Antiviral Research 101 (2014) 12–25

Contents lists available at ScienceDirect

Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

Review

Resistance of human cytomegalovirus to ganciclovir/valganciclovir: A

comprehensive review of putative resistance pathways q
Takashi E. Komatsu a,⇑, Andreas Pikis a,b, Lisa K. Naeger a, Patrick R. Harrington a
a
Division of Antiviral Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA
b
Microbial Biochemistry and Genetics Section, Laboratory of Cell and Developmental Biology, NIDCR, National Institutes of Health, Bethesda, MD 20892, USA

a r t i c l e i n f o a b s t r a c t

Article history: Human cytomegalovirus (HCMV) is a pathogen that can be life-threatening in immunocompromised
Received 3 June 2013 individuals. Valganciclovir and its parent drug ganciclovir are currently the principle drugs used for
Revised 20 October 2013 the treatment or prevention of HCMV disease. The development of HCMV resistance to ganciclovir/val-
Accepted 21 October 2013
ganciclovir has been documented in treated patients and is associated with the emergence of amino acid
Available online 31 October 2013
substitutions in the viral proteins pUL97, pUL54 or both. Generally, single amino acid substitutions asso-
ciated with clinical resistance that alone do not confer decreased ganciclovir susceptibility in cell culture
Keywords:
have been disregarded as causative or clinically significant. This review focuses on the analysis and mech-
Cytomegalovirus
Ganciclovir
anisms of antiviral drug resistance to HCMV. We also conducted a review of publicly available clinical and
Valganciclovir nonclinical data to construct a comprehensive list of pUL97 and pUL54 amino acid substitutions that are
Resistance associated with a poor clinical response to the first line therapies ganciclovir and valganciclovir, or asso-
Cidofovir ciated with reduced HCMV ganciclovir susceptibility in cell culture. Over 40 putative ganciclovir/val-
Foscarnet ganciclovir resistance-associated substitutions were identified in this analysis. These include the
commonly reported substitutions M460I/V and C592G in pUL97. There were additional substitutions that
are not widely considered as ganciclovir/valganciclovir resistance-associated substitutions, including
V466M in pUL97 and E315D in pUL54. Some of these ganciclovir/valganciclovir resistance-associated
substitutions may confer cross-resistance to other HCMV therapies, such as cidofovir and foscarnet. Based
on this review, we propose that there are more potential HCMV ganciclovir/valganciclovir resistance
pathways than generally appreciated. The resulting comprehensive list of putative ganciclovir/valganci-
clovir resistance-associated substitutions provides a foundation for future investigations to characterize
the role of specific substitutions or combinations of substitutions, which will enhance our understanding
of HCMV mechanisms of ganciclovir/valganciclovir resistance and also provide insight regarding the
potential for cross-resistance to other HCMV therapies.
Published by Elsevier B.V.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2. HCMV infection and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1. Congenital HCMV infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2. HCMV infection in AIDS patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3. HCMV infection in transplant recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3. Antiviral agents for HCMV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.1. Ganciclovir/valganciclovir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2. Other agents used for HCMV therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3. Investigational anti-HCMV agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4. HCMV gene targets of antiviral drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.1. HCMV replication components involved in current treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.2. Mechanisms of resistance to HCMV drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

q
The views expressed in this article are those of the authors and do not necessarily reflect the official position of the FDA.
⇑ Corresponding author. Address: Division of Antiviral Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire
Avenue, Bldg 22/Room 6323, Silver Spring, MD 20993-0002, USA. Tel.: +1 301 796 4195.
E-mail address: [email protected] (T.E. Komatsu).

0166-3542/$ - see front matter Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.antiviral.2013.10.011
 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25 13

5. Methodologies used for resistance analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

5.1. Phenotypic methods used to characterize resistance to HCMV drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5.2. Genotypic methods used to characterize resistance to HCMV drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
6. Analysis of ganciclovir/valganciclovir resistance pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
6.1. pUL97 kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.2. pUL54 DNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
7. Cross-resistance between ganciclovir/valganciclovir and other drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
8. Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

1. Introduction of a potentially indirect mechanism of drug resistance for HCMV,

the pUL54 K805Q substitution improved the fitness of a virus har-
As antiviral therapy has become widely used in various viral boring the pUL54 T821I substitution associated with resistance to
diseases, including disease associated with human cytomegalovi- foscarnet (Martin et al., 2010a), although the clinical relevance of
rus (HCMV) infection, it has also become crucial to understand this particular observation remains unclear.
the mechanisms and clinical consequences of antiviral drug resis- While many of the concepts and approaches to studying antivi-
tance, including the relevant virus, host and drug-related factors. ral drug resistance generally apply to all viruses, HCMV brings
Knowledge of the mechanisms of antiviral drug resistance has al- along several unique challenges. In this article we review the cur-
lowed for development of genotypic and phenotypic techniques rent state of the field regarding the treatment of HCMV disease,
for the timely diagnosis of resistance. Consequences of drug resis- specifically focusing on the study, mechanisms and clinical conse-
tance for any viral infection may range from toxicity inherent in quences of antiviral drug resistance. We also conducted a review of
the use of antivirals designated as second-line in treatment guide- publicly available non-clinical and clinical data to construct a com-
lines to severe disease and even death from progressive viral infec- prehensive list of amino acid substitutions that are associated with
tion when no effective alternative treatments are available. While resistance to the most commonly used treatments for HCMV infec-
the mechanisms and clinical impact of antiviral drug resistance tion, ganciclovir and valganciclovir, and we discuss the potential
have been well described for human immunodeficiency virus for cross-resistance to other HCMV therapies. Finally, we highlight
(HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV) and influ- some of the major knowledge gaps in HCMV drug resistance to
enza virus, they have been less well characterized for HCMV even help stimulate further studies, which may ultimately help guide
though drug-resistant HCMV is also a major clinical concern. the development of new therapies and rational strategies that ad-
Cytomegaloviruses are members of the Herpesviridae family, dress the challenges of HCMV drug resistance.
and are capable of causing a variety of acute, latent and recurrent
infections in humans and animals. HCMV, also designated as the 2. HCMV infection and disease
human herpesvirus 5, is the prototype of the betaherpesvirus
group (Roizman et al., 1981). The HCMV genome is about 230 kb The prevalence of HCMV infection is estimated to vary from
and encodes more than 250 open reading frames. HCMV infection about 45% in developed countries to near 100% in developing coun-
can be primary or secondary. Primary infection occurs in a HCMV tries. Primary infection usually occurs during the first decades of
seronegative susceptible host, whereas secondary infection repre- life and humans are believed to be the only host of the virus. Trans-
sents reactivation of a latent infection or reinfection of a seroposi- mission sources of HCMV include saliva, urine, semen, cervical and
tive host. vaginal secretions, milk, stool, blood, cells, tissues, and organ trans-
As a general rule for all viral infections and antiviral drugs, anti- plants. Primary infection in immunocompetent adults is mainly
viral drug pressure can select for the emergence of pre-existing asymptomatic or it may be associated with a self-limited mononu-
drug resistant variants by conferring a survival advantage to those cleosis-like syndrome and leads to a life-long latent infection.
subpopulations that are relatively less susceptible to the antiviral However, in patients with immature or compromised immune sys-
agent. Alternatively, spontaneous changes in the viral genome gen- tems (i.e., congenitally infected newborns, patients with AIDS and
erated during viral replication in the presence of drug selection can transplant patients) HCMV infections are often symptomatic, caus-
lead to the emergence of a drug resistant viral population. In either ing a range of symptoms such as colitis and pneumonitis, and are
case, secondary consequences of resistance-associated nucleotide associated with increased morbidity and mortality.
or amino acid substitutions can include alterations in viral patho-
genicity, transmissibility, and genetic stability. Development of 2.1. Congenital HCMV infection
antiviral resistance can often increase in frequency and signifi-
cance as antiviral agents are utilized more widely. Great strides In the United States, it is estimated that approximately 44,000
have been made in the methods to characterize the mechanisms infants are born each year with congenital HCMV infection (Stagno
and development of antiviral drug resistance using biochemical as- and Britt, 2006). Approximately 10% of infected newborns are
says or cell culture models, but there are still many limitations to symptomatic at birth. Mortality in symptomatic infants is about
these approaches. While these assays have become more sensitive, 12% and approximately 90% of survivors experience significant
they often do not identify certain indirect mechanisms that may morbidity from the infection, including mental retardation, senso-
contribute towards clinically relevant drug resistance, such as rineural hearing loss, microcephaly, seizures, or other medical
compensatory viral fitness amino acid substitutions (Bloom et al., problems. No drugs are FDA-approved for antiviral treatment of
2010), viral substitutions that only confer resistance when present congenital HCMV infection. However, a report by Kimberlin and
in combination with other substitutions (Kobayashi et al., 2011; his colleagues (Kimberlin et al., 2003) suggests that 6 weeks intra-
Sun et al., 2012), or viral- or host-specific (e.g., ribavirin, (Ibarra venous ganciclovir may have a role in treatment of neonates with
et al., 2011)) changes that impact drug metabolism. As an example symptomatic HCMV infection.
14 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25

2.2. HCMV infection in AIDS patients is approved for treatment of HCMV retinitis by intraocular
injection.
HCMV disease is common in patients with advanced HIV dis-
ease. Before the availability of highly active antiretroviral regimen 3.1. Ganciclovir/valganciclovir
(HAART), up to 45% of patients with AIDS acquired HCMV disease
(Masur et al., 1996). HCMV retinitis is by far the most common Ganciclovir (CytoveneÒ), in its intravenous formulation, was the
manifestation of HCMV disease in patients with AIDS, accounting first antiviral agent approved by FDA for the treatment of HCMV
for 85% of the cases (Gallant et al., 1992). The introduction of disease. It is an acyclic nucleoside analogue of 20 -deoxyguanosine,
HAART decreased the incidence of HCMV disease in patients with and must be phosphorylated to the triphosphate form to exert its
AIDS by more than 80% (Jabs et al., 2007). Currently FDA-approved antiviral activity by inhibiting viral DNA replication. It is not effi-
drugs for the treatment of HCMV retinitis in patients with AIDS in- ciently phosphorylated in most uninfected cells, thereby reducing
clude intravenous ganciclovir, oral ganciclovir, ganciclovir intraoc- toxicity, but is phosphorylated to ganciclovir monophosphate in
ular implant, oral valganciclovir, intravenous foscarnet, and HCMV-infected cells by the viral protein kinase (pUL97). Further
intravenous cidofovir. Oral valganciclovir is frequently used for ini- phosphorylation to the triphosphate form occurs by cellular
tial treatment (Stewart, 2010). kinases.
Safety risks coupled with the inconvenience of administering
2.3. HCMV infection in transplant recipients the drug for a prolonged period of time led to the development
of oral ganciclovir. Oral ganciclovir is approved for the prevention
HCMV is the most frequent opportunistic pathogen in trans- of HCMV disease in solid organ transplant recipients and in indi-
plant patients contributing significantly both to patient morbidity viduals with advanced HIV-1 disease at risk for developing HCMV
and mortality. The risk of developing HCMV disease after trans- disease. Oral ganciclovir is also approved for maintenance treat-
plantation depends on a number of different factors, including ment only for HCMV retinitis in immunocompromised patients
the HCMV serologic status of both the donor and recipient, the type including patients with AIDS, in whom retinitis is stable following
of transplant, and the level of immunosuppression. The clinical appropriate induction therapy. The poor bioavailability of the oral
symptoms of HCMV infection range from asymptomatic HCMV formulation raised concerns that the lower systemic exposure
viremia to tissue invasive HCMV disease. Because of the increased could be suboptimal and lead to emergence of drug resistance. In
morbidity and mortality associated with HCMV infection in trans- addition, the high pill burden for prophylaxis (four capsules three
plant patients, prevention of HCMV disease is a preferred strategy times daily) makes compliance challenging. These limitations lead
over waiting for established disease to occur and treating. Prophy- to the development of valganciclovir (ValcyteÒ), a more orally bio-
lactic therapy and pre-emptive therapy are the two major strate- available form of ganciclovir.
gies used for prevention of HCMV disease (Boeckh and Ljungman, Valganciclovir is an L-valyl ester of ganciclovir. After oral
2009; Tomblyn et al., 2009; Kotton et al., 2010; Razonable et al., administration, valganciclovir is rapidly absorbed and hydrolyzed
2013). by intestinal and hepatic esterases to ganciclovir. The absolute bio-
Although there have been no large studies comparing the two availability of ganciclovir from oral valganciclovir tablets (60%) is
approaches, prophylaxis with oral valganciclovir has emerged as up to 10 times greater than the bioavailability of oral ganciclovir
the most common strategy in solid organ transplant recipients (6–9%) (Brown et al., 1999). Valganciclovir is approved for the
due in part to the once daily dosing with this drug (Kotton, treatment of HCMV retinitis in adults with AIDS, the prevention
2013). Valganciclovir has also emerged as the drug of choice for of HCMV disease in adult solid organ transplant (SOT) patients at
pre-emptive therapy and treatment of mild-to-moderate HCMV high risk for HCMV disease (Paya et al., 2004), and the prevention
disease in solid organ transplant recipients. Intravenous ganciclo- of HCMV disease in pediatric kidney and heart transplant patients
vir, with or without administration of intravenous immunoglobu- P4 months of age. Valganciclovir has not only replaced oral ganci-
lin, remains the standard for treatment of established severe clovir for the prevention of HCMV disease but has also become the
HCMV disease (Kotton, 2013). recommended and most commonly used agent to treat solid organ
HCMV disease appears to be more severe in allogeneic stem cell transplant recipients with mild-to-moderate HCMV disease (As-
recipients compared to the solid organ transplant recipients be- berg et al., 2007).
cause of the more potent immunosuppressive therapy. Pneumonia
is the most serious manifestation of HCMV disease, which is asso- 3.2. Other agents used for HCMV therapy
ciated with a >50% mortality rate (Ljungman, 1995; Boeckh et al.,
1996). Although prophylaxis is the preferred approach in prevent- Foscarnet (FoscavirÒ) was the second drug approved for the
ing HCMV disease in solid organ transplant patients, pre-emptive treatment of HCMV retinitis in AIDS patients. Foscarnet sodium
therapy is more widely used in stem cell transplant patients be- is the trisodium salt of phosphormophonic acid, a pyrophospho-
cause of the bone marrow toxicities of the current anti-HCMV nate analogue. Foscarnet inhibits the activity of the viral DNA poly-
drugs (Boeckh and Ljungman, 2009). Intravenous ganciclovir is merase by binding to the pyrophosphate binding site and blocking
the recommended drug of choice for pre-emptive therapy cleavage of pyrophosphate from the terminal nucleoside triphos-
(Ljungman et al., 2011). Intravenous ganciclovir with or without phate added to the growing DNA chain. Foscarnet does not require
administration of intravenous immunoglobulin is also the first-line phosphorylation by kinases and is therefore active against pUL97
treatment for established HCMV disease (Boeckh and Ljungman, kinase variants that confer resistance to ganciclovir/valganciclovir
2009). (mechanism of drug resistance is described in greater detail be-
low). Foscarnet is considered a second-line therapy due to its rela-
3. Antiviral agents for HCMV tively toxic safety profile. Its role in HCMV infections has been
limited to patients who are failing ganciclovir therapy, presumably
There are limited therapeutic options for the treatment or due to drug resistance, or those who cannot be treated with ganci-
prevention of HCMV disease in immunocompromised patients. clovir due to dose-limiting neutropenia (Razonable and Emery,
Currently, four drugs have received FDA approval for the systemic 2004).
treatment of HCMV disease: ganciclovir and its prodrug Cidofovir (VistideÒ, CDV) is an acyclic monophosphate nucleo-
valganciclovir, foscarnet, and cidofovir. Another dug, fomivirsen, tide analogue of 20 -deoxycytidine with cell culture antiviral
 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25 15

