Antiviral Chemistry & Chemotherapy 12:233–239
Effect of long-term, low-dose acyclovir suppressive
therapy on susceptibility to acyclovir and frequency of
acyclovir resistance of herpes simplex virus type 2
Mariko Honda1*, Tomoko Okuda2, Tomomi Hasegawa2, Masahiko Kurokawa2, Kimiyasu Shiraki2,
Koma Matsuo1, Makoto Komatsuzaki1 and Michihito Niimura1
1
Department of Dermatology, The Jikei University School of Medicine, Tokyo, Japan;
2
Department of Virology, Toyama Medical and Pharmaceutical University, Toyama, Japan
*Corresponding author: Tel: +813 3433 1111 (ext. 3341); Fax: 813 5401 0125; E-mail: m-honda@jikei.ac.jp
We have examined the susceptibility to acyclovir
and frequency of acyclovir-resistant viruses in
herpes simplex virus type (HSV) 2 clones isolated
directly from genital lesions of 11 patients who
had taken suppressive therapy (200 mg/day) for
1–9 years and 15 patients naive to acyclovir.
Suppressive therapy significantly reduced the
incidence of recurrence and the severity of the
skin lesions. HSV samples from genital lesions
were directly inoculated into Vero cells, and viral
clones were isolated in the absence and presence
of 10 µg/ml acyclovir. Five-hundred-and-ninety-
two clones, isolated in the absence of acyclovir,
were subjected to the acyclovir susceptibility
test, and 155 clones isolated in the presence of
acyclovir were analysed for the mechanisms of
resistance to acyclovir. There were no significant
Introduction
Acyclovir (Elion et al., 1977) is one of the current treatments
of choice for herpes simplex virus (HSV) infection and is
used mainly for short-term treatment in acute or recurrent
infections. Long-term continuous acyclovir therapy for
recurrent herpes simplex is considered effective and safe
(Straus et al., 1984; Mostrow et al., 1988; Kaplowitz et al.,
1991; Fife et al., 1994; Kimberlin et al., 1995; Centers for
Disease Control and Prevention, 1998). However, an
increasing number of chronic HSV infections in immuno-
compromised hosts, such as patients in the late phase of
HIV-infection, require long-term treatment. In such cases,
the emergence of drug-resistant viruses might occur (Straus
et al., 1988; Erlich et al., 1989; Gray et al., 1989; Englund et
al., 1990; Kaplowitz et al., 1991; Balfour et al., 1994;
Chistophers et al., 1998; Swetter et al., 1998).
An oral acyclovir was licensed for the first time in Japan
in 1988, but suppressive and preventive administration for
genital herpes is not permitted. The only recommended reg-
imen for episodes of recurrent infection is acyclovir 200mg
orally, five times a day, for 5 days. In our 379 Japanese
©2001 International Medical Press 0956-3202/01/$17.00
differences in the susceptibility to acyclovir, the
frequency of acyclovir-resistant virus and the
ratio of thymidine kinase-deficient viruses in acy-
clovir-resistant viruses between the two groups.
The frequency of acyclovir-resistant clones was
about three per 10000 plaque forming units
(PFU), and genital lesions contained up to 3x106
PFU of replicating virus in the specimens from the
patients with genital herpes with or without
acyclovir-suppressive therapy. Thus, the low dose
of acyclovir suppressive therapy did not affect
the susceptibility to acyclovir or increase the fre-
quency of acyclovir-resistant viruses in the geni-
tal lesions.
Keywords: genital herpes, herpes simplex virus,
acyclovir, susceptibility
patients with genital herpes the mean rate of recurrences
caused by HSV-1 is 1.0 per year, while that for HSV-2 is 9.7
per year. We use 200 mg oral acyclovir once a day for daily
low-dose acyclovir suppressive therapy in our patients with
genital herpes caused by HSV-2 who have six or more recur-
rences per year. Thus, this study is designed to characterize
the affect of long-term, low-dose acyclovir suppressive ther-
apy on susceptibility to acyclovir and frequency of acyclovir-
resistance for HSV-2 isolated from genital lesions. In order
to perform this investigation, HSV-2 was isolated as clones
directly from a smear of genital lesions in the presence and
absence of 10 µg/ml acyclovir. Then the amount of total
virus and the frequency of acyclovir resistant virus in the
smear samples was determined in patients on suppressive
therapy and in those naive to acyclovir therapy. We could not
find any affect of long-term, low-dose acyclovir suppressive
therapy on the susceptibility to acyclovir and on the frequen-
cy of acyclovir-resistance of the virus population isolated
directly from lesions of genital herpes. This finding suggests
that long-term, low-dose acyclovir suppressive therapy for
1
M Honda et al.

