJczak, H., Kats, A , and Stark, B C (1993) mternal cRNA stan-
dards for quantitative northern analysts. BzoTechnrques 14,935-94 1.
32. Stroop, W G , Rock, D. L , and Fraser, N. W (1984) Location of herpes simplex
vn-us m the trtgemmal and olfactory system of the mouse central nervous system
during acute and latent infections by m situ hybrtdtzatton. Lab Znvest 51,27-38.
33. Stroop, W G. and Banks, M. C. (1992) The weakly virulent herpes srmplex vtrus
type 1 strain KOS-63 establishes peripheral and central nervous system latency
followmg intranasal mfection of rabbits, but poorly reacttvates m viva J
Neuropathol Exp Neurol. 51,55&559
34. Stroop, W. G. and Banks, M. C (1994) Herpes simplex virus type 1 strain KOS-63
dose not cause acute or recurrent ocular disease and does not reactivate qanghomc
latency in vwo Acta Neuropathol 87, 14-22.
35. Gordon, Y. J , Johnson, B , Romanowskt, E., and Araullo-Cruz, T (1988) RNA
complementary to herpes simplex vu-us type-l ICPO gene demonstrated in neu-
rons of human trtgemmal ganglta. J. Vzrol. 62, 1832-l 834.
36 Stmmonns, D M , Arrtza, J. L , and Swanson, C. F (1989) A complete protocol
m situ hybridization of messenger RNAs in brain and other tissues wtth radiola-
beled single-stranded RNA probes J Htstotechnol 12, 169-l 8 1,
37 Tenser, R B, Edris, W A., Gaydos, A., and Hay, K. A (1994) Secondary herpes
simplex vu-us latent infection m transplanted gangha J Vu-01 68, 72 12-7220
38. Montone, K. T and Brtgati, D J. (1994) In situ molecular pathology. instrumenta-
tion, oligonucleottdes, and viral nucleic acid detectton J. Hlstotechnol 17, 195-201 I
39 Slobedman, B , Efstathtou, S , and Sunmons, A. (1994) Quantitative analysts of
herpes simplex virus DNA and transcriptional activity in ganglia of mice latently
infected with wild-type and thymtdme kmase-deficient viral strams. J Gen Vwol
40. Wagner, E K., Flanagan, W. M., Devi-Rao, G., Zhang, Y , Hill, J M , Anderson,
K P., and Stevens, J. G. (1988) The herpes stmplex virus latency associated tran-
script 1s spliced durmg the latent phase of mfectton. J VwoI 62,4577-4585
4 1 Stevens, J G. (1989) Herpes simplex virus latency analyzed by m situ hybridiza-
tion Curr Topics Mcrobtol. Immunol 143, 1-8.
42. Hill, J. M , Gebhardt, B M , Wen, R , Bouterie, A M , Thompson, H. W , Halford,
W P., Oâ€™Callaghan, R. J., and Kaufman, H E. (1996) Quantificatton of herpes
simplex virus type 1 DNA and latency-associated transcripts m rabbit trigemmal
ganglia demonstrates a stable reservoir of viral nucleic acids during latency.
J Vzrol 70,3137-3141.
43 Stroop, W G. (1986) Herpes simplex vtrus encephahtts of the human adult: reac-
HSV Latency 313
tivation of latency brain mfectton Pathol. Immunopathol. Res 5, 156-169
44 Hill, J. M., Oâ€™Callaghan, R J., Hobden, J. A., and Kaufman, H. E (1993) Oph-
thalmic Drug Delivery Systems, Ocular iontophoresis (Mitra, A. K., ed.), Marcel
Dekker, NY, 331-354.
45. Shtmomura, Y , Gangarosa, L P. Sr., Kataoka, M., and Hill, J. M (1983) HSV- 1
shedding by rontophoresis of 6-hydroxydopamine followed by topical epineph-
rine Invest Ophthalmol Vis SCL 24, 1588-1594.
