Kinase Human Serum Antibodies to Varicella-Zoster Virus Thymidine - Infection and Immunity
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INFECTION AND IMMUNITY, Apr. 1982, p. 30-37 Vol. 36, No. 1
0019-9567/82/040030-08$02.00/0
Human Serum Antibodies to Varicella-Zoster Virus Thymidine
Kinase
CLAS F. R. KALLANDER,* J. SIMON GRONOWITZ, AND EINAR G. TORFASON
Department of Medical Virology, University of Uppsala, Biomedical Center, 751 23 Uppsala, Sweden
Received 1 June 1981/Accepted 12 November 1981
The conditions required for the production of varicella-zoster virus (VSV)-
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induced deoxythymidine kinase (dTk) have been studied. Extracts from Vero
cells harvested 62 h after VZV infection were found to contain VZV-induced dTk
activity, with a minimal contribution from the cellular dTk activity. VZV dTk was
shown to have a broad substrate specificity phosphorylating both deoxythymi-
dine, deoxycytidine, and iododeoxyuridine. Deoxythymidine triphosphate inhibi-
tion studies revealed an intermediate deoxythymidine triphosphate sensitivity
when compared with that of the cellular cytosolar enzyme and the deoxythymi-
dine triphosphate-insensitive herpes simplex virus dTk. An assay for VZV dTk-
blocking antibodies was developed, with [1251]iododeoxyuridine as a substrate in
the presence of a deoxythymidine triphosphate concentration which selectively
blocked the dTk of host cell origin. A total of 79 serum samples were studied;
these included serum pairs from patients with varicella or herpes zoster and single
sera from immune and nonimmune adults. VZV dTk blocking antibodies were
detected exclusively in sera from patients with herpes zoster. All serum pairs
showing VZV dTk seroconversion also showed a parallel conversion of comple-
ment fixation titers. The VZV dTk antibodies were found to be of the immuno-
globulin G class. The immunological specificity of VZV dTk was investigated, and
no cross-reactivity with herpes simplex virus type 1 or 2 dTk was found.
Herpes simplex virus (HSV) and varicella- ase, and dTk by immunizing animals with par-
zoster virus (VZV) are known to induce virus- tially purified extracts from infected cells (11,
specific deoxythymidine kinase (dTk), whereas 13, 17, 20, 23, 30). The occurrence of HSV dTk-
cytomegalovirus apparently lacks dTk, and the and deoxyribonuclease-blocking antibodies in
existence of an Epstein-Barr virus dTk remains human sera have been reported (4, 9, 25), and
to be proven (18). The HSV dTk is well charac- the correlation between the presence of dTk-
terized, and the enzyme has been used as a blocking antibodies and the different stages of
marker in a variety of studies on transformation human HSV infections of both types have been
and gene expression (1, 2, 24, 29). Due to the described elsewhere (9a). In contrast to comple-
cell-associated nature of VZV and the problems ment-fixing (CF) antibodies, detectable amounts
associated with large-scale virus propagation, of dTk-blocking antibodies appeared late after
comparatively few studies have been reported primary infection and exhibited good type speci-
on the VZV dTk. Dobersen et al. (7) reported ficity. No immunological studies have yet been
induction of elevated levels of a dTk with a reported regarding VZV dTk.
broad substrate specificity in VZV-infected hu- The intention of the current study was to
man embryonic lung cells. The enzyme has been examine the occurrence of VZV dTk antibodies
characterized with regard to molecular weight, in relation to human VZV infections and to
pH optimum, electrophoretic mobility, and study the properties of the enzyme.
deoxythymidine triphosphate (dTTP) inhibition
(7, 10, 22). Recently the enzyme has been puri-
fied by affinity chromatography, and kinetic MATERIALS AND METHODS
studies made with different phosphate donors
and acceptors (5). The enzyme kinetics of VZV Cells. Green monkey kidney cells (GMK) and pri-
dTk were found to be different from human mary human fibroblasts (HF) were grown in Eagle
minimum essential medium. Vero cells were grown in
cytoplasmic and mitochondrial dTks; they also medium 199 supplemented with Earle salts. The
varied from those of HSV type 1 (HSV-1) and growth media for uninfected cells contained 10% calf
HSV-2 dTks. serum, whereas media used for VZV propagation
Enzyme-blocking antibodies have been raised contained 2% calf serum.
