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1. The mice are injected mtraperitoneally with 0.2 mL of a 7.5 mg/mL solution of
sodium pentobarbital m groups of 10, and placed m their cages until they are
completely asleep (see Note 11).
2. Mice are removed from their cages and laid on then backs.
3. With an emery board, the feet are hghtly abraded with l-2 strokes only to remove
the surface layer of skin.
4. With a plpetman, 25-50 pL are applied to each foot and rubbed over the surface
with the side of the plpet tip The animals are then arranged on their backs feet up
and flat m the cage with the bedding material to prop then up.
5 An additional 25-50 pL are applied to each foot. Care should be taken so that the
feet are level and the moculum stays on the feet.
6 This 1s allowed to adsorb for 30-60 mm (when the mace begin wakmg) (see
Note 12).
7. Watch the mice to ensure that they are awakening
8 Return them to the vlvarium for 2-3 wk. Removal of Spinal Ganglia. The ability of the constructs to express
the desired transgene is assessedfollowing removal of the spinal ganglia. Typi-
cally, expression IS assessedat acute times (4 d) and latent times (21 d or
longer). The procedure presented here is for the preparation of the tissue for
1. Mice are killed by ether inhalation.
2. The feet of the mice are then quickly pinned to a Styrofoam board.
3. Two Incisions are made from the sternum anteriorly to open up the rib cage and
expose the heart.
4. The sternum IS reflected back to the left of the head, and pinned to provide easy
access to the heart.
5. A 28-gage needle on a 12-mL syringe filled with PBS 1sinserted mto the apex of
the left ventricle.
6. Pressure applied, andsahne1sInjectedInto the heart at aslow rate (3 mL/mm).
An mclsion is made Immediately at the right atrium to provide a release.

7 After the saline has been injected, the barrel of the syringe is removed from the
needle, and another syringe that has been filled with 4% paraformaldehyde IS
placed on the needle. The mouse 1sperfused with the formaldehyde at the same
rate as the salme.
8 The spine is then removed by first wetting the fur with 70% ethanol.
9 A dorsal mclslon IS made starting near the tall and proceeding up the midline
anteriorly to the base of the neck The skm 1sfolded back and the spine 1sexposed.
10 The spine is removed by making two lateral mcistons on either side of the spme,
starting at the tall and moving anteriorly
11 The spine is grasped with a pair of forceps and lifted up The vessels and adher-
mg tissue are trimmed away as the spine 1sremoved from the carcass
12. The spme IS placed m a Petri dish, ventral side up It IS grasped firmly on its side
with a pair of forceps. Usmg small scissors, a medial Incision IS made exactly on
the midline cuttmg through the vertebrae
13 Usmg two pairs of forceps, the back IS pried open to expose the spinal cord and
the dorsal root ganglia on the sides The ganglia are small round white-clear trans-
lucent buttons located m small lacunae on the sides of the spinal column They
are attached to the roots, which connect them to the spinal cord The ganglia are
removed by chppmg with cornea1 scissors (see Fig 4)
14 They are then placed m 4% paraformaldehyde solution, and incubated for 12 h at
4°C to postfix
15 The ganglia are then placed m 40% sucrose solution overnight
16 The ganglia are then blotted dry on a paper towel. combined, and rolled mto a
ball using forceps
17 The ganglia are then placed on a bead of 0 C T compound and flash-frozen m
liquid nitrogen.
18 The tissue can then be stored m Eppendorftubes at -70°C or colder until sectlonmg
19. The ganglia are sectioned on a cryostat and mounted on slides The tissue can
then be processed for lmmunohlstochemlstry or zn sztu hybrldlzatlon