Table 1. Isolation of acyclovir-resistant virus from the artificial mixtures of acyclovir-sensitive and thymidine
kinase (TK)-deficient acyclovir-resistant viruses
Test sample No ACV(PFU) 10 µg/ml ACV Ratio
ACV-sensitive 7401H strain 3.19×107 1.10×103 3.45×10-4
TK-deficient strain 2.60×106 1.90×106 0.730
7
Mixture 1 2.82×10 1.51×105 5.35×10–3
Mixture 2 2.58×107 7.00×104 2.71×10–3
7 4
Mixture 3 2.93×10 2.70×10 0.92×10–3

Diluted TK-deficient acyclovir-resistant virus was mixed with acyclovir-sensitive virus at three different concentrations as indicated: mixture 1
(undiluted TK-deficient virus), 2 (twice dilution) and 3 (5 times dilution). Virus titre was determined in the presence and absence of acyclovir
(10 mg/ml) and the ratio indicates the comparative values of the virus titre with acyclovir over the titre without acyclovir. ACV, acyclovir; PFU,
plaque forming unit.

1–9 years has not altered the nature of the latent virus in the
ganglia, even using highly sensitive assay methods to detect
acyclovir-resistant virus in the genital lesions.
Materials and Methods
Patients
Sixty-two patients (25 women, 37 men; mean age 44 years,
range 26–73 years) who had given informed consent, had
HSV-2 isolated from the genital lesions and had six or more
recurrences per year, were taken from a cohort of Japanese
patients with recurrent genital herpes who had received acy-
clovir therapy and had been followed between 1990 and
1998. The patients had been assigned a dosage of 200 mg
once daily between 1990 and 1998. Single daily doses pro-
duced less complete suppression of genital herpes. Eighteen
of the 62 patients (29%) had not experienced a herpes
episode in 1998, and 18 (29%) had a single episode, 12 (19%)
two episodes, seven (11%) three episodes, five (8%) four
episodes, one (2%) five episodes and one (2%) six episodes.
Therefore, among 62 patients who have been receiving sup-
pressive therapy in 1998, 44 (71%) had one and more recur-
rences. Eighteen of the 44 patients who revealed sympto-
matic recurrences could visit our hospital, and specimens for
HSV culture were obtained from their genital lesions.
We also selected 16 patients (12 women and four men:
mean age 49 years, range 25–77 years) with genital herpes
and a history of six or more recurrences per year. The 16
patients had HSV-2 isolated from genital lesions, and had
not received acyclovir therapy. All patients were healthy and
received no immunosuppressive medications. Thus, we
obtained specimens from 18 patients treated with acyclovir
suppressive therapy, and from an additional 16 patients who
had not received treatment with antiviral agents.
Isolation of acyclovir-resistant virus from the
artificial mixtures of acyclovir-sensitive and
thymidine kinase-deficient, acyclovir-resistant
viruses
We have performed the quantitation of acyclovir-resistant
virus from the artificial mixtures of acyclovir-sensitive and
thymidine kinase (TK)-deficient, acyclovir-resistant viruses.
Acyclovir-resistant and -sensitive viruses were mixed at the
different ratios and inoculated into Vero cell cultures. One
set of monolayers was overlaid with methylcellulose without
acyclovir and another with methylcellulose containing 10
µg/ml of acyclovir. When the plaques appeared, the number
of plaques was counted under a dissecting microscope.
Direct isolation of HSV-2 as clones from the
lesions of genital herpes
Vero cells were grown in Eagle’s minimum essential medi-
um supplemented with 5% bovine serum and maintained
with 2% bovine serum. The procedures for the isolation of
virus clones and their susceptibility assay have been report-
ed previously (Hasegawa et al., 2001). Smears from genial
lesions were suspended in 1 ml of PBS and centrifuged at
12 000 rpm for 5 min at 4°C. The supernatant was 10-fold
serially diluted with PBS, and 0.2 ml from each dilution
was inoculated into two sets of Vero cell monolayers in
60mm plastic dishes for 1 h. One set of monolayers was
overlaid with methylcellulose without acyclovir and anoth-
er with methylcellulose containing 10 µg/ml of acyclovir.
When the plaques appeared, they were counted under a
dissecting microscope, and clones were directly isolated
from the monolayers with and without acyclovir. The iso-
lated clones were grown in Vero cell cultures and subjected
to a susceptibility assay to antiviral agents. Virus titres in
the samples were determined from the cultures without
acyclovir, and the numbers of acyclovir-resistant viruses was
determined from the cultures with acyclovir. The frequen-
cy of acyclovir-resistant viruses in the sample was defined
as the number of resistant virus per 104 viruses.
Determination of susceptibility to antiviral
drugs
The susceptibilities of viruses to antiviral drugs were deter-
mined by examining the inhibitory concentration for 50%
plaque reduction (IC50) (Crumpacker et al., 1979; Biron &
Elion, 1980; Kurokawa et al., 1993, 1998; Hasegawa et al.,

