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7 Kodak fixer and Kodak developer D- 19 (Eastman)
8. 2% Epinephrine (Epifren, Allergan, Irvine, CA).
9 0 5% Timoptic (trmoloi maleate; 0 5%, Merck, Sharp and Dohme, West Point,
PA)
10. Mineral oil-light white (Sigma, St. Louis, MO)
11. DNA dtgestion buffer 100 mM NaCl, 10 mM Trts-HCI, pH 8.0,25 mA4 EDTA,
0 5% SDS, and 0.2 mg/mL Protemase-K.
12 10X PCR buffer: 500 mA4 KCl, 100 mA4 Tris-HCl, pH 9 0 (at 25”C), 1% glyc-
erol, 25 mM MgCl, (Promega, Madison, WI)
13 PCR assay buffer 2.5 U Z™aqpolymerase, 10 pL of 10X PCR buffer, 50 PM
dNTPs, 50 ng of each HSV-1 primer, and 100 ng samples of gangliomc DNA.
14. RNase-free water To prepare RNase-free water, distilled water is added to
RNase-free glass bottles and diethylpyrocarbonate (DEPC) is added to a concen-
tration of 0 Ol%, after which the bottles are allowed to stand overnight and then
autoclaved
15 10X RT buffer. 100 rnA4Tris-HCl, pH 8 8, at 25™C, 500 mM KCl, and 1% Triton
X- 100 (Promega).
16. RT reaction buffer. 5 mMMgC12, 0 5 ug ohgo (dT) primer, 1 mMeach dNTP, 2 5
U rtbonuclease mhibitor (RNasm), 15 U AMV reverse transcriptase, m 20 pL 1X
RT buffer (Promega).
17. 2X Denhardt™s solution: 0.22% Ficoll, 0 02% polyvmyl pyrrohdone, 0.02% BSA
(fraction V)
18. 2X Hybridization buffer “mix”: 4X SSC, 0.2M sodium phosphate buffer (pH
6.5), and 2X Denhardt™s solution.
19. Terminal deoxynucleotidyl transferase (Promega)
20. TuqDNA polymerase (Promega)
2 1. T, promoter and T7 polymerase (Stratagene, La Jolla, CA).
22 RQl RNase-free DNase (Promega).
23. Rabbit DNA primers, Actin 1: S™AAGATCTGGCACCACACCTT-3™, and
Actin 2: S™CGAACATGATCTGGGTCATC-3™ The actin 1 and actm 2 primer
pair amplify a 125-bp product from the rabbit a-actm gene Probe Actm 1 5:
S™CTCGGTGAGCAGAGTGGGGTG-3™ HSV DNA primers and probes for
latency-associated transcript (LAT), thymrdme kmase (TK), and rlbonucleotlde
reductase (RR) genes are shown m Table 1.
Hill, Wen, and Ha/ford
294
Table 1
HSV Gene, Primer Pairs, Probe,
Sequence Location, Size After PCR, and References
Sue
of PCR
HSV-1 Sequence product,
gene Primer pairs (5 + 3™)” locattonb bp Ref.
LAT
5™-GACAGCAAAAATCCCCTGAG-3™ 120702+ 195 26
SK-l.
6120896
SK-2, 5™-ACGAGGGAAAACAATAAGGG-3™
t 120762
Probe 5™-CACACCAGCGGGTCTTTTGTGTTGG-3™
TK
TK-1. 5™-CTGCAGATACCGCTCCGTATT-3™ 47053+ 273 27
TK-2 5™-CATCTTCGACCGCCATCCCAT-3™ t47326
Probe. 5™-GTCAAGCTGCCCATAAGGTAT-3™ t47255
RR
RR-l: 5™-ATGCCAGACCTGTTTTTCAA-3™ 88517+ 243 15
RR-2. 5™-GTCTTTGAACATGACGAAGG-3™ t88759
Probe 5™-GGACACCAGCATGTCGCTCGCCGACTTTCA-3™ 88594+
“The topmostprimer m eachpau ISthe upstream (mRNA sense) prtmer, the bottom ISthe down-
stream(mRNA antlsense) primer Primers are chosen by use of a computer algorithm
hLocatlon IS based upon the complete sequence of the 17 Syn™ strain of HSV-1 Only the location m the
long internal repeat 1s indicated for LAT gene Arrows designate orientation of ohgonucleotlde 3™ end



24. 96-Well dot blot apparatus(Ltfe Technologtes)
25. Phosphortmager(Molecular Dynamics, Sunnyvale, CA)
26 Programmable PTC-100 thermal cycler (MJ Research,Watertown, MA)
27 Storage phosphor screen (Molecular Dynamics).

