Journal
of Virological
Methods,
7 (1983) 199-206
199
Elsevier
SEROTYPING OF HERPES OF BVDU SENSITIVITIES,
SIMPLEX INDIRECT
VIRUS ISOLATES: A COMPARISON IMMUNOFLUORESCENCE WITH
MONOCLONAL ANTIBODIES, AND INDIRECT IMMUNOFLUORESCENCE WITH CROSS-ADSORBED RABBIT ANTIBODIES*
STEPHEN
L. SACKS,
Division
Infectious Diseases, Department of Medicine, Health Sciences
of
British Columbia,
(Accepted
CHONG-ZE
Vancouver.
21 June
Four methods
for typing
monoclonal
deoxyuridine
of herpes simplex
antibodies
(BVDU)
rabbit
with HSV-1 monoclonals
antisera.
were compared
University
of
for 43 recent clinical
to indirect immunofluorescence Sensitivity
using
to E-5-(2-bromovinyl)-2’-
accurate,
When dilutions
fluorescence
fluorescence
endpoints
dilution at a greater
Results of typing
were predetermined
22 isolates (14%) of HSV- 1 were correctly (92%).When
were found to be sensitive to BVDU with ID,,‘s
All isolates labelled as HSV-2 using monoclonal
from 0.4 to 3.5 ug/ml.
herpes
Hospital,
was also examined.
to BVDU ranging
also correctly
virus (HSV) isolates
and cross-adsorbed
from 0.001 to 0.006 pg/ml. however.
Centre
Canada
strains of HSV. These isolates were subjected
All isolates which fluoresced ranging
LEMCHUK-FAVEL
1983)
isolates and 3 reference both
B.C.,
TEH and LAUREL
according
to manufacturer’s
typed. HSV-2 isolates were correctly
endpoints dilution
antibodies
with rabbit cross-adsorbed
were compared,
however,
with HSV-1 antiserum.
had ID,,‘s
antisera
were less
instructions,
only 3 of
labelled in 24 of 26 situations
21 of 22 (95%) HSV-1 isolates had
Twenty-three
of 24 HSV-2 isolates were
typed (96%).
simplex
virus
indirect
immunofluorescence
BVDU
monoclonal
antibodies
antiviral
assay
INTRODUCTION
Nahmias and Dowdle (1968a) demonstrated the antigenic and biological differences between HSV-1 and HSV-2. HSV-1 is isolated most often from nongenital sites, while HSV-2 is most often isolated from genital sites (Josey et al., 1972; Rawls and Gardner, 1972). Furthermore, HSV-1 and HSV-2 are most commonly recoverable from their latent ganglionic sites in the trigeminal and sacral ganglia, respectively (Baringer, 1974; Baringer and Swovel, 1973). However, the site -of origin is only suggestive of the HSV type. Furthermore, mixed infections have been seen (Fife et al., 1983). In the near future, the clinical virology laboratory may need to type HSV *This work was supported
01660934/83/$03.00
by a grant
from the British Columbia
@ 1983 Elsevier Science Publishers
B.V
Health
Care Research
Foundation
200
because
the risk of genital
recurrence
may be less with HSV-1 than with HSV-2
(Reeves et al., 1981). Furthermore, type-specific or type-preferential anti-herpes agents are likely to play an active role in the therapy of HSV diseases. HSV-1 is selectively and specifically inhibited by BVDU (De Clerq et al., 1980; Mayo, 1982) and may be more sensitive to acyclovir (ACV) (Crumpacker et al., 1979), altough this latter observation is variable (Schaeffer et al., 1978; De Clerq et al., 1980). HSV-2 is preferentially inactivated by 5-ethyl-2’-deoxyuridine (EDU) (Teh and Sacks, 1983). HSV-1 and HSV-2 are closely related since over 50% of their genomes are homologous (Kieff et al., 1972). They are morphologically identical. Hence, they have not been easily distinguished in the laboratory by serological or other means. To date, methods that have been used to differentiate the two types of HSV include the size of pocks produced on the chorioallantoic membranes of hen’s eggs (Nahmias, 1968b), fluorescent antibody staining (Nahmias et al., indirect immunoperoxidase staining (Benjamin,
