Journal of Medical Virology 25259-270 (1988)

Detection of Herpes Simplex Virus Type 1

in Herpetic Ocular Diseases by DNA-DNA
Hybridization Using a Biotinylated DNA
Probe
Ryosuke Nago, Kyoko Hayashi, Hiroshi Ochiai, Yasuo Kubota, and
Seihachiro Niwayama
Department of Ophthalmology (R.N., Y K.) and Department of Virology (K.H., H. O.,
S.N.), Toyama Medical and Pharmaceutical University, Toyama, Japan

A diagnostic hybridization assay for detecting herpes simplex virus type 1 (HSV-
1) in ocular specimens was developed using cloned viral DNA as a probe. This
hybridization assay is based on visualizing a biotinylated probe that is hybridized
to the target DNA by a streptavididalkaline phosphatase system. The time re-
quired for performing this assay system is only two days. This assay system could
detect a probe which had been hybridized to as little as 1 pg of homologous DNA
and did not cross-react with DNA of other human herpes viruses except that of
herpes simplex virus type 2 (HSV-2) which showed weak cross=reactivity. The
assay system was applied to experimental keratitis in albino rabbits and clinical
specimens. In experimental keratitis in rabbits it was possible to detect HSV-1
DNA in the eye swab samples at least until the ninth day after virus inoculation.
Five clinical specimens collected from patients with corneal ulcer or blepharitis
contained HSV-1 DNA in spite of the failure of demonstration of viral antigen
and/or virus isolation in two cases.

Key words: slot-blot, herpetic keratitis, sensitivity of detection system, specificity

INTRODUCTION
Herpes simplex virus (HSV) is a major cause of ocular infections such as
keratitis, conjunctivitis, and uveitis, and it may even cause acute retinal necrosis
[Culbertson et al., 1982; Sarkies et al., 19861. Moreover, ocular herpes is a leading
cause of corneal blindness, of which approximately 300,000 cases are reported
annually in the United States [Notluns et al., 19731. Therefore, accurate diagnosis

Accepted for publication January 15,1988.

Please send reprint requests to R. Nago, Department of Ophthalmology, Toyama Medical and Pharma-
ceutical University, 2630 Sugitani, 930-01 Toyama, Japan.

0 1988 Alan R. Liss, Inc.

260 Nagoetal.

and immediate treatment of herpetic eye diseases are vitally important. Various
laboratory procedures such as the detection of the significant rise of serum antibody
titers, cytological procedures, virus isolations [walpita et al., 19851, and the immu-
nological assay by immunoperoxidase method [Herbort et al., 19851 have been used
so far for diagnosis of HSV infections. However, each method has some disadvan-
tages. Almost all adults have already contracted a primary HSV infection during their
childhood [Longson, 19781; thus, diagnostic rises in antibody titers over a four-week
interval can not be expected [wilhelmus et al. , 19861. The cytological procedures can
not be decisive because of their lack of specificity, and virus recovery by cultural
assay is sometimes time-consuming . Assays based on immunological procedures such
as the immunofluorescent method and the peroxidase anti-peroxidase method are
apparently easily performed, but their specificity and sensitivity still remain question-
able [Nahmias et al., 19821.
On the other hand, the hybridization assay relies on rhe requirement that strands
be complementary as a precondition for strand reassociation; so, denatured DNA
from HSV-infected cells that are immobilized on nitrocellulose filters can be detected
by hybridization using a specific and labeled probe. The probe DNA is typically
labeled with radioactive nucleotides, hybridized to the immobilized target DNA, and
identified by autoradiography . The assay, using a radioactive probe, can detect
specifically as little as 5 pg of homologous HSV type 1 (HSV-1) DNA [Schuster et
al., 1986aI. Applications of this radioactive system for the detection of various kinds
of viruses have been reported from various fields [Andiman et al., 1983; Barker et
al., 1986; Berger et al., 1985; Brandsma and Miller, 1980; Engleberg and Eisenstein,
1984; Gibson et al., 1986; Roy et al., 1985; Schuster et al., 1986b; Seidlin et al.,
1984; Spector et al., 1984; Taylor et al., 1984; Virtanen et al., 19831. However,
radioactive probes are inconvenient for clinical use because they require special
handling, due to their short half-life and the risk of exposure hazard.
To overcome these disadvantages, an alternative approach, a biotin-avidin
system, had been developed [Leary et al., 19831. This method makes use of the high
affinity between biotin and avidin. The biotinylated probe, hybridized to the target
DNA, can be detected with an avidin-enzyme conjugate and color producing sub-
strate. With recent improvements, this can be as sensitive a method as autoradiogra-
phy. Moreover, the advantages of safety and long probe life make this method a likely
candidate to use in clinical laboratories. Consequently, we investigated the specificity
and sensitivity of this system using HSV-infected ocular cultured cell lines and
experimental keratitis in albino rabbits. We also applied this assay to detect HSV-1
DNA in clinical specimens and demonstrated that this procedure was useful for
screening clinical specimens.