activity against various DNA viruses including herpesviruses and of HCMV by a novel mechanism, presumably by targeting the
adenovirus (De Clercq and Holy, 2005). Viral kinases are not pUL56 subunit of the viral terminase complex (Goldner et al.,
needed for the initial phosphorylation because a single phosphate 2011; Lischka et al., 2010). Cyclopropavir (MBX-400) is a methyl-
group is present in its native form. Cidofovir inhibits DNA replica- enecyclopropane guanosine nucleoside analog with a mechanism
tion by viral DNA polymerase after phosphorylation to its active of action similar to that of ganciclovir/valgancyclovir (Gentry
diphosphate form by cellular kinases. Cidofovir is approved for et al., 2010). Artesunate (ART) is a derivative of the Chinese herb
the treatment of HCMV retinits in AIDS patients. It is available only Artemisia annua and has been used to treat malaria (Reyburn,
as an intravenous formulation because of its poor oral bioavailabil- 2010), but has also been reported to have anti-HCMV activity in
ity, although the relatively long half-life of the active metabolite al- nonclinical and clinical studies (Efferth et al., 2002; Efferth et al.,
lows for an intermittent dosing schedule (Yang and Datema, 1991). 2008; Kaptein et al., 2006; Schnepf et al., 2011; Wolf et al., 2011;
The clinical use of cidofovir is limited because of adverse events Shapira et al., 2008).
such as nephrotoxicity and neutropenia. Additionally, cidofovir
has been shown to be carcinogenic, teratogenic, and to cause hyp-
4. HCMV gene targets of antiviral drugs
ospermia in animal studies (Vistide Package Insert, 1996). In clin-
ical practice, cidofovir is generally used in patients with
4.1. HCMV replication components involved in current treatment
established HCMV disease who are failing treatment with ganciclo-
vir/valganciclovir, foscarnet or both.
In general, HCMV gene expression is divided into three sequen-
Fomivirsen (VitraveneÒ) is an antisense phosphorothioate oli-
tial kinetic classes: a (immediate early), b1 and b2 (delayed early),
gonucleotide that blocks the replication of HCMV mRNA. It is 21
and c1 and c2 (late), based on the time of expression after virus
nucleotides long, comprised of a sequence that is complementary
infection (Grant et al., 1981; Stinski et al., 1991). Expression of b
to the mRNA transcribed from the major immediate-early tran-
and c gene products requires the synthesis of functional a gene
scriptional unit of HCMV (Mulamba et al., 1998). Fomivirsen was
products that act as transcriptional trans-activators. Delayed early
approved by FDA in 1998 for treatment of HCMV retinitis, but is
b1 genes are transcribed between 4 and 8 h post-infection, while
no longer marketed in the United States.
b2 genes are expressed approximately between 8 and 24 h post-
Acyclovir (ZoviraxÒ, ACV) is a guanosine analogue with cell cul-
infection. Most of the b2 gene products, such as viral DNA polymer-
ture antiviral activity against various DNA viruses including herpe-
ase, are involved in viral DNA synthesis (Wright et al., 1964).
sviruses (O’Brien and Campoli-Richards, 1989). It is converted into
The HCMV DNA polymerase, pUL54, which is a b2 delayed early
acyclo-guanosine monophosphate (acyclo-GMP) by viral thymi-
product encoded by the UL54 gene (also referred as the HCMV pol
dine kinase. Subsequently, the monophosphate form is further
gene), is the central enzyme involved in viral DNA replication and
phosphorylated into the active triphosphate form, acyclo-guano-
the primary drug target for current therapies. It is a relatively large,
sine triphosphate (acyclo-GTP), by cellular kinases. As a substrate,
140 kD protein, consisting of 1242 amino acids based on the AD169
acyclo-GTP is incorporated into viral DNA, resulting in premature
HCMV reference strain. Its range of activities includes viral DNA
chain termination. Acyclovir is approved for the treatment of her-
synthesis, as well as a 30 - to 50 -exonuclease proof-reading activity
pes simplex virus (HSV) and varicella zoster virus (VZV) infections.
to increase replication fidelity (Kouzarides et al., 1987; Pari and
High doses of acyclovir and its prodrug valacyclovir have been used
Anders, 1993). Both DNA and amino acid sequence analyses have
off label for HCMV prophylaxis. However, due to the high pill bur-
revealed that the UL54 gene has significant homology to the pol
den and the risk of neurologic adverse events, valganciclovir has
genes of other herpesviruses, such as herpes simplex virus type 1
become the preferred drug for prophylaxis.
(HSV-1) and Epstein–Barr virus (EBV) (Shepp et al., 1985). In addi-
Leflunomide (AravaÒ) is an immunomodulatory drug inhibiting
tion, herpesvirus DNA polymerases share striking sequence simi-
mitochondrial enzyme dihydroorotate dehydrogenase which plays
larities with eukaryotic DNA polymerases a and d and many
a key role in the de novo synthesis of the pyrimidine ribonucleo-
other DNA polymerases of viruses and bacteriophages in several
tide uridine monophosphate (rUMP) (Fukushima et al., 2007).
discrete regions that define a family of a-like polymerases (Wong
There are published reports of a beneficial effect of leflunomide
et al., 1988; Sullivan et al., 1993). This homology has been used
in some cases (Levi et al., 2006; Mossad and Avery, 2007), but fail-
to define seven conserved regions of the pUL54 protein, labeled
ures (in haemopoietic transplantation) in other cases (Battiwalla
I–VII in order of decreasing homology.
et al., 2007).
The UL97 gene, which is also a b2 delayed early gene and has
conserved homologues in all herpes sub-families (Michel et al.,
3.3. Investigational anti-HCMV agents
1998), encodes pUL97. This protein has homologies with protein
kinases, and may impair HCMV growth (Michel et al., 1996; Prich-
A number of other anti-HCMV agents are in development and in
ard et al., 1999; Wolf et al., 2001; Gill et al., 2012). The pUL97 ki-
the future may provide alternatives to currently approved drugs.
nase activity plays a key role in the phosphorylation of the
New agents with greater antiviral potency or novel mechanisms
nucleoside ganciclovir, which is necessary for generation of the ac-
of action may be particularly important for the treatment of HCMV
tive triphosphate polymerase inhibitor (Littler et al., 1992; Sullivan
infections that are resistant to one or more of the currently avail-
et al., 1992). Experiments with recombinant vaccinia viruses
able therapies.
expressing a range of mutant pUL97 proteins have indicated the
Investigational anti-HCMV drugs currently include maribavir,
C-terminal region of pUL97 (amino acids 292–707) binds and
CMX001, letermovir, cycloprapavir, and artesunate. Maribavir is a
phosphorylates ganciclovir, while the central portion (amino acids
novel inhibitor of the pUL97 kinase and has been investigated for
305–365) has a role in phosphorylating both ganciclovir and pUL97
prophylaxis of HCMV. Unfortunately, the drug failed to demon-
itself (Michel et al., 1998). The N-terminal region of pUL97 (amino
strate efficacy in two Phase III trials; one in allogeneic hematopoi-
acids 1–199) contains a nuclear localization signal.
etic stem cell transplant recipients (Marty et al., 2011) and the
other in liver transplant recipients at high risk for HCMV disease
(Winston et al., 2012). CMX001 is a cidofovir analog with enhanced 4.2. Mechanisms of resistance to HCMV drugs
oral bioavailability and activity active against a broad range of
herpes viruses including many drug-resistant HCMV isolates Variants with reduced susceptibility to ganciclovir/valganciclo-
(Quenelle et al., 2010). Letermovir (AIC246) inhibits the replication vir occur naturally in circulating HCMV (Drew et al., 1993).
16 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25

Furthermore, antiviral resistance develops in a stepwise fashion DNA polymerase in HSV and HHV-6 (Freifeld and Ostrove, 1991;
(Muller and Krausslich, 2009; Nijhuis et al., 2009). Prior to treat- Nakano et al., 2009; Isegawa et al., 2009; Baldwin, 2011).
ment, the entire virus population contains a small fraction of less Resistance to foscarnet in the laboratory or clinical setting has
drug susceptible variants due to error-prone replication by the vir- been mapped to amino acid substitutions in pUL54. Cross-resis-
al DNA polymerase. When subsequent antiviral treatment does not tance has been observed between ganciclovir and foscarnet in sev-
suppress viral replication completely, for example due to subopti- eral laboratory and clinical isolates with both genotypic and
mal levels of antiviral drugs or extreme immunosuppression of the phenotypic resistance. Resistance to cidofovir has been difficult
host, a selection process favoring these variants occurs. For exam- to select in the laboratory (Cihlar et al., 1998a), and has not been
ple, ganciclovir-resistant pUL97 mutants may have some subtle reported in clinical isolates, although this may be due to the short-
loss of growth fitness, as supported by in vivo viral population dy- er treatment duration and limited use of this agent. Nevertheless,
namic studies (Emery et al., 1999). Since they can replicate in the several ganciclovir-resistant HCMV clinical isolates or laboratory-
presence of the antiviral drug, they are able to evolve and acquire selected strains with specific pUL54 amino acid substitutions are
additional amino acid substitutions that enhance replicative fitness clearly cross-resistant to cidofovir (Gilbert and Boivin, 2005).
and possibly increase resistance, ultimately resulting in a large
population of highly resistant and replication competent viruses.
This model is supported by the observation that major risk factors 5. Methodologies used for resistance analysis
for HCMV drug resistance are the residual capacity of the host’s im-
mune system to control viral replication and the overall level and 5.1. Phenotypic methods used to characterize resistance to HCMV
duration of viremia in the presence of drug (Drew, 2000). Clinicians drugs
should keep these selection mechanisms in mind when monitoring
viral loads in treated patients. Furthermore, the timing of samples Phenotypic testing of clinical isolates was the primary method
obtained for antiviral drug susceptibility testing combined with for HCMV drug susceptibility testing before the establishment of
the sensitivity of applied assays has to be considered. For example, genotypic testing. The gold standard for phenotypic characteriza-
analyzing phenotypic susceptibility early during antiviral treat- tion of HCMV drug susceptibility has historically been the plaque
ment may not predict eventual treatment failure due to an under- reduction assay (PRA), which measures viral spread in cell culture
lying drug resistant viral population that has yet to predominate in based on the quantity of newly formed viral plaques that emerge in
the viral population. the presence of different antiviral drug concentrations (Table 1).
The development of resistance to ganciclovir has been docu- However, inter-assay and especially inter-laboratory standardiza-
mented during the treatment of HCMV retinitis in patients with tion of the PRA has been hampered by technical challenges and of-
AIDS. Before HAART was introduced, HCMV antiviral treatment ten poor reproducibility of the assay (Landry et al., 2000).
was usually administered in the presence of overt disease, followed Consequently, efforts have been made to develop new assays that
by lifelong maintenance therapy. The incidence of HCMV-related are more amenable to standardization.
complications and death in patients with AIDS has declined due Several other phenotypic or combined genotypic/phenotypic
to the introduction of HAART (Palella et al., 1998; Kedhar and Jabs, methods have been used to characterize HCMV drug susceptibility
2007), but still remains a concern in patients with low CD4+ T cell and resistance (Table 1). Reporter cell lines have been developed to
counts. Therefore, diagnosis and monitoring of active HCMV vire- measure HCMV spread in cell culture reflected by cell-expressed
mia and, in many cases, long-term antiviral therapy against HCMV luciferase activity (Gilbert and Boivin, 2005), green fluorescent
are life-saving for patients at risk for severe HCMV disease. Unfor- protein (GFP) (Ueno et al., 2006; Ueno and Ogawa-Goto, 2009),
tunately, the likelihood of drug resistance emergence in patients or enhanced green fluorescent protein (EGFP) (Luganini et al.,
with AIDS appears to increase with duration of therapy. Jabs 2008). Another approach uses quantitative real-time PCR to mea-
et al. (1998) reported that approximately 2–7% of patients with sure the declining number of HCMV genome copies in cell culture
AIDS have ganciclovir resistant HCMV after 2–3 months of drug due to different concentrations of the antiviral agent (Schnepf
therapy, while a study by Boivin et al. (2001) demonstrated that et al., 2009). Marker transfer approaches have been developed to
up to 15–28% of patients have drug resistant virus after 9 or generate recombinant HCMV viruses and characterize the impact
18 months of therapy. Ganciclovir-resistant HCMV is a clinical of specific genetic changes or entire gene cassettes on viral suscep-
problem in organ transplant recipients as well (Bhorade et al., tibility and resistance to antiviral drugs. These recombinant viruses
2002; Limaye, 2002; Boivin et al., 2009). Patients with drug-resis- may express various reporter proteins in infected cells, such as se-
tant HCMV strains often have virological failure and might have creted alkaline phosphatase (Chou et al., 2005), GFP (Marschall
unfavorable clinical outcomes (Boivin et al., 2001). et al., 2000), enhanced yellow fluorescent protein (Dal et al.,
Pathways leading to the development of resistance to ganciclo- 2008) or EGFP (Chevillotte et al., 2009). Finally, a biochemical assay
vir have been reported from selection of resistant virus in cell cul- has been developed to measure the incorporation of digoxigenin-
ture passage experiments, as well as treatment studies of HCMV labeled nucleotides by recombinant pUL54 polymerase enzymes
retinitis in patients with AIDS (Lurain and Chou, 2010; James and (Ducancelle et al., 2007). These relatively newer methods of pheno-
Prichard, 2011). Amino acid substitutions conferring resistance to typic analysis are often easier to perform and better standardized
ganciclovir have been detected in the pUL97 kinase (decreasing than the PRA, and are important tools to identify and characterize
phosphorylation of ganciclovir) and in the pUL54 DNA polymerase resistance-associated amino acid substitutions. However, all as-
(decreasing inhibition of the viral DNA polymerase by ganciclovir says, including the PRA, generally have not been in widespread
triphosphate), sometimes occurring in both within a single resis- use for routine clinical decision making as they lack clinically val-
tant variant. The passage of laboratory HCMV strain AD169 in cell idated data defining cutoffs in EC50 values that likely reflect clinical
culture in the presence of increasing concentrations of ganciclovir drug resistance.
(from 1.5 to 3000 lM) resulted in the selection of amino acid sub- An important limitation of phenotypic analysis of clinical iso-
stitutions in pUL97 (M460I and L595S) and in pUL54 (L545S, lates is the impact of mixed populations of wild-type and drug
V812L, P829S and D879G) (Gilbert and Boivin, 2005). Resistance resistant virus and the potential bias of the assays for detecting
to ganciclovir has also been studied in other herpesviruses. Similar wildtype virus phenotypic susceptibility. Detection of mixed viral
to what was found in HCMV, amino acid substitutions conferring populations has been reported in patients with HCMV viremia
resistance to ganciclovir have been detected in the kinase and (Eckle et al., 2004; Jabs et al., 2006; Ross et al., 2011), and while
 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25 17

Table 1
Phenotypic assays used to characterize resistance to HCMV drugs.

