Biochemical Pharmacology 206 (2022) 115322

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Biochemical Pharmacology
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Review

Acyclovir resistance in herpes simplex viruses: Prevalence and

therapeutic alternatives
Hanna H. Schalkwijk , Robert Snoeck , Graciela Andrei *
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Herpes simplex viruses (HSV), the causative agents of recurrent orofacial and anogenital infections, can cause
Immunocompromised significant morbidity and mortality in both immunocompetent and immunocompromised individuals. In
Immune-privileged sites immunocompromised patients, HSV tends to be more persistent with chance of dissemination. The nucleoside
Herpes simplex virus
analogue acyclovir has drastically improved the management of HSV infections although acyclovir resistant
Acyclovir
strains have been reported in the clinic. We performed a systematic search to summarize the prevalence data
Acyclovir resistance
Prevalence reported in both the immunocompetent and immunocompromised populations. Defining the global prevalence of
acyclovir resistance in HSV infections is hampered by the high variability in methodology, patient selection,
study design, and treatment history among the studies. Acyclovir resistant HSV is infrequent in the immuno­
competent population (generally below 1%), irrespective of treatment history. Exceptions are infections at
immune-privileged sites such as the cornea, where frequent recurrences and extensive acyclovir therapy favor
the emergence of acyclovir resistance. Higher frequencies of acyclovir resistant HSV infections are reported
among immunocompromised individuals, with the highest prevalence seen among hematopoietic stem cell
transplant recipients. All antivirals approved for the treatment of HSV infections have the same target, i.e. the
viral DNA polymerase, and cross-resistance to different antivirals has been described, complicating therapy of
acyclovir resistant strains. In this review we will discuss acyclovir mode of action, mechanisms of resistance,
prevalence of resistance, and alternative antiviral treatments for acyclovir resistant HSV infections.

1. Introduction human cytomegalovirus (CMV), HHV-6A, HHV-6B, and HHV-7. The

The Herpesviridae family includes a large group of double-stranded
DNA viruses that can infect a wide range of animal species [1]. Her­
pesviruses show narrow host-specificity indicating evolution with their
hosts over long periods of time [2]. A common characteristic is their
ability to establish lifelong latent infections, followed by periods of
reactivation upon diverse stimuli. Nine human herpesviruses (HHV)
have been identified which are classified into three subfamilies based on
their genome organization, cell tropism, and growth characteristics: the
alphaherpesvirinae, betaherpesvirinae, and gammaherpesvirinae. The
alphaherpesvirinae comprises herpes simplex virus 1 (HSV-1) and 2 (HSV-
2), and varicella zoster virus (VZV). The betaherpesvirinae includes
gammaherpesvirinae contains Epstein-Barr virus (EBV) and HHV-8 or
Kaposi’s sarcoma-associated herpesvirus (KSHV) [3]. All HHV infections
can cause severe disease in immunocompromised hosts such as lym­
phoproliferative disorders associated with EBV or KSHV and dissemi­
nated infections (VZV, CMV, or HSV) [4,5]. HSV-1 and HSV-2 can also
cause substantial morbidity during primary and recurrent infections in
immunocompetent individuals [6]. Infections of the central nervous
system (CNS) with herpesviruses are an important cause of morbidity
and mortality and can lead to long-term neurologic sequelae. The most
significant CNS infections are due to HSV and CMV. CNS infection with
CMV occur mainly perinatally, though it may also be seen seldom in
children and adults, particularly in the immunocompromised

Abbreviations: ACV, acyclovir; ACV-MP, acyclovir monophosphate; ACVr, acyclovir resistant; CMV, human cytomegalovirus; CNS, central nervous system; CPE,
cytopathic effect; DNA pol, DNA polymerase; dT, thymidine; dTMP, thymidine monophosphate; EC50, concentration inhibiting 50% of virus growth in cell culture;
HHV, human herpesvirus; HIV, human immunodeficiency virus; HSCT, hematopoietic stem cell transplant; HSE, herpes simplex encephalitis; HSV, herpes simplex
virus; HSV-1, herpes simplex virus 1; HSV-2, herpes simplex virus 2; SEM, skin, eye and/or mouth; TK, thymidine kinase; VACV, valacyclovir; VZV, varicella zoster
virus.
* Corresponding author at: Rega institute for Medical Research, Herestraat 49 bus 1043, Leuven, Belgium.
E-mail address: graciela.andrei@kuleuven.be (G. Andrei).

https://doi.org/10.1016/j.bcp.2022.115322
Received 31 August 2022; Received in revised form 20 October 2022; Accepted 20 October 2022
Available online 26 October 2022
0006-2952/© 2022 Elsevier Inc. All rights reserved.
H.H. Schalkwijk et al.
population. CNS infection with HSV can occur in neonates as well as in
older children and adults [7].
The discovery of the nucleoside analogue acyclovir (ACV) was a
milestone in the management of HSV infections. However, frequent use
of ACV increased the incidence of ACV resistant (ACVr) HSV infections.
Drug resistance in herpes simplex viruses poses a major concern in the
immunocompromised host as they often require long-term antiviral
therapy, and this, in combination with ongoing viral replication in­
creases the risk of drug resistance emergence. The limited number of
antivirals available for the therapy of HSV infections, all having the
same drug target (i.e., the viral DNA polymerase), makes the manage­
ment of HSV drug resistance in the clinic challenging. In this review we
summarize the prevalence data on ACV resistance in herpes simplex
viruses. We also describe clinical manifestations of HSV infections,
mechanisms of ACV resistance, and therapeutic alternatives.
2. Herpes simplex virus infections
HSV infections can have a variety of clinical manifestations. HSV-1 is
typically transmitted during childhood and predominantly causes oro­
facial herpes but can also cause anogenital herpes. HSV-2, mostly
sexually transmitted, causes anogenital herpes and infrequently causes
orofacial herpes. In 2016, the global seroprevalence of HSV-1 was esti­
mated at 66.6 % in the population aged 0–49 years and seroprevalence
of HSV-2 in 15–49-year-olds was estimated at 13.2 % [8]. Seropreva­
lence increases with age and the highest seroprevalence of HSV-1 and
HSV-2 is reported in Africa [8–10]. Women are more likely to become
infected with HSV-2, possibly due to a greater biological susceptibility
[8,10,11]. Several clinical manifestations of HSV infections will be dis­
cussed in more detail in the following sections.
2.1. Orofacial herpes
Primary orofacial HSV infections are often asymptomatic, but
symptomatic infection is characterized by fever and lesions at the buccal
and gingival mucosa. Intraoral lesions are mostly associated with pri­
mary infection while lip lesions (cold sores) are suggestive for re­
currences [12]. Recurrences with HSV-1 occur in approximately 40 % of
infected individuals, while orofacial HSV-2 recurs in only 4 % of the
cases [13]. Recurrent orofacial herpes is preceded by pain, burning,
tingling, or itching after which vesicles appear and is often self-limiting,
with lesions usually healing after 8 to 10 days [12].
2.2. Anogenital herpes
Anogenital herpes is mostly caused by HSV-2, and less frequently by
HSV-1 although the proportion of anogenital herpes caused by HSV-1 is
increasing, particularly among young people in the Americas and Eu­
ropean region [8,14,15]. Possible explanations are the decrease in HSV-
1 acquisition during childhood and an increased frequency of oral sex
[16,17]. Anogenital HSV shedding and transmission to sexual partners
can take place even in the absence of symptoms [12]. Anogenital herpes
infections with HSV-1 and HSV-2 are clinically indistinguishable. The
clinical presentation is characterized by painful, vesicular, and ulcera­
tive lesions on the external genitalia, perianal region, buttocks, or upper
thighs. Lesions cause pain, itching, burning, and dysuria. Patients with
primary infection can additionally present with fever, headache, malaise
and myalgia, although primary infections usually remain asymptomatic
[18]. HSV-2 causes frequent recurrent anogenital outbreaks, especially
in the first year, typically lasting for 8 to 10 days, whereas HSV-1 re­
currences tend to be milder and less frequent [19–21]. In most patients,
the rate of reactivation decreases with time, but some patients continue
to have recurrences for>10 years [19,22].
2
Biochemical Pharmacology 206 (2022) 115322
2.3. Neonatal herpes
Although neonatal herpes is relatively rare, it is a potentially
devastating complication of anogenital herpes during pregnancy. HSV
transmission to the fetus commonly occurs through exposure of the
neonate to HSV-1 or HSV-2, shed in the genital tract during delivery (85
%). Besides perinatal infection, HSV may be acquired by intrauterine (5
%) or post-partum infection (10 %) [23]. HSV-2 is responsible for about
two-thirds of the cases with HSV-1 accounting for the remains [24]. Risk
of transmission is the highest when primary anogenital herpes develops
late during pregnancy [22,24]. The global rate of neonatal herpes is
estimated at 10 per 100.000 live births [24].
Neonatal herpes can be classified as (i) localized skin, eye and/or
mouth (SEM) disease accounting for 45 % of neonatal herpes, (ii) CNS
disease, accounting for 30 % of cases, and as (iii) disseminated disease
that accounts for 25 % of cases [25]. In SEM disease, lesions are confined
to the skin, eye, and/or mouth, whereas neonates with CNS disease
present irritability, poor feeding, seizures, and/or temperature insta­
bility with or without SEM involvement. Disseminated disease involves
multiple organs including the CNS, lungs, liver, skin, eye, and/or mouth
[26]. SEM disease has no attributable mortality, whereas the mortality
rate due to disseminated disease is 85 % when left untreated [27,28].
Due to antiviral treatment, mortality rates have been reduced to ~ 5 %
in neonates with CNS disease and to ~ 30 % in neonates with dissemi­
nated disease [29,30].
2.4. Herpetic keratitis
Herpetic eye infections can have different manifestations including
blepharitis, conjunctivitis, retinitis, and herpetic keratitis [31]. Herpetic
keratitis, mostly associated with HSV-1, manifests predominantly as
epithelial keratitis, limited to the superficial cornea, or stromal keratitis
[31,32]. In 2016, the incidence of herpetic keratitis was estimated at 24
cases per 100.000 person-years, giving an estimated 1.7 million cases of
herpetic keratitis worldwide [33]. Herpetic keratitis is an important
cause of visual impairment due to its recurrent nature, and annually
causes 40.000 new cases of monocular visual impairment [34,35]. HSV-
1 is considered the leading cause of infectious blindness in Western
countries.
2.5. Herpes simplex encephalitis (HSE)
Herpes simplex encephalitis, mostly caused by HSV-1, has an esti­
mated incidence of one case in 250.000–500.000 individuals per year
and is the most common cause of sporadic infectious encephalitis
[36–39]. Common symptoms of HSE are changes in mental status,
abnormal behavior, fever, headache, and seizures but clinical presen­
tation differs among patients, making diagnosis difficult [40,41].
Without treatment, the mortality rate of HSE is 70 % and most survivors
suffer from severe neurologic sequelae [12]. Neurologic sequelae remain
common despite antiviral treatment, especially when treatment is
delayed [40].
2.6. HSV infection in the immunocompromised host
Both innate and adaptive immune responses play a pivotal role in
determining the severity of HSV infections and frequency of reactivation
[42]. Immunocompromised patients are at risk for developing severe
and potentially life-threatening HSV infections, particularly when cell-
mediated immune responses are reduced, as is the case in hematopoi­
etic stem cell transplant (HSCT) recipients and in human immunodefi­
ciency virus (HIV) infected individuals [43]. Latent HSV frequently
reactivates in immunocompromised individuals, particularly in HSCT
recipients, of whom 80 % experience HSV reactivation [43–45]. Most
HSV infections in immunocompromised hosts locate to the orofacial and
anogenital sites and may have an atypical appearance with extensive
H.H. Schalkwijk et al. Biochemical Pharmacology 206 (2022) 115322