MATERIALS AND METHODS

Cell Culture and HSV-1 Virus Infection
HeLa 229 cells and SIRC cells, a cell line from rabbit corneal stromal fibroblasts
established by M. Volkert (Statens Seruminstitut, Copenhagen, Denmark), were
grown in Eagle's minimum essential medium (MEM, Flow Laboratories, Rockville,
MD, USA) supplemented with 10% fetal calf serum (Filtron, Victoria, Australia).
Chang cells, a cell line derived from human conjunctival cells, were grown in
Dulbecco's modified Eagle's medium (D-MEM, Flow Laboratories) supplemented
 Detection of HSV-1 DNA in Ocular Tissues 261

with 10% fetal calf serum. Human embryonic lung fibroblast (HEL) cells were
cultured in MEM supplemented with nonessential amino acids (Flow Laboratories),
0.1 % lactoalbumin hydrosate, 1 mM sodium pyruvate, and 10% fetal calf serum, and
human B lymphocyte (P3HR-1) was cultured in RPMI-1640 (Flow Laboratories)
supplemented with 10% fetal calf serum. These cell lines were cultured at 37°C in a
humidified atmosphere of 5 % C 0 2 and 95% air.
Virus Infection
HeLa 229, Chang, and SIRC cells were grown to confluence in 24-well plastic
plate (Corning No. 25820), washed with phosphate buffered saline (PBS), and
infected with HSV-1 HF strain at a multiplicity of infection (MOI) of 2 plaque-
forming units (PFU)/cell followed by adsorption at 37°C for 2 hr. After washing
three times with prewarmed PBS, the prewarmed maintenance medium (medium
supplemented with 2 % fetal calf serum) was added and the cells were incubated at
37°C. This time corresponded to zero time after infection. At the indicated times,
cytopathic effect was observed using phase-contrast microscopy and the virus yield
was determined by plaque assay on HeLa 229 cells.
Monolayers of HeLa 229 cells in 24-well plastic plate were washed with PBS
and infected with HSV type 2 (HSV-2), adsorbed for 2 hr at 37°C and refed with
maintenance medium (MEM plus 2 % fetal calf serum). The infection of varicella
zoster virus (VZV) and human cytomegalovirus (HCMV) to HEL cells was treated
in the same manner as that of HSV-2.
Preparation of DNA Samples From HSV-1 Infected Cell Lines
When cultures showed advanced cytopathic effect (CPE) after infection with
HSV-1, viral DNA samples were prepared by three different procedures: (a) the
boiling method in which harvested cells were suspended in PBS and processed by
boiling for 3-5 min followed by cooling on ice. After centrifugation (1,500 rpm, 10
rnin), the supernatant was sequentially diluted and applied to slot-blot apparatus
(HYBRI-SLOT'" manifold, BRL, Gaithersburg, MD, USA); (b) the Nonidet P-40
method in which cells were washed twice in PBS and suspended in 10 mM Tris, pH
7.5, 1 mM EDTA, 150 mM NaCl and 0.6% Nonidet P-40. After 10 min incubation
on ice, the suspension was vortexed and clarified by low speed centrifugation. The
supernatant was then subjected to (c), the proteinase K method, in which, after
treatment with Nonidet P-40 and Centrifugation as described in (b), the supernatant
was treated with proteinase K (0.1 mg/ml) at 37°C for 3 hr and then applied. All the
samples were boiled for 10 min immediately before being applied to slot-blot
apparatus.
Host DNA samples from noninfected HeLa cells were prepared by method (b)
mentioned above. DNA samples of other human herpes viruses were also prepared
by method (b).
Preparation of a Biotinylated DNA Probe
HSV-1 (F strain) DNA Bam HI Q fragment cloned in pBR 322 was received
from Dr. Y.-C. Chang, University of North Carolina, USA. Escherichia coli HB 101
cells were then transformed, amplified, and the recombinant DNA extracted and
purified as described by Maniatis et al. [1982]. This fragment represents a portion of
the long, unique sequence of the genome and is part of the region coding for thymidine
 262 Nagoet al.