1969, Geder and Skinner, 1971), 1974), enzyme linked immunosorbent assay (Mills et al., 1978), microneutralization (Pauls and Dowdle, 1967; Stalder et al., 1975) indirect hemagglutination (Bernstein and Stewart, 1971, mixed agglutibtion (Ito and Barron, 1974), and solid phase radioimmunoassay (Forghani et al., 1974). Other less commonly used techniques are immunoelectro-osmophoresis (Jeansson, 1972), counter-immunoelectrophoresis (Jeanson, 1974), complement-dependent cytotoxicity (Olofson et al., 1978) and the ‘251-labelled staphylococcal protein A immunofiltration technique (Cleveland et al., 1982). More elaborate techniques are nucleic acid hybridization (Brautigam et al., 1980) and restriction enzyme analysis of viral DNA (Lonsdale, 1974; Buchman et al., 1979). However, many of these methods remain impractical for the clinical laboratory. In order to evaluate the speed and accuracy of available
methods,
we compared
four
typing methods for three reference strains and 43 recent clinical isolates of HSV. Each isolate was subjected to sensitivity testing with BVDU (De Clerq et al., 1980; Mayo, 1982), IFA with monoclonal antibodies (IFA-MC) (Balachandran et al., 1982; Pereira et al., 1982) and IFA using commercially available rabbit cross-adsorbed antisera (IFA-DAKO) (Geder and Skinner, 197 1). Rabbit antisera was used for testing isolates by two methods. Dilutions were made according to the manufacturer’s suggestions and endpoint fluorescence was determined, in parallel, for each antisera, with each isolate. MATERIALS
AND METHODS
Virus and cells
Initial HSV isolations were obtained in human foreskin fibroblast (HFF) cultures from patients with oral, genital or central nervous system infection. The McIntyre strain of HSV-1 was obtained from the American Type Culture Collection. The F strain of HSV-1 and the G strain of NSV-2 were provided by Dr. B. Roizmam (University of Chicago, Chicago, IL). Virus stocks from passage 2 in baby hamster
201
kidney (BHK-21)
cells were titred and stored at -70°C for use in the BVDU assay. HFF
cells were obtained
locally
methods
described
previously
from routine
ded by Dr. Lorne Kastrukoff,
(Reigner
circumcisions
and processed
according
to
et al., 1977). BHK-21 cells were kindly provi-
University
of British Columbia,
Vancouver,
Canada.
Cells were maintained in Earle’s minimal essential medium (MEM) supplemented with 200 mM L-glutamine, 10% (v/v) fetal bovine serum and 2.2 ml of 7.5% sodium bicarbonate per 100 ml (Gibco Canada, Calgary, Alberta). Drug
BVDU was a gift from Dr. E. De Clercq, Katholieke Universiteit, Leuven, Belgium. Indirect
immunofluorescence
Rega Institute
for Medical
Research,
techniques
Infected HFF cells coded by random number assignment, showing approximately 80% cytopathic effect (CPE) were liberated from the growth surface of a culture tube by scraping with a sterile Pasteur pipette. One drop of each cell suspension was placed on a g,lass slide and allowed to dry. Slides were rinsed in acetone at room temperature for 1 min, followed by fixing in cold acetone at -20°C for 10 min and dried. The process was c’ontinued in either of the three following ways. (I) Indirect
immunofluorescence
with rabbit cross-adsorbed
antibody
(predetermined
dilutions)
Separate cell areas were each flooded with a drop of rabbit antiserum (DAKO, Copenhagen, Denmark) to HSV- 1 (1 : 2048). A second cell area was flooded with a drop of the corresponding dilution of rabbit antiserum to HSV-2 (1 : 512), following the manufacturer’s recommendations. Slides were incubated at 37’C for 45 min in a moist environment, and washed in three 5-min changes of PBS (pH 7.4) at room temperature and allowed to air dry. Cell areas were again flooded with swine fluorescein-conjugated mark).