against HSV deoxyribonuclease, DNA polymer- Virus. Our VZV strain (pE) originated from a 3-
30VOL. 36, 1982 VZV dTk AND HUMAN ENZYME-BLOCKING ANTIBODIES 31
year-old female with typical varicellae. The original correction, given as a percentage of the normal control
isolation was made on HF cells, and the strain was (the control being enzyme incubated with a negative
then transferred to GMK and Vero cells after eight reference serum), was plotted against serum dilutions,
passages. The infection could be serially maintained and the reciprocal log2 dilution giving 30% inhibition
only by transfer of infected cells, and the virus was was defined as the titer (see Fig. 4). Sera giving less
subcultivated every 72 to % h. The pE isolate was than 30%o inhibition in a 1:1 dilution were considered to
typed as VZV by immunofluorescence (28), the HSV be negative.
strains C42 (HSV-1) and C168 (HSV-2) having been Polyacrylamide gel electrophoresis. The polyacryl-
characterized previously (9). amide gel electrophoresis method of Kit et al. (19) was
Enzyme preparation. Cultures infected with VZV used, with the following modifications. Thymidine was
exhibiting 70 to 90%o cytopathogenic effect were omitted from all buffers. The samples were layered
washed in physiological NaCl (48 to 72 h after infec- directly on top of the gel in a buffer containing 0.25 M
tion) and harvested by trypsinization. After washing Tris-hydrochloride buffer (pH 7.5), 2 mM Mg2+, 2 mM
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once again in NaCl, the cells were pelleted by low- ATP, 3 mM dithiothreitol, and 15% sucrose. The
speed centrifugation and resuspended in stabilizing electrophoresis was performed in 4- by 75-mm glass
buffer (9). After freeze-thawing three times, the sus- tubes. The positions of dTk activity were determined
pension was ultracentrifuged for 2 h at 200,000 x g. by slicing the gel in 2.3-mm sections and incubating the
The supernatant was collected, diluted, and frozen at slices in 100 ,u1 of assay mixture for 2 h at 37°C with
-70°C in 100-pl samples. Enzyme quantities given in IUdR (1.0 x 10-7 M; specific activity, 40 Ci/mmol),
the text are related to the number of cells from which [3H]thymidine (1.6 x 10-7 M; specific activity, 70 Ci/
the enzyme preparation was derived. The HSV-1 and mmol), and [3H]deoxycytidine (6.0 x 10-' M; specific
HSV-2 preparations used for comparison were made activity, 26.8 Ci/mmol) as substrates. The phosphory-
as previously described (9). lation was terminated as described above. The relative
Serum specimens. Acute-convalescent serum pairs fraction of migration (Rf) was calculated with bromo-
with clinical indications of varicella or zoster, con- phenol blue as a reference.
firmed by conversions of VZV CF titers, were ob- Serum fractionation. The immunoglobulin G (IgG)
tained from the diagnostic laboratory at our depart- fraction was adsorbed to protein A-Sepharose (Phar-
ment. Sera (10 VZV positive and 10 VZV negative, macia Fine Chemicals); 250 ,ul of gel suspension
according to the VZV fluorescence immunity test [8]) containing 37.5 mg of gel (binding capacity, 2.5 mg of
were provided by National Bacteriological Labora- IgG) was used with 250 p.l of serum diluted 1/2. All
tory, Stockholm. The HSV dTk antisera were selected components were prepared in 5 x 10-' M Tris-
from a large collection of human sera investigated hydrochloride buffer (pH 7.6)-0.09 M NaCl. The mix-
recently (9a). Titers of CF antibodies were determined ture was incubated for 60 min at 37°C. The sample was
by standard methods (12). All sera used in this study then centrifuged at low speed, the supernatant was
were heat inactivated at 56°C for 30 min. collected, and the gel was washed three times with 5 x
Enzyme and antibody assay. The enzyme and anti- 10-4 M Tris-hydrochloride buffer (pH 7.6)-0.09 M
body assays were essentially performed as described NaCl. To recover bound IgG at the same concentra-
previously (9), with 125I-labeled iododeoxyuridine tion as in the original serum specimens, the gel was
(IUdR) as a substrate (final concentration, 10-7 M; mixed with 125 p.l of 0.01 M glycin buffer (pH 2.3), and
specific activity, approximately 200 Ci/mmol) at 37°C. after S min the supernatant was removed and mixed
The 2-1-mercaptoethanol concentration was raised to with 40 p.l of Tris-hydrochloride buffer (pH 9.3) to
10 mM, instead of 3 mM which was used before. To restore the pH. The recovery of IgG was checked with
minimize the contribution of cell dTk in antibody Oxford Diffu-gen immune diffusion plates, and anti-
assays, dTTP with a final concentration of 1.5 x 10-5 body titers in the IgG fractions were calculated with
M was included in the reaction solution (see Fig. 3). reference to the amount of IgG in the original serum.
