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are analyzed with ImageQuant software (Molecular Dynamics) to quantltate the
[32P]-HSVand [32P]-a-actm probe hybridizationto their respectivePCR products.
3.3.3 2.4. Calculation of HSV- 7 Genome Copy Number
1 The ratio of [32P]-HSV gene probe hybrldlzation to [32P]-,-actm probe hybnd-
lzatlon m the HSV DNA standards are calculated.
2 A straight line is generated by plotting the log of these ratios vs the log of the
HSV- 1 copy number obtained from the standards.
3. The HSV-1 genome copy number per 100 ng of genomlc DNA m the trlgemmal
ganglion or superior cervical ganglion DNA samples is determined by fitting
their measured ratios to the standard curve.
3.3.4. Determination of HSV RNA
in Trigeminal Ganglia and Superior Cervical Ganglia
3 3.4.1. RNA EXTRACTION
1 TRIzol reagent is used for RNA extraction as described m the manufacturer™s
protocol The samples are homogenized in 1 mL of TRIzol reagent/5&100 mg of
tissue and incubated for 5 mm at room temperature
2. Chloroform (0.2 mL/l mL of TRIzol reagent) is added.
3. Then, the samples are shaken for 15 s, incubated at room temperature for 2-3
mm, and centrifuged at 12,000g for 15 mm at 4°C.
4. The mixture separates into a lower red, phenol-chloroform phase, an interphase,
and a colorless upper aqueous phase. RNA remains m the aqueous phase
5. The aqueous is transferred to 2-mL tubes and lsopropanol (0 5 mL/l mL of
TRIzol reagent) 1s added.
6. The samples are incubated at room temperature for 10 mm and centrifuged at
12,000g for 10 min at 4°C. The RNA precipitate forms a gel-like pellet on the
bottom of the tube.
7. The supernate is removed and the RNA pellet is washed once using 1 mL of 75%
ethanol per 1 mL of TRIzol reagent.
8. The samples are mixed and centrifuged at 7500g for 5 mm at 4°C.
9. The RNA pellet 1sair-dried for 5-10 min. Do not let the pellet dry completely as
the solubility will greatly decrease.
10 The RNA is dissolved m 100 pL RNase-free water or 0.5% SDS solution by
mixing, and then incubated for 10 min at 55-60°C The resulting solution of
RNA has an A 260.280 ratio between 1.8 and 2.1.
HI//, Wen, and Ha/ford
300
11. The solution can be stored at -70°C by adding 5M NaCl solution (8 pL) and
100% ethanol (500 pL)
3 3 4.2. REVERSE TRANSCRIPTION AND PCR ANALYSIS
1 Immediately prior to the reverse transcription reaction, the RNA standards are
diluted to 10slo* molecules/yL m 5 0 pL rlbonuclease(RNase)-free water
2. For each sample, approx 1 pg RNA m 5 0 pL RNase-free water IS needed
3 The RNA is denatured at 90°C and cooled to 67°C
4 The samples and standards are reverse-transcribed m 20 pL RT reaction buffer
5 The reaction mixtures are incubated at 23°C for 10 mm followed by 40 mm at 42™C
6 The reaction mix 1s extracted by the phenol-chloroform method and quantified
spectrophotometrically at 260 nm.
3 3.4.3. QUANTITATIVE DETERMINATION
OF HSV LATENCY ASSOCIATED TRANSCRIPTS (LAT)

The concentration of the cDNA, which contains the LAT and actm prod-
ucts, 1s adjusted to 10 ng/pL for quantitative PCR analysis. The same proce-
dure as 1s described above for quantltation of copy number of the HSV genome
can be used to calculate the number of LAT per cellular genome. For another
approach to quantltatlon of LAT, see ref. 30 for a detailed description of com-
petitive quantltatlve RT-PCR.
3.4. Identification of Latency Associated Transcripts (LAT)
3.4.1. Northern Analysis
Northern blot analysis 1sused widely to determine the molecular size, dlstn-
bution, and concentratton of RNA. Relative quantities of RNA often are calcu-
lated from optical density by densltometrlc scanning of Northern blot
autoradlograms. The method estimates the amount of a specific RNA by com-
paring the intensity of RNA hybridization in a test sample to that of an internal
standard conslstmg of a homologous copy RNA (cRNA)
1. After coelectrophoresls and transfer to nylon membrane, the Internal standard
cRNA and targeted RNA are detected by a radiolabeled nucleic acid probe.
2 In each case, variable amounts of internal cRNA control can be added to create a
standard curve (31)
3. The internal standard RNA can be synthesized from the specific DNA template
using T, RNA polymerase by the standard m Wro transcnptlon protocol (Promega)
4 The transcriptlon mix is treated with RQ 1 RNase-free DNase to remove the DNA
template and 1squantltated spectrophotometrlcally.
In Situ Hybridization
3.42
The production of two types of 3H probes IS described: one by PCR (29) and
the other by random hexamer priming (32-34). No other In sztu probes are
307
HSV Latency