Phenotypic assay/ Summary of methodology Technical/practical challenges

readout
Plaque reduction assay a. Clinical or recombinant isolates grown in presence of a. Inter-assay and inter-laboratory standardization very difficult.
varying drug concentrations. b. Technically challenging and time consuming, and not
b. Viral spread in culture quantified by plaque assay. amenable to automated or high throughput analysis.
c. Drug effective concentrations (e.g., EC50 values) calculated c. Potential bias to wild-type phenotype when clinical isolates
and compared for different isolates. include a mixture of wild-type and drug resistant variants.
Reporter cell lines a. Clinical or recombinant isolates grown in presence of a. No standardization for different reporter cell lines.
varying drug concentrations. b. Although genotypic data may be informative, construction and
b. Cell line used for infection carries a reporter gene (e.g., phenotypic analysis of recombinant viruses not amenable to
luciferase) under the control of HCMV promoter. routine clinical use.
c. Viral spread in culture quantified by reporter assay (e.g., c. Potential bias to wild-type phenotype when clinical isolates
plate reader, flow cytometry). include a mixture of wild-type and drug resistant variants.
d. Drug effective concentrations (e.g., EC50 values) calculated
and compared for different isolates.
Real-time PCR a. Clinical or recombinant isolates grown in presence of a. Relatively expensive.
varying drug concentrations. b. DNA readout does not distinguish between infectious and
b. Viral spread in culture quantified by measuring the noninfectious virus particles.
declining number of genome copies. c. Potential bias to wild-type phenotype when clinical isolates
c. Drug effective concentrations (e.g., EC50 values) calculated include a mixture of wild-type and drug resistant variants.
and compared for different isolates.
Marker transfer a. Recombinant viruses are generated to encode specific a. Most direct method to understand impact of specific genetic
genetic changes or entire gene cassettes. changes on HCMV drug susceptibility, but mutant strains
b. Viral spread in culture quantified by reporter or other assay. technically challenging to generate.
c. Drug effective concentrations (e.g., EC50 values) calculated b. Although genotypic data may be informative, construction and
and compared for different isolates, or for parental and phenotypic analysis of recombinant viruses not amenable to
recombinant HCMV constructs. routine clinical use.
c. Results may be impacted by background sequence.
Polymerase a. HCMV UL54 DNA polymerase gene amplified by PCR. a. Does not recapitulate entire viral life cycle.
Biochemical assays b. Construct expression vectors harboring the HCMV DNA b. Cannot characterize impact of other viral proteins (pUL97
polymerase. kinase).Cannot account for impact on viral fitness.
c. DNA polymerase activity assayed by measuring the c. Require cidofovir diphosphate/ganciclovir triphosphate
incorporation of digoxigenin deoxyuridine triphosphate (active forms) for conducting assay.
(DIG-dUTP) into the growing DNA chain.

Abbreviation: EC50, 50% effective concentration.

HCMV phenotypic assays can sometimes detect minority drug sequences in the plasmid and the digested parental DNA in
resistant populations, the sensitivity of these assays likely varies cotransfected cells results in viable recombinant virus harboring
depending on the specific viral variants. Drew et al. (2001) state the desired gene cassette or amino acid substitution(s) (Chou,
that a phenotypic assay probably only exceeds the cut-off level 2008; Chou and Marousek, 2008; Chou et al., 2003, 2005; Drew,
for resistance when at least 50% of the viral population is resistant. 2006; Iwasenko et al., 2009; Marfori et al., 2007; Scott et al.,
Poor sensitivity of phenotypic assays for detecting mixed popula- 2007; Springer et al., 2005; Weinberg et al., 2003). This method
tions of drug resistant and drug susceptible virus has been noted highly increases the selective pressure for the formation of recom-
for other viruses including influenza (Wetherall et al., 2003) and binant virus compared to wild-type virus, but plaque purification is
HCV (Verbinnen et al., 2012). still necessary. Of note, the problem of wild-type virus contamina-
Marker transfer is generally the method of choice to provide a tion is circumvented using a biochemical assay, where only the
link between viral genetic changes and phenotypic drug suscepti- mutated enzymes, and not viruses, are generated and tested for
bility. In these experiments, amino acid substitutions suspected drug susceptibility (Ducancelle et al., 2005, 2007).
to confer resistance are introduced into a well-defined, drug-sensi- More recently, bacterial artificial chromosome (BAC) technol-
tive viral background, and the recombinant viruses are tested phe- ogy has been applied to generate clonal recombinant viruses for
notypically. Early protocols for marker transfer were based on marker transfer experiments. Since all progeny viruses start from
either co-transfection of HCMV DNA or transfection of HCMV in- BAC DNA from one single bacterial colony, clonal virus populations
fected cells with linear DNA containing the mutated sequence. are generally obtained (although the potential for other changes
After recombination of the DNA in the transfected cell, recombi- during viral amplication can still apply). Several protocols have
nant viruses bearing the desired amino acid substitution could be been published for the generation of recombinant BACs for HCMV
reconstituted and then plaque purified in order to avoid wild-type marker transfer purposes, based on a flp recombinase (Martin
virus contamination (Lurain et al., 1992; Sullivan et al., 1992; Bal- et al., 2006), RED recombinase (Chou, 2009), or RED-GAM recombi-
danti et al., 1996; Bourgeois et al., 1997; Cihlar et al., 1998a; Faizi nase system (Chevillotte et al., 2009). Only the latter two are mark-
et al., 1998; Chou et al., 2000; Mousavi-Jazi et al., 2001a, 2001b). erless mutagenesis methods, meaning that they leave no trace of
These approaches do not always produce a clonal virus population the recombination procedure in the BAC sequence (Tischer et al.,
due to residual wild-type virus contamination or the introduction 2006; Warming et al., 2005). BAC mutagenesis is efficient and rel-
of other substitutions during the viral amplification process, influ- atively fast, and is now becoming the method of choice for the
encing the results of drug susceptibility testing. characterization of the many still uncharacterized resistance-asso-
Other methods use a drug-sensitive parental HCMV strain that ciated substitutions.
bears unique restriction sites in the target gene. The parental Although marker transfer experiments offer a powerful ap-
DNA is digested with restriction enzymes and cotransfected with proach to better understand the mechanisms of HCMV drug resis-
a plasmid encoding the amino acid substitution(s) of interest in tance, several limitations remain. One concern is that the genetic
the respective gene. Recombination between overlapping background may influence the ability of a particular substitution
18 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25

to confer resistance. Furthermore, the number of needed marker stage, prior to the predominance of drug resistant viral populations
transfers has increased as it is becoming clearer that combinations (Sahoo et al., 2013).
of substitutions play an important role in HCMV drug resistance. A major advantage with the use of genotypic assays is the po-
For example, certain combinations of resistance-associated substi- tential to identify novel mechanisms of HCMV drug resistance. Vir-
tutions in the UL97 and UL54 gene products have been shown to al genetic changes associated with treatment or prophylaxis failure
have a synergistic effect on drug resistance (Ducancelle et al., can be identified, reported and further characterized as appropriate
2007; Mousavi-Jazi et al., 2001a, 2001b; Scott et al., 2007), while to determine their potential impact on HCMV drug susceptibility,
others have been shown to partially compensate for replicative fit- including cross-resistance to other drugs. Continually monitoring
ness defects caused by individual substitutions (Ijichi et al., 2002; HCMV genetic changes for potentially novel resistance mecha-
Martin et al., 2010a). However, these results have been conflicting. nisms could lead to further refinement of genotypic and pheno-
In the case of the pUL54 D605E substitution, there are also reports typic drug resistance assays, which in turn could help optimize
that show it does not have compensatory effects (Chou et al., treatment decision making.
2005). Another important limitation of marker transfer-based as-
says, and phenotypic assays in general, is that the length of drug
exposure is generally not representative of the drug exposure in 6. Analysis of ganciclovir/valganciclovir resistance pathways
treated patients. For these and other reasons noted above, substi-
tutions associated with clinical drug resistance do not always con- Amino acid changes that confer reduced HCMV susceptibility to
fer obvious reductions in phenotypic susceptibility in cell culture ganciclovir/valganciclovir are believed to map entirely to the
or biochemical assays (Chou et al., 2002). Therefore, the lack of a pUL97 kinase and pUL54 DNA polymerase (Lurain and Chou,
phenotypic fold-shift by itself does not necessarily prove that a 2010; James and Prichard, 2011). There are numerous pathways
particular genetic variant has no impact on drug resistance. De- for HCMV to become resistant to ganciclovir/valganciclovir and
spite the numerous challenges and lack of standardization of phe- many different resistance-associated substitutions have been re-
notypic assays, a >5-fold reduction in HCMV phenotypic ported. Multiple resistance pathways are possible complicating
susceptibility to GCV has been proposed as a basis for switching the identification of resistance pathways from clinical samples.
therapy (Kotton et al., 2010). An additional complication is the lack of a baseline comparator
for resistance analyses in isolates from patients experiencing fail-
ure in prophylaxis studies.
5.2. Genotypic methods used to characterize resistance to HCMV drugs To better understand the full breadth of potential mechanisms
of ganciclovir/valganciclovir resistance, we conducted a compre-
Genotypic assays take advantage of knowing specific resis- hensive review of the literature to identify and compile specific
tance-associated substitutions in the drug target gene(s). Geno- amino acid substitutions potentially associated with resistance to
typic drug resistance testing is used extensively for guiding ganciclovir/valganciclovir. Although both clinical efficacy and phe-
treatment decisions for other viruses, particularly HIV and hepati- notype data were considered, our analysis focused primarily on
tis B virus, but are used relatively less frequently for HCMV. As an amino acid substitutions that have been clinically associated with
example for ganciclovir genotypic resistance testing, the UL97 and resistance to ganciclovir/valganciclovir. We use the term ‘‘clinically
UL54 genes can be amplified by PCR, the PCR product analyzed by associated’’ to refer to substitutions that have been observed in
Sanger DNA sequencing, and amino acid coding substitutions al- subjects who failed ganciclovir/valganciclovir treatment.
ready known to be associated with ganciclovir resistance can be The list of resistance-associated amino acid substitutions were
identified and reported. Other HCMV genotypic methods that have constructed based on the following criteria:
been used include restriction fragment length polymorphism of
PCR products (Novak et al., 2008; Pignatelli et al., 2003), and geno-  single substitution with supporting phenotypic data (P2-fold
type-specific PCR assays (Mattick et al., 2004). Genotypic assays reduction in susceptibility to ganciclovir),
can be directly applied to clinical samples if there is sufficient  substitutions at positions identified in cell culture selection
HCMV DNA in, for example, plasma or cerebrospinal fluid, or can studies,
be used on cultured isolates. Several commercial laboratories can  substitutions reported at conserved positions from multiple
perform HCMV genotypic assays, which have a typical turnaround independent studies in literature (P2 patients failing ganciclo-
time of <3 days. vir or valganciclovir treatment or prophylaxis),
The main limitation with genotypic assays used in clinical prac-  substitutions at conserved positions with other known resis-
tice is that they require prior knowledge of key drug resistance- tance-associated substitutions,
associated substitutions to distinguish them from other sequence  codon usage (e.g., multiple nucleotide changes within a codon
substitutions/polymorphisms believed to have no impact on drug required for a substitution),
activity. The known drug resistance-associated substitutions are  clear treatment-emergent amino acid substitution in P2
typically those that have been demonstrated to confer reduced patients.
HCMV drug susceptibility in phenotypic assays, and substitutions
that may contribute less directly towards drug resistance (as dis- ‘‘Conserved’’ positions are defined as any position having 100%
cussed above) are generally disregarded as being clinically relevant identity based on a BLAST search from GenBank using the AD169
and often are not even reported. Another limitation of some cur- pUL97 or pUL54 sequences. A potential limitation of this definition
rent HCMV genotypic analysis methods is they are not capable of is that unlike other viruses like HIV-1 and influenza, there are not
detecting and quantifying minority drug resistant viral popula- as many HCMV sequences (200) deposited to GenBank, which
tions. Next-generation sequencing (NGS) technologies (Gorzer could lead to underestimation of codon variability. For example,
et al., 2010) can enable detection of far smaller viral subpopula- a few amino acid positions, such as A692, the collection of com-
tions, and have been explored in the context of HIV-1, HBV, and plete UL54 putative wildtype sequences from GenBank did not re-
HCV (Margeridon-Thermet et al., 2009; Simen et al., 2009; Svarovs- veal any heterogeneity and therefore the percent identity is listed
kaia et al., 2012). While the NGS technology has not been fully as 100% (i.e. all sequences were A692). Nonetheless, both A692S
developed for routine HCMV diagnostic purposes, in the future it and A692V have been reported in the literature (Lurain and Chou,
may provide the ability to identify drug resistance at an earlier 2010). The sequences containing A692S and A692V may not have
 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25 19

been deposited to GenBank or may have been partial pUL54 se- Table 2
quences. Nevertheless, there are some clear examples of conserved Ganciclovir resistance-associated amino acid substitutions in the pUL97 kinase.