lesions, slow healing, more invasive disease, and risk of viral dissemi­
nation [43,46,47]. Therefore, prophylactic or therapeutic antiviral
treatment is often required in immunocompromised patients to prevent
morbidity and mortality.

3. Acyclovir

3.1. Mode of action

Since its introduction in the 1980 s, the nucleoside analogue

acyclovir [9-(2-hydroxyethoxymethyl)guanine] has become the first
line antiviral for prophylaxis and treatment of HSV infections. Acyclovir
is an acyclic guanosine analogue that lacks both the 2′ - and 3′ -OH
moieties and the 2′ - and 3′ -carbons [48] (Fig. 1). Three phosphorylation
steps are required to obtain the active (triphosphate) form of ACV, the
first one being performed by the virus-encoded thymidine kinase (TK)
[49]. The two successive phosphorylation steps are carried out by the
cellular guanosine monophosphate (GMP) kinase and nucleoside
diphosphate (NDP) kinase, respectively [50–52] (Fig. 1). Acyclovir
triphosphate (ACV-TP) competes with deoxyguanosine triphosphate
(dGTP) as substrate of the viral DNA polymerase (DNA pol) and is
incorporated into the elongating DNA chain [53]. Since incorporated
ACV monophosphate (ACV-MP) lacks a 3′ -hydroxyl moiety, its incor­
poration results in chain termination [54]. ACV-MP cannot be efficiently
excised by the 3′ -5′ exonuclease activity of the viral DNA pol due to its
altered sugar structure that impedes the hydrolysis reaction [55].
Acyclovir is highly selective due to its dependence on viral TK activity
for activation and a 100-fold more efficient recognition and incorpora­
tion of ACV-TP by the HSV DNA pol in comparison to cellular DNA
polymerases [56–58]. The major limitation of ACV is its poor oral
bioavailability (10 to 20 %), likely due to inadequate absorption of the
drug. Therefore, the prodrug valacyclovir (VACV), an L-valyl-ester of
ACV, has been developed with a three- to fivefold higher bioavailability
than ACV [59,60] (Fig. 1).

3.2. Clinical use of acyclovir

Topical formulations of ACV are available for the treatment of

recurrent herpes labialis (cold sores) and herpetic keratitis [61,62]. Oral
ACV and VACV are used for treatment of primary and recurrent ano­
genital HSV infections and as chronic suppressive therapy to prevent
recurrent anogenital herpes [63,64]. Therapy with ACV or VACV
effectively reduces symptomatic recurrences of anogenital herpes and
asymptomatic shedding, although the suppression of viral replication is
incomplete [65,66]. Prophylactic therapy is frequently used in severe Fig. 1. Mode of acyclovir activation. Hydrolysis converts the prodrug valacy­
immunosuppressed patients, including HSCT recipients, and is highly clovir to acyclovir. Three phosphorylation steps are required for acyclovir
effective in preventing reactivation [67–70]. Oral (V)ACV is often activation, the first one performed by the herpes simplex virus (HSV) thymidine
indicated for therapy of mucocutaneous HSV infections in immuno­ kinase (TK), confining its action to viral-infected cells. The two subsequent
compromised individuals. Intravenous formulations of ACV are used to phosphorylations are ensured by the cellular guanosine monophosphate (GMP)
treat severe HSV infections, including neonatal herpes, encephalitis, and kinase and nucleoside diphosphate (NDP) kinase. In its triphosphate form
infections in immunocompromised patients [68]. (acyclovir-TP), acyclovir targets the viral DNA polymerase by inhibiting viral
DNA synthesis. Abbreviations: acyclovir-MP, acyclovir monophosphate;
acyclovir-DP, acyclovir diphosphate.
4. Acyclovir resistance

A lack of clinical response to ACV treatment a week after beginning
of therapy can be suggestive of acquisition of drug resistance [63]. There
are several risk factors for the development of ACV resistance and those
include prolonged use of ACV, suboptimal dosing of ACV, (degree of)
immunosuppression, and ongoing viral replication [4,71,72]. Acyclovir
resistance arises due to the acquisition of mutations in the UL23 gene,
encoding for the viral TK, and/or the UL30 gene, encoding for the DNA
pol. Acyclovir resistant HSV infections are most frequently associated
with mutations in the TK gene [73,74].
4.1. Viral thymidine kinase
The UL23 genes of HSV-1 and HSV-2 encode for the 376 amino acid
long TK proteins which share approximately 74 % sequence identity. Six
highly conserved regions (site 1 to site 6) have been identified among
Herpesviridae thymidine kinases [75] (Fig. 2). The most important sites
for enzyme activity are the ATP-binding site (site 1), nucleoside binding
site (site 4), and the highly conserved cysteine at codon 336 (HSV-1) and
337 (HSV-2), which is essential to maintain the three-dimensional
conformation of the active site and contributes to the phosphorylation
efficiency of the viral enzyme [63,76].
Viral TK activity is dispensable in dividing cells but is essential for