3. Methods
3.1. Rabbit Ocular Inoculation
All rabbit studies should conform to the ARVO Resolution on the Use of
Animals m Research.
3.1.1. Topical Inoculation
1 Scarified or unscarified eyes of New Zealand white rabbits (1 5-2 5 kg) are
infected with a suspenstonof HSV at 0 5 x lo6 plaque forming units (PFU) m
25 pL
2 Before scarification, rabbits are anesthetized with mtramuscular ketamme
(20 mg/kg) and xylazme (4 mg/kg), and topical 1% proparacame 1sapplied
to the eye.
295
HSV Latency
3 Scarrtication IS done with a sterrle needle, a double cross-hatch on the epithebum
is made with care to avoid woundmg the stroma
4. After virus 1sapphed to the eye, the eye is held open by pulling up on the eyelrds;
then the eye is closed and gently rubbed
3.1.2. lntrastromal Injection
1, Rabbits are anesthetized as described earlier.
2. With observation by means of an operating microscope, the central cornea1 IS
inoculated mtrastromally with a 32-gage needle to form five focal blebs (drce
pattern, total volume 50 pL containing 4 x lo5 PFU of vnus)
3. The cornea is then scarified superficially with eight needle scratches, forming a
square around the central intrastromal injection
4. Finally, the lids are closed and the eye is massaged
3.1.3. Superior Cervical Gang/ionic Injection
1. Rabbits are anesthetized, the neck area IS shaved, and the skin 1s washed with
70% alcohol.
2 A midline neck mctsron IS made, followed by blunt drssectron to separate the
neck muscles
3 The cervical sympathetic trunk is identified adjacent to the common carottd artery
and vagus nerve By following the sympathetic trunk to the bifurcatton of the
carotid artery, the superior cervical ganglion 1sidentified as a fusiform structure
4. HSV-1 ( lo5 PFU/mL) is injected (25 pL) into the superior cervical ganglion wtth
a 30-gage needle
5. Subcutaneous tissue and skin are closed separately with 4-O silk sutures.
6 An antibiotic powder (ampicillin) is placed m the wound area before the skin IS
closed (19). Aseptic technique 1sused for all procedures.
3.2. ldentificeflon of Viral hfection
3.2.1. S//t Lamp Biomicroscopic Examination
1. Primary cornea1 infection 1s verified by slit lamp examination wrth fluorescem
staimng on postmoculatron d 4-8.
2 Cornea1 epithehal disease is graded on a O-4 scale following fluorescem staining
(0, no stain; 1,25% of the corneas surface stained, 2,50% staining, 3, 75% stam-
ing; 4, 100% staining)
3. The eprthelial lesions generally are characterized as deep epithehal punctate, den-
drttic, or geographic.
4 The severity of cornea1 stromal disease can be graded on a O-4 scale (0, clear
cornea with iris detarls drstmctly visible, 1, detectable edema with u-1sdetails
clearly visible, 2, gross edema with stromal swelling, errs details still vrstble, 3,
gross edema with stromal swelling, puprllary border no longer distmctly visible,
some cellular mfiltratron possibly present; 4, an opaque cornea, anterior chamber
structures not vrsrble) (20-22).
Hill, Wen, and Ha/ford
296