kinase. For the hybridization experiment, the whole plasmid DNA (pTK-1) was
labeled with biotin-ll-dUTP (BRL) by nick translation [Rigby et al., 19771 employing
commercially available reagents (Nick Translation Reagent Kit, No. 8160SP, BRL).
The biotin-labeled DNA was separated from unincorporated nucleotides by gel filtra-
tion on Sephadex G-50.
Purification of Homologous Viral DNA
HSV-1 DNA was isolated and purified according to Yanagi et al. [1979]. The
yields of cell-associated and non-cell-associated DNA from 2 x lo8 infected HeLa
cells were about 50 pg and 30 pg, respectively.
Prehybridization Treatment and HybridizationConditions
Nitrocellulose filters (Schleicher and Schuell, BA-85, Keene, NH, USA) were
rinsed with distilled water, then fixed to the HYBRI-SLOT manifold. Cell specimens
in volume of 100 pl were applied to each well of the manifold. Each well was then
washed successively at room temperature twice, first with: 200 pl of 6x SSC (1 x
SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate) and then with 200
p1 of 2 x SSC. The nitrocellulose filters were air-dried and then baked at 80°C for
3 hr in an oven. The filters were prehybridized in thermally sealed plastic bags for at
least 2 hr at 42°C in 5 ml of a solution containing 50% deionized formamide, 5X
SSC, 5 x Denhardt’s solution, 25 mM sodium phosphate buffer (pH 6.5) and 250 pg
heat-denatured salmon sperm DNA/ml. The prehybridization solution was then dis-
carded and replaced with 1 pg of heat-denatured, nick-translated HSV-1 cloned probe
in 5 ml of 45% deionized formamide, 4X SSC, 1 X Denhardt’s solution, 20 mM
sodium phosphate (pH 6.5), 10% (w/v) sodium dextran sulfate and 200 pg of heat-
denatured salmon sperm DNA/ml. Hybridization was carried out at 42°C in thermally
sealed plastic bags for 16-24 hr with gentle shaking. A series of slots containing
known amounts of HSV-1 DNA was prepared on each filter as a control.
Detection of HSV-1 DNA Probe Binding
The post-hybridization washes were performed as follows:
(a) Wash filters twice in 50 ml of 2 x SSC and 0.1 % SDS for 3 min at room
temperature with gentle agitation;
(b) Wash filters twice in 50 ml of 0.2 X SSC and 0.1 % SDS for 3 min at room
temperature with gentle agitation;
(c) Wash filters twice in 50 ml of 0.16 X SSC and 0.1 % SDS for 15 min at
50°C;
(d) Briefly rinse filters in 2 x SSC at room temperature.
After the final post-hybridization wash, biotinylated probes were detected by
means of a streptavididalkaline phosphatase kit (BluGENE, No. 8279SA, BRL)
under instructions provided by the manufacturers.
Detection of HSV-1 DNA in Experimental Keratitis in Albino Rabbits
After one drop of 0.4% oxybuprocaine HCl, three New Zealand albino rabbits
(1.5-2.0 kg) were inoculated with 10 pl of lo8 PFU/ml suspension of HSV-1 HF
strain in both eyes following corneal epithelial scarification (ten vertical and horizon-
tal strokes with a #25 needle). After confirming establishment of infections with the
aid of slitlamp biomicroscopy using fluorescein staining, the corneas were gently
 Detection of HSV-1 DNA in Ocular Tissues 263