3
H.H. Schalkwijk et al.
Fig. 2. Schematic overview of the A) herpes simplex virus 1 (HSV-1) and B) herpes simplex virus 2 (HSV-2) thymidine kinase protein. Conserved regions are
represented by grey boxes. Number under the boxes indicate the codon position of these regions. Abbreviations: ATP, ATP-binding site; NBS, nucleoside-binding site.
viral replication and reactivation in non-proliferating cells, such as
neurons [77–79]. The HSV-1 and HSV-2 TK have a broad range of ac­
tivity and can phosphorylate thymidine (dT), deoxycytidine (dC),
deoxyguanosine (dG), and ACV, into their respective monophosphate
forms dTMP, dCMP, dGMP, and ACV-MP [77,80]. Furthermore, HSV
TKs exhibit thymidylate activity and can convert dTMP into its
diphosphate dTDP, although HSV-2 TK exhibits a much lower rate of
thymidylate activity than HSV-1 TK [81].
Mutations in the TK that confer ACV resistance affect its enzymatic
activity and are associated with the following phenotypes: TK-negative
mutants which completely lack TK activity (ACV- / dT-), TK-altered
mutants (ACV- / dT 15–100 %) that retain the ability to phosphorylate
dT but have an impaired ability to phosphorylate ACV, TK-low mutants
(ACV- / dT 1–15 %) that show decreased TK activity or production, and
TK-partial mutants (ACV 1–15 % / dT 100 %), exhibiting full dT phos­
phorylation capacity and reduced ACV phosphorylation activity
[82–84]. Most ACVr TK mutants in the clinic exhibit a TK-negative or
TK-low phenotype, but TK-partial and TK-altered mutant strains have
also been reported [82–85].
Resistance associated mutations are located throughout the entire
UL23 gene and have been extensively described in several reviews
[74,82,86,87]. The HSV TK gene presents several G- and C- homopoly­
mer stretches that are hot-spots for nucleotide insertions and deletions
resulting in frameshifts and can potentially introduce premature stop
codons. Approximately 50 % and up to 80 % of the ACVr TK mutants in
the clinic harbor insertions or deletions in these homopolymer stretches,
while others carry nucleotide substitutions in conserved and non-
conserved regions introducing amino acid changes or stop codons (see
section 5.8) [73,83,88,89].
4.2. Viral DNA polymerase
The UL30 genes of HSV-1 and HSV-2 encode for a DNA pol of 1235
and 1240 amino acids long, respectively, that have a protein sequence
identity of approximately 90 % [90]. They share several functional
domains with the type B DNA polymerase family, including the 3′ -5′
Fig. 3. Schematic overview of the A) herpes simplex virus 1 (HSV-1) and B) herpes simplex virus 2 (HSV-2) DNA polymerase protein. Proposed functions and
subdomains are shown. Conserved regions are represented by grey boxes. The numbers under the boxes indicate the codon position of these regions.
4
Biochemical Pharmacology 206 (2022) 115322
exonuclease, palm, fingers, and thumb domains (Fig. 3). Herpesvirus
polymerases additionally possess a pre-NH2-terminal domain which is
required for efficient viral replication and latency establishment
although the precise role has not been established yet [91–94]. The palm
and finger domains are involved in the catalytic activity of the enzyme
and binding of incoming nucleotides, while the thumb domain interacts
with the primer-template complex [95]. The 3′ -5′ -exonuclease domain
exhibits proofreading activity allowing the excision and correction of
mismatched nucleotides during DNA replication, and is responsible for
the low mutation frequency of herpesviruses (1x10-7 to 1x10-8 muta­
tions/base/infectious cycle) [96,97]. Seven regions of conserved se­
quences have been identified in herpesvirus DNA pols that are numbered
I to VII based on their degree of conservation among the DNA pol, region
I being the most conserved [91] (Fig. 3). Besides, the HSV DNA pol also
contains the δ-region C, which is shared by polymerases having ho­
mology with the eukaryotic DNA polymerases δ.
Acyclovir resistance associated mutations in the DNA pol are pri­
marily located in the conserved regions of the palm, finger, and thumb
domains, favoring less ACV incorporation into the viral DNA [95].
Mutations in the exonuclease domain are found less frequently and it is
proposed that they confer resistance by enhancing the nucleoside
analogue excision rate [96]. An overview of resistance mutations
detected in the HSV DNA polymerases is available in several reviews
[74,86,95]. Mutations in the DNA pol conferring ACV resistance
commonly show cross-resistance to the second-line treatment foscarnet
(see section 6.2) [95,98].
4.3. Diagnosis of antiviral drug-resistance
Laboratory diagnosis of antiviral resistance is essential to guide cli­
nicians towards different treatment options in case of therapy failure.
Both phenotypical and genotypical assays are available for the diagnosis
of antiviral drug resistance. Genotypical analysis is based on the iden­
tification of resistance mutations in the UL23 and/or UL30 gene and is
almost exclusively performed by Sanger sequencing. Sequencing of both
the UL23 and UL30 genes yields the most reliable results. Sanger
H.H. Schalkwijk et al.
sequencing lacks sensitivity to detect minor resistant variants with a
frequency below 20 %. Recently an ultra-deep sequencing approach
using the Miseq Illumina platform has been implemented for HSV
resistance testing, enabling the detection of emerging resistant sub­
populations at an earlier stage [99]. Although genotypic analysis is a fast
approach that does not require the growth of virus in cell culture,
interpretation can be complicated due to the high variability of the TK
and DNA pol [74]. Novel mutations are continuously being identified in
the clinic, and phenotypical characterization or functional assays are
then required to confirm their effect on antiviral susceptibility. In the
study by Burrel et al. [89], 36 % of the clinical samples that were
genotypically characterized for ACV resistance required additional
phenotyping to interpret drug susceptibility due to the detection of
formerly unreported or undefined mutations in the UL23 and/or UL30
genes.
Phenotypical assays are used to determine the effective concentra­
tion of an antiviral inhibiting 50 % of virus growth in cell culture (EC50).
As it is dependent on the propagation of the virus in cell culture,
phenotypical assays are often time-consuming and can only be per­
formed if the virus is successfully isolated in cell culture. The presence of
minor resistant subpopulations can be detected if the resistant virus
exceeds 20 % and is not lost during growth in cell culture [100]. A va­
riety of phenotypical assays have been used to determine HSV multi­
plication in the presence of antivirals, including plaque reduction assays,
cytopathic effect (CPE) reduction assays, luciferase reporter assays, real-
time quantitative polymerase chain reaction (qPCR), colorimetric as­
says, virus yield inhibition assays, and DNA hybridization assays
[101–108]. The plaque reduction assay is considered the gold standard
for HSV drug susceptibility testing and uses the number of viral plaques
formed under various antiviral concentrations as readout, which is
somewhat subjective when counting very small plaques. The DNA hy­
bridization method quantifies the viral DNA produced in infected cells
under ACV pressure as an assessment of antiviral susceptibility and the
obtained EC50 values correlate well with those obtained by plaque
reduction assays [71,104]. The dye uptake assay is a modification of the
neutral red dye cell viability assay and measures the ability of an anti­
viral to inhibit cell damage induced by HSV replication [102]. The dye
uptake assay generally gives higher EC50 values than plaque reduction
assays and its limitation is the high false-positive rate for resistance of 3
% [109].
Many variables can influence the phenotypical results, such as the
used assay system, cell line, viral inoculum concentration, range of
antiviral concentrations, and resistance breakpoint but efforts have been
made to standardize the results [74,105]. The breakpoint used to define
drug resistance differs between different laboratories, although an EC50
of ≥ 2 µg/mL (8.9 µM) in plaque reduction assays is widely accepted as a
breakpoint for resistance [110]. Other studies have used breakpoints
between 1 and 3 µg/mL (4.4 to 13.3 µM), ≥3-fold increase in EC50 values
compared to the mean EC50 of all isolates, or EC50 values > 10-fold
higher than sensitive control strains [105]. HSV-1 is more susceptible to
ACV than HSV-2 and it is therefore advisable to use different resistance
cutoff values for each HSV species [73,111–113].
5. Prevalence of acyclovir resistance
5.1. Systematic search and article inclusion
As discussed in section 4, several factors can increase the risk for
emergence of ACV resistance, including long-term ACV use and immune
status. Hence, the prevalence of ACVr HSV infections varies among
different population groups. It is generally accepted that the prevalence
of ACVr HSV infections is higher in immunocompromised patients than
in immunocompetent patients. We reviewed the prevalence of ACV
resistance reported in different populations by performing a systematic
search in PubMed, Embase, Web of Science, and Cochrane with several
related keywords including herpes simplex, acyclovir, and resistance or
5
Biochemical Pharmacology 206 (2022) 115322
susceptibility. Articles published until May 2022 written in English,
reporting prevalence data on ACVr HSV infections based on a minimum
of 5 patients with specified immune status were selected. All included
studies used phenotypical and/or genotypical assays to confirm ACV
resistance. Clinical trials and studies where patient selection was based
on suspected resistance were excluded.
After deduplication, 5276 articles were screened based on title and
abstract and 174 articles were assessed for eligibility based on full-text.
Prevalence data from 43 included articles was extracted and subdivided
into immunocompetent individuals (21 articles), infections at immune-
privileged sites such as the eye and CNS (see section 5.3, 6 articles), and
the general immunocompromised population (12 articles). When spec­
ified, immunocompromised patients were subdivided into solid organ
transplant recipients (7 articles), HSCT recipients (16 articles), and HIV
infected individuals (9 articles).
5.2. Immunocompetent population
Twenty-one studies reported prevalence data on ACVr HSV infections
in the immunocompetent population [71,73,108,109,111–127]
(Table 1). The used methods and resistance cutoffs varied widely be­
tween those studies and it is therefore difficult to define the true inci­
dence of ACV resistance. The reported prevalence of ACVr HSV
infections in the immunocompetent population ranged from 0 % to 6.2
%. However, most studies, including multiple extensive screening sur­
veys, reported a prevalence under 1 %.
Four studies reported frequencies of ACV resistance above 1 % with
only the study from Collins et al. [109] reporting over 5 % prevalence in
the United States survey [108,109,123,125]. The studies from Collins
et al. and Varella et al. [125], reporting 6.2 %, and 4.2 % resistance,
respectively, used the dye uptake assay that has a high false-positive rate
(section 4.3). Additionally, all patients included in the studies from
Collins et al. received ACV therapy, although the incidence of ACVr virus
was similar to that observed in untreated patients. The study from Wang
et al. [108] reported ACVr HSV strains in 3 out of 68 (4.4 %) immuno­
competent children when using the phenotypical method mVILA but
only one case (1.4 %) could be confirmed by Sanger sequencing.
Schmidt et al. [123] reported a prevalence of 4.3 %, but scarce infor­
mation was available on clinical diagnosis, immunocompetence, and
treatment status of the tested patients.
Acyclovir resistance occurred at similar frequencies in ACV treated
and treatment naive patients (Table 1). Christophers et al. [116] re­
ported a prevalence of 0.67 % in ACV treated patients versus 0.42 % in
untreated patients. Fife et al. [117] reported similar frequencies of ACV
resistance before and after six years of suppressive ACV therapy in
immunocompetent patients with recurrent anogenital herpes. Thus,
despite the widespread availability of ACV over the past decades, ACV
resistance remains infrequent in the immunocompetent population.
Acyclovir resistant HSV infections in the immunocompetent popu­
lation are usually self-limiting as the natural immune response can
eliminate the virus that is not eradicated by antiviral therapy. Conse­
quently, antiviral resistance often has a limited effect on disease
outcome in immunocompetent individuals, although exceptions have
been reported [111,128,129]. Cases of herpetic keratitis were included
in some studies [112,116]. As the cornea is an immune-privileged site,
there is a high risk for development of drug resistance in patients
suffering from herpetic keratitis even though they do not have an
impaired immunity.
5.3. Immune-privileged sites
Immune-privileged sites such as the cornea and CNS show locally
reduced immune responses. HSV infections at these sites are not self-
limiting and ACV susceptibility correlates with a clinical response.
Therapy failure due to ACVr HSV infections can therefore ultimately
lead to blindness in case of ocular infections and to neurologic sequelae
 H.H. Schalkwijk et al.
Table 1
Characteristics of studies reporting on acyclovir resistance in herpes simplex virus among the immunocompetent population.
First author Publication Country HSV disease Treatment HSV N◦ patients N◦ N◦ patients N◦ N◦ HSV N◦ Method (cutoff
year status species patients with ACV samples positive resistant resistance)
tested resistant HSV isolates HSV
(%) (tested) isolates

Bacon [114] 2002 United Recurrent herpes Untreated & HSV-1 1803 1002a 2 (0.20 %) 1803 1087 (1002) 2 Plaque reduction assay
States labialis ACV treated (EC50 ≥ 2 µg/mL)
Boon [115] 2000 United Recurrent herpes Untreated or HSV-1 & 1304 924 1 (0.11 %) 1297 920 HSV-1 1 Plaque reduction assay
Kingdom labialis previously ACV HSV-2 2 HSV-2 0 (EC50 ≥ 2 µg/mL)
treated 2 HSV-1 & 0
HSV-2
Christophers 1998 United Genital herpes Untreated Untyped 708 708 3 (0.42 %) 760 760 3 Plaque reduction assay
[116] Kingdom Genital herpes ACV treated 149 149 1 (0.67 %) 162 162 1 (EC50 ≥ 3 µM)
Genital herpes, Unknown 918 918 1c (0.11 %) 944 944 1
herpetic keratitis, or
unknownb
Collins [109] 1993 Europe Unspecifiedb ACV treated Untyped 287 287 2 (0.70 %) 420 420 2 Plaque reduction assay
(EC50 ≥ 3 µM)
United 663 663 41 (6.2 %) 1448 1448 44 Dye uptake assay (EC50 ≥
States 3 µg/mL)
Danve- 2004 France Different herpetic Untreated & HSV-1 1237 1237 3 (0.24 %) 1326 1326 3 Dye uptake assay (EC50 ≥
Szatanekd infectionsb ACV treated 6.5 µM)
[111] HSV-2 618 618 3 (0.49 %) 664 664 13 Dye uptake assay (EC50 ≥
13.5 µM)
Englund [71] 1990 United Herpetic infections at Untreated & Untyped 59 59 0 (0 %) -e 61 0 DNA hybridization (EC50
States oral site or genital ACV treated ≥ 9 µM)
tract
Fife [117] 1994 United Recurrent genital History of ≥ 6 Untyped 239 4 113 4 Dye uptake assay (EC50 ≥
6