46. Hill, J. M , Haruta, Y , and Rootman, D. S. (1987) Adrenergrcally induced recur-
rent HSV-1 cornea1 eptthehal lesion, Curr Eye Res 6, 1065-1071
47. Haruta, Y., Rootman, D S., and Hill, J. M (1988) Recurrent HSV-1 cornea1 epr-
thehal lesions induced by timolol iontophoresis m latently infected rabbits Invest
Ophthalmol Vls Scl 29,387-392
48. Stroop, W. G. and Schaefer, D. C. (1987) Severrty of experlmentally reactivated
herpetrc eye disease is related to the neurovirulence of the latent virus. Invest
Ophthalmol. Vu. Scl 28,229-237.
49. Haruta, Y , Rootman, D. S , Xie, L , Kmtoshi, A., and HIII, J. M (1989) Recur-
rent HSV-I cornea1 lesions m rabbits Induced by cyclophosphamide and dexam-
ethasone Invest Ophthalmol Vls SCI 30, 371-376
50 Beyer, C. F., Hrll, J M., Reldy, J. J , and Beuerman, R. W (1990) Cornea1 nerve
dtsruption reactivates virus m rabbits latently infected wtth HSV-1 Invest
Ophthalmol Vu SCI 31,925-932
51 Beyer, C F , Tepper, D J., and Hill, J. M. (1989) Cryogenic induced ocular
HSV-1 reacttvatron IS enhanced by an mhtbitor of the hpoxygenase pathway.
Curr. Eye Res 8, 1287-1292
52. Gordon, Y. J., Romanowski, E , and Araullo-Cruz, T (1990) A fast, simple reac-
trvatton method for the study of HSV- 1 latency m the rabbrt ocular model Invest
Ophthalmol Vzs Scz 31,921-924.
53 Htll, J. M., Shrmomura, Y., Kwon, B. S., and Gangarosa, L P (1985) Ionto-
phoresis of epinephrme isomers to rabbit eyes Induce HSV-1 ocular sheddmg
Invest. Ophthalmol Vu Scl 26, 1299-1303.
54 Stevens, J. G (1994) Overview of herpesvirus latency Sem Vwol 5, 19 l-l 96
55. Speck, P. G and Simmons, A (1991) Divergent molecular pathways of produc-
trve and latent infection wrth a virulent strain of herpes stmplex vn-us type 1 J
56 Margolis, T. P., Sedaratt, F., Doboson, A. T., Feldman, L. T., and Stevens, J G
(1992) Pathway of viral gene expression during acute neuronal mfectton with
HSV-1. Vzrology 189, 150-160.
57 Rock, D L., Nesburn, A. B., Ghiasi, H., Ong, J., Lewis, T , Lokensgard, J R., and
Wechsler, S. L (1987) Detection of latency-related viral RNAs m trigemmal gan-
glia of rabbits latently infected with herpes stmplex vnus type 1. J Vu-01 61,
58. Stevens, J. G., Wagner, E. K , Devi-Rao, G. B., Cook, M. L , and Feldman, L. T
(1987) RNA complementary to a herpesvnus CLgene mRNA is prominent m la-
tently infected neurons. Science 235, 1056-1059
Hill, Wen, and Ha/ford
59 Perng, G C., Dunkel, E. C , Geary, P. A., Slamna, S M., Ghlasl, H., Kalwar, R ,
Nesburn, A B , and Wechsler, S L (1994) The Latency-Associated Transcript
gene of HSV-1 1srequired for efficient m vtvo spontaneous reactivation of HSV-1
from latency. J Vwol 68, 8045-8055
60 Trousdale, M. D , Steiner, I, Spivack, J. G , Deshmane, S L , Brown, S M ,
MacLean, A R., Subak-Sharpe, J. H , and Fraser, N W (1991) In vivo and m
vitro reactivatton lmpanment of a herpes simplex virus type 1 latency-associated
transcript variant m a rabbit eye mode1 J Vrrol 65
61 Farrell, M. J , Hill, J M., Margohs, T. P , Stevens, J. G , Wagner, E K , and
Feldman, L T. (1993) The herpes simplex virus type 1 reactivation for lies out-
side the latency-associated transcript open reading frame ORF-2 J Vzrol 67,
62. Javler, R. T., Steven, J G , Dlssette, V B., and Wagner, E K (1988) An HSV
transcript abundant m latently infected neurons 1s dispensable for establishment
of the latent state. Vzrology 166, 254-257.