swabbed with sterile cotton tips on days 5 , 7, 9, and 21 postinoculation. Cotton tips
were then washed with PBS in sterile polyethylene tubes followed by centrifugation
at 1,OOO rpm for 10 min, and the pellets were used for detecting HSV-1 DNA. At the
same time, smears were prepared for detection of virus antigen by indirect fluorescent
antibody technique.

Detection of HSV-1 DNA in Clinical Specimens

The clinical specimens were collected from six patients who had lesions of
suspected HSV infections, and from two additional patients who suffered from
bacterial infections. They were processed in the same manner as described above in
order to detect HSV-1 DNA. The sample sources and clinical features of eight patients
are summarized in Table 11.

lrnmunofluorescence(IF)
Smears were prepared on glass slides from biopsy, then air-dried and fixed in
cold acetone for 10 min. The fixed smears were stored at -20°C until examined for
the presence of HSV-1 antigens using indirect immunofluorescence with anti-HSV- 1
antiserum.

RESULTS
Establishment of Optimum Conditions for DNA Extraction in
Respect of Sensitivity
First, to estimate the sensitivity of the biotinylated probe used in this assay, the
hybridization between the probe and the appropriate concentrations of homologous
HSV-1 DNA extracted from purified viruses was tested. As shown in Figure 1, this
biotinylated probe could detect as little as 1 pg of homologous HSV-1 DNA.
The three different methods for DNA extraction, the boiling method, the
Nonidet P-40 method and the proteinase K method, were compared with respect to
sensitivity. As shown in Table I, the Nonidet P-40 and proteinase K methods were
excellent because they were five times more sensitive than the boiling method in
Chang and HeLa cell lines, and 10 times, in SIRC cell line. Thereafter, we adopted
the Nonidet P-40 method for DNA extraction. Figure 1 shows representative data
obtained by the Nonidet P-40 method. As the result of Nonidet P-40 method the assay
could detect DNA from at least I d infected cells in both SIRC and HeLa 229 cells,
and from at least lo3 infected cells in the case of Chang cells. The virus yields at 36
hr postinfection in Chang, SIRC, and HeLa 229 cells were 6, 28, and 31 PFU/cell,
respectively (see Discussion).

Specificity of the Probe

Biotinylated pTK- 1 probes were examined for specificity by comparing their
reaction with: HSV-1 infected HeLa 229 cells, HSV-2 infected HeLa 229 cells, VZV-
infected HEL, Burkitt lymphoblastoid cells (P3HR-1), and HCMV-infected HEL
cells (Fig. 2). The number of the infected cells applied was lo6 cells in each cell line.
The results revealed that the probe did not react with DNA from cells infected with
any other human herpes viruses except HSV-2. HSV-2 DNA showed weak cross-
reactivity with the HSV-1 recombinant DNA probe (Fig. 2).
264 Nagoet al.