anti-rabbit
immunoglobulin
(1 : 40 dilution,
DAKO,
Den-
(2) Indirect immunojluorescence with rabbit cross-adsorbed antibody (endpoint dilutions) The above method was repeated using serial two-fold dilutions of each antiserum. Fluorescence endpoint (last dilution showing distinct cytoplasmic rimming) was scored for each isolate for each antiserum. (3) Indirect immunojluorescence with monoclonal antibodies Slides were flooded with a drop of 1 : 20,OOOdilution of monoclonal antibody (HC-1) to HSV-1 directed against a glycoprotein L; a second cell area with a drop of 1 : 20,000 dilution of three monoclonal antibodies (H222, H368, H379) to HSV-2. These antibodies are directed against glycoprotein E (H222) and other as yet undefined antigens (Pereira et al., 1982). Slides were processed at 37°C for 45 min in a moist environment and washed in three 5-min changes of PBS (pH 7.4) at room temperature and then allowed to air dry. Goat
202
fluorescein-conjugated
anti-mouse
(Cappel
Cochranville,
Laboratories,
Slides were then mounted
immunoglobulin PA) was added
with FA mounting
sauga, Ontario) and examined Vancouver. B.C.).
under
mercury
(1 : 100 dilution,
Fab fragment)
to cell areas.
medium
(Flow Laboratories,
vapor fluorescence
(Nikon
MissisCanada,
Antiviral assays Newly confluent BHK-21 cell monolayers in flat-bottomed microtitre trays (NUNC, Roskilde, Denmark) were washed once with PBS. Each well was infected with 100 TCID,, of virus in 50 ul of medium 199. Virus was adsorbed for 1 h at 37°C. The overlay was then removed and each well was replaced with 100 ul of drug in serial two-fold dilutions ranging from 0.00078 to 25 ug/ml. Trays were then incubated for 48 h at 37°C in the presence of 5% CO,. Assays were halted when microscopic examination showed 65 to 85% CPE (48-72 h) post infection. The antiviral activity was expressed as ID,, or the concentration of antiviral drug required to reduce the viral CPE to 50% of control, calculated according to the method of Reed and Muench (1938). Each assay was repeated at least four times. RESULTS
Twenty clinical isolates were typed as HSV-1 by the IFA-MC method. All HSV-1 isolates were sensitive to BVDU with ID,,‘s of 0.006 pg/ml or less. Twenty-three clinical isolates were typed as HSV-2 by the IFA-MC method. All HSV-2 isolates had ID,,‘s to BVDU of 0.4 pg/ml or more. Table 1 displays the results of all typing assays for isolates categorized as HSV-1 by both IFA-MC and BVDU sensitivity. Commercial antisera were first assessed by diluting according to package instructions followed by scoring of fluorescence. Three of 22 (14%) HSV- 1 isolates were correctly typed using this method. Two isolates were ambiguously typed and the remaining isolates (17/22) actually gave stronger fluorescence with HSV-2 antisera. The isolates were then typed by establishing an endpoint of fluorescence
by serial dilutions
with each antiserum.
Twenty-one
of 22 HSV-1
isolates gave fluorescence at greater dilutions of HSV- 1 antisera compared to HSV-2 antisera (95% accuracy). One isolate (strain 801) had identical endpoint staining with both antisera. No HSV-1 isolates were incorrectly typed by this method as HSV-2. Table 2 displays the same parameters for the HSV-2 isolates. Twenty-one of 23 isolates were typed as HSV-2 by scoring of fluorescence using predetermined antisera dilutions. However, two isolates gave stronger fluorescence with the HSV-1 antiserum than with HSV-2 antiserum and were thus typed incorrectly. Twenty-three of 24 strains were correctly typed as HSV-2 by this commercial antisera. When the endpoint fluorescence method was used (96% accuracy), one isolate showed fluorescence at the same dilution (strain 202) using both antisera and therefore could not be typed by this method.