3.2.2. Assay of Infectious Virus (Qualitative and Quantitative)
3.2.2.1. SwA0S
1 The eye IS opened by holding upper and lower eyelids as close to the hairless
border as possible
2. A sterile Dacron-tipped swab IS used to collect the tear film. The swab is gently
rotated in the upper cul-de-sac and then mto the lower formx, where the swab 1s
allowed to rest m the nasal fornix to absorb the tear film
3 Each swab is placed mto a screw-cap test tube containing 1 0 mL Eagle™s mmr-
mum essential medium (EMEM) with 2% fetal bovine serum (FBS)
4 The tubes are shaken m a water bath (37°C) for 90 mm and the swabs are trans-
ferred to tubes contammg confluent PRK cells.
5 The remaining solution m the original tube IS frozen (-7O™C) for later use m viral
trtratron Followmg a 1g-24 h mcubatlon at 37™C m a CO2 incubator, the swabs are
removed from the PRK cell-contammg tubes and EMEM with 2% FBS IS added
6 The tubes are monitored daily for 14 d for cytopathrc effect consistent with
HSV- 1 infection.
7 The frozen ahquots from all the samples that are positive for cytopathrc effect are
titrated on CV- 1 cells (23,24)
3.2.2 2 EYEWASH
1. The eye IS opened by holding the upper and lower eyelids as close to the hairless
border as possrble
2 Wash medium (0.1 mL) contammg EMEM with 2% FBS IS msttlled into the eye.
The eyelids are then closed and lightly massaged for 30 s
3 The eyelids are opened and the wash medium IS collected
4 Next, 0 3 mL of wash medium IS used to rinse the cornea and upper and lower
cul-de-sacs for viral collectron.
5. The eyes are massaged after each collectton
6. The sequence of wash, collection, and massage is repeated until a total of 1.5 mL
per eye is collected
7. An ahquot of the collected wash medium IS maculated onto mdtcator cells (24,25)
(Table 2)
3.3. Identification of Latency
3.3.7. Removal of Neural Tissue
3.3.1 1 RABBIT TRIGEMINAL GANGLIA
Aseptic technique is used to perform these procedures.
1. Rabbits are sacrtficed by intravenous injection of sodmm pentobarbttal, the hair
and skin are removed from the head, and a bone cutter IS used to break away the
skull and expose the brain
2 The brain is removed and the trrgemmal ganglia are exposed
297
HSV Latency

Table 2
Frequency and Titer of Ocular HSV-1 Shedding
After Adrenergic Induction
Days after EP/TEa (%) Ttteti EP/TE (%) Titer,
induction swab onlyb log,, PFU swab onlyC log0 PFU™
None o/12
o/10 None
(0)
(0)
1.2
7/10 (70) 8/12 (67) 14
9/10 (90) 1.7 10/12 (83) 3.0
2.6 9/12 (75) 33
8/10 (80)
11/12 (92)
7/10 (70) 2.2 3.7
4/10 (40) 2.0 10/12 (83) 3.4
l/10 (10) 2.5 8/12 (67) 27
None Not done
o/10 Not done
(0)
aEP/TE = eyes posltwekotal eyes
hData are from ref. 2.5.
CData are from ref 24 (avg Figs 1 and 2)


3 The large centrally located ophthalmic nerve IS severed from the opttc chrasma
4. The third (oculomotor), fourth, and sixth cranial nerve pans are laterally located and cut
5. The trtgeminal nerve IS cut and the trtgeminal ganglia are exposed and removed,
and then either frozen immedrately in liquid nitrogen for nuclerc acrd extractron,
or put in 4 0% paraformaldehyde for zn sztu hybridization (ISH), or cut into sev-
eral pieces and cocultured m the medium contammg PRK cell monolayers
3 3.1.2. RABBITSUPERIORCERVICALGANGLIA
1. The ventral neck area IS shaved and the skin IS washed with 70% alcohol
2. Make a midline neck mctslon and blunt dissectton to separate the neck muscles.
3 Followmg the sympathetic trunk to the bifurcation of the carotid artery, the supe-
rior cervical ganghon is identified and removed as noted.
4. The superior cervical ganglion is processed tn the sameway asthe trlgemmal ganglion.
3.3.2. Coculture of Trigeminal Ganglia and Superior Cervical Ganglia
1. The trtgeminal ganglia and superior cervical ganglia are removed, washed in cold
(4°C) EMEM, and then outer sheaths are removed.
2. The ganglia are cut into several pieces and cocultured on PRK cell monolayers.
3. The cultures are monitored for cytopathic effect for up to 28 d
3.3.3. Determination of HSV DNA
in Trigeminal Ganglia and Superior Cervical Ganglia