2 ©2001 International Medical Press

 Safety of long-term, low-dose acyclovir suppressive therapy

Table 2. Characterization of isolated clones from genital lesions of patients who had suppressive therapy with
acyclovir
Suppressive Frequency
Patient age period of acyclovir-
Sample [years (sex)] (years) IC50 (µg/ml) Virus titre (PFU/ml) resistant virus
2 44 (M) 9 0.42± 0.11 (10) 1.6×106 0.1
3 54 (F) 4 0.42 (1) 1.7×100 ND
5
4 30 (F) 3 0.81± 0.16 (10) 5.1×10 0.0
6 50 (F) 6 0.50± 0.08 (10) 5.0×101 ND
5
6 6 0.97± 0.22 (14) 2.5×10 9.9
7 66 (M) 7 0.66± 0.09 (10) 2.7×103 0.0
6
7 7 0.67± 0.10 (10) 1.1×10 0.1
10 33 (M) 3 0.63± 0.09 (2) 3.3×106 0.0
11 36 (F) 6 0.76± 0.06 (3) 5.0×100 ND
1
13 37 (M) 3 0.80± 0.16 (10) 2.5×10 ND

15 44 (M) 9 0.71± 0.11 (10) 2.3×106 1.1

21 60 (F) 2 0.68± 0.08 (10) 5.2×102 ND

22 28 (M) 1 0.83± 0.12 (13) 3.9×103 6.4

Mean± SD 0.68± 0.16 3.38± 2.35 (log10) 2.51± 4.0

IC50, inhibitory concentration for 50% plaque reduction; ND, not determined; PFU, plaque forming units; M, male; F, female. IC50 is expressed
as the mean ± SD of the clones isolated directly from lesions. The figures in parentheses indicate the numbers of the isolated clones examined.