The amount of enzyme used was standardized to give Gel filtration was carried out on a Sephacryl 300
20 x 103 to 40 x 103 cpm of incorporated radioactivity (Pharmacia) column, and the sample was eluted with
during a 30-min assay. For VZV this required enzyme 0.05 M Tris-hydrochloride buffer (pH 8.0)-0.5 M
from 2,000 cells, whereas for HSV-1 and HSV-2 250 NaCl-0.02% NaN3. Samples of undiluted serum (400
cells sufficed. Starting with a 1:1 dilution, 25-p.l sam- p.l) were fractionated after addition of a small amount
ples of serial twofold dilutions of test sera were added of "25I-labeled sheep anti-rabbit IgG. This tracer was
to microtiter plates, after which 5 p.l of enzyme and 10 shown to coelute with human IgG in a separate experi-
of complement (guinea pig serum diluted 1/5) were ment. "25I-labeled adenovirus type 5 DNA (2.3 X 107
added. (Complement is not necessary for the blocking daltons) was used as a marker for the void volume.
of viral dTk; however, we have found that the use of Fractions of 2.5 ml were collected. The protein con-
complement improves the blocking effect of a few sera tent of each fraction was determined spectrophotomet-
with flat titration profiles and that, in addition, it helps rically at 280 nm.
to diminish the variation of total serum concentration.)
The mixture was then incubated at 37°C for 90 min, RESULTS
and the residual enzyme activity was determined by Induction of dTk activity by VZV. The time
the addition of 21 of substrate solution and a further course for the increase in dTk activity relative to
30-min incubation at 37°C. The enzyme reactions were the number of intact cells per culture is de-
terminated by adsorbing duplicate 25-,ul samples to
Whatman DEAE 81 filter disks kept at 90 to 100°C. scribed in Fig. 1. In infected cell cultures the
Before counting in a gamma spectrometer, the disks dTk activity increased and reached a peak value
were washed extensively to remove the unused sub- at about 62 h. The following decrease in absolute
strate. Residual enzyme activity after background dTk activity was related to diminishing cell32 KALLANDER, GRONOWITZ, AND TORFASON INFECT. IMMUN.
Electrophoretic analysis of dTk activities from
VZV-infected cells and noninfected cells. Extracts
prepared from three different cell lines mock
infected or infected with VZV were analyzed by
gel electrophoresis as described above. All ex-
tracts from infected cells revealed a prominent
El new peak, not present in the control extracts
(Fig. 2). The peaks of the new activity coincided
10
u~~~~0
5 for all investigated cell extracts, giving Rf values
in the range of 0.29 to 0.32. Enzyme activity
originating from the HSV-2 strain C168 had an
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Rf value of 0.39. Figure 2 also shows that the
0 10 20 30 40 50
hours
100 VZV-induced enzyme activity had a broad sub-
strate specificity, phosphorylating IUdR (Fig.