amino acid motifs that are identical throughout the herpesvirus AA Fold shift in susceptibility to Reference(s)
polymerases, such as 30 –50 exonuclease domain (Exo I through GCV
III) and polymerization domain (I through VII) (Lurain et al., L405P 2.5–2.7 1, 2
1992; Ito and Braithwaite, 1991). There are also some conserved M460I/V/ I: 4 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
subdomains and residues present in all serine-threonine kinases, T V: 5–10 36
T: 9.3
including the three glycines in the P-loop, the lysine amino acid V466G/M G: 3.5–7 2, 14, 15
position 355, and some other residues in the catalytic and activa- M: 1.3
tion loops (Hanks et al., 1988; Wilks et al., 1989; He et al., 1997; C518Y 12 38
Marschall et al., 2001; Gershburg and Pagano, 2008). H520Q 5–10 16, 17, 18, 19
del 590– 3–10 20
593
6.1. pUL97 kinase A591D/V D: NA 8, 18, 21, 22, 23
V: 1.3
Using the resistance analysis criteria outlined above, there were C592G 2–3 3, 12, 22, 24
33 amino acid substitutions and 6 amino acid deletions, all at A594E/G/ E: 3 2, 3, 5, 6, 8, 11, 12, 13, 18, 24,
T/V/P G: 13.5 25, 26. 36, 37
either conserved positions or at same positions at well-established
T: 2.7
resistance-associated substitutions, which were identified as being V: 8.6
associated with resistance to valganciclovir or ganciclovir (Table 2). P: 2.9
Most of these amino acid substitutions were reported in multiple L595F/S/ F: no EC50 ratio available, 2, 3, 5, 9, 11, 12, 13, 18, 22, 36
W/T EC50 = 31.9 uM
studies in the literature, including the well established resistance
S: 8.5
substitutions M460V/I, H520Q, C592G, A594V, L595S, C603W. W: 5.1
Amino acid substitutions L405P, M460I/V/T, V466G, C518Y, T: no EC50 ratio available,
H520Q, C592G, A594E/G/T/V/P, L595F/S/W, E596G, K599T, EC50 = 17.6 lM
C603W/R, C607Y, and all of the deletion variants were reported del 595 13 27
del 595– 8.4 28
to confer at least a 2-fold reduction in susceptibility to ganciclovir
603
in phenotypic assays. E596D/G D: NA 5, 18, 23
Amino acid substitutions V466M, A591V, L595T, C603S, and G: 2.3
C607F have been observed in patients failing treatment or prophy- K599E/M/ E: NA 23, 29
Ta M: NA
laxis, but have been shown to confer a <2-fold reduction in suscep-
T: 5.9
tibility to ganciclovir in phenotypic assays. Despite the lack of a del 597– NA 35
clear resistance phenotype, each of these substitutions occurs at 600
a conserved site where other substitutions have been shown to del 600– NA 24
be associated with ganciclovir/valganciclovir resistance. Addition- 601
del 601– 15 7
ally, reductions in susceptibility of <2-fold have been reported to
603
be clinically significant (Chou et al., 2002). C603W/R/ W: 5–10 2, 5, 12, 13, 15, 23, 30
We are not aware of any published phenotypic data to charac- S/Y R: 3.6
terize amino acid substitutions A591D, E596D, K599E/M, C603Y, S: 1.9
Y: NA
and C607S. However, each substitution occurs at the same position
C607F/S/Y F: 1.6 14, 18, 31, 32, 33, 34, 35
as a confirmed resistance-associated amino acid substitution, and S: NA
all have been observed in patients failing treatment or prophylaxis. Y: 12.5
Amino acid substitution G598S does not have a ganciclovir EC50
1: Martin et al. (2006); 2: Chou (2010); 3: Chou et al. (2005); 4: Lurain et al. (1994);
value reported directly in comparison to a wild-type reference, 5: Boutolleau et al. (2009); 6: Scott et al. (2004); 7: Marfori et al. (2007); 8: Wolf
although based on recombinant phenotyping it appears to confer et al. (1995); 9: Gilbert and Boivin (2005); 10: Palmer et al. (2010); 11: Arslan et al.
significant ganciclovir resistance (Baldanti et al., 2002a, 2002b). (2010); 12: Hantz et al. (2010); 13: Myhre et al. (2011); 14: Boivin et al. (2001); 15:
Other less common resistance-associated substitutions would Martin et al. (2010b); 16: Hanson et al. (1995); 17: Li et al. (2007); 18: Chou et al.
(2002); 19: Schreiber et al. (2009); 20: Sullivan et al. (1992); 21: Erice (1999); 22:
be relatively difficult to generate at the molecular level. For exam-
Smith et al. (1997); 23: Wolf et al. (1998); 24: Hu et al. (2002); 25: Bourgeois et al.
ple, A594E, L595T and L595W require either a transversion muta- (1997); 26: Kuo et al. (2003); 27: Baldanti et al. (1995); 28: Chou and Meichsner
tion or 2 nucleotide mutations to generate the specific amino acid (2000); 29: Faizi et al. (1998); 30: Chou et al. (1997); 31: Baldanti et al. (1998); 32:
substitution, which would not be enriched frequently by DNA rep- Reddy et al. (2007); 33: Lurain et al. (2002); 34: Baldanti et al. (2002a,b); 35:
lication error alone in the absence of drug selection. Campanini et al. (2012); 36: Chou and Bowlin (2011); 37: Ijichi et al. (2002); 38:
Zhang et al. (2013).
a
Developed in cell culture studies only.
6.2. pUL54 DNA polymerase

There were 32 amino acid substitutions and 1 amino acid dele- P2-fold reduction in susceptibility to ganciclovir (Chou et al.,
tion that were identified as being associated with resistance to val- 2003; Cihlar et al., 1998b; Gilbert and Boivin, 2005). Other single
ganciclovir or ganciclovir (Table 3). All of these substitutions/ amino acid substitutions selected in cell culture have been re-
deletions occur at conserved sites or at the same positions as ported to confer <2-fold reductions in ganciclovir susceptibility
well-established resistance-associated substitutions. Amino acid (F595I, L862F, V946L), or have an unclear phenotype (P829S)
substitutions D301N, N408D/K/S, N410K, F412C/L/S, D413A/E, (Gilbert et al., 2011).
L501F/I, T503I, K513E/N/R, I521T, P522A/S, L545S/W, Q578H, Amino acid substitutions E315D, P522L, Q578L, D588E, S695T,
D588N, E756K, V781I, V787L, L802M, A809V, T813S, T821I, I726T/V, D879G, and A972V have been shown to confer a <2-fold
A834P, G841A, A987G, and del 981–982 are reported to confer a reduction in susceptibility to ganciclovir in phenotypic assays.
P2-fold reduction in susceptibility to ganciclovir. Amino acid sub- However, most of these substitutions occur at a conserved site.
stitutions F412V, P488R, L516R, C539R, and V812L were identified Substitutions P522L, Q578L, D588E, and A972V are located at
only in cell culture selection studies and were reported to confer a the same position as confirmed resistance-associated amino acid
20 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25

Table 3
Ganciclovir resistance-associated amino acid substitutions in the pUL54 DNA polymerase.

AA Fold Shift in Susceptibility to GCV Cross-Resistance to CDV/FOS Reference

D301N 2.6 (viral fitness defect) CDV: 3X, FOS: 0.5X 1
E315D 0.8 2, 3
N408D/K/S D: 4.9 D: CDV: 5.6X, FOS: 1.3X 4, 5, 6, 7, 8, 39
K: 5.3 K: CDV: 5.4X, FOS: 1.6X
S: 3.1 S: CDV: 7.5
N410K 2.9 CDV: 3X, FOS: 0.8X 1
F412C/L/S/Va C: 3.6 C: CDV: 10X, FOS: 1.2–2.5X 5, 7, 9, 10, 11, 12, 35
L: 4.6 L: CDV: 9.4X, FOS: 1.1X
S: 5.3 S: CDV: 13X, FOS: 0.8X
V: 4.3 V: CDV: 15.5, FOS: 1.1
D413A/E A: 6.5 A: CDV: 10.9X, FOS: 0.8X 1, 8, 13, 14, 15
E 4.8 E: CDV: 4.3X, FOS: 0.8X
P488Ra 3.5 CDV: 7.9 12
L501F/I F: no EC50 ratio available, EC50 = 80.9 uM F: CDV: NA, FOS: no EC50 ratio available, EC50 = 234.1 uM 5, 7, 11, 14, 16, 17, 18, 19
I: 6–20 I: CDV: 9X, FOS: 1.4X
T503I 2.9 CDV: 6.1X, FOS: 0.5X 1, 5
K513E/N/R E: 5 E: CDV: 9.1, FOS: 1.4 5, 7, 9, 20, 21, 22
N: 6 N: CDV: 12.5, FOS: 1.1
R: no EC50 ratio available, EC50 = 36.7 lM R: no EC50 ratio available, CDV EC50 = 7.2 lM, FOS EC50 = 200 lM
L516Ra 2.1 CDV: 5.1X, FOS: 0.8X 1

I521T 3 CDV: 3.9X, FOS: 0.9X 23, 24

P522A/L/S A: 3 A: CDV: 4.1X, FOS: 1.0X 7, 14, 17, 23, 25, 26
L: 1.3 L: CDV: 1.4X, FOS: 1.2X
S: 3 S: CDV: 3.6, FOS: 1.1
C539Ra 3.2 CDV: 13.3 12
L545S/W S: 3–7.4 S: CDV: 9X, FOS: 1.2X 7, 12, 27, 28, 29
W: 4.9 W: CDV: 6.3, FOS: 1.3X
Q578H/L H: 3.3 H: CDV: 2.3, FOS: 4.5 12, 38
L: 1.9 L: CDV: 0.8, FOS: 3.0
b
D588E /N E: 1.3 E: CDV: 1.1X, FOS: 2.3X 5, 7, 30
N: 4 N: CDV: 2.7X, FOS: 3.2X
a
F595I 1.3 CDV: 1.2X, FOS: 2.0 12, 36
G629S NA NA 2, 27
S695T 1.1 CDV: 0.5X, FOS: 1.2 27, 37
I726T/V T: 1.9 T: CDV: 1.8, FOS: 1.1 5, 38
V: 1.8 V: CDV: 1.9, FOS: 1.2
E756K K 3.5 K:CDV: 2.2X, FOS: >8X 1, 14
V781I 1–4 CDV: 1.2X, FOS: 5.2X 5, 7, 9
V787L 2–3 CDV: 1X, FOS: 4.1X 25, 31
L802M 4 CDV: 1.8X, FOS: 10.8X 5, 8, 9, 10
A809V 2.4 CDV: 1.9X, FOS: 3.3X 32, 33
V812Ia 2.5 CDV: 3.2X, FOS: 2.9X 22, 29
T813S 2.5 CDV: 2.7X, FOS: 4.9X 32
T821I 4.5 CDV: 1.9X, FOS: 21X 5, 7
P829Sa Unclear, too many other aa substitutions 29
A834P 6.1 CDV: 3.0, FOS: 6.4 4, 8, 17
G841A/S A:3.2 A: CDV: 2.6X, FOS: 4.3X 14, 32, 38
S: NA S: NA
L862Fa 1.7 CDV: 0.9X, FOS: 1.1X 12
D879G 1.2 CDV: 0.9X, FOS: 0.9X 29, 36
V946La 1.1 CDV: 0.9X, FOS: 2.4 12
A972V 1 CDV: 0.9X, FOS: 0.9X 9, 18, 37
del 981–982 8.3 CDV: 2.8X, FOS: 3.6X 1, 20
A987G 5.3 CDV: 11.3X, FOS: 1.2X 6, 7, 8, 20, 25, 34

1: Chou et al. (2003); 2: Boutolleau et al. (2009); 3: Chou et al. (2010); 4: Scott et al. (2007); 5: Smith et al. (1997); 6: Kuo et al. (2003); 7: Cihlar et al. (1998a); 8: Hantz et al.
(2010); 9: Mousavi-Jazi et al. (2001a,b); 10: Chou et al. (1997); 11: Lurain et al. (1992); 12: Hakki and Chou (2011); 13: Marfori et al. (2007); 14: Erice et al. (1997); 15: Seo
et al. (2001); 16: Ducancelle et al. (2004); 17: Reddy et al. (2007); 18: Scott et al. (2004); 19: Lurain et al. (2002); 20: Drew et al. (2006); 21: Smith et al. (1998); 22: Cihlar
et al. (1998b); 23: Chou et al. (2008); 24: Eckle et al. (2000); 25: Jabs et al. (2001); 26: Foulongne et al. (2004); 27: Boivin et al. (2005); 28: Erice (1999); 29: Gilbert and Boivin
(2005); 30: Springer et al. (2005); 31: Levi et al. (2006); 32: Chou et al. (2007a,b); 33: Chou et al. (1998); 34: Sullivan et al. (1993); 35: Chou (2011); 36: Gilbert et al. (2011);
37: Chevillotte et al. (2010); 38: (Valcyte Package Insert, 2013); 39: Hantz et al. (2013).
a
Developed in cell culture studies only.
b
Neither emerged in cell culture nor in vivo but subjected to recombinant phenotyping.