– –
States herpes years of chronic 3 µg/mL)
ACV therapy
Frobertd [73] 2014 France Different herpetic Unspecified HSV-1 & – – – 703 703 1 Dye uptake assay (EC50 ≥
infections in adultsb HSV-2 6.5 µM HSV-1, ≥13.5 µM
Different herpetic – – – 324 324 0 HSV-2) & Sanger
infections in childrenb sequencing of UL23 &
UL30
Gray [118] 1989 United Herpetic infections of Unspecified HSV-1 155 155 0 (0 %) 97 97 0 Dye uptake assay
Kingdom skin, mucous or HSV-2 66 66 0
genitals Untyped 4 4 0
Hasegawa 2001 Japan Genital herpes in Untreated & HSV-1 & 38 38 0 (0 %) 56 56 0 Plaque reduction assay
[119] women ACV treated HSV-2
Hashido [120] 1988 Japan Genital herpes in Unspecified HSV-1 & – – – 56 56 0 Plaque reduction assay
women HSV-2 (EC50 ≥ 13.32 µM)
Nugier [112] 1992 France Unspecifiedb Untreated & HSV-1 459 459 1c (0.22 %) 520 520 1 Dye uptake assay (EC50 ≥

Biochemical Pharmacology 206 (2022) 115322

ACV treated 1.5 µg/mL)
HSV-2 142 142 1 (0.70 %) 146 146 2 Dye uptake assay (EC50 ≥
3 µg/mL)
Reyes [121] 2003 United (suspected) Genital Untreated & HSV-1 – 189 0 (0 %) – 189 0 Plaque reduction assay
States herpes ACV treated HSV-2 – 1644 3 (0.18 %) – 1644 3 (EC50 ≥ 2 µg/mL)
Sangdara 2008 Thailand (suspected) Herpetic Unspecified HSV-1 & 148 – 0 155 155 (1 3 0) 0 Plaque reduction assay
[122] infections in genital or HSV-2 (EC50 ≥ 3 µg/mL)
non-genital areasb
Schmidtd 2015 Germany Different herpetic Untreated & HSV-1 276 276 12 (4.3 %) 276 276 12 Sanger sequencing of
[123] infectionsb ACV treated UL23 & UL30, Plaque
reduction assay (≥5x
EC50 control strain)
2005 368 368 1 (0.27 %) 389 389 1
(continued on next page)
H.H. Schalkwijk et al. Biochemical Pharmacology 206 (2022) 115322

or mortality in case of CNS infections [28,130].

ACV: acyclovir, EC50: effective concentration inhibiting 50% of virus growth in cell culture, HSV: herpes simplex virus. a includes 37 patients potentially immunocompromised based on subjects’ reported medication and
medical histories, b may include infections at immune-privileged sites, c resistant sample isolated from ocular swab, d Samples collected in laboratories from routine diagnostic services, e data not available, f only 1 out of
Six studies reporting prevalence data at immune-privileged sites

mVILA (˃3x EC50 control

control strain) & Sanger

Dye uptake assay (EC50

Plaque reduction assay

Plaque reduction assay

Plaque reduction assay

were included: five related to ocular infections [35,107,118,130,131],

Sanger sequencing of
Sequencing of UL23
ELVIRA (≥3x EC50

(EC50 ≥ 3 µg/mL)

(EC50 ≥ 2 µg/mL)
and one to CNS infections [132] (Table 2). The study of Mitterreiter et al.
Method (cutoff

[132] reported on 12 HSE patients, including 7 immunocompromised

UL23 & UL30

resistance)

˃2 µg/mL)
individuals, of which 11 received antiviral treatment. No ACV resistance
was reported but only sequencing of the UL23 (TK) gene was performed

strain)
due to limited availability of HSV DNA. Although ACVr HSV infections of
the CNS have been described in several case reports [133–135], other
HSE patient cohorts reporting on the prevalence of ACV resistance are
1 (EC50 =
resistant

2.29 µg/
lacking.
isolates
HSV

The prevalence of ACV resistance in ocular HSV infections ranged

mL)
N◦

0
from 0 % to 34.6 %. Menage et al. [107] and Gray et al. [118] reported
no resistance but included only limited number of patients (35, and 6,
260 (2 3 3)
respectively), and Gray et al. sampled patients before start of ACV
positive

(tested)
isolates
N◦ HSV

treatment. Acyclovir resistance was detected more frequently (6.4 % to

102
34.6 %) in patients with (recurrent) herpetic keratitis, most of whom
27

24

68

were receiving ACV treatment or had a history of ACV treatment

samples

[35,130]. Thus, ACV resistance seems more prevalent in the immuno­

1000

1191

competent population in case of herpetic keratitis, in particular in pa­

102
36

68
N◦

tients with recurrent disease and a history of ACV treatment.

resistant HSV
N◦ patients

5.4. General immunocompromised population

with ACV

3f (4.4 %)
1 (4.2 %)

1 (1.4 %)
0 (0 %)

0 (0 %)

0 (0 %)

In immunocompromised patients, infections with resistant HSV

(%)

strains can be associated with persistent and disseminated disease and

are potentially life-threatening [82]. It is therefore essential to test
the 3 isolates had a known drug-resistance genotype (G insertion in homopolymer stretch at positions 430–436 in the HSV-1 TK).
patients

clinically unresponsive infections in immunocompromised patients for

tested

ACV resistance to rationally adapt antiviral therapy. The prevalence of

233

102
27

24

68
N◦

ACVr HSV infections in the general immunocompromised population

ranged from 0 % to 28.8 % (Table 3), which was higher than that found
asymptomatic)

in the immunocompetent population. The study from Carrega et al. was

N◦ patients

1191 (702

the only study reporting no ACV resistance in immunocompromised

individuals, but tested only 10 patients, all children with cancer [136].
1000

102
36

68

The 11 other studies all reported a prevalence of ACV resistance above 3

%.
HSV-1 &

HSV-1 &

HSV-1 &

HSV-1 &

The immunocompromised population is a highly diverse group as

species

HSV-2

HSV-1

HSV-2

HSV-1

HSV-2

HSV-2

several conditions can lie at the base of immunosuppression, including

HSV

HIV infection, primary immunodeficiency, chemotherapy, and immu­

nosuppressive therapy following transplantation, inflammation or im­
mune diseases [71,111,118]. Patients with profound
Unspecified &
Untreated &
ACV treated

ACV treated
Unspecified

Unspecified

Unspecified

Unspecified
Treatment

immunosuppression, in particular those with suppressed cell-mediated

immunity, may have prolonged viral replication associated with more
status

extensive lesions and often require lengthy or multiple courses of ACV

treatment. Accordingly, severely immunocompromised individuals are
more likely to carry ACVr HSV than moderately immunosuppressed in­
Oral mucositis or skin

Oral HSV infection in

(asymptomatic) Oral

dividuals. Hence, the prevalence data presented in each study is largely

herpetic infections
Different herpetic

Orofacial lesionsb
(A)symptomatic
genital herpetic

defined by the underlying morbidities of the included patients. Evalu­

HSV disease

ating the prevalence of ACVr HSV infections in patients grouped ac­

infectionsb

infections
children

cording to the pathology causing immunosuppression is therefore more

lesion

informative.

5.5. Solid organ transplant recipients

Netherlands

Thailand
Country

Japan

Herpesviruses are the most common viral pathogens causing disease

China
Brazil

Iran
The

after solid organ transplantation. Infections with HSV occur most

commonly in the first month after transplantation and ACV prophylaxis
for at least 1 month after solid organ transplantation is therefore rec­
Publication

ommended [137,138]. The prevalence of ACVr HSV infections in the

1994

2012

2011

1989

2007

seven studies reporting on solid organ transplant recipients ranged from

year
Table 1 (continued )

0 % to 10 %, with only 1 study reporting a prevalence above 3.5 %

(Table 4). The highest prevalence was seen in patients receiving heart
Tateishi [124]

Varella [125]
First author

and/or lung transplantations [111,116].