4. Notes
1 Probably the most important single parameter m determnung the efficiency of transfec-
tlon 1sthe quality of the transfectmg viral DNA. In order to work, the HSV transfectlon
DNA needs to be unit 1engtMhat IS, not sheared or degraded Care should be taken at
all steps after the SDS/Pronase digestion not to vortex or plpet the DNA vigorously
2 The exact amount of HSV-DNA used/transfectlon 1s generally m the range of
l-l 0 yg/60-mm dish of cells The optimal amount for a given DNA prep should
be determined emplrlcally by transfectmg dllutlons of the stock DNA and deter-
mmmg the concentration that yields the highest efficiency. Once this is deter-
mined for a particular stock of DNA, the proper amount of DNA should not vary
apprecrably from experiment to experiment
3 Although the DNA can be crosshnked to nylon membranes by UV n-radiation, we
have found that the baking method ts more efficient when performing dot-blots
4 When neutral red staining, it 1simportant not to leave the neutral red on too long,
US V Vectors


Camfully open spine
by cutting on ventral
midline to expose

- spinal cord

The position of ganglia is
remove these ganglia
delwmined by counting down
(from both sides)
2 vertebrae from the position
of the last rib. The Ll ganglia
am located on both sides of
this position.

- position of vertebrae

ventral wew 01 open spine

Fig. 4. Removal of mouse spinal ganglia.

since it will eventually kill the cells (rendering the plaques invisible).
5. Plaques can be screened by limiting dilution, but, in our hands, picking the
plaques and screening them directly speeds up the screening process and has
allowed us to identify some recombinants that we were unable to detect by limit-
ing dilutions. This may be owing to the fact that wild-type virus may have a
significant growth advantage over some recombinants.
6. Transfections can be performed using LipofectinTM (Life Technologies,
Gaithersburg, MD) or similar reagent. This method is quicker and yields results
comparable the CaP04 method presented here. However, we have noted greater
variability in the transfection efficiencies with Lipofectin. It should also be noted
that the optimal amount of transfection DNA should be determined separately for
the specific transfection protocol employed.
7. The plasmid DNA used for transfection should be from a CsCl preparation, since
impurities from minipreps seem to reduce transfection efficiencies.