1 2 3 4 5 6

Fig. 1. The determination of the minimum cell number to identify HSV infection by slot-blot hybridiza-
tion with the biotinylated HSV plasmid thymidine kinase-l DNA probe. The various cell lines (A:Chang,
B:SIRC, and C:HeLa 229) infected with HSV-1 at a MOI of 2 PFU/cell were treated with the solution
containing Nonidet P-40. The applied cell numbers were lo5 (lane l), lo4 (lane 2), lo3 (lane 3 ) , lo2
(lane 4), 10' (lane 5 ) , and lo6 (lane 6: uninfected HeLa 229 cell control). Row D shows titration of
homologous HSV-1 DNA: 100 pg (Dl), 10 pg (D2), 5 pg (D3), 1 pg (D4), 0.1 pg (D5), 0 pg (D6).

TABLE I. The Difference in Sensitivity Among Three Cell Lines

Number of infected cellsa
Nonidet and
Boiling Nonidet proteinase K
Cell line method method method
Chang 5 ,OOo 1,OOo 1,Ooo
SIRC 1,OOo 100 100
HeLa 229 500 100 100
'Cells were infected with HSV-I at a MOI of 2 PFU/cell. At an advanced CPE, 36 h postinfection, cells
were harvested and subjected to three different treatments. The number of cells represent the limit of
detection of viral DNA.

The probe was also examined with DNA from the uninfected cells (1 X lo6
cells) with no detection of hybridization (Fig. 1, slots A6, B6 and C6).
Detection of HSV-1 DNA in Experimental Keratitis and Clinical Specimens
The high sensitivity of this method in cultured cells suggested that this procedure
might be useful for detecting HSV-1 DNA in clinical specimens. We first investigated
specimens from experimental keratitis in albino rabbits. Viral DNA could be detected
in all six samples from rabbit corneas collected on days 5 , 7, and 9 postinoculation.
Samples collected from two rabbits on day 21 postinoculation were also examined.
 Detection of HSV-I DNA in Ocular Tissues 265

Fig. 2. Slot-blot hybridization with the biotinylated HSV-1 plasmid thymidine kinase-I DNA probe.
After treatment with Nonidet P-40and proteinase K, samples (1 x lo6 cells) were applied to each well.
1, HSV-I infected HeLa cells; 2, HSV-2 infected HeLa 229 cells; 3, varicella-zoster virus infected
human embryonic lung fibroblasts (HEL); 4, Burkitt lymphoblastoid cells (P3HR-1); 5, human cytomeg-
alovirus infected-HEL cells; and 6 , uninfected cell control (HeLa 229 cells).

Fig. 3. The detection of HSV-1 DNA from corneal swabs of experimental keratitis in albino rabbits
with the biotinylated HSV-I plasmid thymidine kinase-I DNA probe. Lane 1, 5 pg of homologous DNA
from HSV-1 infected HeLa 229 cells; lane 2, 20 pg of homologous DNA; lane 3, corneal swab from
the right eye of rabbit (#1) at 3 weeks after infection with HSV-1; lane 4, corneal swab from the left eye
of the same rabbit as in lane 3; lane 5, corneal swab from the right eye of rabbit (#2) at 3 weeks after
infection with HSV-1; lane 6, corneal swab from the left eye of the same rabbit as in lane 5 ; lane 7,
uninfected HeLa 229 cells (1 X lo5 cells).

HSV-1 DNA was detected in both eyes of one rabbit and the right eye of another
rabbit, while HSV-1 DNA was not detected in the left eye of the latter rabbit (Fig.
3). When the HSV-1 antigen in smear preparations were examined by IF, the DNA
assay agreed with antigen detection by the IF method.
In addition, six clinical specimens collected from six patients who had lesions
with suspected herpetic infections and two specimens from control patients with
bacterial keratitis and conjunctivitis were examined. Table I1 shows patient profiles
and the results of the DNA assay. Case 1 had previously received 0.5% idoxuridine
(IDU) ophthalmic solutions for therapy of corneal ulcers. After antiviral therapy had
been discontinued for two weeks, the sample was collected from the ulcer with a
cotton tip (Fig. 4). Although we could not detect HSV-1 antigen by IF, we succeeded
in demonstrating the presence of HSV-1 DNA (Fig. 5). Treatment with 9-(2-hydroxy-
ethoxymethy1)-guanine (acyclovir, ACV) was then started, and the ulcer had almost
disappeared within a month. In case 2, the ACV therapy for herpetic stromal keratitis
was continued for a month. When the patient came to our clinic, skin vesicles were
partially ruptured and crusts had formed. These crusts were examined for HSV-1
266 Nag0 et al.