substitutions and have been observed in patients failing treat- Although we expect some of these single amino acid substitu-
ment or prophylaxis. Substitutions E315D and D879G have also tions in Tables 2 and 3 to have little direct impact on HCMV sus-
been observed in multiple patients failing treatment or ceptibility to ganciclovir, they may augment the effect of other
prophylaxis. substitutions in HCMV, either by directly enhancing resistance or
We are not aware of any published phenotypic data for amino indirectly by increasing fitness. For example, the HCMV DNA poly-
acid substitutions G629S, and G841S. Substitution G629S has been merase amino acid substitution K805Q improved the fitness of the
observed in multiple patients failing prophylaxis. Substitution T821I amino acid substitution associated with high levels of resis-
G841S occurs at the same position as the established resistance- tance to foscarnet (Martin et al., 2010a). It has also been demon-
associated substitution G841A. strated that combination of amino acid substitutions in pUL97
 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25 21

and pUL54 resulted in greater reduction in susceptibility to ganci- HCMV drug resistance studies have been limited by technical chal-
clovir than in HCMV with either amino substitution alone (Chou lenges and the lack of standardization of antiviral susceptibility as-
et al., 2007a,b; Gilbert et al., 2011). In one recent study, a recombi- says. The recognition that specific amino acid substitutions in
nant virus with pUL54 amino acid substitution D413A exhibited pUL97 and pUL54 are associated with different antiviral suscepti-
ganciclovir and cidofovir resistance but remained susceptible to bility patterns has prompted the development of molecular labora-
foscarnet (Chou and Marousek, 2008). Moreover, after a limited tory methods for the detection of drug resistant strains in clinical
number of passages in the absence of selective pressure, the plat- specimens.
ing efficiency of the unpurified D413A recombinant virus under Our review and analysis of the literature indicate there are
400 lM foscarnet was about 30-fold greater than that of the paren- potentially more HCMV ganciclovir/valganciclovir resistance path-
tal strain passaged under the same conditions. These results indi- ways than generally appreciated. We compiled a listing of 85 resis-
cate that at least some amino acid substitutions listed in Table 3 tance-associated amino acid substitutions and 7 deletions in
could facilitate the subsequent development of other substitutions pUL97 and pUL54. In clinical isolates, seven pUL97 substitutions,
associated with drug resistance. (M460V/I, H520Q, C592G, A594V, L595S, C603W) are the most fre-
quently reported and validated ganciclovir resistance-associated
substitutions. Although we identified several potentially novel
7. Cross-resistance between ganciclovir/valganciclovir and
amino acid substitutions that appear to be clinically associated
other drugs
with ganciclovir/valganciclovir resistance, and potentially associ-
ated with cross-resistance to other drugs, we anticipate that with
Antiviral drugs with similar targets and mechanisms of action
further characterization not all substitutions will necessarily be
often have overlapping resistance pathways. Given that all ap-
proven causally related to ganciclovir/valganciclovir resistance.
proved HCMV drugs target the pUL54 polymerase to inhibit viral
Questions that remain to be addressed include: What is the precise
DNA replication, it is conceivable that some degree of cross-resis-
clinical impact of these resistance-associated substitutions and
tance will occur between all of the drugs. Cross-resistance between
deletions alone or in combination? Does their impact vary in the
ganciclovir/valganciclovir and cidofovir or foscarnet has been re-
context of prophylaxis versus treatment? Why do some substitu-
ported for several amino acid substitutions in the DNA polymerase
tions that are clinically associated with resistance have no obvious
(Table 3). Amino acid substitutions D301N, N408D, N410K, F412C/
effect on HCMV phenotypic resistance to ganciclovir, and are there
V, D413A/E, L501I, T503I, L516R, I521T, P522A, L545S, D588N,
other reasons (e.g., unrelated to drug resistance or replicative fit-
E756K, V812L, T813S, G841A, A987G, and del 981–982 all confer
ness) for the detection of these?
a P2-fold reduction in susceptibility to cidofovir in phenotypic as-
Because of these remaining questions the listings included in
says. Amino acid substitutions D588N, V781I, V787L, L802M,
this review are not intended to guide treatment decisions at this
A809V, V812L, T813S, T821I, and G841A all confer a P2-fold
time, as additional studies are clearly needed, particularly for
reduction in susceptibility to foscarnet. There are multiple reports
potentially novel resistance substitutions and in the context of
of treatment failure associated with some of these amino acid sub-
both available and investigational therapies. These listings are in-
stitutions, indicating possible cross-resistance (Smith et al., 1997;
tended to generate hypotheses and guide future studies aimed at
Eckle et al., 2000; Scott et al., 2004). There are no controlled clinical
better understanding how to detect and minimize the impact of
trials that have been conducted to support the use of specific treat-
drug resistance in clinical practice. Examples of next steps include
ment options where HCMV resistance to ganciclovir/valganciclovir
the continued phenotypic evaluation of clinical isolates, systematic
is suspected. Clinical management guidelines have been proposed
phenotypic characterization of complex combinations of amino
based largely on expert opinion assessing published case reports
acid substitutions observed in clinical isolates, and evaluations of
and anecdotal experience (Boeckh and Ljungman, 2009; Kotton
specific amino acid substitutions on HCMV replication capacity/fit-
et al., 2010; Preiksaitis et al., 2005; Schreiber et al., 2009).
ness. Additionally, genotypic analyses of clinical isolates should
Two independent genes have been reported to be associated
continue to be conducted to substantiate uncommon but clinically
with maribavir drug resistance in cell culture. The first gene is
relevant drug resistance pathways, and also to identify potentially
UL97 (Biron et al., 2002; Chou et al., 2007a,b; Chou and Marousek,
novel resistance pathways for further characterization. As detec-
2008) and the second is UL27 (Komazin et al., 2003; Chou et al.,
tion of antiviral resistance improves and new treatment options
2004). No cross-resistance has been detected between most of
are introduced, additional strategies to combat the emergence of
the viruses resistant to maribavir and those resistant to one or
antiviral resistance can be developed and existing strategies
more of the current HCMV antiviral drugs (Drew et al., 2006). How-
refined.
ever, there was a recent report of amino acid substitutions F342S,
V356G, V466G, and P521L in the pUL97 conferred reduced suscep-
tibility to ganciclovir, maribavir and cyclopropavir (Chou et al., Acknowledgements
2013). There are currently ongoing Phase 2 studies evaluating
the activity of high doses of maribavir in subjects with HCMV We thank Debra Birnkrant, Mike Bray, Edward Cox, Jeffrey Mur-
refractory or resistant to ganciclovir/valganciclovir or foscarnet. ray, Jules O’Rear, and Kuate Seraphin for helpful manuscript sug-
The frequency with which prior treatment with acyclovir elicits gestions and discussion. A.P. is supported in part by the
ganciclovir resistance remains unclear. In one study, subjects Intramural Research Program of the National Institute of Dental
receiving prolonged high-dose acyclovir did not have evidence of and Craniofacial Research, National Institutes of Health, Depart-
ganciclovir resistance (Drew et al., 1995). However, there is a re- ment of Health and Human Services, Bethesda, Maryland.
port of preexisting resistance before receiving any ganciclovir but
after receiving acyclovir (Erice et al., 1989). References

Arslan, F., Tabak, F., Avsar, E., Midilli, K., Mert, A., Ozaras, R., Soysal, T., Ozturk, R.,
8. Conclusions and future directions Ferhanoglu, B., 2010. Ganciclovir-resistant cytomegalovirus encephalitis in a
hematopoietic stem cell transplant recipient. J. Neurovirol. 16, 174–178.
There are five drugs that have received FDA approval for the Asberg, A., Humar, A., Rollag, H., Jardine, A.G., Mouas, H., Pescovitz, M.D., Sgarabotto,
D., Tuncer, M., Noronha, I.L., Hartmann, A., 2007. Oral valganciclovir is
treatment of systemic or localized HCMV disease: ganciclovir and noninferior to intravenous ganciclovir for the treatment of cytomegalovirus
its prodrug valganciclovir, foscarnet, cidofovir, and fomivirsen. disease in solid organ transplant recipients. Am. J. Transplant. 7, 2106–2113.
22 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25

Baldanti, F., Michel, D., Simoncini, L., Heuschmid, M., Zimmermann, A., Minisini, R., Chou, S., 2011. Phenotypic diversity of cytomegalovirus DNA polymerase gene
Schaarschmidt, P., Schmid, T., Gerna, G., Mertens, T., 2002a. Mutations in the variants observed after antiviral therapy. J. Clin. Virol. 50, 287–291.
UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates Chou, S., Bowlin, T.L., 2011. Cytomegalovirus UL97 mutations affecting
differentially affect GCV phosphorylation as determined in a recombinant cyclopropavir and ganciclovir susceptibility. Antimicrob. Agents Chemother.
vaccinia virus system. Antiviral Res. 54, 59–67. 55, 382–384.
Baldanti, F., Sarasini, A., Drach, J.C., Zemlicka, J., Gerna, G., 2002b. Z-isomers of 2- Chou, S., Lurain, N.S., Thompson, K.D., Miner, R.C., Drew, W.L., 2003. Viral DNA
hydroxymethylcyclopropylidenemethyl adenine (synadenol) and guanine polymerase mutations associated with drug resistance in human
(synguanol) are active against ganciclovir- and foscarnet-resistant human cytomegalovirus. J. Infect. Dis. 188, 32–39.
cytomegalovirus UL97 mutants. Antiviral Res. 56, 273–278. Chou, S., Marousek, G., Boivin, G., Goyette, N., Farhan, M., Ives, J.A., Elston, R., 2010.
Baldanti, F., Silini, E., Sarasini, A., Talarico, C.L., Stanat, S.C., Biron, K.K., Furione, M., Recombinant phenotyping of cytomegalovirus sequence variants detected
Bono, F., Palu, G., Gerna, G., 1995. A three-nucleotide deletion in the UL97 open after 200 or 100 days of valganciclovir prophylaxis. Transplantation 90, 1409–
reading frame is responsible for the ganciclovir resistance of a human 1413.
cytomegalovirus clinical isolate. J. Virol. 69, 796–800. Chou, S., Marousek, G., Guentzel, S., Follansbee, S.E., Poscher, M.E., Lalezari, J.P.,
Baldanti, F., Underwood, M.R., Stanat, S.C., Biron, K.K., Chou, S., Sarasini, A., Silini, E., Miner, R.C., Drew, W.L., 1997. Evolution of mutations conferring multidrug
Gerna, G., 1996. Single amino acid changes in the DNA polymerase confer resistance during prophylaxis and therapy for cytomegalovirus disease. J. Infect.
foscarnet resistance and slow-growth phenotype, while mutations in the UL97- Dis. 176, 786–789.
encoded phosphotransferase confer ganciclovir resistance in three double- Chou, S., Marousek, G., Li, S., Weinberg, A., 2008. Contrasting drug resistance
resistant human cytomegalovirus strains recovered from patients with AIDS. J. phenotypes resulting from cytomegalovirus DNA polymerase mutations at the
Virol. 70, 1390–1395. same exonuclease locus. J. Clin. Virol. 43, 107–109.
Baldanti, F., Underwood, M.R., Talarico, C.L., Simoncini, L., Sarasini, A., Biron, K.K., Chou, S., Marousek, G., Parenti, D.M., Gordon, S.M., LaVoy, A.G., Ross, J.G., Miner, R.C.,
Gerna, G., 1998. The Cys607 Tyr change in the UL97 phosphotransferase confers Drew, W.L., 1998. Mutation in region III of the DNA polymerase gene conferring
ganciclovir resistance to two human cytomegalovirus strains recovered from foscarnet resistance in cytomegalovirus isolates from 3 subjects receiving
two immunocompromised patients. Antimicrob. Agents Chemother. 42, 444– prolonged antiviral therapy. J. Infect. Dis. 178, 526–530.
446. Chou, S., Marousek, G.I., 2008. Accelerated evolution of maribavir resistance in a
Baldwin, K., 2011. Ganciclovir-resistant human herpesvirus-6 encephalitis in a liver cytomegalovirus exonuclease domain II mutant. J. Virol. 82, 246–253.
transplant patient: a case report. J. Neurovirol. 17, 193–195. Chou, S., Marousek, G.I., Senters, A.E., Davis, M.G., Biron, K.K., 2004. Mutations in the
Battiwalla, M., Paplham, P., Almyroudis, N.G., McCarthy, A., Abdelhalim, A., Elefante, human cytomegalovirus UL27 gene that confer resistance to maribavir. J. Virol.
A., Smith, P., Becker, J., McCarthy, P.L., Segal, B.H., 2007. Leflunomide failure to 78, 7124–7130.
control recurrent cytomegalovirus infection in the setting of renal failure after Chou, S., Marousek, G.I., Van Wechel, L.C., Li, S., Weinberg, A., 2007a. Growth and
allogeneic stem cell transplantation. Transpl. Infect. Dis. 9, 28–32. drug resistance phenotypes resulting from cytomegalovirus DNA polymerase
Bhorade, S.M., Lurain, N.S., Jordan, A., Leischner, J., Villanueva, J., Durazo, R., Creech, region III mutations observed in clinical specimens. Antimicrob. Agents
S., Vigneswaran, W.T., Garrity, E.R., 2002. Emergence of ganciclovir-resistant Chemother. 51, 4160–4162.
cytomegalovirus in lung transplant recipients. J. Heart Lung Transplant. 21, Chou, S., Meichsner, C.L., 2000. A nine-codon deletion mutation in the
1274–1282. cytomegalovirus UL97 phosphotransferase gene confers resistance to
Biron, K.K., Harvey, R.J., Chamberlain, S.C., Good, S.S., Smith III, A.A., Davis, M.G., ganciclovir. Antimicrob. Agents Chemother. 44, 183–185.
Talarico, C.L., Miller, W.H., Ferris, R., Dornsife, R.E., Stanat, S.C., Drach, J.C., Chou, S., Miner, R.C., Drew, W.L., 2000. A deletion mutation in region V of the
Townsend, L.B., Koszalka, G.W., 2002. Potent and selective inhibition of human cytomegalovirus DNA polymerase sequence confers multidrug resistance. J.
cytomegalovirus replication by 1263W94, a benzimidazole L-riboside with a Infect. Dis. 182, 1765–1768.
unique mode of action. Antimicrob. Agents Chemother. 46, 2365–2372. Chou, S., Van Wechel, L.C., Lichy, H.M., Marousek, G.I., 2005. Phenotyping of
Bloom, J.D., Gong, L.I., Baltimore, D., 2010. Permissive secondary mutations enable cytomegalovirus drug resistance mutations by using recombinant viruses
the evolution of influenza oseltamivir resistance. Science 328, 1272–1275. incorporating a reporter gene. Antimicrob. Agents Chemother. 49, 2710–2715.
Boeckh, M., Ljungman, P., 2009. How we treat cytomegalovirus in hematopoietic Chou, S., Waldemer, R.H., Senters, A.E., Michels, K.S., Kemble, G.W., Miner, R.C.,
cell transplant recipients. Blood 113, 5711–5719. Drew, W.L., 2002. Cytomegalovirus UL97 phosphotransferase mutations that
Boeckh, M., Stevens-Ayers, T., Bowden, R.A., 1996. Cytomegalovirus pp65 affect susceptibility to ganciclovir. J. Infect. Dis. 185, 162–169.
antigenemia after autologous marrow and peripheral blood stem cell Chou, S., Wechel, L.C., Marousek, G.I., 2007b. Cytomegalovirus UL97 kinase
transplantation. J. Infect. Dis. 174, 907–912. mutations that confer maribavir resistance. J. Infect. Dis. 196, 91–94.
Boivin, G., Gilbert, C., Gaudreau, A., Greenfield, I., Sudlow, R., Roberts, N.A., 2001. Chou, S., Ercolani, R.J., Marousek, G., Bowlin, T.L., 2013. Cytomegalovirus UL97
Rate of emergence of cytomegalovirus (CMV) mutations in leukocytes of kinase catalytic domain mutations that confer multidrug resistance.
patients with acquired immunodeficiency syndrome who are receiving Antimicrob. Agents Chemother. 57, 3375–3379.
valganciclovir as induction and maintenance therapy for CMV retinitis. J. Cihlar, T., Fuller, M.D., Cherrington, J.M., 1998a. Characterization of drug resistance-
Infect. Dis. 184, 1598–1602. associated mutations in the human cytomegalovirus DNA polymerase gene by
Boivin, G., Goyette, N., Gilbert, C., Covington, E., 2005. Analysis of cytomegalovirus using recombinant mutant viruses generated from overlapping DNA fragments.
DNA polymerase (UL54) mutations in solid organ transplant patients receiving J. Virol. 72, 5927–5936.
valganciclovir or ganciclovir prophylaxis. J. Med. Virol. 77, 425–429. Cihlar, T., Fuller, M.D., Mulato, A.S., Cherrington, J.M., 1998b. A point mutation in the
Boivin, G., Goyette, N., Rollag, H., Jardine, A.G., Pescovitz, M.D., Asberg, A., Ives, J., human cytomegalovirus DNA polymerase gene selected in vitro by cidofovir
Hartmann, A., Humar, A., 2009. Cytomegalovirus resistance in solid organ confers a slow replication phenotype in cell culture. Virology 248, 382–393.
transplant recipients treated with intravenous ganciclovir or oral valganciclovir. Dal, P.F., Andrei, G., Daelemans, D., Winkler, M., Piette, J., de Clercq, E, Snoeck, R.,
Antivir. Ther. 14, 697–704. 2008. Fluorescence-based antiviral assay for the evaluation of compounds
Bourgeois, C., Sixt, N., Bour, J.B., Pothier, P., 1997. Value of a ligase chain reaction against vaccinia virus, varicella zoster virus and human cytomegalovirus. J.
assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene Virol. Methods 151, 66–73.
of human cytomegalovirus. J. Virol. Methods 67, 167–175. De Clercq, E., Holy, A., 2005. Acyclic nucleoside phosphonates: a key class of
Boutolleau, D., Deback, C., Bressollette-Bodin, C., Varnous, S., Dhedin, N., Barrou, B., antiviral drugs. Nat. Rev. Drug Discov. 4, 928–940.
Vernant, J.P., Gandjbakhch, I., Imbert-Marcille, B.M., Agut, H., 2009. Resistance Drew, W.L., 2000. Ganciclovir resistance: a matter of time and titre. Lancet 356,
pattern of cytomegalovirus (CMV) after oral valganciclovir therapy in transplant 609–610.
recipients at high-risk for CMV infection. Antiviral Res. 81, 174–179. Drew, W.L., 2006. Is combination antiviral therapy for CMV superior to
Brown, F., Banken, L., Saywell, K., Arum, I., 1999. Pharmacokinetics of valganciclovir monotherapy? J. Clin. Virol. 35, 485–488.
and ganciclovir following multiple oral dosages of valganciclovir in HIV- and Drew, W.L., Anderson, R., Lang, W., Miner, R.C., Davis, G., Lalezari, J., 1995. Failure of
CMV-seropositive volunteers. Clin. Pharmacokinet. 37, 167–176. high-dose oral acyclovir to suppress CMV viruria or induce ganciclovir-resistant
Campanini, G., Zavattoni, M., Cristina, E., Gazzolo, D., Stronati, M., Baldanti, F., 2012. CMV in HIV antibody positive patients. J. Acquir. Immune Defic. Syndr. Hum.
Multiple ganciclovir-resistant strains in a newborn with symptomatic Retrovirol. 8, 289–291.
congenital human cytomegalovirus infection. J. Clin. Virol. 54, 86–88. Drew, W.L., Miner, R., Saleh, E., 1993. Antiviral susceptibility testing of
Chevillotte, M., Ersing, I., Mertens, T., von Einem, J., 2010. Differentiation between cytomegalovirus: criteria for detecting resistance to antivirals. Clin Diagn.
polymorphisms and resistance-associated mutations in human Virol. 1, 179–185.
cytomegalovirus DNA polymerase. Antimicrob. Agents Chemother. 54, 5004– Drew, W.L., Miner, R.C., Marousek, G.I., Chou, S., 2006. Maribavir sensitivity of
5011. cytomegalovirus isolates resistant to ganciclovir, cidofovir or foscarnet. J. Clin.
Chevillotte, M., Schubert, A., Mertens, T., von Einem, J., 2009. Fluorescence-based Virol. 37, 124–127.
assay for phenotypic characterization of human cytomegalovirus polymerase Drew, W.L., Paya, C.V., Emery, V., 2001. Cytomegalovirus (CMV) resistance to
mutations regarding drug susceptibility and viral replicative fitness. antivirals. Am. J. Transplant. 1, 307–312.
Antimicrob. Agents Chemother. 53, 3752–3761. Ducancelle, A., Alain, S., Petit, F., Sanson Le Pors, M.J., Mazeron, M.C., 2007.
Chou, S., 2008. Cytomegalovirus UL97 mutations in the era of ganciclovir and Development and validation of a non-radioactive DNA polymerase assay for
maribavir. Rev. Med. Virol. 18, 233–246. studying cytomegalovirus resistance to foscarnet. J. Virol. Methods 141, 212–
Chou, S., 2009. Diverse cytomegalovirus UL27 mutations adapt to loss of viral UL97 215.
kinase activity under maribavir. Antimicrob. Agents Chemother. 53, 81–85. Ducancelle, A., Belloc, S., Alain, S., Scieux, C., Malphettes, M., Petit, F., Brouet, J.C.,
Chou, S., 2010. Recombinant phenotyping of cytomegalovirus UL97 kinase sequence Sanson Le Pors, M.J., Mazeron, M.C., 2004. Comparison of sequential
variants for ganciclovir resistance. Antimicrob. Agents Chemother. 54, 2371– cytomegalovirus isolates in a patient with lymphoma and failing antiviral
2378. therapy. J. Clin. Virol. 29, 241–247.
 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25 23