8 Saline pretreatment of the feet increases the efficiency of uptake of the virus by some
loo-fold. Nonrephcatmg viruses can be inoculated by directly inJecting the virus mto
the sciatic nerve. Although this route is more efficient, only 10 uL can be injected. In
contrast, if the vuus is applied to saline-treated feet directly (80 uL), the estabhsh-
ment of latent infection is comparable to that obtained followmg sciatrc nerve mfec-
tions In addition, the footpad method of inoculation is much easier to perform
Technically, a nonrephcatmg virus has no acute phase though low level tran-
scription of some immediate early genes can be detected for several days after
maculation in mice (3) For this reason, a waiting period is still required prior to
evaluation of latent-phase gene expression.
It is important to infect a large enough number of mice to make the assay statisti-
cally significant. Remember-with animals, there is always some degree of varia-
tion to be expected Therefore, one should plan to infect at least 4 ammals/assay
point It is often helpful to infect some additional ones m case some die. Also
remember, since it takes 2 wk to establish a latent infection, it is useful to plan ahead
11 The mice should be completely anesthetized-not quivering or movmg One
wants them to remain under anesthesia to allow the moculum to stay on the feet
long enough for rt to adsorb efficiently If the animal IS quivering or rolls over,
give it 0 1 mL more Nembutol
12 If the ammals look as though they are under too deeply (shallow), a heat lamp
placed 3 ft from them to keep the cages warm during recovery is often helpful
13 You want Just to abrade the foot surface enough to be able to peel the layer of
skm off. Do not do it so forcefully that the feet bleed, however.
14 The LTR from the MoMuLV was removed from the plasmid pBAG obtained
from Conme Cepko (28).
15 Depending on the procedures used, the ganglia can simply be “fresh-frozen”
instead of perfused. In general, if the desired antigen survives the fixation pro-
cess, the tissue morphology is much better m fixed tissue. Even greater preserva-
tion of cell morphology can be obtained by paraffin embedding of the tissue.
It should be noted that some variation m the virulence properties of KOS strains
used m different laboratories has been reported This is likely because of repeated
passage of these stocks outside of the animal. Care should be taken m selectmg a
source for KOS that has demonstrated the desired virulence characteristics.
1. Stevens, J. G. (1989) Human herpesviruses a consideration of the latent state.
Muzroblol. Rev 53,3 18-332.
2. Rock, D. L. and Fraser, N. W. (1985) Latent herpes simplex virus type 1 DNA
contains two copies of the virion DNAJomt region. J Vzrol. 55, 849-852.
3. Sedarati, F , Margolis, T. P , and Stevens, J. G. (1993) Latent mfection can be
established with drastically restricted transcription and replication of the HSVl
genome. Virology 192,687-69 1.
4. Glorioso, J C., Goins, W F , and Fink, D. J (1992) HSV-based vectors. Sem
Vwol 3,265-276.
US V Vectors 385
5 Andersen, J. K., Garber, D. A., Meaney, C. A., and Breakefield, X. 0. (1992)
Gene transfer into mammalian central nervous system using herpes virus vectors:
extended expression of bacterial 1ucZ in neurons using the neuron-specific eno-
lase promoter. Hum Gene Ther. 3,487-499.
6. Davar, G., Kramer, M. F., Garber, D., Rota, A. L., Andersen, J. K., Bebrm, W.,
Coen, D. M., Kosz-Venechak, M., Knipe, D. M., and Breakefield, X 0 (1994)
Comparative efficacy of expression of genes delivered to mouse sensory neurons
with herpes virus vectors, J Comp Neurol. 339,3-l 1.
7. Dobson, A. T., Margolis, T. P., Sedarati, F., Stevens, J G., and Feldman, L. T.
(1990) A latent, nonpathogemc HSVl-derived vector stably expresses beta-
galactosidase in mouse neurons. Neuron 5,353-360.
8. Fink, D. J., Sternberg, L. R , Weber, P. C., Mata, M., Goms, W. F., and Glorioso,
J. C. (1992) In viva expression of beta-galactostdase m hippocampal neurons by
HSV-mediated gene transfer. Hum Gene Ther 3, 1l-19.
9. Wolfe, J. H., Deshmane, S. L., and Fraser, N W. (1992) Herpesvirus vector gene
transfer and expression of beta-glucuromdase m the central nervous system of
MPS VII mice. Nature Gen 1,379-384.
10. During, M. J., Naegele, J. R., O™Malley, K L., and Geller, A. I. (1994) Long-term
behavioral recovery m Parkinsonian rats by an HSV vector expressing tyrosme
hydroxylase. Sctence 266, 1399-1403.
11. Federoff, H J , Geschwind, M. D , Geller, A. I., and Kessler, J A. (1992) Expres-
ston of NGF in VIVOfrom a defective herpes srmplex virus 1 vector prevents effects
of axotomy on sympathetic ganglia Proc Natl. Acad Scl USA 89, 1636-1640
12. Margolis, T. P., Bloom, D. C., Dobson, A. T., Feldman, L. T , and Stevens, J. G.
(1993) LAT promoter activity decreases dramatically during the latent phase of
gangliaonic infection with HSV. Vzrology 197, 585-592.
13, Stevens, J. G., Wagner, E. K., Devi, R. G. B., Cook, M. L., and Feldman, L T.
(1987) RNA complementary to a herpesvirus alpha gene mRNA is prominent in
latently infected neurons. Sczence 235, 1056-1059
14. Bloom, D. C., Lokensgard, J. R., Maidment, N. T., Feldman, L. T., and Stevens, J.
G. (1994) Long-term expression of genes in vivo usmg nonreplicatmg HSV vec-
tors. Gene Ther. 1, S-36-38
15. Bloom, D. C., Maidment, N. T., Tan, A., Dissette, V. B., Feldman, L T., and
Stevens, J. G. (1995) Long-term expresston of a reporter gene from latent herpes
simplex vnus in the rat hippocampus. Mol. Brain Res. 31,48-60.
16. Lokensgard, J. R., Bloom, D. C., Dobson, A. T., and Feldman, L. T. (1994) Long-
term promoter activity during herpes simplex virus latency. J Viral 68,7 148-7 158.
17. Stevens, J G. (1987) Defining herpes simplex genes involved m neurovnulence
and neuroinvasiveness. Curr. Eye Res. 6,63-67
18. Efstathiou, S., Kemp, S , Darby, G., and Mmson, A. C. (1989) The role of herpes
simplex type 1 thymidine kinase m pathogenesis. J Gen Vwol 70, 869-879
19. Chang, J. Y., Johnson, E. M. J., and 011˜0, P. D. (1991) A gene dehvery/recall
system for neurons which utrhzes ribonucleottde reductase-negative herpes sim-
plex viruses. Virology 185,437-440.
386 Bloom