TABLE 11. Results of HSV-1 DNA Hybridization in Eight Patients

Serum antibody Immunofluorescence Clinical
Case Age Sex Source titer (CF) DNA assay (and/or virus isolation) diagnosis
1 61 F" Corneal 16 Herpetic
swab keratitis
2 21 Ma Palpebral 64 Herpetic
CNStS & blepharitis
vesicles
3 49 M Corneal 16 Herpetic
swab keratitis
4 57 M Corneal 16 Herpetic
swab keratitis
5 35 F Corneal 32 Herpetic
swab keratitis
6 42 F Corneal <4 Herpetic
swab keratitis
suspected
7 66 F Corneal 16 Bacterial
swab keratitis
8 82 M Coniunctival 16 Bacterial
swab conjunctivitis
"F, female; M, male.

Fig. 4. Case 1 corneal ulcers stained with fluorescein. The corneal swab was taken from the ulcer at 8
o'clock position near the limbus.

DNA. As shown in Table 11, a positive result was obtained; therefore, ACV ophthalmic
ointments were applied to the eye lid, and the lesion was cured completely in a week.
The positive sample in case 3 was collected from the typical dendritic corneal ulcer.
In case 4, after the patient had an episode of acute pancreatitis, corneal ulcers
appeared in both his eyes. Case 5 had previously undergone successive ACV therapy
for herpetic stromal keratitis for 2 months; HSV-1 DNA was detected in this case but
demonstration of HSV-1 antigen failed. Case 6 was given steroid ophthalmic solution
 Detection of HSV-1 DNA in Ocular Tissues 267

Fig. 5. The detection of HSV-1 DNA from case 1 corneal swab. Lane 1, HSV-1 DNA 20 pg; lane 2,
samples from case 1 corneal swab; lane 3, uninfected HeLa 229 cells (1 X lo5).

therapy for scleritis. After about 3 weeks of steroid therapy, a geographical-like ulcer
appeared in the right eye. The patient had a definite decrease of corneal sensation in
the affected eye; thus, the diagnosis of herpetic keratitis was made only from clinical
signs. Corneal swabs were collected to examine HSV-1 DNA; however, the result
was negative. The complement-fixing (CF) antibody titer was also not detectable
( < 1:4) days 14 after the appearance of ulcer. In cases 7 and 8, both of whom had
bacterial infections caused by alpha streptococcus and nonfermentative gram negative
bacilli, viral DNA could not be detected from the samples.

DISCUSSION
A diagnostic hybridization assay for detecting HSV-1 in clinical specimens was
developed based on the results obtained from infected ocular cell lines and experimen-
tal keratitis in albino rabbits.
The hybridization and enzymatic detection protocol reported here offer several
advantages over conventional procedures in which radioactive probes and autoradio-
graphic detection are used. From a practical standpoint, in addition to being free from
special handling and risk of the exposure hazard, chemical stability of probes is
considered to be the most advantageous. In fact, it has been reported that biotinylated
probes can be used for at least one year and still yield reproducible hybridization
results [Leary et al., 19831. Furthermore, this assay system saves the time required
for the exposure that is necessary in autoradiographic detection.
To improve the sensitivity of this assay system, some attempts were made with
reference to previous papers [McKeating et al., 1985; Schuster et al., 1986aI. Because
McKeating et al. [1985] reported that the assay sensitivity could be increased when
plasmid sequences were removed before labeling, the difference in sensitivity between
the two DNA probes (pTK-1, which contains plasmid sequences, and the purified
Barn HI Q fragment) was also investigated. However, the results did not show any
increase in its sensitivity when a purified Bam HI Q fragment was used as a probe
(data not shown). Therefore, the whole plasmid DNA probe was used throughout in
this study. In this connection, Schuster et al. [1986a] reported that gel impurities used
for the isolation of restriction enzyme-digested DNA fragment and contaminated
during electrophoresis for separating DNA, might decrease the nucleotide incorpora-
 268 Nagoetal.