3.3.3.1. DNA EXTRACTION
1. The frozen trigeminal ganglion or supertor cervical ganglion is rapidly thawed,
298 Hill, Wen, and Halford
ground with a pestle, and homogenized m a specrfic volume (600 uL for trigemi-
nal ganglion, 300 uL for superior cervrcal ganglion) of digestron buffer
2 After an 18-h mcubatron at 50°C the DNA IS phenol extracted and the DNA
concentration IS adjusted to 10 ng/uL
3 3.3.2 PCR AMPLIFICATION OF HSV-1 AND RABBIT a-ACTIN DNA
3.3.3.2 1 Standard PCR
1 Three pans of ohgonucleotrde primers are chosen from regions of HSV-1 The
primer pairs correspond to regions located m the latency-associated transcript
(LAT), thymrdme kmase (TK), and ribonucleotrde reductase (RR) genes (Table 2)
2 100 uL of PCR assay buffer IS overlaid with mineral or1 and heated for 5 mm at
97°C Tuq polymerase (2 5 U) are then added under the mineral or1 layer
3 PCR is performed for 40 cycles of 75 s at 94™C, 75 s at 56°C and 30 s at 72™C
4. After the PCR, 20 uL of each reaction IS electrophoresed m a 2 0% Tns-borate-EDTA
agarose gel.
5 The PCR products can be vtsuahzed on the gel, or can be transferred to a nylon
membrane for dot or Southern blot analysis.
3 3 3 2 2. Quantitative PCR. This procedure mvolves the simultaneous
amplification of a target sequence and a control sequence from the same DNA
sample. The target is the sequence whose concentration is to be measured. The
control is a sequence whose concentratron IS constant in all DNA samples
assayed. The yield of control PCR product provides a measure of the amphti-
catton efficiency and consrstency of each reaction.
The HSV-1 genes, RR, TK, and LAT (Table 2), are amphtied to determine
the concentratron of vu-al genomes m DNA samples. One genome equivalent
of HSV DNA was calculated to be 0.166 fg, assuming a massof I O* Dalton for
HSV DNA. The rabbit a-actm gene IS used as the internal control sequence.
The primers used to amplify portions of these sequencesare Actm 1 and Actin 2.
The pair of primers amplifies a 110-bp product from the rabbit a-actm gene.
A standard PCR procedure rs modified to permit quantrficatron of HSV DNA.
1. Umnfected rabbtt ganghomc DNA (100 ng) spiked, respectively, wrth known amounts
(5, 10,20,40,80 copy numbers) of HSV DNA (as standards to determme gene copy
numbers) are rmxed with 50 pmol each of actm pnmers and HSV DNA prtmers
2. For precision, the assay must be repeated using very drlute HSV DNA samples
(i.e., 0.1,0.5, 1,2,5 copy numbers). This establishes the sensmvny and precrsron
of the reactron.ThesePCR samples,referred to asHSV DNA standards, the
are
standard curve for each quantrtatrve PCR assay
3 Then, infected rabbit ganghomc DNA (100 ng) IS mixed with 50 pmol each of
actm primers and HSV DNA prrmers
4. All these PCR samples are assembled as described
5. The samples are heated at 97°C for 5 mm, after which 2.5 U of Tag polymerase
are added.
299
HS V Latency
6 The quantitative PCR assay is performed for 30 cycles of 1 mm 15 s at 94”C, 1
min 15 s at 56”C, and 10 s at 72°C (28,29) These optimal condltlons have been
determined by standard procedures (29)
The Zeta-Probe GT nylon membrane
3 3 3.2 3. Analyszs of PCR Products
(Bio-Rad, Hercules, CA) and a 96-well dot blot apparatusare used for dot blotting.
HSV-specific and a&n-specific [32P]-end-labeledoligonucleotlde probes are used for
hybndizatlon.The radioactive membranes scanned aPhosphorlmager.Images
are with

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