Ducancelle, A., Gravisse, J., Alain, S., Fillet, A.M., Petit, F., Pors, M.J., Mazeron, M.C., He, Z., He, Y.S., Kim, Y., Chu, L., Ohmstede, C., Biron, K.K., Coen, D.M., 1997. The
2005. Phenotypic characterisation of cytomegalovirus DNA polymerase: a human cytomegalovirus UL97 protein is a protein kinase that
method to study cytomegalovirus isolates resistant to foscarnet. J. Virol. autophosphorylates on serines and threonines. J. Virol. 71, 405–411.
Methods 125, 145–151. Hu, H., Jabs, D.A., Forman, M.S., Martin, B.K., Dunn, J.P., Weinberg, D.V., Davis, J.L.,
Eckle, T., Jahn, G., Hamprecht, K., 2004. The influence of mixed HCMV UL97 wildtype 2002. Comparison of cytomegalovirus (CMV) UL97 gene sequences in the blood
and mutant strains on ganciclovir susceptibility in a cell associated plaque and vitreous of patients with acquired immunodeficiency syndrome and CMV
reduction assay. J. Clin. Virol. 30, 50–56. retinitis. J. Infect. Dis. 185, 861–867.
Eckle, T., Prix, L., Jahn, G., Klingebiel, T., Handgretinger, R., Selle, B., Hamprecht, K., Ibarra, K.D., Jain, M.K., Pfeiffer, J.K., 2011. Host-based ribavirin resistance
2000. Drug-resistant human cytomegalovirus infection in children after influences hepatitis C virus replication and treatment response. J. Virol. 85,
allogeneic stem cell transplantation may have different clinical outcomes. 7273–7283.
Blood 96, 3286–3289. Ijichi, O., Michel, D., Mertens, T., Miyata, K., Eizuru, Y., 2002. GCV resistance due to
Efferth, T., Marschall, M., Wang, X., Huong, S.M., Hauber, I., Olbrich, A., Kronschnabl, the mutation A594P in the cytomegalovirus protein UL97 is partially
M., Stamminger, T., Huang, E.S., 2002. Antiviral activity of artesunate towards reconstituted by a second mutation at D605E. Antiviral Res. 53, 135–142.
wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses. J. Isegawa, Y., Hara, J., Amo, K., Osugi, Y., Takemoto, M., Yamanishi, K., Fukunaga, R.,
Mol. Med. (Berl.) 80, 233–242. Shibata, M., Ohshima, A., Horiguchi, Y., Sugimoto, N., 2009. Human herpesvirus
Efferth, T., Romero, M.R., Wolf, D.G., Stamminger, T., Marin, J.J., Marschall, M., 2008. 6 ganciclovir-resistant strain with amino acid substitutions associated with the
The antiviral activities of artemisinin and artesunate. Clin. Infect. Dis. 47, 804– death of an allogeneic stem cell transplant recipient. J. Clin. Virol. 44, 15–19.
811. Ito, J., Braithwaite, D.K., 1991. Compilation and alignment of DNA polymerase
Emery, V.C., Cope, A.V., Bowen, E.F., Gor, D., Griffiths, P.D., 1999. The dynamics of sequences. Nucleic Acids Res. 19, 4045–4057.
human cytomegalovirus replication in vivo. J. Exp. Med. 190, 177–182. Iwasenko, J.M., Scott, G.M., Rawlinson, W.D., Keogh, A., Mitchell, D., Chou, S., 2009.
Erice, A., 1999. Resistance of human cytomegalovirus to antiviral drugs. Clin. Successful valganciclovir treatment of post-transplant cytomegalovirus
Microbiol. Rev. 12, 286–297. infection in the presence of UL97 mutation N597D. J. Med. Virol. 81, 507–510.
Erice, A., Chou, S., Biron, K.K., Stanat, S.C., Balfour Jr., H.H., Jordan, M.C., 1989. Jabs, D.A., Enger, C., Dunn, J.P., Forman, M., 1998. Cytomegalovirus retinitis and viral
Progressive disease due to ganciclovir-resistant cytomegalovirus in resistance: ganciclovir resistance. CMV Retinitis and Viral Resistance Study
immunocompromised patients. N. Engl. J. Med. 320, 289–293. Group. J. Infect. Dis. 177, 770–773.
Erice, A., Gil-Roda, C., Perez, J.L., Balfour Jr., H.H., Sannerud, K.J., Hanson, M.N., Jabs, D.A., Martin, B.K., Forman, M.S., Dunn, J.P., Davis, J.L., Weinberg, D.V., Biron,
Boivin, G., Chou, S., 1997. Antiviral susceptibilities and analysis of UL97 and K.K., Baldanti, F., 2001. Mutations conferring ganciclovir resistance in a cohort
DNA polymerase sequences of clinical cytomegalovirus isolates from of patients with acquired immunodeficiency syndrome and cytomegalovirus
immunocompromised patients. J. Infect. Dis. 175, 1087–1092. retinitis. J. Infect. Dis. 183, 333–337.
Faizi, K.R., Mori, S., Eizuru, Y., Kumura, I.K., Minamishima, Y., 1998. Genetic analysis Jabs, D.A., Martin, B.K., Ricks, M.O., Forman, M.S., 2006. Detection of ganciclovir
of a ganciclovir-resistant human cytomegalovirus mutant. Antiviral Res. 40, 95– resistance in patients with AIDS and cytomegalovirus retinitis: correlation of
103. genotypic methods with viral phenotype and clinical outcome. J. Infect. Dis.
Foulongne, V., Turriere, C., Diafouka, F., Abraham, B., Lastere, S., Segondy, M., 2004. 193, 1728–1737.
Ganciclovir resistance mutations in UL97 and UL54 genes of Human Jabs, D.A., van Natta, M.L., Holbrook, J.T., Kempen, J.H., Meinert, C.L., Davis, M.D.,
cytomegalovirus isolates resistant to ganciclovir. Acta Virol. 48, 51–55. 2007. Longitudinal study of the ocular complications of AIDS: 2. Ocular
Freifeld, A.G., Ostrove, J.M., 1991. Resistance of viruses to antiviral drugs. Annu. Rev. examination results at enrollment. Ophthalmology 114, 787–793.
Med. 42, 247–259. James, S.H., Prichard, M.N., 2011. The genetic basis of human cytomegalovirus
Fukushima, R., Kanamori, S., Hirashiba, M., Hishikawa, A., Muranaka, R.I., Kaneto, M., resistance and current trends in antiviral resistance analysis. Infect. Disord.
Nakamura, K., Kato, I., 2007. Teratogenicity study of the dihydroorotate- Drug Targets 11, 504–513.
dehydrogenase inhibitor and protein tyrosine kinase inhibitor Leflunomide in Kaptein, S.J., Efferth, T., Leis, M., Rechter, S., Auerochs, S., Kalmer, M., Bruggeman,
mice. Reprod. Toxicol. 24, 310–316. C.A., Vink, C., Stamminger, T., Marschall, M., 2006. The anti-malaria drug
Gallant, J.E., Moore, R.D., Richman, D.D., Keruly, J., Chaisson, R.E., 1992. Incidence artesunate inhibits replication of cytomegalovirus in vitro and in vivo. Antiviral
and natural history of cytomegalovirus disease in patients with advanced Res. 69, 60–69.
human immunodeficiency virus disease treated with zidovudine. The Kedhar, S.R., Jabs, D.A., 2007. Cytomegalovirus retinitis in the era of highly active
Zidovudine Epidemiology Study Group. J. Infect. Dis. 166, 1223–1227. antiretroviral therapy. Herpes 14, 66–71.
Gentry, B.G., Kamil, J.P., Coen, D.M., Zemlicka, J., Drach, J.C., 2010. Stereoselective Kimberlin, D.W., Lin, C.Y., Sanchez, P.J., Demmler, G.J., Dankner, W., Shelton, M.,
phosphorylation of cyclopropavir by pUL97 and competitive inhibition by Jacobs, R.F., Vaudry, W., Pass, R.F., Kiell, J.M., Soong, S.J., Whitley, R.J., 2003.
maribavir. Antimicrob. Agents Chemother. 54, 3093–3098. Effect of ganciclovir therapy on hearing in symptomatic congenital
Gershburg, E., Pagano, J.S., 2008. Conserved herpesvirus protein kinases. Biochim. cytomegalovirus disease involving the central nervous system: a randomized,
Biophys. Acta 1784, 203–212. controlled trial. J. Pediatr. 143, 16–25.
Gilbert, C., Azzi, A., Goyette, N., Lin, S.X., Boivin, G., 2011. Recombinant phenotyping Kobayashi, M., Yoshinaga, T., Seki, T., Wakasa-Morimoto, C., Brown, K.W., Ferris, R.,
of cytomegalovirus UL54 mutations that emerged during cell passages in the Foster, S.A., Hazen, R.J., Miki, S., Suyama-Kagitani, A., Kawauchi-Miki, S., Taishi,
presence of either ganciclovir or foscarnet. Antimicrob. Agents Chemother. 55, T., Kawasuji, T., Johns, B.A., Underwood, M.R., Garvey, E.P., Sato, A., Fujiwara, T.,
4019–4027. 2011. In Vitro antiretroviral properties of S/GSK1349572, a next-generation HIV
Gilbert, C., Boivin, G., 2005. New reporter cell line to evaluate the sequential integrase inhibitor. Antimicrob. Agents Chemother. 55, 813–821.
emergence of multiple human cytomegalovirus mutations during in vitro drug Komazin, G., Ptak, R.G., Emmer, B.T., Townsend, L.B., Drach, J.C., 2003. Resistance of
exposure. Antimicrob. Agents Chemother. 49, 4860–4866. human cytomegalovirus to the benzimidazole L-ribonucleoside maribavir maps
Gill, R.B., James, S.H., Prichard, M.N., 2012. Human cytomegalovirus UL97 kinase to UL27. J. Virol. 77, 11499–11506.
alters the accumulation of CDK1. J. Gen. Virol. 93, 1743–1755. Kotton, C.N., 2013. CMV: Prevention, Diagnosis and Therapy. Am. J. Transplant. 13
Goldner, T., Hewlett, G., Ettischer, N., Ruebsamen-Schaeff, H., Zimmermann, H., (Suppl. 3), 24–40.
Lischka, P., 2011. The novel anticytomegalovirus compound AIC246 Kotton, C.N., Kumar, D., Caliendo, A.M., Asberg, A., Chou, S., Snydman, D.R., Allen, U.,
(Letermovir) inhibits human cytomegalovirus replication through a specific Humar, A., 2010. International consensus guidelines on the management of
antiviral mechanism that involves the viral terminase. J. Virol. 85, 10884– cytomegalovirus in solid organ transplantation. Transplantation 89, 779–795.
10893. Kouzarides, T., Bankier, A.T., Satchwell, S.C., Weston, K., Tomlinson, P., Barrell, B.G.,
Gorzer, I., Guelly, C., Trajanoski, S., Puchhammer-Stockl, E., 2010. Deep sequencing 1987. Sequence and transcription analysis of the human cytomegalovirus DNA
reveals highly complex dynamics of human cytomegalovirus genotypes in polymerase gene. J. Virol. 61, 125–133.
transplant patients over time. J. Virol. 84, 7195–7203. Kuo, I.C., Imai, Y., Shum, C., Martin, D.F., Kuppermann, B.D., Margolis, T.P., 2003.
Grant, S., Edmond, E., Syme, J., 1981. A prospective study of cytomegalovirus Genotypic analysis of cytomegalovirus retinitis poorly responsive to
infection in pregnancy. I. Laboratory evidence of congenital infection following intravenous ganciclovir but responsive to the ganciclovir implant. Am. J.
maternal primary and reactivated infection. J. Infect. 3, 24–31. Ophthalmol. 135, 20–25.
Hakki, M., Chou, S., 2011. The biology of cytomegalovirus drug resistance. Curr. Landry, M.L., Stanat, S., Biron, K., Brambilla, D., Britt, W., Jokela, J., Chou, S., Drew,
Opin. Infect. Dis. 24, 605–611. W.L., Erice, A., Gilliam, B., Lurain, N., Manischewitz, J., Miner, R., Nokta, M.,
Hanks, S.K., Quinn, A.M., Hunter, T., 1988. The protein kinase family: conserved Reichelderfer, P., Spector, S., Weinberg, A., Yen-Lieberman, B., Crumpacker, C.,
features and deduced phylogeny of the catalytic domains. Science 241, 42–52. 2000. A standardized plaque reduction assay for determination of drug
Hanson, M.N., Preheim, L.C., Chou, S., Talarico, C.L., Biron, K.K., Erice, A., 1995. Novel susceptibilities of cytomegalovirus clinical isolates. Antimicrob. Agents
mutation in the UL97 gene of a clinical cytomegalovirus strain conferring Chemother. 44, 688–692.
resistance to ganciclovir. Antimicrob. Agents Chemother. 39, 1204–1205. Levi, M.E., Mandava, N., Chan, L.K., Weinberg, A., Olson, J.L., 2006. Treatment of
Hantz, S., Cotin, S., Borst, E., Couvreux, A., Salmier, A., Garrigue, I., Merville, P., multidrug-resistant cytomegalovirus retinitis with systemically administered
Mengelle, C., Attal, M., Messerle, M., Alain, S., 2013. Novel DNA polymerase leflunomide. Transpl. Infect. Dis. 8, 38–43.
mutations conferring cytomegalovirus resistance: input of BAC-recombinant Li, F., Kenyon, K.W., Kirby, K.A., Fishbein, D.P., Boeckh, M., Limaye, A.P., 2007.
phenotyping and 3D model. Antiviral Res. 98, 130–134. Incidence and clinical features of ganciclovir-resistant cytomegalovirus disease
Hantz, S., Garnier-Geoffroy, F., Mazeron, M.C., Garrigue, I., Merville, P., Mengelle, C., in heart transplant recipients. Clin. Infect. Dis. 45, 439–447.
Rostaing, L., Saint, M.F., Essig, M., Rerolle, J.P., Cotin, S., Germi, R., Pillet, S., Limaye, A.P., 2002. Ganciclovir-resistant cytomegalovirus in organ transplant
Lebranchu, Y., Turlure, P., Alain, S., 2010. Drug-resistant cytomegalovirus in recipients. Clin. Infect. Dis. 35, 866–872.
transplant recipients: a French cohort study. J. Antimicrob. Chemother. 65, Lischka, P., Hewlett, G., Wunberg, T., Baumeister, J., Paulsen, D., Goldner, T.,
2628–2640. Ruebsamen-Schaeff, H., Zimmermann, H., 2010. In vitro and in vivo activities of
24 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25

