Serotyping of herpes simplex virus isolates: A comparison of BVDU sensitivities, indirect immunofluorescence with monoclonal antibodies, and indirect immunofluorescence with cross-adsorbed rabbit antibodies

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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