described. Other Investigators have used [35S]-labeled nucleotldes (35-37) or
blotin-labeled nucleotides (38,39) for ISH.
3.4.2.1. I-AT-SPECIFIC PROBES
The PCR-created [3H]-LAT probe (29) 1sprepared as follows.
1. The dNTPs (dGTP, [3H]-dCTP, [3H]-dTTP, [3H]-dATP) are combined and dried.
2. LAT-probe primer (SKI, SKl.5) and LAT-probe DNA template (SKI, 2, 195 bp
post-PCR product) are added to the dried dNTPs.
3. PCR is performed as described except that 45 cycles are used.
4 The PCR products are transferred to a Mtlhpore Ultrafree-MC unit
5 TE buffer is added and the solutton 1scentrifuged at 7000g for 20 mm
6 The [3H]-DNA probe in the upper cup of the microcentrator is removed and tested
for purity in a 2.0% agarose gel. The 6 1-bp probe lies between bases 120702 and
120762 in the genome (40)
7. Stevens (41) and Hill et al (I 7) used a single-stranded [3H]-RNA probe prepared
by random hexamer priming.
8 The LAT probe IS prepared from a truncated Sal-&l fragment spanning 0 79%
0.798 p on the HSV genome This fragment IS cloned into pGEM and transcribed
by a T, promoter using a T, polymerase
9. The same [3H]-dNTPs are used as noted. The probe is degraded to 50-200 nucle-
otide fragments by mcubatlon in 200 mA4 NaOH prior to hybridization
3.4.2.2. TISSUE PROCESSING
1. The tissues (trtgemmal ganglia and superior cervical ganglia) are immersed m
4% paraformaldehyde and fixed at 4°C for 18-24 h.
2. Immediately after the fixation period, the tissue is dehydrated and embedded m
paraffin according to the following schedule: Ethanol at concentrations of 70, 80,
95, 100, 100, 100, each for 30 mm at room temperature; chloroform for 45 mm at
room temperature, followed by a change of chloroform for another 45 mm at
room temperature; paraffin for 30 min at 60°C, followed by a change of paraffin
for another 30 mm at room temperature.

3.4.2.3. PARAFFIN EMBEDDING
1 A metal mold is heated briefly over an open flame and filled with hot paraffin.
2. The ganglion is taken from the paraffin bath and oriented as destred.
3 The metal mold is placed on ice to sohdify the bottom of the paraffin.
4 The mold is filled with hot paraffin, and the embedding ring (Fisher, Pittsburgh,
PA) is placed on top.
5. The plastic ring is tilled completely to the top wtth more hot paraffin and refilled
two to three more times as the paraffin sohdtfies.
6. When the paraffin IS solid, the specimen IS kept overnight at 4°C. The resultmg
tissue blocks are stored at room temperature.
Hill, Wen, and Ha/ford
302
3 4.2.4. PARAFFIN SECTIONING
1 The paraffin block is trimmed mto a rectangular shape, mounted on the mtcro-
tome, and cut mto a ribbon of 7-pm secttons
2. The sections are separated m a water bath (45™C) and floated mdtvtdually, shiny
side down
3 When the sections have smoothed out, they are placed on Ftsher “plus” slides and
oriented
4. The slides are dried vertically and stored m a slide box containing desiccant

3.4.2 5. IN SW HYBRIDIZATION
1 Xylene or toluene can be used to deparaflimze the sections
2. Then the sections are washed with 100% ethanol for 5 min
3. The slides are mcubated m a protemase-K solution for 3 mm at 37°C and washed
three times in 1X PBS for 5 mm each time
4 The sections are refixed m 100% acetone for 2 mm and an-dried
5 Circles are drawn around each section with a PAP pen (KIYOTA International,
Elk Grove, IL)
The probe IS prepared as follows:
1. The amount of probe needed 1s calculated:
Vol (probe) = 86,400 dpm x nldpm/uL of probe
where n = the number of sections to be processed
2 This amount IS added to a sterile Eppendorf tube with an equal amount of 4X
hybridization buffer “mix ” The 4X solution and a solution of 20% dextran sul-
fate m formamide (42) are heated to 65°C
3. The total volume of diluted probe needed is calculated
Total vol of diluted probe = IZ x 25 pL
4. The volume of 2X hybridization buffer “mix” needed is calculated,
Vol = l/2 (total vol of diluted probe) - 2 (vol of probe)
and this volume is added to the probe m the Eppendorf tube.
5 The volume of 20% dextran sulfate needed 1s calculated
Vol = l/2 (total vol of dtluted probe)
6 Aliquots of the diluted probe (25 uL/sectlon) are added to each slide
7 The section is covered with a stlicomzed cover slip.
8 Au bubbles over sections are eliminated
9 The slides are heated to 100°C for 5 min to denature the DNA, after which the
slides are placed m moist chambers, incubated overnight at 42°C and washed m
0.2X SSC three times for 15 mm each.
10 The slides are completely dried before autoradtography
303
HSV Latency