the novel anticytomegalovirus compound AIC246. Antimicrob. Agents Mulamba, G.B., Hu, A., Azad, R.F., Anderson, K.P., Coen, D.M., 1998. Human
Chemother. 54, 1290–1297. cytomegalovirus mutant with sequence-dependent resistance to the
Littler, E., Stuart, A.D., Chee, M.S., 1992. Human cytomegalovirus UL97 open reading phosphorothioate oligonucleotide fomivirsen (ISIS 2922). Antimicrob. Agents
frame encodes a protein that phosphorylates the antiviral nucleoside analogue Chemother. 42, 971–973.
ganciclovir. Nature 358, 160–162. Muller, B., Krausslich, H.G., 2009. Antiviral strategies. Handb. Exp. Pharmacol., 1–24.
Ljungman, P., 1995. Cytomegalovirus pneumonia: presentation, diagnosis, and Myhre, H.A., Haug, D.D., Kristiansen, K.I., Rollag, H., Leivestad, T., Asberg, A.,
treatment. Semin. Respir. Infect. 10, 209–215. Hartmann, A., 2011. Incidence and outcomes of ganciclovir-resistant
Ljungman, P., Hakki, M., Boeckh, M., 2011. Cytomegalovirus in hematopoietic stem cytomegalovirus infections in 1244 kidney transplant recipients.
cell transplant recipients. Hematol. Oncol. Clin. North Am. 25, 151–169. Transplantation 92, 217–223.
Luganini, A., Caposio, P., Landolfo, S., Gribaudo, G., 2008. Phosphorothioate- Nakano, K., Nishinaka, K., Tanaka, T., Ohshima, A., Sugimoto, N., Isegawa, Y., 2009.
modified oligodeoxynucleotides inhibit human cytomegalovirus replication by Detection and identification of U69 gene mutations encoded by ganciclovir-
blocking virus entry. Antimicrob. Agents Chemother. 52, 1111–1120. resistant human herpesvirus 6 using denaturing high-performance liquid
Lurain, N.S., Bhorade, S.M., Pursell, K.J., Avery, R.K., Yeldandi, V.V., Isada, C.M., chromatography. J. Virol. Methods 161, 223–230.
Robert, E.S., Kohn, D.J., Arens, M.Q., Garrity, E.R., Taege, A.J., Mullen, M.G., Todd, Nijhuis, M., van Maarseveen, N.M., Boucher, C.A., 2009. Antiviral resistance and
K.M., Bremer, J.W., Yen-Lieberman, B., 2002. Analysis and characterization of impact on viral replication capacity: evolution of viruses under antiviral
antiviral drug-resistant cytomegalovirus isolates from solid organ transplant pressure occurs in three phases. Handb. Exp. Pharmacol., 299–320.
recipients. J. Infect. Dis. 186, 760–768. Novak, Z., Ross, S.A., Patro, R.K., Pati, S.K., Kumbla, R.A., Brice, S., Boppana, S.B., 2008.
Lurain, N.S., Chou, S., 2010. Antiviral drug resistance of human cytomegalovirus. Cytomegalovirus strain diversity in seropositive women. J. Clin. Microbiol. 46,
Clin. Microbiol. Rev. 23, 689–712. 882–886.
Lurain, N.S., Spafford, L.E., Thompson, K.D., 1994. Mutation in the UL97 open reading O’Brien, J.J., Campoli-Richards, D.M., 1989. Acyclovir. An updated review of its
frame of human cytomegalovirus strains resistant to ganciclovir. J. Virol. 68, antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs
4427–4431. 37, 233–309.
Lurain, N.S., Thompson, K.D., Holmes, E.W., Read, G.S., 1992. Point mutations in the Palella Jr., F.J., Delaney, K.M., Moorman, A.C., Loveless, M.O., Fuhrer, J., Satten, G.A.,
DNA polymerase gene of human cytomegalovirus that result in resistance to Aschman, D.J., Holmberg, S.D., 1998. Declining morbidity and mortality among
antiviral agents. J. Virol. 66, 7146–7152. patients with advanced human immunodeficiency virus infection. HIV
Marfori, J.E., Exner, M.M., Marousek, G.I., Chou, S., Drew, W.L., 2007. Development of Outpatient Study Investigators. N. Engl. J. Med. 338, 853–860.
new cytomegalovirus UL97 and DNA polymerase mutations conferring drug Palmer, S.M., Limaye, A.P., Banks, M., Gallup, D., Chapman, J., Lawrence, E.C., Dunitz,
resistance after valganciclovir therapy in allogeneic stem cell recipients. J. Clin. J., Milstone, A., Reynolds, J., Yung, G.L., Chan, K.M., Aris, R., Garrity, E., Valentine,
Virol. 38, 120–125. V., McCall, J., Chow, S.C., Davis, R.D., Avery, R., 2010. Extended valganciclovir
Margeridon-Thermet, S., Shulman, N.S., Ahmed, A., Shahriar, R., Liu, T., Wang, C., prophylaxis to prevent cytomegalovirus after lung transplantation: a
Holmes, S.P., Babrzadeh, F., Gharizadeh, B., Hanczaruk, B., Simen, B.B., Egholm, randomized, controlled trial. Ann. Intern. Med. 152, 761–769.
M., Shafer, R.W., 2009. Ultra-deep pyrosequencing of hepatitis B virus Pari, G.S., Anders, D.G., 1993. Eleven loci encoding trans-acting factors are required
quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor for transient complementation of human cytomegalovirus oriLyt-dependent
(NRTI)-treated patients and NRTI-naive patients. J. Infect. Dis. 199, 1275–1285. DNA replication. J. Virol. 67, 6979–6988.
Marschall, M., Freitag, M., Weiler, S., Sorg, G., Stamminger, T., 2000. Recombinant Paya, C., Humar, A., Dominguez, E., Washburn, K., Blumberg, E., Alexander, B.,
green fluorescent protein-expressing human cytomegalovirus as a tool for Freeman, R., Heaton, N., Pescovitz, M.D., 2004. Efficacy and safety of
screening antiviral agents. Antimicrob. Agents Chemother. 44, 1588–1597. valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in
Marschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van Den, B.M., Stamminger, solid organ transplant recipients. Am. J. Transplant. 4, 611–620.
T., 2001. Inhibitors of human cytomegalovirus replication drastically reduce the Pignatelli, S., Dal, M.P., Rossini, G., Chou, S., Gojobori, T., Hanada, K., Guo, J.J.,
activity of the viral protein kinase pUL97. J. Gen. Virol. 82, 1439–1450. Rawlinson, W., Britt, W., Mach, M., Landini, M.P., 2003. Human cytomegalovirus
Martin, M., Azzi, A., Lin, S.X., Boivin, G., 2010a. Opposite effect of two glycoprotein N (gpUL73-gN) genomic variants: identification of a novel
cytomegalovirus DNA polymerase mutations on replicative capacity and subgroup, geographical distribution and evidence of positive selective
polymerase activity. Antivir. Ther. 15, 579–586. pressure. J. Gen. Virol. 84, 647–655.
Martin, M., Gilbert, C., Covington, E., Boivin, G., 2006. Characterization of human Preiksaitis, J.K., Brennan, D.C., Fishman, J., Allen, U., 2005. Canadian society of
cytomegalovirus (HCMV) UL97 mutations found in a valganciclovir/oral transplantation consensus workshop on cytomegalovirus management in solid
ganciclovir prophylactic trial by use of a bacterial artificial chromosome organ transplantation final report. Am. J. Transplant. 5, 218–227.
containing the HCMV genome. J. Infect. Dis. 194, 579–583. Prichard, M.N., Gao, N., Jairath, S., Mulamba, G., Krosky, P., Coen, D.M., Parker, B.O.,
Martin, M., Goyette, N., Ives, J., Boivin, G., 2010b. Incidence and characterization of Pari, G.S., 1999. A recombinant human cytomegalovirus with a large deletion in
cytomegalovirus resistance mutations among pediatric solid organ transplant UL97 has a severe replication deficiency. J. Virol. 73, 5663–5670.
patients who received valganciclovir prophylaxis. J. Clin. Virol. 47, 321–324. Quenelle, D.C., Lampert, B., Collins, D.J., Rice, T.L., Painter, G.R., Kern, E.R., 2010.
Marty, F.M., Ljungman, P., Papanicolaou, G.A., Winston, D.J., Chemaly, R.F., Strasfeld, Efficacy of CMX001 against herpes simplex virus infections in mice and
L., Young, J.A., Rodriguez, T., Maertens, J., Schmitt, M., Einsele, H., Ferrant, A., correlations with drug distribution studies. J. Infect. Dis. 202, 1492–1499.
Lipton, J.H., Villano, S.A., Chen, H., Boeckh, M., 2011. Maribavir prophylaxis for Razonable, R.R., Asberg, A., Rollag, H., Duncan, J., Boisvert, D., Yao, J.D., Caliendo,
prevention of cytomegalovirus disease in recipients of allogeneic stem-cell A.M., Humar, A., Do, T.D., 2013. Virologic suppression measured by a
transplants: a phase 3, double-blind, placebo-controlled, randomised trial. cytomegalovirus (CMV) DNA test calibrated to the world health organization
Lancet Infect. Dis 11, 284–292. international standard is predictive of CMV disease resolution in transplant
Masur, H., Whitcup, S.M., Cartwright, C., Polis, M., Nussenblatt, R., 1996. Advances in recipients. Clin. Infect. Dis. 56, 1546–1553.
the management of AIDS-related cytomegalovirus retinitis. Ann. Intern. Med. Razonable, R.R., Emery, V.C., 2004. Management of CMV infection and disease in
125, 126–136. transplant patients. 27–29 February 2004. Herpes 11, 77–86.
Mattick, C., Dewin, D., Polley, S., Sevilla-Reyes, E., Pignatelli, S., Rawlinson, W., Reddy, A.J., Zaas, A.K., Hanson, K.E., Palmer, S.M., 2007. A single-center experience
Wilkinson, G., Dal, M.P., Gompels, U.A., 2004. Linkage of human with ganciclovir-resistant cytomegalovirus in lung transplant recipients:
cytomegalovirus glycoprotein gO variant groups identified from worldwide treatment and outcome. J. Heart Lung Transplant. 26, 1286–1292.
clinical isolates with gN genotypes, implications for disease associations and Reyburn, H., 2010. New WHO guidelines for the treatment of malaria. BMJ 340,
evidence for N-terminal sites of positive selection. Virology 318, 582–597. c2637.
Michel, D., Pavic, I., Zimmermann, A., Haupt, E., Wunderlich, K., Heuschmid, M., Roizman, B., Carmichael, L.E., Deinhardt, F., de-The, G., Nahmias, A.J., Plowright, W.,
Mertens, T., 1996. The UL97 gene product of human cytomegalovirus is an Rapp, F., Sheldrick, P., Takahashi, M., Wolf, K., 1981. Herpesviridae. Definition,
early-late protein with a nuclear localization but is not a nucleoside kinase. J. provisional nomenclature, and taxonomy. The Herpesvirus Study Group, the
Virol. 70, 6340–6346. International Committee on Taxonomy of Viruses. Intervirology 16, 201–217.
Michel, D., Schaarschmidt, P., Wunderlich, K., Heuschmid, M., Simoncini, L., Ross, S.A., Novak, Z., Pati, S., Patro, R.K., Blumenthal, J., Danthuluri, V.R., Ahmed, A.,
Muhlberger, D., Zimmermann, A., Pavic, I., Mertens, T., 1998. Functional Michaels, M.G., Sanchez, P.J., Bernstein, D.I., Tolan, R.W., Palmer, A.L., Britt, W.J.,
regions of the human cytomegalovirus protein pUL97 involved in nuclear Fowler, K.B., Boppana, S.B., 2011. Mixed infection and strain diversity in
localization and phosphorylation of ganciclovir and pUL97 itself. J. Gen. Virol. congenital cytomegalovirus infection. J. Infect. Dis. 204, 1003–1007.
79 (Pt 9), 2105–2112. Sahoo, M.K., Lefterova, M.I., Yamamoto, F., Waggoner, J.J., Chou, S., Holmes, S.P.,
Mossad, S.B., Avery, R.K., 2007. Second look at leflunomide ‘‘failure’’ to control Anderson, M.W., Pinsky, B.A., 2013. Detection of cytomegalovirus drug
cytomegalovirus infection in the setting of renal failure. Transpl. Infect. Dis. 9, resistance mutations by next-generation sequencing. J. Clin. Microbiol. 51,
260–261. 1605–1613.
Mousavi-Jazi, M., Hokeberg, I., Schloss, L., Zweygberg-Wirgart, B., Grillner, L., Linde, Schnepf, N., Boiteau, N., Petit, F., Alain, S., Sanson-Le Pors, M.J., Mazeron, M.C., 2009.
A., Brytting, M., 2001a. Sequence analysis of UL54 and UL97 genes and Rapid determination of antiviral drug susceptibility of human cytomegalovirus
evaluation of antiviral susceptibility of human cytomegalovirus isolates by real-time PCR. Antiviral Res. 81, 64–67.
obtained from kidney allograft recipients before and after treatment. Transpl. Schnepf, N., Corvo, J., Pors, M.J., Mazeron, M.C., 2011. Antiviral activity of ganciclovir
Infect. Dis. 3, 195–202. and artesunate towards human cytomegalovirus in astrocytoma cells. Antiviral
Mousavi-Jazi, M., Schloss, L., Drew, W.L., Linde, A., Miner, R.C., Harmenberg, J., Res. 89, 186–188.
Wahren, B., Brytting, M., 2001b. Variations in the cytomegalovirus DNA Schreiber, A., Harter, G., Schubert, A., Bunjes, D., Mertens, T., Michel, D., 2009.
polymerase and phosphotransferase genes in relation to foscarnet and Antiviral treatment of cytomegalovirus infection and resistant strains. Expert
ganciclovir sensitivity. J. Clin. Virol. 23, 1–15. Opin. Pharmacother. 10, 191–209.
 T.E. Komatsu et al. / Antiviral Research 101 (2014) 12–25 25