Wang [108]
Stránskád

Ziyaeyan
Yoosook
[113]

[126]

[127]

7
H.H. Schalkwijk et al. Biochemical Pharmacology 206 (2022) 115322

5.6. Hematopoietic stem cell transplant recipients

ACV: acyclovir, EC50: effective concentration inhibiting 50% of virus growth in cell culture, HSV: herpes simplex virus, PCR: polymerase chain reaction, a study included 5 immunocompetent and 7 immunocompromised
Dye uptake assay (˃10x mean

Plaque reduction assay (EC50

˃2µM), Sanger sequencing of
Real time PCR (EC50 ˃1µM),
Method (cutoff resistance)

Plating efficiency, Real time

Sanger sequencing of UL23
CPE reduction assay (≥5x
Nowadays, most HSCT recipients receive prophylactic ACV and this

EC50 of sensitive isolates)

has considerably reduced the number of patients suffering from HSV

EC50 control strains)

infections posttransplantation [12,139]. The use of ACV prophylaxis

PCR (EC50 ˃1µM)

Dye uptake assay
reduces the risk of resistance development in comparison to acute
treatment, as less viral replication is allowed.
In the study from Wade et al. [140], patients received acute ACV

UL23
therapy and ACV resistance was reported in three out of 52 patients (5.8
%). High levels of ACV resistance were reported in HSCT recipients
receiving prophylaxis (Table 5). However, prevalence data in most
HSV isolates
N◦ resistant

studies was based on the number of patients tested (i.e., patients with
HSV infections while receiving prophylaxis), and this might over­
11

44
estimate the prevalence of HSV resistance in this population (range from
1

0
0 % to 100 %). When assessing ACV resistance in all patients receiving
HSCTs, lower but clinically significant rates of ACVr HSV infections were
positive
isolates

observed, generally within the 0–10 % range (Table 5, column preva­

N◦ HSV

lence of ACV resistant HSV) [141–143]. This shows that a substantial

173

169
40

35

15
6

portion of all patients receiving stem cell transplants, irrespective of

their HSV serostatus, experience ACVr breakthrough infections.
samples

Two studies reported high levels of ACV resistance in HSV-1 in­

173

169

fections while no ACVr HSV-2 infections were detected among the HSCT
40

35

15
N◦

recipients [111,144]. Additionally, the origin of the transplant (alloge­

neic or autologous donor) might impact the emergence of ACV resis­
resistant HSV

tance. Although HSV excretion is detected at equal rates after

N◦ patients

27 (34.6 %)
11 (6.4 %)
with ACV

1 (2.9 %)

autologous (10 %) and allogeneic (9.7 %) transplantation, ACVr HSV

0 (0 %)

0 (0 %)

0 (0 %)

infections seem to occur more frequently in allogeneic transplant re­

(%)

cipients [111,139]. Suboptimal dosing of ACV prophylaxis might also

dispose for resistance development. The study from Langston et al. [145]
demonstrated HSV reactivation in all 7 HSV seropositive patients
patients
Characteristics of studies reporting on acyclovir resistance in herpes simplex virus infections at immune-privileged sites.

tested

receiving ACV prophylaxis (5 mg/kg intravenous every 8 h), of whom 5

173
35

35

12

78

patients developed ACVr infections. Increasing the dose of prophylaxis

N◦

to 10 mg/kg was associated with a reduction in HSV reactivation and

patients

ACV resistance.
A rise in the frequency of ACVr HSV infections among HSCT re­
173

12a
35

35

78
N◦

cipients was described by several studies [73,144]. This might be

explained by an increased number of HLA-mismatched donors or
HSV-2 (1)

HSV-2 (8)
HSV-1 &

HSV-1 &
species

improved protocols for stem cell transplantation and induction chemo­

HSV-1

HSV-1

HSV-1

HSV-1
HSV

therapy regimens that induce longer and deeper immunosuppression.

5.7. HIV infected individuals

ACV treated & history

ACV treated & history

Unknown, untreated,

of ACV treatment (n
Treatment status

treatment (n = 8)
of ACV treatment
Untreated & ACV

or history of ACV

Untreated & ACV

treated (n = 10)
treated (n = 5)

HSV-2 infected patients have an increased risk of HIV acquisition

Unspecified

[146]. When left untreated, HIV infected individuals can show a marked
reduction of their CD4+ T cell count and become severely immunosup­
= 19)

pressed. This advanced state of HIV infection, often referred to as ac­

quired immunodeficiency syndrome (AIDS), is associated with
opportunistic infections and malignancies [147]. Low CD4+ T cell
Herpes simplex
Ocular herpes
HSV disease

Herpetic eye

encephalitis

counts seem to correlate with a higher risk of symptomatic HSV in­

Recurrent
infection
Herpetic

herpetic
keratitis

keratitis

fections [148]. The implementation of combination antiretroviral

lesions
Ocular

treatment (tri-therapy) resulted in an increase in CD4+ T cell count and

in low HIV viral loads associated with less opportunistic infections
[149]. However, HSV-2 is shed more frequently in HIV infected in­
Netherlands

Netherlands

Netherlands

dividuals, even when receiving antiretroviral therapy and when CD4+ T

Kingdom

Kingdom

Kingdom
Country

cell counts are high (>400cells/mm3) [150,151]. Acyclovir resistance

United

United

United
The

The

The

was detected at a marked higher frequency in HSV-2 infections than in

HSV-1 infections by Danve-Szatanek et al. (7.3 % versus 0 %), Reyes
et al. (5.3 % versus 0 %), and Ziyaeyan et al. (25 % versus 4.3 %)
Publication

[111,121,127] (Table 6). The reported prevalence of ACVr HSV in­

fections in HIV infected individuals ranged from 0 to 25 %, with most
1990

2008

1989

1990

2016

2013
year

studies reporting a prevalence between 3.4 % and 7.3 %.

First author

5.8. Prevalence of ACV resistance mutations

Duan [130]

Mitterreiter

Van Velzen
Gray [118]
[131]

[107]

[132]
Menage

patients.
Charles
Table 2

[35]

Nine of the included studies performed genotyping of the UL23 and/

or UL30 genes to characterize ACV resistance

8
 H.H. Schalkwijk et al.
Table 3
Characteristics of studies reporting on acyclovir resistance in herpes simplex virus among the general immunocompromised population.
First author Publication Country HSV disease Treatment HSV N◦ N◦ N◦ patients N◦ N◦ HSV N◦ resistant Method (cutoff resistance)
year status species patients patients with ACV samples positive HSV isolates
tested resistant HSV isolates
(%)

Carrega [136] 1994 Italy Oral mucositis in ACV treated HSV-1 & 20 10 0 (0 %) 20 10 0 Plaque reduction assay
children with HSV-2 (2)
cancer
Christophers 1998 United Different herpetic ACV treated Untyped 95 95 6 (6.3 %) 146 146 6 Plaque reduction assay (EC50
[116] Kingdom infectionsa ≥ 3 µM)
Collins [109] 1993 Europe Unspecifieda ACV treated Untyped 81 81 18 (22.2 %) 206 206 64 Plaque reduction assay (EC50
≥ 3 µM)
United 208 208 60 (28.8 %) 582 582 105 Dye uptake assay (EC50 ≥ 3
States µg/mL)
Danve- 2004 France Different herpetic Untreated & ACV HSV-1 1168 1168 36 (3.1 %) 1523 1523 -b Dye uptake assay (EC50 ≥ 6.5
Szatanek infectionsa treated µM)
[111] HSV-2 334 334 18 (5.4 %) 411 411 - Dye uptake assay (EC50 ≥
13.5 µM)
Englund [71] 1990 United Herpetic infections Untreated & ACV Untyped 148 148 7 (4.7 %) - 218 23 DNA hybridization (EC50 ≥ 9
States at oral site & treated (1 2 8) µM)
genital tract
Frobert [73] 2014 France Different herpetic Unspecified HSV-1 & 360 360 35 (9.7 %) 360 360 35 Dye uptake assay (EC50 ≥ 6.5
infections in adultsa HSV-2 µM HSV-1, ≥13.5 µM HSV-2),
9

Different herpetic - - - 141 141 7 Sanger sequencing of UL23 &

infections in UL30
childrena
Gray [118] 1989 United Herpetic infections Untreated & ACV HSV-1 49 49 2 (4.1 %) - 101 2 Dye uptake assay
Kingdom of skin or mucous treated
Labrunie 2019 France Different herpetic Unknown, HSV-1 239 200 36 (18 %) 239 200 36 Dye uptake assay (EC50 ≥ 6.5
[218] infections in adults untreated & ACV µM)
& childrena treated (46) 239 35 (14.6 %) 239 UL23 35 Sanger sequencing of UL23 &
148 UL30 UL30
Nugier [112] 1992 France Different herpetic Untreated & ACV HSV-1 156 156 6 (3.8 %) 186 186 13 Dye uptake assay (EC50 ≥ 1.5
infectionsa treated µg/mL)
HSV-2 28 28 4 (14.3 %) 37 37 4 Dye uptake assay (EC50 ≥ 3
µg/mL)
Rabella [219] 2002 Spain Different herpetic Untreated, HSV-1 165 165 8 (4.8 %) 204 131 1 CPE reduction assay (EC50
infectionsa unknown & ACV HSV-2 73 8 ˃3µg/mL)

Biochemical Pharmacology 206 (2022) 115322

treated
Schmidt [123] 2015 Germany Different herpetic Untreated & ACV HSV-1 24 24 6 (25 %) 24 24 6 Sanger sequencing of UL23 &
infectionsa treated UL30 Plaque reduction assay
(≥5x EC50 control strain)
Stránská [113] 2005 The Different herpetic Untreated & ACV HSV-1 & 128 128 9 (7.0 %) 153 153 12 ELVIRA (≥3x EC50 control
Netherlands infectionsa treated HSV-2 strain), Sanger Sequencing of
UL23

ACV: acyclovir, EC50: effective concentration inhibiting 50% of virus growth in cell culture, HSV: herpes simplex virus. a may include infections at immune-privileged sites, b
data not available.
 H.H. Schalkwijk et al.
Table 4
Characteristics of studies reporting on acyclovir resistance in herpes simplex virus among solid organ transplant recipients.
First author Publication Country HSV disease Treatment HSV N◦ N◦ N◦ patients N◦ N◦ HSV N◦ resistant Method (cutoff resistance)
year status species patients patients with ACV samples positive isolates
tested resistant HSV isolates
(%)

Boivin [220] 1993 United Different herpetic ACV or GCV HSV-1 & 14 14 0 (0 %) 18 18 0 DNA hybridization assay (EC50
States infectionsa treated HSV-2 ≥ 9 µM)
b c
Christophers 1998 United Different herpetic ACV treated untyped 20 20 2 (10 %) - - 2 Plaque reduction assay (EC50 ≥
[116] Kingdom infectionsa 3 µM)
Danve- 2004 France Different herpetic Untreated & HSV-1 168 168 4b (2.4 %) - - - Dye uptake assay (EC50 ≥ 6.5
Szatanek infectionsa ACV treated µM)
[111] HSV-2 33 33 1b (3.0 %) - - - Dye uptake assay (EC50 ≥ 13.5
µM)
Englund [71] 1990 United Herpetic Untreated (4) & untyped 58 58 2 (3.4 %) - - - DNA hybridization (EC50 ≥ 9
States infections at oral ACV treated µM)
10

site & genital (54)

tract
Frobert [73] 2014 France Different herpetic Unspecified HSV-1 & – – – 94 94 1 Dye uptake assay (EC50 ≥ 6.5
infections in HSV-2 µM HSV-1, ≥13.5 µM HSV-2) &
adultsa Sanger sequencing of UL23 &
Different herpetic – – – 38 38 1 UL30
infections in
childrena
Gray [118] 1989 United Herpetic Unspecified HSV-1 10 10 0 (0 %) 17 17 0 Dye uptake assay
Kingdom infections of skin
or mucous
Stránská [113] 2005 The Different herpetic Untreated & HSV-1 & 17 17 0 (0 %) 17 17 0 ELVIRA (≥3x EC50 control
Netherlands infectionsa ACV treated HSV-2 strain), Sanger sequencing of
UL23