63 Fraser, N. W., Block, T M., and Splvack, J. G. (1992) The latency-associated
transcripts of herpes simplex virus* RNA m search of function Vzrology 191, l-8
64. Ward, P. L. and Rolzman, B (1994) Herpes simplex genes the blueprint of a
successful human pathogen Trends Genet 10,267-274
65. Feldman, L. T (1994) Transcription of the HSV-1 genome m neurons in vlvo
Sem Vwol 5,207-2 12
66 Hill, J M , Helmy, M F , Osborne, P A., Johnson, E M , Jr, and Gebhardt, B
(1990) Antibodies to nerve growth factor induce HSV-1 ocular shedding in la-
tently infected rabbits. Invest Ophthalmol VESSci 3l(suppl.), 3 14.
67. Gerdes, J C. and Smith, D S (1983) Recurrence phenotypes and establishment
of latency followmg rabbit keratltrs produced by multiple herpes simplex strains.
J Gen Vwol 64,2441-2454
68. Hill, J M , Rayfield, M A., and Haruta, Y (1987) Strwn specificity of spontane-
ous and adrenerglcally induced HSV-1 ocular reactlvatlon in latently infected rab-
bits Curr Eye Res. 6, 91-97
69 Kwon, B. S , Gangarosa, L. P , Burch, K. D , de Back, J., and Hill, J M (1981)
Induction of ocular herpes simplex virus shedding by lontophoresls of epmeph-
rme mto rabbit cornea Invest Ophthalmol Vzs Sci 21,442-449.
70. Gordon, Y J., Romanowskl, E., Araullo-Cruz, T , and McKmght, J L. C. (1991)
HSV-1 cornea1 latency Invest Ophthalmol VLY Scl 32,663-665
71. Sabbara, E M H., Pavan-Langston, D , Bean, K M , and Dunkel, E. C. (1988)
Detection of HSV nucleic acid sequences in the cornea during acute and latent
ocular disease. Exp Eye Res 47,545-553
72. Cook, S. D., Hill, J M , Lynas, C , and Maltland, N J (199 1) Latency-associated
transcripts m corneas and ganglia of HSV- 1 infected rabbits Br J Ophthalmol
73. Oâ€™Brien, W. J and Taylor, J. L. (1989) The isolation of herpes simplex virus from
rabbtt corneas during latency Invest. Ophthalmol Vzs Scz 30,357-364
74 Cantm, E , Chen, J , Wllley, D E , Taylor, J L , and Oâ€™Brien, W J (1992) Persls-
HSV Latency 315
tence of herpes simplex virus DNA m rabbit cornea1 cells Invest. Ophthalmol
VW. SCL 33, 2470-2475
75. Brown, D C. and Kaufman, H E. (1969) Chronic herpes simplex infection of the
ocular adnexa. Arch Ophthalmol 81,837˜839.
76 Rootman, D S , Haruta, Y , Hill, J. M , and Kaufman, H. E (1988) Cornea1 nerves
are necessary for adrenerglc reactlvatlon of ocular herpes Invest Ophthalmol
Vis Sci 29,351-356.