FIG. 1. Induction of VZV dTk in Vero cells. 2A, B, and C), thymidine (Fig. 2D), and deoxy-
Monolayer cultures of Vero cells were infected by cytidine (Fig. 2D). The detection of VZV dTk
transfer of infected Vero cells. Cell number and dTk was in fact even better with [125I]IUdR than with
activity per 10 min were determined at the indicated [3Hlthymidine. The analysis presented in Fig.
times. Symbols: cell number per culture (E); dTk 2D, with [3H]thymidine as a substrate, was
activity proportional to yield per culture (0); dTk
activity originating from 1,800 intact cells (A). performed with double the amount of cell ex-
tract, 1.75 times higher specific activity of the
substrate, and the same incubation time as the
amounts, depending on cell lysis. The dTk activ- corresponding analysis with IUdR (Fig. 2A).
ity per infected cell continued to increase during The latter still revealed 30 times more incorpo-
the late period. In the mock-infected cultures the rated radioactivity. The cellular dTk was only
level of dTk activity remained low during the detected in extracts from mock-infected GMK
investigated time period. cells (Fig. 2C) and migrated with an Rf value of
FIG. 2. Electrophoretic separation of dTk activities from uninfected and virus-infected cells. Extracts were
prepared as described in the text and analyzed for dTk activity by using different substrates. A, Extract from 2.5
x 10i VZV-infected Vero cells (0) and 2.5 x 105 uninfected cells (E), assayed with [1251]IUdR as a substrate. B,
Extract from 2.5 x 104 VZV-infected HF cells (0) and 2.5 x 105 uninfected cells (E), assayed with [1251]IUdR as
substrate. C, Extract from 3 x 104 VZV-infected GMK cells (0) and 3 x 105 uninfected GMK cells (E), assayed
with [125 ]IUdR. D, Extract from 5 x 104 VZV-infected Vero cells assayed with [3H]thymidine (@) and
[3H]deoxycytidine (A). E, Extract from 2.5 x 104 HSV-2-infected BHK cells (0), assayed with [1251]IUdR.VOL. 36, 1982 VZV dTk AND HUMAN ENZYME-BLOCKING ANTIBODIES 33
/. (Enz Act) activity with the expected Rf of 0.60 to 0.80 (19,
100 21) was detected under these conditions.
[1251]IUdR is obviously a preferable substrate
for the VZV dTk assay, due to the higher
sensitivity, thus permitting the detection of
small amounts of enzyme, which is essential for
studies of enzyme-blocking antibodies.
dTrP inhibition of viral and cellular dTk. The
50 A VZV dTk preparation from the infected Vero
cells analyzed above, exhibiting minimal
amounts of cellular dTk, was used as a source of
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VZV enzyme. Since the mock-infected Vero
preparation had too low levels of host enzyme,
enzymes from GMK (Fig. 2B) and HF cells were
used as sources of cellular cytosol dTk. The HF
-6 -5 -4 -3 dTk preparation originated from proliferating
Log10 Concn of TTP cells and mainly contained cytosol dTk. The
FIG. 3. dTTP inhibition of viral and cellular cyto-
HSV enzymes used have been characterized
solar dTk enzyme extracts. Enzyme activities were previously (9). The experiments were performed
determined by a separate assay for each dTTP molar by assaying the different enzyme preparations at
concentration. Enzyme activity in percentage of con- enzyme concentrations giving linear turnover of
trol without dTTP is plotted against loglo of the [125I]IUdR. To avoid competition for Mg2' be-
different dTTP concentrations. Symbols: human cyto- tween ATP and dTTP our standard conditions
sol enzyme (0), GMK cytosol enzyme (0), VZV include an excess of Mg2+ (17 mM, compared
enzyme (O), HSV-2 enzyme (A) and HSV-1 enzyme with 9.3 mM ATP). Reaction velocities were
(A). determined in the presence of different dTTP
concentrations and were calculated relative to a
0.17. However, this activity was not detected in control without dTTP. The cellular enzymes
the extract from VZV-infected GMK cells. This exhibited similar properties, whereas each viral
probably represents a shut-off of the cellular dTk enzyme gave its own characteristic inhibition
due to the VZV infection. No mitochondrial dTk profile (Fig. 3). The cellular enzymes, being the
a-
2-
a:
4,on
13 11 9 7 5 3 1 11 9 7 5 3 1
Log 2 dilution
FIG. 4. dTk blocking efficiency of different human antisera. Extracts from 2 x 103 VZV-infected cells (O), 2.5
x 10' HSV-1-infected cells (0), and 2.5 x 102 HSV-2-infected cells (0) were used as enzyme sources. A,
Normal human immunoglobulin (Kabi). B, Zoster acute-convalescent pair with VZV CF titers of34 KALLANDER, GRONOWITZ, AND TORFASON INFECT. IMMUN.