ACV: acyclovir, EC50: effective concentration inhibiting 50% of virus growth in cell culture, GCV: ganciclovir, HSV: herpes simplex virus. a may include infections at immune-privileged sites, b heart or lung transplant

Biochemical Pharmacology 206 (2022) 115322

recipients, c data not available.
 H.H. Schalkwijk et al.
Table 5
Characteristics of studies reporting on acyclovir resistance in herpes simplex virus among hematopoietic stem cell transplant recipients.
First author Publication Country HSV disease Treatment HSV N◦ N◦ N◦ patients Prevalence N◦ N◦ HSV N◦ Method (cutoff
year status species patients patients with ACV ACV resistant samples positive resistant resistance)
tested resistant HSVa isolates HSV
HSV (%) isolates

Anton- 2020 United Suspected HSV ACV treated HSV-1 532b 48 8 (16.7 %) 1.5 % -c – – Plaque reduction assay
Vazquez Kingdom infection HSV-2 18 0 (0 %) 0% – – – (EC50 ˃40 µM), Sanger
[144] sequencing of UL23 &
UL30
Ariza Heredia 2018 United Orolabial or ACV treated HSV-1 4028b – 12 0.30 % – – – Plaque reduction assay
[141] States anogenital HSV HSV-2 – 6 0.15 % – – –
infection
Chakrabarti 2000 United Infection of mouth, ACV treated HSV-1 75b 16 4 (25 %) 5.3 % – – – Plaque reduction assay
[221] Kingdom eye, skin, or genitals (EC50 ≥ 1.62 µM)
b
Chen [142] 2000 France Different herpetic ACV treated HSV-1 196 – 14 7.1 % – – – Dye uptake assay (EC50
infectionsd ≥ 6.5 µM)
Danve- 2004 France Different herpetic Untreated & HSV-1 182 182 22 (12.1 %) 12.1 % – – – Dye uptake assay (EC50
Szatanek infectionsd ACV treated ≥ 6.5 µM)
[111] HSV-2 19 19 0 (0 %) 0% – – – Dye uptake assay (EC50
≥ 13.5 µM)
Englund [71] 1990 United Herpetic infections ACV treated Untyped 29 29 4 (13.8 %) 13.8 % – – – DNA hybridization
States at oral site & genital assay (EC50 ≥ 9 µM)
tract
Frobert [73] 2014 France Different herpetic Unspecified HSV-1 & – – – – 90 90 30 Dye uptake assay (EC50
infections in adultsd HSV-2 ≥ 6.5 µM HSV-1, ≥13.5
Different herpetic – – – – 30 30 5 µM HSV-2), Sanger
infections in sequencing of UL23 &
childrend UL30
11

Heidenreich 2020 Germany Different herpetic ACV treated HSV-1 214b -e 9 4.2 % – – – Sanger sequencing,
[143] infectionsd plaque reduction assay
Kakiuchi 2017 Japan (asymptomatic) ACV treated HSV-1 251b 39 11 (28.2 %) 4.4 % 2747 103 35 Plaque reduction assay
[152] oropharyngeal (EC50 > 2 µg/mL),
infection Sanger sequencing of
UL23 & UL30
Langston 2002 United Infection at mucosa, ACV treated HSV 14b 5e 5 (100 %) 35.7 % – – – Plaque reduction assay
[145] States esophagus &
anogenital area
Morfin [139] 2004 France (asymptomatic) Untreated & HSV-1 283b 28 3 (10.7 %) 1.1 % – – – Dye uptake assay (EC50
HSV mucositis ACV treated ≥ 6.5 µM)
Nichols [222] 2003 United Different herpetic ACV treated HSV-1 436b – 14 3.2 % – – – DNA hybridization
States infectionsd HSV-2 – 1 0.23 % – – – assay (EC50 > 4 µg/mL)
Schmidt 2015 Germany Different herpetic Untreated & HSV-1 13 13 6 (46.2 %) 46.2 % 13 13 6 Sanger sequencing of
[123] infectionsd ACV treated UL23 & UL30, Plaque

Biochemical Pharmacology 206 (2022) 115322

reduction assay (≥5x
EC50 control strain)
Stránská 2005 The Different herpetic Untreated & HSV-1 & 28 28 4 (14.3 %) 14.3 % 34 34 5 ELVIRA (≥3x EC50
[113] Netherlands infectionsd ACV treated HSV-2 control strain), Sanger
sequencing of UL23
Van der Beek 2012 The Herpetic oral Untreated & HSV-1 49b 12e 5 (41.7 %) 10.2 % – 191 – Sanger sequencing of
[153] Netherlands ulcerations ACV treated UL23 & UL30, DNA
reduction assay
Wade [140] 1983 United Oropharyngeal & ACV treated HSV-1 & 52 52 3 (5.8 %) 5.8 % – – – Dye uptake assay
States genital herpetic HSV-2
infections

ACV: acyclovir, EC50: effective concentration inhibiting 50% of virus growth in cell culture, HSV: herpes simplex virus. a prevalence was calculated based on the total number of patients included in the study, b
total
number of transplanted patients, not all patients experienced HSV infections c data not available, d may include infections at immune-privileged sites, e only patients refractory to therapy were tested.
 H.H. Schalkwijk et al.
Table 6
Characteristics of studies reporting on acyclovir resistance in herpes simplex virus among HIV infected individuals.
First author Publication Country HSV disease Treatment HSV N◦ N◦ N◦ patients N◦ N◦ HSV N◦ Method (cutoff resistance)
year status species patients patients infected with samples positive resistant
tested ACV resistant isolates isolates
HSV (%)

Christophers 1998 United Different herpetic ACV treated Untyped 29b 29 1 (3.4 %) -c – 1 Plaque reduction assay (EC50
[116] Kingdom infectionsa ≥ 3 µM)
Danve- 2004 France Different herpetic Untreated & HSV-1 133 133 0 (0 %) – – – Dye uptake assay (EC50 ≥ 6.5
Szatanek infectionsa ACV treated µM)
[111] HSV-2 177 177 13 (7.3 %) – – – Dye uptake assay (EC50 ≥ 13.5
µM)
Englund [71] 1990 United Herpetic infections Untreated (5) & untyped 14d 14 1 (7.1 %) – – – DNA hybridization (EC50 ≥ 9
States at oral site & genital ACV treated (9) µM)
tract
Frobert [73] 2014 France Herpetic infections Unspecified HSV-1 & – – – 37 37 3 Dye uptake assay (EC50 ≥ 6.5
at oral site, genital HSV-2 µM HSV-1, ≥13.5 µM HSV-2)
tract in adults & Sanger sequencing of UL23
12

& UL30
Muller [223] 2022 South Africa Genital ulcer ACV treated HSV-2 27 27 0 (0 %) 27 27 0 Sanger sequencing of UL23,
disease plaque reduction assay (EC50
≥ 2 µg/mL)
Nag [224] 2021 India Genital or oral Unspecified HSV-1 & 52 8 2 (25 %) 52 8 2 CPE reduction assay
herpetic lesions HSV-2
Reyes [121] 2003 United (Suspected) genital Untreated & HSV-1 – 12 0 (0 %) – 12 0 Plaque reduction assay (EC50
States herpes ACV treated ≥ 2 µg/mL)
HSV-2 – 226 12 (5.3 %) – 226 12 Plaque reduction assay (EC50
≥ 2 µg/mL)
Stránská [113] 2005 The Different herpetic Untreated & HSV-1 & 51 51 2 (3.9 %) 59 59 3 ELVIRA (≥3x EC50 control
Netherlands infectionsa ACV treated HSV-2 strain), Sanger sequencing of
UL23
Ziyaeyan 2007 Iran Orofacial lesionsa Unspecified HSV-1 23e 23 1 (4.3 %) 23 23 1 Plaque reduction assay (EC50
[127] HSV-2 8e 8 2 (25 %) 8 8 2 ≥ 2 µg/mL)

Biochemical Pharmacology 206 (2022) 115322

ACV: acyclovir, AIDS: acquired immunodeficiency syndrome, EC50: effective concentration inhibiting 50% of virus growth in cell culture, HSV: herpes simplex virus. a May include infections at immune-privileged sites, b
17 HIV infected individuals were diagnosed with AIDS, c data not available, d 12 patients with AIDS, 2 with AIDS-related complex, e all patients were diagnosed with AIDS.
 H.H. Schalkwijk et al. Biochemical Pharmacology 206 (2022) 115322

[73,108,113,123,130,132,144,152,153]. We evaluated the prevalence

The occurrence of mutations conferring acyclovir resistance in articles included in this review. Novel mutations associated with acyclovir resistance are underlined and mutations conferring cross resistance to foscarnet are

of different mutations conferring ACV resistance among the 87 ACVr

[73,108,113,123,130,132,144,152,153]
HSV-1 infected patients and 9 ACVr HSV-2 infected patients included in
these nine studies. An overview of insertions, deletions, and amino acid
changes detected in these patients is available in Table 7.

[73,108,123,144,152,153]
In the samples derived from 87 patients with ACVr HSV-1 infections,
75 mutations conferring ACV resistance were reported in the UL23
Included studies

gene, of which the majority (54.7 %) were insertions or deletions

(Table 7). The G insertion in the homopolymer stretch at nucleotides
433–436 was reported most frequently (15 patients). The G200D mu­

[73,113]
tation was the most frequently reported point mutation (3 patients).