2001). Antiviral drugs used were acyclovir (kindly supplied
by Nippon Wellcome KK, Osaka, Japan), iododeoxyuridine
(IDU) and phosphonoacetic acid (PAA) (purchased from
Seikagakukogyo, Japan). Briefly, confluent cell monolayers
in 60 mm plastic dishes in duplicate or triplicate were
infected with 100 plaque forming units (PFU) of virus for
1 h, and incubated in a nutrient methylcellulose medium
containing different concentrations of the drugs. After the
appearance of cytopathic effect, the cells were fixed with
5% neutral formalin and stained with methylene blue, and
the number of plaques was counted with a dissecting
microscope. The IC50 was determined graphically.
Statistical analysis
The Student’s t-test and χ2 Fisher test was used to evaluate
the statistical significance of the difference in the IC50 val-
ues among the isolates, and probability values (P) less than
0.05 were considered to be statistically significant.
Results
Isolation of acyclovir-resistant virus from the
artificial mixtures of acyclovir-sensitive and TK-
deficient, acyclovir-resistant viruses
The quantitation of acyclovir-resistant virus from the arti-
ficial mixtures of acyclovir-sensitive and TK-deficient, acy-
clovir-resistant viruses is shown in Table 1. Acyclovir-
resistant viruses showed 73% of virus titre in the presence
of 10 µg/ml acyclovir. Diluted TK-deficient virus was
mixed with acyclovir-sensitive virus at three different mix-
tures: (1) undiluted TK-deficient virus; (2) twice dilution;
and (3) five times dilution. Thus, the artificial mixtures
were prepared at the various mixtures of acyclovir-sensitive
and -resistant viruses, and the ratio of acyclovir-resistant
viruses per total virus titre was consistent with the mixing
ratio of the two viruses. The artificial mixture experiment
verified the quantitative assay system for detecting acy-
clovir-resistant virus in the samples containing acyclovir-
sensitive and -resistant virus.
Efficacy of low-dose suppressive therapy
Virus titre and frequency of acyclovir-resistant virus in
genital samples. HSV-2 clones of 13 samples were isolat-
ed from the genital lesions of 11 out of 18 patients on acy-
clovir treatment and from 15 out of 16 patients without
treatment. Table 2 summarizes the results of virus isolation,
the susceptibility to acyclovir and the frequency of acy-
clovir-resistant virus in the samples directly isolated from
genital lesions of patients on suppressive therapy. The mean
IC50 of clones from genital lesions was similar among 13
samples from the 11 patients on acyclovir therapy. Total
virus amount, expressed as PFU per ml per smear, from
skin lesions ranged from 1 to 2×l06 PFU per smear, and six
samples of 13 contained more than 104 PFU. This variation
in virus titre may be mainly due to the stage of the skin
lesions. Acyclovir-resistant virus was defined as viruses
with an IC50 of more than 10 µg/ml of acyclovir. The fre-
quency of acyclovir-resistant viruses in the samples ranged

Antiviral Chemistry & Chemotherapy 12:4 3

M Honda et al.

Table 3. Characterization of isolated clones from genital lesions of patients naive to acyclovir therapy
Patient No./age Frequency of
Sample (years)/sex IC50 (µg/ml) Virus titre (PFU/ml) acyclovir-resistant virus
12 46 (M) 0.83± 0.11 (10) 1.6×105 7.8
18 66 (F) 0.71± 0.18 (14) 3.5×101 ND

19 64 (M) 0.59± 0.06 (10) 1.8×102 ND

4
20 77 (F) 0.54± 0.10 (15) 4.0×10 0
23 42 (F) 0.63± 0.11 (10) 1.7×102 ND

24 31 (F) 0.69± 0.06 (2) 5.0×100 ND

25 68 (F) 0.67± 0.09 (10) 2.8×103 ND

3
26 56 (F) 0.45± 0.08 (10) 7.1×10 ND

27 25 (F) 0.48± 0.05 (10) 1.6×106 1.0

28 28 (F) 0.48± 0.15 (10) 2.5×101 ND

29 52 (F) 0.57± 0.09 (10) 8.9×103 ND

4
30 33 (F) 0.71± 0.12 (14) 1.2×10 13.5
31 31 (M) 0.58± 0.07 (10) 6.4×105 0.5
32 47 (F) 0.57± 0.04 (10) 3.0×106 0.2
1
34 69 (F) 0.45± 0.00 (2) 1.0×10 ND

Mean± SD 0.60± 0.11 3.52± 1.92 (log10) 3.38± 5.59

IC50, inhibitory concentration for 50% plaque reduction; ND, not determined; PFU, plaque forming units; M, male; F, female. IC50 is expressed
as the mean ± SD of the clones isolated directly from lesions. The figures in parentheses indicate the numbers of the isolated clones examined.