HSV Latency In Vitro
In Situ Hybridization Methods
Christine L. Wilcox and R. L. Smith
We have developed an in vitro model of herpes simplex virus (HSV) latency
in primary neurons that mimics many aspectsof HSV latency m animal models
and the human disease (I-3). Using this model, we demonstrated that HSV- 1
and HSV-2 establish latent infections in vitro in the same neuronal cell types
that are shown to harbor latent HSV in humans (3). Latent HSV mfectlons can
be produced m neuronal cultures from ganglia of rodents and primates with
similar results (3). In all casesexamined, the neurotrophm, nerve growth factor
(NGF), 1srequired to mamtam the latent infections. Depletion of NGF results
m the reactivation of latent vn-us (l-3). Depending upon the condltlons and the
use of a high multiphclty of infection, latent HSV-1 infections are established
m the majority of primary sensory or sympathetic neurons m tissue culture
(2,4). To achieve high efficiency of establishment of latency with little or no
evidence of lytic infection, an antiviral agent (e.g., acyclovir) is added to the
neuronal cultures during the first week after inoculation with vn-us. However,
latency can be established in the absence of antiviral treatment provided that
the multlphcity of infection (MOI) is very low (1,2). At least one of the actions
of the antiviral treatment 1sto prevent amplification of the input virus in the
nonneuronal cells that are present in the culture at the outset of the infection.
These nonneuronal cells are destroyed m the presence of acyclovlr and virus
(4). Latency is maintained m neurons in culture for as long as 10 wk in the
presence of NGF. Viral transcripts and antigens associated with the productive
infection are not detected during the latent infection (2,3,5). Viral transcription
1srestricted to the latency-associated transcripts (LAT) during the latent infec-
From Methods in Molecular Medrone, Vol 10 Herpes Bmplex Wrus Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ
Wilcox and Smith
non and is present m the nuclei of 80-90% of the neurons by 3 wk postmfection
(4,.5) Upon removal of NGF from the culture medium, for as brief as 1 h, reactiva-
tion of latent virus is induced (3), and viral antigens associatedwith the productive
mfection and mfectious virus are detectedbetween 48-72 h after NGF deprivation.
The m vitro mode1 is well suited for molecular studies of the regulation of
latency and cellular stgnalmg since the model can produce a population of
neurons with a very high frequency of latent virus, and the establishment and
reactivation of latency are controlled, synchronous events. The m vitro mode1
of latency m neurons also allows mampulations using pharmacologtcal meth-
ods (6) and has the potential for exammation of signaling pathways m mdi-
vidual neurons using micromJection methods.
The in vitro neuronal latency system that is currently used m our respective
laboratories and that has been best characterized is prepared m sensory neu-
rons from dorsal root ganglia (DRG) obtained from embryonic d 15 rats DRG
neurons are used because they are a natural site of HSV latency m humans (7)
and they are relatively easy to isolate and mamtam m tissue culture. Recently
we described m detatl the methods for the preparation of the neuronal cultures
for the establishment of latent infections and the Induction of reactivation (8)
To further understand the regulation HSV latency, examination of the pat-
tern of viral transcription during the establishment of latency m the in vitro
neuronal model has proved information that could not be readtly obtained m
animal models (4). In this chapter, we have focused on a specialized method
for the analyses of HSV latency in vitro that we feel is not well documented by
other sources: ln situ hybridization (ISH). For neurons m culture, radiolabeled
probes and emulsion methods are not well-suited. The techniques that we have
found work well for ISH m neurons in culture are somewhat unique. In some
cases relatively subtle changes m techniques result m significant changes in
the successof the methods. The techmques presented here have evolved and
have been simplified over the years of working with the in vitro neuronal system.
1.1. Unique Problems with ISH Methods in Neurons In Vitro
ISH data have been invaluable for correct interpretation of transcrtption
detected by other methods in the neuronal cultures. A significant population of
quiescent nonneuronal cells (fibroblasts and Schwann cells) remam in the neu-
ronal cultures even after extensive exposure to antimitotic agents and may be a
significant source of viral transcription during the establishment of latency (4).
Furthermore, only ISH data can provide mformation about the population of
cells expressmg a transcript. However, the characteristtcs of the neuronal cul-
ture system imposes several unique problems. The neurons m vitro requtre a
substrate for attachment. We have found collagen to work the best. The neu-
rons grow on and extend their neuronal processes through the 3D matrix pro-
HSV Latency In Vitro
duced by collagen-coating of cover slips. Upon establishment of DRG neurons
m vitro, the cell bodies of the neurons are relatively large compared to many
cell lmes. Neuronal cultures, even with appropriate dlssoclatlon of the ganglia,