most sensitive, were inhibited by 50% at a dTTP dTTP, thereby being more sensitive than the
concentration of 4 x 10-6 M. In contrast, dTk HSV enzymes but significantly less sensitive
from the HSV-1 and HSV-2 strains required high than the host enzymes. To increase the sensitiv-
dTTP concentrations, being blocked to 50% ity of assays for enzyme-blocking antibodies 1.5
activity by 4.2 x 10-4 and 1.8 x 10-4 M, x 10-5 M dTTP was routinely included in our
respectively. The VZV dTk gave an intermedi- reaction solutions, regardless of the actual con-
ate picture, being inhibited 50% by 4.6 x 10-5 M tent of host dTk in the preparations used.
TABLE 1. Antibodies against viral dTk in serum specimens from patients with varicella or herpes zoster and
in serum specimens from VZV-immune and nonimmune healthy individuals
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Group Age (yr) CF VZV CF HSV VZV dTk Days between
(10g2) specimens
Typical varicella (8 pairs) 33VOL. 36, 1982 VZV dTk AND HUMAN ENZYME-BLOCKING ANTIBODIES 35
Occurrence of VZV dTk-blocking antibodies in of HSV dTk antisera had no effect on VZV dTk,
human sera. To find out whether any VZV dTk- despite high titers against HSV dTk. Only one
blocking antibodies are produced in humans, HSV-1 dTk antiserum had a slight blocking
commercially obtained normal human immuno- effect on VZV dTk (Table 2).
globulin was analyzed first. The results (Fig. 4) Nature of VZV dTk-blocking activity. To dem-
showed that the normal human immunoglobulin onstrate that the blocking activity in the positive
preparation contained VZV dTk-blocking activi- sera is confined to the immunoglobulin fraction,
ty, with a blocking titer of 4 as compared with a selected antisera were fractionated before being
titer of 12 and 8 against HSV-1 and HSV-2 dTk, used in the dTk inhibition assay. Two methods
respectively. The following serum groups were were used for serum fractionation: exclusion
selected to elucidate the correlation between chromatography on Sephacryl 300 and affinity
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clinical VZV picture and the occurrence of VZV chromatography on protein A-Sepharose. Two
dTk-blocking antibodies: (i) pairs of sera from VZV dTk-blocking sera were fractionated on a
patients with clinical indications of varicella or Sephacryl 300 column, with 125I-labeled sheep
herpes zoster, showing a significant CF titer anti-rabbit IgG as a marker. The peak of dTk-
conversion; and (ii) VZV CF-negative sera from blocking activity coincided with the peak of the
10 varicella-immune and 10 nonimmune individ- IgG fraction (Fig. 5). In serum B1210, which has
uals (8). an exceptionally high blocking activity (Fig. 5),
All sera were investigated for VZV dTk- we in addition found a small blocking activity in
blocking antibodies as described above, and the the void fractions, which could represent either
results are summarized in Table 1. In the varicel- IgM or aggregated IgG. After affinity chroma-
la-positive material, consisting of eight serum tography separation, the protein A-Sepharose
pairs with VZV-negative acute sera and 9 to 22 was found to absorb the blocking activity, con-
days between serum samplings, no dTk-blocking firming that the blocking factor consists of IgG,
antibodies were found. None of the serum sam- since protein A-Sepharose is known to absorb
ples from the varicella-immune and nonimmune this immunoglobulin class (Table 3). The block-
individuals contained detectable VZV dTk- ing activity was also eluted by low pH, giving a
blocking antibodies. recovery proportional to the IgG recovery,
The herpes zoster material consisted of 10 which again shows that the VZV dTk-blocking
patients with a clear clinical diagnosis and 5 activity in zoster sera is associated with antibod-
atypical cases. Of the clinically verified cases, ies, mainly the IgG class.