tion rate in nick translation, and thereby prevent the increase in sensitivity of the
assay.
In addition, it was demonstrated that the extraction methods of DNA from the
infected cells acted as a potential factor influencing the sensitivity of hybridization
assay, as reported by others [McKeating et al., 19851; that is, a significant difference
was shown between the boiling method and both Nonidet P-40 and proteinase K
methods, but not between the two latter methods. Therefore, the treatment that used
only Nonidet P-40 seems to yield sufficiently good results.
According to the standardized protocol described above, streptavidin-alkaline
phosphatase conjugate, in combination with the NBTIBCIP substrate, was sufficiently
sensitive to visualize 1 pg of homologous DNA (the equivalent of about 5,000 viral
genomes). In the experiments using infected cells, this assay system could detect viral
DNA from lo3 infected Chang cells, 102 SIRC, and lo2 HeLa cells. The virus yield
per cell in Chang cells was about one-fifth of that in the two latter cell lines, when
determined by plaque assay on HeLa cells. This might be the reason why many more
cells were required for the detection of HSV-1 DNA in Chang cells, compared with
the other cell lines. When the viral genome number per infected cell was calculated-
taking into account the fact that 1 pg of viral DNA is equivalent to about 5,000 viral
genomes-on average 50 viral genomes per infected SIRC or HeLa 229 cell were
present at the limits of detection.
This assay system also provided a very specific probe which did not hybridize
to human cellular DNA or to the DNA of other herpes viruses, except that it faintly
bound to HSV-2 DNA. The nucleotides encoding thymidine kinase gene were re-
ported to vary 19% between HSV-1 and HSV-2 [Swain et al., 19831. Thus, the cross-
reaction between HSV-1 and HSV-2 is considered to be due to the partial homology
of the DNA sequences between HSV-1 and HSV-2 DNA, as reported by others
[Ludwig et al., 1972; Stalhandske and Pettersson, 19821.
The application for detection of viruses in clinical specimens from various
tissues using a biotinylated DNA probe has been reported by Gomes et al. [1985].
However, we do not have data concerning the application of biotinylated probes for
the detection of HSV-1 DNA in ocular tissues. From the data described above, this
assay system seems to be sensitive enough to detect HSV-1 DNA in clinical speci-
mens. In fact, we could detect DNA not only in experimental keratitis in albino
rabbits, but also in ocular tissues. Although there was a discrepancy between the
results of antigen detection by IF and/or virus recovery and DNA detection by
hybridization assay in cases 1 and 5, these patients could be successfully treated
according to DNA assay. In case 6, the failure to detect significant antibody titers, as
well as the negative result obtained from DNA-DNA hybridization, suggested that
the corneal ulcer might have been caused by different infectious agents or mecha-
nisms. In addition, the negative results in cases 7 and 8 proved that this method is
very specific, reliable, and valuable in the clinical field.
This method has enabled us to show quite easily the existence of the viral DNA
in clinical specimens without the excessive time consumption and disadvantages of
radioactive probes; furthermore, it can help us achieve the correct diagnosis and
initiate prompt, proper therapy in severe cases. Today we can use very powerful and
specific antiviral reagents to herpetic eye diseases; correct and prompt diagnosis of
HSV-1 infections by DNA-DNA hybridization in routine clinical uses may be there-
fore useful in improving the prognosis of herpetic infections.
 Detection of HSV-1 DNA in Ocular Tissues 269

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