1
Mouth
413
Lip
Lip
302
x49
Perineum
280
Lip
Vulva
279
Vulva
Penis
229
837
Penis
224
801
Vulva
221
Vulva
Brain
u- 1000
717
Brain
u-900
Chin
Brain
U-800
768
Vulva
s-303
Lip
Vulva
S-82
Vulva
Brain
R-1272
623
Reference
454
Reference
F
methods
McIntyre
Strain
Isolate
Results of typing
TABLE
applied
++
++
+++
+++
++
+++
++
+++
+++
i-t+
+++
++
+++
++
++++
+++
++
+++
++
++
+++
+++
HC-1
IFA-MC
to
to HSV-1 isolates to
_
_
_
_
H368, H379
H222,
IFA-MC BVDU
0.004
0.001
0.002
0.004
0.003
0.006
0.002
0.005
0.006
0.005
0.004
0.005
0.001
0.003
0.002
0.002
0.001
0.002
0.001
0.002
0.002
0.004
ID,, b/ml)
IFA-DAK0
++
++
++
+++
++
+++
++
++
+++
++
+++
++
++
++
++
++
++
++
++
+++
++
+++
HSV-I
dilution)
to
+++
+++
+++
+++
++++
+
+++
+++
++
+++
+
+++
++++
+++
+++
+++
+++
+++
+++
+++
+++
++++
HSV-2
(fixed
15
15
14
15
15
15
15
16
16
15
I6
16
15
16
14
15
15
14
16
15
15
15
HSV-1
points
(l/2”)
end-
IFA-DAK0 dilution
13
I4
14
13
13
13
13
13
14
13
14
15
13
I4
12
13
13
12
13
13
13
13
HSV-2
with
serial
E
2
+++ +++ ++++
+++ +++ ++++ ++i +++ +++ ++ +++ +++ +++ +++ Cfi++++ +++ +++ +++
Penis
Leg Penis
Vulva
Penis
Vulva
Vulva
Penis
Vulva
Vulva
Vulva
Cervix
Scrotum
Vulva
Penis
Vulva
Vulva
Vulva
Vulva
Cervix
Penis
Penis
S-48
124
129
138
150
159
170
176
180
182
183
184
191
202
203
210
228
253
216
381
418
431
to
++++
++++
+++
+++
+++
H368, H379
H222,
Brain
IFA-MC
Reference
to
u-1300
WC-I
IFA-MC
to HSV-2 isolates
Strain G
applied
source
methods
Isolate
Results of typing
TABLE
0.5
0.4
0.7
1.3
0.8
I.2
1.4
1.0
0.8
0.9
2.2
1.9
0.8
3.5
0.7
0.7
0.5
1.0
0.8
1.8
0.6
1.3
1.7
0.6
ID (itg/mU
BVDU
IFA-DAK0
I-+++
+
13
++
++
++++
i-+-i-
ii-++
++-t-+
?-i--b+
f + _
ii-++
13
13
15
13
13
13
14
t+i-+ ++++
14
14
14
13
13
13
14
++++
+-t-++
+
+
++
++
+
++++
++++
+ +
++++
++++
+
+
+++
++
14
13
14
+
++++
+
12
13
++
+++t
+
14
14
14
HSV-1
13
++++
+
serial
15
15
15
15
15
14
16
15
14
16
16
16
15
15
1s
16
16
15
I5
16
15
16
1.5
16
HSV-2
points (112”) with
end-
IFA-DAK0 diIution
Sfff
+
++
t-t++
i+++
++
+
(fixed
HSV-2
to
HSV-1
dilution)
205
DISCUSSION
Recently, monoclonal antibodies have been shown to be accurate tools for typing of clinical HSV isolates (Balachandran et al., 1982; Pereira et al., 1982). We have confirmed the accuracy of monoclonal antibodies and have compared three other readily available typing methods. The use of cross-adsorbed
the results to antisera for
typing of HSV was first described by Geder and Skinner (197 1). It was suggested in this original description that dilutions of each ‘type-specific’ antisera could be made which showed little if any overlap. We have confirmed that cross-adsorbed rabbit antisera can be made relatively accurate, but that this requires serial fluorescence endpoint titration. Fixed dilutions may cross-react with HSV of the opposite type. This may be misleading and lead to errors in clinical strain typing. Alternatively, monoclonal antibodies to HSV may be selected which are 100% specific. This lack of cross-reaction means that predetermined dilutions may be used for typing by IFA, immunoperoxidase (Richman et al., 1982) or other methods. Monoclonal antibodies retain their specificity when used directly on clinical specimens. Sensitivity, however, is decreased compared with polyclonal antibody preparations and background (nonspecific) fluorescence is a minimal problem. With the isolates used in this study, sensitivity to BVDU was also 100% accurate. The method was somewhat more cumbersome than IFA since stock growth and titration may be required. In addition, resistance to BVDU has been reported (Darby et al., 1981). Since naturally occurring resistance to other thymidine analogs has been reported (Parris and Harrington, 1982) this might be expected to lead to rare cases of mistyping. The theoretical possibility that the highly specific nature of monoclonals could lead to errors in typing has not been found to be a problem after experience with several hundred clinical isolates. REFERENCES
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