from 0.0 to 9.9 PFU per 104 PFU of the isolated clones in
the samples with more than 104 PFU and with acyclovir-
resistant virus. Table 3 shows the results of virus isolation,
the susceptibility to acyclovir and the frequency of acyclovir
resistant virus in the samples directly isolated from genital
lesions of patients naive to acyclovir therapy. Similar results
from patients on suppressive therapy were observed for
each item in Table 3.
Comparison of the results between patients on suppres-
sive therapy and those naive to acyclovir is summarized in
Table 4. There was no significant difference in the virus
amount and frequency of acyclovir-resistant virus per I04
PFU of the sample between the two groups.
Characterization of acyclovir-resistant viruses in
the genital lesions
In order to characterize the mechanism of acyclovir resist-
ance in the viruses from the skin lesions, we categorized
these acyclovir-resistant viruses by the pattern of suscepti-
bility and resistance to IDU and PAA as shown in Table 4.
The acyclovir-resistant viruses were defined as those with an
IC50 value of more than 10 µg/ml of acyclovir. TK-deficient

Table 4. Summary of virus isolation and resistant viruses in the lesions of genital herpes
Patients on suppressive therapy Patients naive to acyclovir

Subjects 18 16
Samples 22 16
Virus isolation 13 15
Virus titre (>104) 6 6
Mean virus titre ±SD (log10) 3.38±2.35 (log10) 3.52±1.92
(1.7×100–3.3×106) (5.0×100–3.0×106)
Cases with resistant virus 5 5
Case without resistant virus 1 1
Resistant viruses cloned 88 72
Frequency of resistant viruses 2.51±4.00 3.83±5.59
(per 10000 PFU in the sample)

PFU, plaque forming units.