[73]
Mutations introducing a stop codon (E95stop, T172stop, R281stop, and
Q342 stop) were detected in samples from 5 patients. Samples from 68
548–553 (4), Ins C nts 548–553 (3), I194del (1), Ins T nt 613 (1), Del G nts 615–619 (1), Del nts 781–793 (1), Del C nt

E70K (1), G191S (1), L359I (1), L448V (1), E671K (1), G691S (1), A748T (1), G841A (1), E860K (1), A874T (1), A910T
430–436 (15), Del G nts 430–436 (1), Ins A nt 438 (1), Del G nt 438 (1), Del C nt 540 (1), Ins G nt 547 (1), Del C nts

G56D (1), G59W (1), I78S (1), E95stop (1), D162H (1), D162N (1), Y172C (1), T172stop (1), R176Q (1), L178R (2),
S181N (1), A189V (1), G200D (3), G200S (1), R216H (1), R220H (1), R222H (2), L249P (1), E257K (1), D258N (1),

patients were analyzed for UL30 mutations, and mutations conferring

E36del (1), Ins C nt 184 (1), Del C nt 184 (1), Ins C nt 185 (1), Del A nts 184–187 (1), Del C nt 269 (1), Ins G nts

ACV resistance were detected in samples from 10 patients (14.7 %). The
R261C (1), R281stop (2), T287M (1), L298P (1), C336Y (1), Q342stop (1), T354P (1), E371D (1), L327R (1)

majority of these mutations (i.e., A605V, R700G, S724N, I890M, and

G901V) conferred cross-resistance to foscarnet. Substitutions of unclear
significance were detected in both the UL23 and UL30 genes of HSV-1.
Q15K (1), E43A (2), A93V (1), A230V (1), L242P (1), P269S (1), S276N (1), I326V (1), A365T (1)

Six out of nine patients infected with an ACVr HSV-2 strain, harbored
mutations in the UL23 gene conferring ACV resistance, with an insertion
(single G in the G-homopolymer stretch at nucleotides 433–439) and a C
deletion (single C at nucleotides 551–556) reported in two patients each
[73,113]. No mutations conferring resistance were detected in the HSV-
2 UL30 gene, and several substitutions of unknown significance were
detected in both the UL23 and UL30 genes.
A605V (3), R700G (1), A719V (2), S724N (2), I890M (1), G901V (1)

The majority of ACV resistance mutations in the UL23 genes of HSV-

G36E (1), M129I (1), T202A (1), D229H (1), R268W (1), R363C (1)

1 and HSV-2 were frameshift mutations, in agreement with the litera­

ture (section 4.1). However, as only a limited number of HSV samples
were genotyped in the included studies, the prevalence of detected
885 (2), Del C nts 896–900 (1), Del C nts 1061–1064 (1)

mutations might not be representative for the total population of HSV

resistant strains. Comprehensive overviews of known HSV resistance
mutations are available in several reviews [74,82,86,87,95].
Ins C nts 433–439 (2), Del C nts 551–556 (2)

6. Antiviral alternatives for acyclovir resistant HSV infections

K458N (1), G506D (1), K592E (1)

Several antiviral treatment options can be considered in case of an

ACVr HSV infection. In some patients, an increased dose or intravenous
(1), T931A (1), P1114H (1)
Mutation (N◦ of patients)

administration of ACV is still effective to treat ACVr infections [28]. A

limited number of antivirals, all targeting the DNA pol, have been
G56E (1), T288M (1)

approved for the treatment of HSV infections. Although numerous an­

tivirals targeting different steps of HSV replication have been investi­
gated, many failed to show sufficient potency, bioavailability, or
acceptable toxicity. Several alternative antiviral compounds, either
approved or under investigation, will be discussed in the following
–

sections.
Insertion or

Insertion or

6.1. Nucleoside analogues

mutation

mutation

mutation

mutation

mutation

mutation

mutation

mutation
deletion

deletion
Type of

Point

Point

Point

Point

Point

Point

Point

In addition to ACV and VACV, other nucleoside analogues are

–

available for the treatment of HSV infections i.e., penciclovir or its

prodrug famciclovir, ganciclovir, brivudine, and trifluridine (tri­
fluorothymidine) (Fig. 4). Penciclovir and ganciclovir are guanosine
significance

significance

significance

significance
Associated
phenotype

analogues similar to ACV but are no obligate chain terminators due to

Resistance

Resistance

Resistance

Resistance
Unclear

Unclear

Unclear

Unclear

the presence of a 3′ hydroxyl on the acyclic sugar moiety. Incorporation

of penciclovir or ganciclovir into the viral DNA allows incorporation of
one additional nucleotide after which DNA elongation is terminated
[95]. Acyclovir and penciclovir differ in their affinity for the viral TK
N◦ resistant

and DNA pol but show a similar spectrum of activity [86]. Ganciclovir is
patients

most frequently used for the treatment of CMV infections but is

approved as topical treatment for herpetic keratitis. Brivudine is a
87

68

deoxyuridine analogue that acts as a competitive dTTP inhibitor and

only shows anti-HSV-1 and anti-VZV activities. Brivudine is dependent
marked in bold.
Gene

UL23

UL30

UL23

UL30

on viral TK activity for conversion to its monophosphate and diphos­

phate form, but the HSV-2 TK is unable to phosphorylate brivudine
Table 7

Virus

HSV-

HSV-

HSV-

HSV-

monophosphate due to its low thymidylate kinase activity [81,154].

1

Penciclovir, famciclovir, ganciclovir, and brivudine are not

13
H.H. Schalkwijk et al. Biochemical Pharmacology 206 (2022) 115322

Fig. 4. Chemical structure of several nucleoside analogues active against herpes simplex viruses.

recommended for the treatment of ACVr HSV infections. As these drugs
have a similar mode of action requiring activation by the viral TK, cross-
resistance between the nucleoside analogues is often observed, espe­
cially when mutations arise in the viral TK [74,155,156]. Exceptionally,
ACVr TK mutants, show no cross-resistance to other nucleoside ana­
logues [134,157,158]. Cross-resistance is less frequently observed in
ACVr DNA pol mutant strains, of which most remain sensitive to
brivudine (HSV-1 only) and ganciclovir. Penciclovir retains activity
against several ACVr DNA pol mutants, including HSV-1 strains bearing
the A605V, A719T, or Y941H mutations, and HSV-2 strains harboring
the A724V, S725G, I731F, Y823C or R964H mutations [74,158–161].
The first activating phosphorylation step of the thymidine analogue
trifluridine can be performed by both viral and cellular kinases. Tri­
fluridine monophosphate irreversibly inhibits the conversion of

Fig. 5. Chemical structures of several antiviral compounds with anti-herpes simplex virus activity. The structures presented are acyclic nucleoside phosphonates
(cidofovir, brincidofovir, adefovir, and tenofovir), pyrophosphate analogue (foscarnet), helicase-primase inhibitors (pritelivir and amenamevir) and n-docosanol.