3.4.2.6. AUTORADIOGRAPHY, DEVELOPING, AND STAINING
1 The radtolabeled slides are dipped m emulsion in a dark room wtth only sodmm hght
2. The slides require 10 d exposure, to ensure dryness, a desiccant 1s placed m the
slide box.
3. The slides are developed by washing m Kodak developer D- 19 (17°C) for 4 mm,
followed by washing m dH20 (18™C) for 1 mitt and immersion m Kodak fixer
(19Y) for 5 mm.
4. The slides are washed again in dH,O (20°C) for 5 mm, and then dried and stained
with Gtll™s H&E (43).
3.5. In Vivo Reactivation of Latent HSV Infection
3.5.7. /on tophoresis of Adrenergic Agents
1. Rabbits are anesthetized as described.
2. A sterile eye-cup 1s centered on the eye within the limits of the corneoscleral
hmbus and filled with epinephrme (0 0 1%).
3 The epmephrme is prepared from commerctal2% epinephrme and must be made
up immediately before use
4 The epmephrine solution is diluted with sterile, distilled, detomzed water
5 The solutton should be protected from heat and light Any tontophorests unit can
be used (I 7,44)
6. The anode (+) makes contact with the epinephrine solution in the eye-cup, and
the cathode (-) 1s attached to the ear over a saline-moistened cloth pad. Ionto-
phorests is at 0 8 mAmp for 8 min once daily and IS done on three consecuttve
days
7 Successful drug delivery 1s verified when pupillary dilation (mydrtasts) occur
within 30-60 mm after tontophorests.
Other soluttons that can be used m tontophoretic reacttvatton include
6-hydroxydopamine and timolol. 6-Hydroxydopamme (1%) must be prepared
immediately before admmtstratton (2445,461.
1. The powdered drug 1sdissolved in sterile, deionized, distilled water and the pH 1s
adjusted to 6 14.8 using sodmm hydroxide.
2. Ttmolol (0.01%) 1s prepared by dilution of 0 5% Ttmoptic with sterile, deion-
ized, dtsttlled water (47).
3. Eye swabs and eye washes are taken to momtor vtrus shedding for 7 consecuttve
days after mttiatton of tontophoresis.
3.5.2. Injection of Cyclophosphamide and Dexamethasone
1 Rabbits are given an tv mjectton of cyclophosphamtde (75 mg/kg) followed 24 h
later by an iv injection of dexamethasone (4 mglkg) (48)
2. Swabs and slit lamp examinations are performed to monitor the virus shedding
and cornea1 lesions for 8 consecutive days begmnmg on the day after injection of
cyclophosphamtde (49).
Hill, Wen, and Halford
304

3.5 3. Surgical Damage to Cornea/ Nerves
1 The rabbits are anesthetized and an anterior superficial keratectomy performed
by partial trephmation mto the superficial cornea1 stroma with a 7 O-mm trephme
2. The edge of the trephmed area IS grasped with forceps and peeled from the cen-
tral surface of the cornea.
3 The transection of the cornea1 nerves is performed with visualization under the
operating microscope Eight to ten cornea1 nerves can be identified as they enter
the cornea near the hmbus where they are cut with the tip of a no 11 Bard-Parker
blade (50)
3 5 4. Cryogenic Inpry
The rabbits are anesthettzed and a cryogemc injury is created by holding a
circular 8- to 9-mrn diameter piece of dry ice against the surface of the cornea
for 30 s This can form an ice ball over the entire cornea that thaws within 1
mm after the ice 1s withdrawn (51).
3 5.5. Intrastromal injection of Water
1 The rabbits are anesthetized and 100 HI sterile, deionized water IS injected
mtrastromally (52)
2 The rabbit eyeball is proptosed with a wooden cotton applicator and stabilized m
that position by an assistant An operating microscope can be used to facilitate all
surgical manipulations
3. A 30-gage short bevel needle attached to a 0.25mL tuberculm syrmge contam-
mg sterile water is mserted mto the central cornea1 sitroma and advanced until
the bevel is beyond the entry wound
4. The water is injected slowly; a grayish white circular bleb appears, which slowly
Increases m size
5. The needle is withdrawn and the proptosed globe is returned to the orbit (52)

4. Notes
1 Molecular mechanisms of latency: Establishment and maintenance™ HSV can
establish a lifelong latent infection in host neuronal tissues. Latent virus can be
reactivated and produce recurrent disease (I 7,18,47,49,.53) Two avenues are
available for the virus to reach a neuronal cell body to establish latent mfection
from the periphery by axonal transport; and from an adJacent cell in which HSV
is replicating Smce HSV is a lytic vuus, initiation of the viral rephcation cycle
eventually will shut off cellular protein synthesis and leads to cell death. This
results m either the establishment of a latent Infection in an adjacent cell or mfec-
tion of an adJacent cell, followed again by viral replication and cell death.
The estabhshment of the latent phase of infection probably IS controlled and
executed by the neuron. Evidence for this idea came from studies showing that
HSV DNA of biologically retrievable vu-us with deletions m the immediate-early
transcriptional regulatory gene ICP4 (which regulates replication) persists m the
305
HSV Latency



A

LAT




B
I
I
117009 2i
Hind -1 8

CE

4 A ˜cpo- Icp34.5
C

D 118801 8.5 kb LAT b 127167

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