4 ©2001 International Medical Press


Table 5. Characterization of acyclovir-resistant clones
isolated directly from skin lesions
Clones from Clones from
patients with patients
suppressive naive to
therapy acyclovir
Clones examined 88 72
TK deficient 84 67
TK altered 1 3
DNA polymerase 4 2
altered
TK, thymidine kinase.
viruses were resistant to IDU but were similarly as sensitive
to PAA as the acyclovir-sensitive viruses. TK-altered virus
was partially resistant to IDU but sensitive to PAA. DNA
polymerase altered virus was sensitive to IDU but resistant
or hypersensitive to PAA. The majority of the acyclovir-
resistant viruses were TK-deficient viruses in both patients
groups, and both groups contained some TK-altered and
DNA polymerase-altered viruses. There was also no signif-
icant difference in the pattern of resistance to acyclovir
between patients with suppressive therapy and those naive
to acyclovir (Table 5). Thus, no significant difference
between the two groups of patients was observed in the fre-
quency of acyclovir-resistant viruses in 104 PFU of the sam-
ple from genital lesions and their pattern of acyclovir resist-
ance in the TK and DNA polymerase genes. This finding
suggested an absence of induction of acyclovir-resistant
viruses in patients with genital herpes during chronic low-
dose suppressive therapy with low-dose acyclovir.
Discussion
Acyclovir is an effective and selective inhibitor of HSV
replication. The mechanism of action of this purine nucleo-
side analogue against HSV implies an initial drug phospho-
rylation by the viral TK. Acyclovir-monophosphate is then
phosphorylated by cellular enzymes to the triphosphate
form. The latter is a competitive inhibitor of viral DNA
polymerase and also acts as a chain terminator. Several
analyses of immunocompetent patients cohorts have been
reported that establish the long-term safety and efficacy of
suppressive acyclovir therapy (Mertz et al., 1988; Kroon et
al., 1990; Wald et al., 1997; Baker et al., 1999). Clinically
significant resistance to acyclovir has been almost exclusive-
ly seen in the immunocompromised population. Acyclovir-
resistant HSV have been reported in patients with immun-
odeficiency in the IC50 assay (Erlich et al., 1989; Englund et
al., 1990; Christophers et al., 1998). The degree of immuno-
suppression, and low doses and prolonged use of acyclovir
are considered important factors for the development of
drug resistance. Phenotypically, viral resistance to acyclovir
Antiviral Chemistry & Chemotherapy 12:4
Safety of long-term, low-dose acyclovir suppressive therapy
is related to one of the following four mechanisms: complete
deficiency in viral TK activity (TK-deficient), viral TK pro-
tein with altered substrate specificity (TK-altered),
decreased activity or production of low amounts of viral TK
(TK-low producer) and viral polymerase with altered sub-
strate specificity (Sasadeusz et al., 1997; Hasegawa et al.,
2001).
In conventional virus isolation, virus is isolated as the
mixture of individual clones in skin lesions. The sensitivity
of the isolated virus to acyclovir is conventionally evaluated
by IC50 and IC90 values, IC90 indicating the presence of less
sensitive virus in the virus inoculum. However, at least 10%
of the inoculum virus sample should exhibit less sensitivity
to acyclovir in order to affect the value of IC90. In this study
we used a highly sensitive method to detect acyclovir-resist-
ant virus in the specimens from the genital lesions. We
directly evaluated the amount acyclovir-resistant virus
clones in 104 PFU of virus in the specimens. In this aspect,
our assay system is at least 1000 times more sensitive than
IC90 in detecting acyclovir-resistant virus in clinical speci-
mens. This highly sensitive method was used to assess and
compare the frequency of acyclovir-resistant virus directly
isolated from genital lesions of patients on acyclovir-sup-
pressive therapy, with the frequency of resistant virus isolat-
ed from patients naive to acyclovir. This method may be
powerful in detecting the increase in acyclovir-resistant
virus in the lesions in the early phase of replacement from
acyclovir-sensitive virus to acyclovir-resistant virus.
The experiment using the artificial mixtures of acyclovir-
resistant and -sensitive viruses verified the assay system for
the quantitative detection of acyclovir-resistant viruses from
their different mixtures. Based on this assay system, we
report the comparison of acyclovir-resistant isolates from
patients who had genital herpes treated with suppressive
acyclovir therapy, and isolates from patients who had not
received treatment with antiviral agents or any other treat-
ment. There was no significant difference between the two
groups in the frequency of resistant virus and their pattern
of resistance to acyclovir. This is consistent with previous
studies concerning the susceptibility of clinical isolates from
patients on acyclovir suppressive therapy in the aspect of
IC50 values (Al-Hasani et al., 1986; Lehrman et al., 1986,
1986; Mertz et al., 1988; Kroon et al., 1990; Molin et al.,
1991; Baker 1994). Furthermore, the pattern of acyclovir
resistance of the acyclovir-resistant viruses was similar for
the TK and DNA polymerase between the two groups.
HSV has widely occurring natural variants, and Parris
and Harrington (1982) reported the presence of acyclovir-
resistant virus in the clinically isolated virus stock at about
10–4 as determined by plating efficiency. Interestingly, most
of them contained alterations in the DNA polymerase gene
locus. In our study, we have directly isolated the acyclovir-
resistant variants as clones in the presence of acyclovir from
5
M Honda et al.
the genital lesions, which contained replicating virus, and
the frequency was about three per 10000 PFU. HSV-2 clin-
ical isolates grown in tissue cultures showed about 11 per
10000 PFU (Hasegawa et al., 2001). Therefore, the fre-
quency of the acyclovir-resistant virus in the genital lesions
was the same as the naturally occurring frequency of acy-
clovir-resistant virus. Furthermore, the proportion of TK-
deficient virus in the acyclovir-resistant viruses coincided
with the frequency of clinical isolates with acyclovir pheno-
typic resistance (Kimberlin et al., 1995; Chistophers et al.,
1998). There was no significant difference between the
patients groups in the susceptibility or frequency of acy-
clovir-resistant virus, and the ratio of TK-deficient virus in
the acyclovir-resistant variants. This finding suggests that
there is no induction of acyclovir-resistant viruses in
patients with genital herpes during chronic suppressive
therapy with acyclovir.
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