20 Pyles, R B , Sawtelle, N. M , and Thompson, R. L (1992) HSV type 1 dUTPase
mutants are attenuated for neurovtrulence, neuromvastveness and reactivatton
from latency J Vzrol. 66, 6706-6713
21 Johnson, D C., McDermott, M. R Chrisp, C , and Glorioso, J C. (1986) Pathoge-
mctty in mice of herpes simplex vu-us type 2 mutants unable to express glycopro-
tem C J Vwol 58, 36-42.
22 DeLuca, N A , McCarthy, A M., and Schaffer, P A (1985) Isolation and charac-
terization of deletion mutants of herpes stmplex vtrus type 1 m the gene encoding
unmedtate-early regulatory protein ICP4. J Virol. 56, 558-570.
23. Izumt, K. M and Stevens, J G. (1988) Two thymtdme kmase deficient herpes stm-
plex viruses exhibit unexpected vtrulence properttes. Mzcrob. Puthog 4, 145-l 53
24. Cameron, J M., McDougall, I , Marsden, H S., Preston, V. G , Ryan, D. M.,
and Subak-Sharp, J. H. (1988) Rtbonucleottde reductase encoded by HSV IS a
determinant of the pathogemctty of the virus in mice and a valid anttvtral target.
J Gen Vzrol 69,2607-2612
25 Yamada, Y , Ktmura, H , Mortshima, T., Datkoku, T., Maeno, K., and Ntshtyama,
Y (199 1) The pathogemctty of rtbonucleottde reductase-null mutants of Herpes
simplex vtrus type 1 in mice. J Infect Dzs. 164, 1091-1097.
26 Sedaratt, F and Stevens, J. G (1987) Btologtcal basis for virulence of three strams
of herpes simplex vtrus type 1 J Gen. Virol 68,2389-2395.
27 McGeoch, D J , Dalrymple, A J , Davtson, A J , Dolan, A , Frame, M. C ,
McNab, D., Perry, L. J., Scott, J E., and Taylor, P. (1988) The complete DNA
sequence of the long umque region m the genome of herpes stmplex virus type 1
J Gen Vzrol 69, 153 1-1574
28 Price, J., Turner, D., and Cepko, C. (1987) Lineage analysis in the vertebrate ner-
vous system by retrovnus-mediated gene transfer Proc Nat1 Acad Scr 84,
Investigation of the Anti-HSV Activity of Candidate
Antiviral Agents
Derrick J. Dargan

1. Introduction
Herpes simplex virus (HSV) 1sa human pathogen that causesdiseases rang-
ing in medical importance from herpes labiahs, through genital herpes and her-
pes keratitis, to herpes encephalitrs-a life-threatenmg disease.HSV types 1
and 2 have the abilrty to enter a latent phase during zn vzvo infectron, durmg
which time the virus IS able to evade immune survetllance, and from which
state it is able to escapefrom time to trme to cause disease,especially m rmmu-
nocompromised individuals. This characteristic makes antrvrral chemotherapy
an mdrspensable weapon m the management of recurrent herpesvirus mfectron.
The search for antivn-al compounds falls mto two patterns: first, random


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