Scott, G.M., Isaacs, M.A., Zeng, F., Kesson, A.M., Rawlinson, W.D., 2004. complications among hematopoietic cell transplantation recipients: a global
Cytomegalovirus antiviral resistance associated with treatment induced UL97 perspective. Biol. Blood Marrow Transplant. 15, 1143–1238.
(protein kinase) and UL54 (DNA polymerase) mutations. J. Med. Virol. 74, 85– Ueno, T., Eizuru, Y., Katano, H., Kurata, T., Sata, T., Irie, S., Ogawa-Goto, K., 2006.
93. Novel real-time monitoring system for human cytomegalovirus-infected cells
Scott, G.M., Weinberg, A., Rawlinson, W.D., Chou, S., 2007. Multidrug resistance in vitro that uses a green fluorescent protein-PML-expressing cell line.
conferred by novel DNA polymerase mutations in human cytomegalovirus Antimicrob. Agents Chemother. 50, 2806–2813.
isolates. Antimicrob. Agents Chemother. 51, 89–94. Ueno, T., Ogawa-Goto, K., 2009. Use of a GFP-PML-expressing cell line as a biosensor
Seo, S.K., Regan, A., Cihlar, T., Lin, D.C., Boulad, F., George, D., Prasad, V.K., Kiehn, T.E., for human cytomegalovirus infection. Methods Mol. Biol. 515, 33–44.
Polsky, B., 2001. Cytomegalovirus ventriculoencephalitis in a bone marrow Valcyte (valgancyclovir hydrochloride tablets) Package Insert, 2013. Roche
transplant recipient receiving antiviral maintenance: clinical and molecular Laboratories Inc.
evidence of drug resistance. Clin. Infect. Dis. 33, e105–e108. Verbinnen, T., Jacobs, T., Vijgen, L., Ceulemans, H., Neyts, J., Fanning, G., Lenz, O.,
Shapira, M.Y., Resnick, I.B., Chou, S., Neumann, A.U., Lurain, N.S., Stamminger, T., 2012. Replication capacity of minority variants in viral populations can affect
Caplan, O., Saleh, N., Efferth, T., Marschall, M., Wolf, D.G., 2008. Artesunate as a the assessment of resistance in HCV chimeric replicon phenotyping assays. J.
potent antiviral agent in a patient with late drug-resistant cytomegalovirus Antimicrob. Chemother. 67, 2327–2337.
infection after hematopoietic stem cell transplantation. Clin. Infect. Dis. 46, Vistide (cidofovir injection) Package Insert, 1996. Gilead Science Inc.
1455–1457. Warming, S., Costantino, N., Court, D.L., Jenkins, N.A., Copeland, N.G., 2005. Simple
Shepp, D.H., Dandliker, P.S., de Miranda, P., Burnette, T.C., Cederberg, D.M., Kirk, L.E., and highly efficient BAC recombineering using galK selection. Nucleic Acids Res.
Meyers, J.D., 1985. Activity of 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl] 33, e36.
guanine in the treatment of cytomegalovirus pneumonia. Ann. Intern. Med 103, Weinberg, A., Jabs, D.A., Chou, S., Martin, B.K., Lurain, N.S., Forman, M.S.,
368–373. Crumpacker, C., 2003. Mutations conferring foscarnet resistance in a cohort of
Simen, B.B., Simons, J.F., Hullsiek, K.H., Novak, R.M., Macarthur, R.D., Baxter, J.D., patients with acquired immunodeficiency syndrome and cytomegalovirus
Huang, C., Lubeski, C., Turenchalk, G.S., Braverman, M.S., Desany, B., Rothberg, retinitis. J. Infect. Dis. 187, 777–784.
J.M., Egholm, M., Kozal, M.J., 2009. Low-abundance drug-resistant viral variants Wetherall, N.T., Trivedi, T., Zeller, J., Hodges-Savola, C., McKimm-Breschkin, J.L.,
in chronically HIV-infected, antiretroviral treatment-naive patients Zambon, M., Hayden, F.G., 2003. Evaluation of neuraminidase enzyme assays
significantly impact treatment outcomes. J. Infect. Dis. 199, 693–701. using different substrates to measure susceptibility of influenza virus clinical
Smith, I.L., Cherrington, J.M., Jiles, R.E., Fuller, M.D., Freeman, W.R., Spector, S.A., isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor
1997. High-level resistance of cytomegalovirus to ganciclovir is associated with susceptibility network. J. Clin. Microbiol. 41, 742–750.
alterations in both the UL97 and DNA polymerase genes. J. Infect. Dis. 176, 69– Wilks, A.F., Kurban, R.R., Hovens, C.M., Ralph, S.J., 1989. The application of the
77. polymerase chain reaction to cloning members of the protein tyrosine kinase
Smith, I.L., Taskintuna, I., Rahhal, F.M., Powell, H.C., Ai, E., Mueller, A.J., Spector, S.A., family. Gene 85, 67–74.
Freeman, W.R., 1998. Clinical failure of CMV retinitis with intravitreal cidofovir Winston, D.J., Saliba, F., Blumberg, E., Abouljoud, M., Garcia-Diaz, J.B., Goss, J.A.,
is associated with antiviral resistance. Arch. Ophthalmol. 116, 178–185. Clough, L., Avery, R., Limaye, A.P., Ericzon, B.G., Navasa, M., Troisi, R.I., Chen, H.,
Springer, K.L., Chou, S., Li, S., Giller, R.H., Quinones, R., Shira, J.E., Weinberg, A., 2005. Villano, S.A., Uknis, M.E., 2012. Efficacy and safety of maribavir dosed at 100 mg
How evolution of mutations conferring drug resistance affects viral dynamics orally twice daily for the prevention of cytomegalovirus disease in liver
and clinical outcomes of cytomegalovirus-infected hematopoietic cell transplant recipients: a randomized, double-blind, multicenter controlled trial.
transplant recipients. J. Clin. Microbiol. 43, 208–213. Am. J. Transplant. 12, 3021–3030.
Stagno, S., Britt, R., 2006. Cytomegalovirus Infections. In: Remingtonm, J., Klein, J., Wolf, D.G., Courcelle, C.T., Prichard, M.N., Mocarski, E.S., 2001. Distinct and separate
Wilson, C., Baker, C. (Eds.), Infectious Diseases of the Fetus and Newborn Infant, roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis
sixth ed. Elsevier Saunders, Philadelphia, pp. 739–781. and encapsidation. Proc. Natl. Acad. Sci. USA 98, 1895–1900.
Stewart, M.W., 2010. Optimal management of cytomegalovirus retinitis in patients Wolf, D.G., Shimoni, A., Resnick, I.B., Stamminger, T., Neumann, A.U., Chou, S.,
with AIDS. Clin. Ophthalmol. 4, 285–299. Efferth, T., Caplan, O., Rose, J., Nagler, A., Marschall, M., 2011. Human
Stinski, M., Malone, C., Hermiston, T., Liu, B., 1991. Regulation of human cytomegalovirus kinetics following institution of artesunate after
cytomegalovirus transcription. In: Wagner, E. (Ed.), Herpesvirus Transcription hematopoietic stem cell transplantation. Antiviral Res. 90, 183–186.
and Its Control. CRC, pp. 246–260. Wolf, D.G., Smith, I.L., Lee, D.J., Freeman, W.R., Flores-Aguilar, M., Spector, S.A., 1995.
Sullivan, V., Biron, K.K., Talarico, C., Stanat, S.C., Davis, M., Pozzi, L.M., Coen, D.M., Mutations in human cytomegalovirus UL97 gene confer clinical resistance to
1993. A point mutation in the human cytomegalovirus DNA polymerase gene ganciclovir and can be detected directly in patient plasma. J. Clin. Invest. 95,
confers resistance to ganciclovir and phosphonylmethoxyalkyl derivatives. 257–263.
Antimicrob. Agents Chemother. 37, 19–25. Wolf, D.G., Yaniv, I., Honigman, A., Kassis, I., Schonfeld, T., Ashkenazi, S., 1998. Early
Sullivan, V., Talarico, C.L., Stanat, S.C., Davis, M., Coen, D.M., Biron, K.K., 1992. A emergence of ganciclovir-resistant human cytomegalovirus strains in children
protein kinase homologue controls phosphorylation of ganciclovir in human with primary combined immunodeficiency. J. Infect. Dis. 178, 535–538.
cytomegalovirus-infected cells. Nature 359, 85. Wong, S.W., Wahl, A.F., Yuan, P.M., Arai, N., Pearson, B.E., Arai, K., Korn, D.,
Sun, J.H., O’Boyle Ii, D.R., Zhang, Y., Wang, C., Nower, P., Valera, L., Roberts, S., Hunkapiller, M.W., Wang, T.S., 1988. Human DNA polymerase alpha gene
Nettles, R.E., Fridell, R.A., Gao, M., 2012. Impact of a baseline polymorphism on expression is cell proliferation dependent and its primary structure is similar to
the emergence of resistance to the hepatitis C virus nonstructural protein 5A both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 7, 37–
replication complex inhibitor, BMS-790052. Hepatology 55, 1692–1699. 47.
Svarovskaia, E.S., Martin, R., McHutchison, J.G., Miller, M.D., Mo, H., 2012. Abundant Wright Jr., H.T., Goodheart, C.R., Lielausis, A., 1964. Human Cytomegalovirus.
drug-resistant NS3 mutants detected by deep sequencing in hepatitis C virus- Morphology by negative staining. Virology 23, 419–424.
infected patients undergoing NS3 protease inhibitor monotherapy. J. Clin. Yang, H., Datema, R., 1991. Prolonged and potent therapeutic and prophylactic
Microbiol. 50, 3267–3274. effects of (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine against
Tischer, B.K., von, Einem, J., Kaufer, B., Osterrieder, N., 2006. Two-step red-mediated herpes simplex virus type 2 infections in mice. Antimicrob. Agents
recombination for versatile high-efficiency markerless DNA manipulation in Chemother. 35, 1596–1600.
Escherichia coli. Biotechniques 40, 191–197. Zhang, Y., Zhao, Z., Sun, J., Cao, G., Zhao, F., Hu, J., Liu, L., Ji, Y., 2013. A new mutation
Tomblyn, M., Chiller, T., Einsele, H., Gress, R., Sepkowitz, K., Storek, J., Wingard, J.R., in the human cytomegalovirus UL97 gene may confer ganciclovir resistance in
Young, J.A., Boeckh, M.J., 2009. Guidelines for preventing infectious Chinese kidney transplant recipients. Arch. Virol. 158, 247–250.