14
H.H. Schalkwijk et al.
deoxyuridine monophosphate (dUMP) to dTMP by the cellular thymi­
dylate synthase. In its triphosphate form, trifluridine can be incorpo­
rated by both the cellular and viral DNA polymerase [162,163]. As a
result, trifluridine exhibits low selectivity and high cytotoxicity and is
therefore only available as topical treatment for herpetic keratitis [164].
Mechanisms of trifluridine drug resistance are not well described but
some studies reported a response to trifluridine treatment in select cases
of clinically ACVr HSV infections [165–167]. Thus, trifluridine therapy
might be beneficial for some patients with ACVr HSV infections.
6.2. Pyrophosphate analogues
The pyrophosphate analogue foscarnet (Fig. 5, phosphonoformic
acid) selectively and reversibly binds to the pyrophosphate binding site
of the viral DNA pol. Binding of foscarnet prevents cleavage of the py­
rophosphate from the elongating viral DNA chain, inhibiting further
nucleotide incorporation [168]. Therapy with foscarnet is complicated
due to its associated nephrotoxicity and poor oral bioavailability, only
allowing intravenous or topical formulation [169]. Foscarnet is used in
immunocompromised patients for the treatment of mucocutaneous
ACVr HSV infections due to changes in the viral TK but not for ACVr
infections having alterations in the DNA pol, since cross-resistance with
foscarnet occurs frequently [74,95,170]. Furthermore, HSV strains
resistant to both ACV and foscarnet due to the acquisition of mutations
in both the TK and DNA pol have been described in the clinic [46,171].
6.3. Acyclic nucleoside phosphonates
Acyclic nucleoside phosphonates are DNA pol inhibitors that have a
phosphonate group attached to the sugar moiety and therefore require
only two activating phosphorylation steps, both executed by cellular
kinases [50,172,173]. Because acyclic nucleoside phosphonates are in­
dependent of viral TK activity, they remain active against most ACVr
HSV strains. Three acyclic nucleoside phosphonates have been licensed
for use in the clinic: cidofovir, adefovir, and tenofovir [50] (Fig. 5).
Cidofovir is a cytosine analogue that selectively inhibits herpesvirus
DNA pols and terminates viral DNA elongation in case of two successive
cidofovir incorporations [172,174,175]. Although cidofovir is only
approved for treatment of CMV retinitis in patients with AIDS, it is used
off-label for the treatment of ACV and/or foscarnet resistant HSV in­
fections. Due to its poor bioavailability, it is solely administered intra­
venously or as topical formulation [50]. Cidofovir treatment is
associated with side effects such as nephrotoxicity and impaired renal
function [169]. Probenecid and intravenous hydration with saline prior
to cidofovir administration are indicated to reduce possible nephrotox­
icity [176]. To improve oral bioavailability and reduce nephrotoxicity,
efforts have been made to develop a prodrug. Brincidofovir (Fig. 5.), a
lipid conjugated prodrug of cidofovir, showed potent in vitro inhibition
of herpesviruses, including HSV-1 and HSV-2 [177]. Although brinci­
dofovir is not nephrotoxic, brincidofovir related gastrointestinal disor­
ders, misdiagnosed as acute graft-versus-host disease, were reported in
clinical trials evaluating the efficacy of brincidofovir against adenovirus
and CMV infections in HSCT recipients and further trials have been
halted [178–180].
Cidofovir was shown effective in the treatment of ACVr HSV in­
fections and is active against TK mutants and most DNA pol mutant
strains [82,181,182]. Acyclovir resistant HSV strains with deficient or
altered TK activity are more susceptible to cidofovir than wild-type
strains as they induce smaller increases in the dCTP pool, enhancing
the inhibitory effect of cidofovir [183]. Resistance to cidofovir is ac­
quired by mutations in the DNA pol, and some mutations, including the
S724N and L778M mutations in the HSV-1 DNA pol, show cross resis­
tance to ACV and/or foscarnet [98,184]. Most cidofovir resistance
associated mutations in the HSV DNA pol selected in vitro show no cross-
resistance to ACV or foscarnet [184]. Only one case of a cidofovir
resistant HSV infection has been reported in the clinic [185]. However,
15
Biochemical Pharmacology 206 (2022) 115322
some patients might experience treatment failure despite phenotypic
susceptibility [142,185]. Thus, cidofovir can be considered to treat ACV
and/or foscarnet resistant HSV infections.
Adefovir, an adenosine analogue highly effective against hepatitis B
virus, and tenofovir, an adenosine analogue that potently inhibits the
HIV and hepatitis B virus reverse transcriptase, show activity against
HSV [159,173]. Most isolates resistant to ACV are susceptible to ade­
fovir and tenofovir although ACVr HSV strains with mutations in the
DNA pol can show cross-resistance to adefovir and tenofovir
[46,159,186]. Furthermore, the activity of adefovir and tenofovir
against HSV is too low to be used therapeutically. Tenofovir 1 % vaginal
microbicide gel has been evaluated for the prevention of HIV and HSV-2
infections. Although tenofovir gel reduced HSV-2 acquisition in the
CAPRISA 004 trial, the VOICE trial was unable to demonstrate an effect,
which was associated with low adherence to antiviral treatment in this
population of patients [187–189].
6.4. Helicase-primase inhibitors
The helicase-primase complex, composed of the helicase, primase,
and cofactor subunits, is essential for viral replication and is targeted by
the helicase-primase inhibitors, a novel class of antivirals. These include
pritelivir (BAY 57–1293) and amenamevir (ASP2151), both showing
potent anti HSV activity [190,191] (Fig. 5).
The thiazolylamide pritelivir shows more potent HSV inhibition than
ACV in vitro and acts by inhibiting the single-stranded DNA-dependent
ATPase activity of the helicase-primase complex [168,192]. Phase I and
II clinical trials in immunocompetent individuals showed effective HSV
inhibition and no safety concerns [193–195]. A phase II/III trial
assessing the efficacy and safety of pritelivir for the treatment of ACVr
mucocutaneous HSV infections is currently ongoing (NCT03073967).
Amenamevir is an oxadiazolephenyl derivative that inhibits the
helicase, primase, and single-stranded DNA-dependent ATPase activities
of the helicase-primase complex [196]. In 2017, amenamevir was
approved in Japan for the treatment of herpes zoster caused by VZV
reactivation [197]. It proved to be a safe and effective treatment for HSV
infections in the immunocompetent population in phase I and II clinical
trials [198,199]. A phase III clinical trial (NCT01959295) evaluating the
efficacy and safety of amenamevir in patients with herpes simplex in­
fections (labial/facial herpes or recurrent genital herpes) has been
completed but results have not been published.
Mutations conferring pritelivir and/or amenamevir resistance map
to the UL5 and UL52 genes encoding the helicase and primase, respec­
tively, and are rapidly selected in vitro [200,201]. However, clinical
trials did not reveal rapid emergence of resistance to helicase-primase
inhibitors [193–195,199]. Amenamevir and pritelivir proved effective
against all ACVr HSV strains as these drugs have a different molecular
target than the DNA pol [190,191]. Three immunocompromised pa­
tients with ACVr HSV infections were effectively treated with pritelivir
without the emergence of pritelivir resistance [202,203].
6.5. n-docosanol
The 22-carbon-long saturated alcohol n-docosanol (Fig. 5) is avail­
able over the counter as 10 % topical cream for labial herpes outbreaks.
It appears that n-docosanol prevents HSV fusion and entry by interfering
with the cell surface phospholipids [204,205]. N-docosanol reduces
symptoms and healing time when therapy is started early during reac­
tivation and is active against ACVr HSV strains in vitro [205,206].
6.6. MBX-2168
A newer class of nucleoside analogues includes the methyl­
enecyclopropane nucleoside analogues that possess potent activity
against various herpesviruses. The third-generation methyl­
enecyclopropane nucleoside analogue MBX-2168 shows convincing
H.H. Schalkwijk et al.
anti-HSV activity in vitro and acts as a competitive inhibitor of viral
DNA pols [207]. MBX-2168 is monophosphorylated, converted to syn­
guanol monophosphate by adenosine deaminase-like protein 1 (ADAL-
1), which is subsequently phosphorylated to the active form synguanol
triphosphate [207] (Fig. 6). Synguanol only shows limited activity
against HSV, likely due to inefficient phosphorylation of this compound
by HSV thymidine kinases. MBX-2168 phosphorylation is, at least in
part, performed by the serine/threonine-protein kinase TAO3 (TAOK3)
and MBX-2168 is therefore active against ACVr HSV with TK mutations
[207,208]. MBX-2168 showed low cytotoxicity in vitro, making it a
promising antiviral for which further development is warranted [209].
6.7. Combination therapy
HSV infections can show heterogeneity of virus populations and
spatial compartmentalization, with different viral strains infecting
distinct body sites [46,135]. When infected with multiple strains
showing dissimilar drug susceptibilities, combination therapy might be
necessary to effectively treat all variants. Combination therapy has the
potential to prevent emergence of drug resistance and to treat resistant
HSV infections. Although combination therapy is a common strategy for
the treatment of HIV and hepatitis C virus infections, it is only sporad­
ically used for treatment of herpesvirus infections [210]. Previous
studies have shown additive or synergistic activity of several antiviral
combinations against HSV, including combinations of helicase primase
inhibitors and nucleoside analogues [162,211–213].
Multiple case reports, have shown the effective use of foscarnet and
ACV combination therapy to treat HSV infections that were ACVr or
refractory to ACV therapy [135,142,214–216]. These include a case
Fig. 6. Proposed mechanism of activation of MBX-2168. Initial phosphorylation is at least in part performed by the serine/threonine-protein kinase TAO3 (TAOK3).
MBX-2168 monophosphate (MBX-2168-MP) is then converted to synguanol monophosphate (synguanol-MP) by adenosine deaminase-like protein 1 (ADAL-1), which
is subsequently phosphorylated to its active form synguanol triphosphate (synguanol-TP) which competitively inhibits the viral DNA polymerase.
16
Biochemical Pharmacology 206 (2022) 115322
report in which heterogeneous virus populations of ACVr and wild-type
virus were detected, and a neonatal herpes infection with spatial
compartmentalization of wild-type and mutant virus [133,135]. Com­
bination therapy of ACV and vidarabine was effective in the treatment of
an HSV-1 infection with mixed populations of ACVr and wild-type
strains [134]. Combination therapy with ganciclovir and cidofovir was
effective in treating a persistent HSV-1 infection that showed both viral
compartmentalization between lesions and mixed virus populations of
(multi)-drug resistant strains [217]. These case reports show the po­
tential of combination therapy and highlight that this strategy should be
considered for treatment of recalcitrant infections and/or for therapy of
infections caused by multiple strains presenting different drug suscep­
tibility patterns. Combinations with other antivirals, in particular of
compounds with different modes of action, might prove effective as well
in the management of ACVr HSV infections, although this requires
further study.
7. Future perspectives and final remarks
In this review we provided an overview of prevalence data on ACV
resistance in HSV infections. The studies reporting prevalence data had a
high variability in study design, methodology, treatment strategies,
treatment histories, immunosuppressive regimens, study population,
and patient selection. This makes it difficult to define the global prev­
alence of ACV resistance in HSV infections. In the immunocompetent
population, the prevalence of ACV resistance in HSV infections did not
seem to be associated with ACV treatment history. However, in in­
fections at immune-privileged sites and in immunocompromised pa­
tients, an impaired immune system allows frequent recurrent infections
H.H. Schalkwijk et al.
and ongoing viral replication, that require long-term ACV exposure,
predisposing for the development of ACVr HSV infections. The immu­
nocompromised population is a highly diverse population, with
different pathologies underlying immunosuppression. Distinguishing
between dissimilar subpopulations of immunocompromised patients,
provides a better indication of the true prevalence of ACVr HSV in­
fections among different groups of immunosuppressed hosts.
Although ACV prophylaxis is effective in reducing HSV reactivation,
resistant infections remain a serious and frequent complication in
herpetic keratitis and in immunocompromised individuals. Acyclovir
should therefore be rationally used to prevent emergence of resistance.
Furthermore, it is beneficial to test patients with unresponsive HSV in­
fections for antiviral resistance, as this can guide clinicians in the
adjustment of antiviral therapy. Ideally both phenotypical and geno­
typical characterization of HSV clinical samples should be performed to
avoid false-positive phenotypical results and misinterpretation of novel
amino acid changes with unknown significance.
The lack of antivirals with a different mechanism of action poses a
problem for the treatment of ACVr HSV infections, in particular when
resistance is caused by mutations in the DNA pol. Although several
promising antivirals are under development, there is still a need for
novel antiviral treatments. Combination therapy has the potential to
treat ACVr infections and should be further investigated.
CRediT authorship contribution statement
Hanna Helena Schalkwijk: Conceptualization, Methodology, Vali­
dation, Visualization, Writing – original draft, Writing – review &
editing. Robert Snoeck: Conceptualization, Methodology, Supervision,
Writing – review & editing, Funding acquisition. Graciela Andrei:
Conceptualization, Methodology, Supervision, Validation, Writing –
review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Financial support was provided by Research Foundation Flanders
(FWO, grant number G08818N). The authors wish to thank Kristel
Paque, Thomas Vandendriessche, and Krizia Tuand, the biomedical
reference librarians of the KU Leuven libraries, 2Bergen learning Centre
Désiré Collen (Leuven, Belgium), for their help in conducting the sys­
tematic literature search.
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