nine patients had sera with VZV dTk-blocking
antibodies, and the remaining patient, who was DISCUSSION
70 years old, had a lymphoma in addition to This report describes the adaptation of the
herpes zoster and was under treatment with sensitive HSV dTk assay we previously de-
cytostatic drugs. Of the five patients with atypi- signed (9) for the detection of VZV dTk and
cal symptoms, only two revealed dTk-blocking antibodies against this enzyme. VZV dTk, like
antibodies. HSV dTk, previously has been shown to have
All individuals positive for VZV dTk-blocking
antibodies had a significant rise in blocking
antibody titer parallel to the rise of the CF TABLE 2. Immunological specificity of antibodies
antibody titer. One individual had a dTk-block- against the viral dTks in a selected material of human
ing acute serum which was negative in the CF sera
test. Several of the titers found were high as Titers of dTk-blocking
compared with the HSV dTk-blocking titers CF HSV CF VZV antibodies (1og2)
found generally in humans (9a). HSV-1 HSV-2 VZV
Immunological specificity of the VZV dTk. To
evaluate possible immunological cross-reactiv-36 KALLANDER, GRONOWITZ, AND TORFASON INFECT. IMMUN. broad substrate specificity, phosphorylating de- TABLE 3. Distribution of dTk-blocking activity in oxythymidine, deoxycytidine, bromodeoxycyti- different serum fractions after affinity dine, and a variety of other substrates (3, 5-7, chromatography with protein A-Sepharose 10, 14, 15, 21). Both cellular and HSV dTk are Reciprocal blocking titer (log2) known to phosphorylate IUdR (3, 6, 9). In our Serum Unfractioned Absorbed Supematant electrophoretic experiments we showed that serum fractiona fraction VZV dTk phosphorylates IUdR efficiently. This prompted us to use 10-7 M [125I]IUdR for detec- D2476 10 10
VOL. 36, 1982 VZV dTk AND H IUMAN ENZYME-BLOCKING ANTIBODIES 37
tion with primary VZV infections, the antibody nucleoside metabolism in varicella-zoster virus-infected
response is directed primarily against the nu- cells. J. Virol. 25:510-517.
11. Halliburton, I. W., and J. C. Andrew. 1976. DNA poly-
cleocapsid antigen, whereas in herpes zoster a merase in Pseudorabies virus infected cells. J. Gen. Virol.
broader antibody response to envelope and solu- 30:145-148.
ble antigens is apparent (26). 12. Hawkes, R. A. 1979. General principles underlying labora-
The human dTk-blocking antibodies seem to tory diagnosis of viral infections, p. 3-48. In E. H.
Lennette and N. J. Schmidt (ed.), Diagnostic procedures
have a good immunological specificity, and of for viral, rickettsial and chlamydial infections, 5th ed.
the 14 dTk-positive antisera investigated only American Public Health Association, Washington, D.C.
one HSV-1 antiserum had a low, seemingly 13. Hoffman, P. J., and Y.-C. Cheng. 1978. The deoxyribonu-
heterologous, VZV dTk-blocking titer. Since clease induced after infection of KB cells by Herpes
simplex virus type 1 or type 2. J. Biol. Chem. 253:3557-
other HSV-1 dTk antisera having much higher 3562.
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titers were devoid of VZV dTk-blocking anti- 14. Jamieson, A. T., G. A. Gentry, and J. H. Subak-Sharpe.
bodies it seems doubtful as to whether this 1974. Induction of both thymidine and deoxycytidine
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480.
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16. Kapsenberg, J. G. 1964. Possible antigenic relationship
ACKNOWLEDGMENTS between varicella zoster virus and herpes simplex virus.
We thank Ulf Pettersson and Hans Diderholm for their Arch. Gesamte Virusforsch. 15:67-73.
critical review and valuable help in preparing this manuscript. 17. Keir, H. M., H. Subak-Sharpe, W. I. H. Shedden, D. H.
We also thank Elisabeth Olding-Stenkvist for help with evalu- Watson, and P. Wildy. 1966. Immunological evidence for
ation of the clinical data. Ann-Kristin Frej provided excellent a specific DNA polymerase produced after infection by
technical assistance. herpes simplex virus. Virology 30:154-157.
18. Kit, S. 1979. Viral-associated and induced enzymes. Phar-
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