M A J O R A R T I C L E
Heterogeneity and Evolution of Thymidine
Kinase and DNA Polymerase Mutants of
Herpes Simplex Virus Type 1: Implications for
Graciela Andrei,1Aspasia Georgala,2Dimitri Topalis,1Pierre Fiten,3Michel Aoun,2Ghislain Opdenakker,3and
1Laboratory of Virology, Rega Institute for Medical Research, KU Leuven,2Jules Bordet Institute, Université Libre de Bruxelles, Brussels, and
3Laboratoty of Immunobiology, Rega Institute for Medical Research, KU Leuven, Belgium
etic stem cell transplantation (HSCT) are an emerging concern. An understanding of the evolutionary aspects of
HSV infection is crucial to the design of effective therapeutic and control strategies.
Methods. Eight sequential HSV-1 isolates were recovered from an HSCT patient who suffered from recurrent
herpetic gingivostomatitis and was treated alternatively with acyclovir, ganciclovir, and foscavir. The diverse
spectra and temporal changes of HSV drug resistance were determined phenotypically (drug-resistance profiling)
and genotypically (sequencing of the viral thymidine kinase and DNA polymerase genes).
Results.Analysis of 60 clones recovered from the different isolates demonstrated that most of these isolates
were heterogeneous mixtures of variants, indicating the simultaneous infection with different drug-resistant
viruses. The phenotype/genotype of several clones associated with resistance to acyclovir and/or foscavir were
identified. Two novel mutations (E798K and I922T) in the viral DNA polymerase could be linked to drug resis-
Conclusions.The heterogeneity within the viral populations and the temporal changes of drug-resistant
viruses found in this HSCT recipient were remarkable, showing a rapid evolution of HSV-1. Drug-resistance sur-
veillance is highly recommended among immunocompromised patients to manage the clinical syndrome and to
avoid the emergence of multidrug-resistant isolates.
Infections caused by acyclovir-resistant isolates of herpes simplex virus (HSV) after hematopoi-
tions; DNA polymerase; thymidine kinase; multiple HSV infections; hematopoietic stem cell transplantation
heterogenicity of HSV populations; drug-resistance dynamics in HSV; evolution of HSV popula-
Diseases due to herpes simplex virus (HSV), although
usually self-limited in the normal population, can be a
major cause of mortality and morbidity in immuno-
compromised patients (ICs). Mucocutaneous HSV re-
activation is very common among individuals with an
impaired immune system, ranging from 15% among
chronic lymphocytic leukemia patients receiving flu-
darabine to 90% among patients with acute leukemia
or allogeneic hematopoietic stem cell transplantation
Among ICs, HSV-associated disease may have an
atypical appearance (more invasive disease, relatively
slow healing, prolonged viral shedding, and risk of dis-
semination) and usually responds to acyclovir (ACV)
treatment within a few days; ACV prophylaxis is highly
efficacious in preventing relapses. Nevertheless, some
of these infections become chronic and require pro-
longed antiviral treatment, favoring the emergence of
drug-resistant viruses . Among ICs, the prevalence
Received 13 August 2012; accepted 15 November 2012; electronically
published 11 January 2013.
Correspondence: Robert Snoeck, Rega Institute for Medical Research, KU
Leuven, Minderbroedersstraat 10. B-3000, Leuven, Belgium (robert.snoeck@rega.
The Journal of Infectious Diseases2013;207:1295–1305
© The Author 2013. Published by Oxford University Press on behalf of the Infectious
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rates of ACV-resistant (ACVr) HSV varies from 3.5% to 10.9%
compared with only 0.3% to 0.7% in immunocompetent indi-
viduals [3–6]. ACV resistance is caused by either mutations in
the viral thymidine kinase (TK; necessary for activation of the
drug) or DNA polymerase (pol; molecular target of ACV-
triphosphate) genes [7–9]. The majority of the clinical ACVr
isolates have mutations in the viral TK gene .
Foscavir (PFA), a direct inhibitor of the virus DNA polymeras-
es, is considered an alternative therapy for the treatment of ACVr
viruses due to alterations in the viral TK gene [10–12]. However,
mutations in the viral DNA pol leading to PFArhave also been
reported. Most of the ACVrstrains that result from changes in
the viral DNA pol gene are also PFAr[13–15]. Furthermore,
HSV drug-resistant mutants associated with alterations in
both the TK and DNA pol genes have been described .
The acyclic nucleoside phosphonate (ANP) cidofovir (CDV,
sine]) is independent of viral TK for its activation. It is consid-
ered an alternative therapy and has been shown efficacious in
the treatment of ACVrand/or PFArHSV infections [17–19].
Infections caused by ACVrHSV have become a concern in
several allogeneic stem cell transplantation centers because their
reported incidence seems to be increasing, they cause major
morbidity, and they can be life threatening , and their man-
agement is challenging [6,21,22]. An understanding of the evo-
lutionary aspects of HSV infection is crucial to the design of
effective therapeutic and control strategies. The purpose of this
study was to investigate HSV dynamics by characterizing multi-
ple temporal strains recovered from a patient with recurrent
HSV type 1 (HSV-1) gingivostomatitis who also had leukemia
and received HSCT. The present study reveals a rapid evolution
of the viral population and the acquisition of multiple drug re-
sistances due to simultaneous infection with strains bearing dif-
ferent mutations in the TK and/or DNA pol genes.
MATERIALS AND METHODS
Patient and Clinical Specimens
A 35-year-old woman was diagnosed with acute myelocytic
leukemia M2 (karyotype 5q-) in April 2006. She was treated
with Cytarabine and Idarubicin as induction chemotherapy.
However, due to therapy failure, she received new induction
chemotherapy with Cytarabine and Amsacrine that resulted in
complete remission. She was given Cytarabine and Amsacrine
as consolidation chemotherapy.
In October 2006, she underwent T-cell–depleted allogeneic
transplantation with stem cells from her HLA-matched sister
(5/8 HLA haploidentical) with alloreactive natural killer cells
(Figure 1). The serological analyses of the donor (D) and re-
cipient (R) were as follows: hepatitis A, Epstein-Barr, and
varicella-zoster viruses: D+/R+; cytomegalovirus (CMV), hepa-
titis B and hepatitis C viruses: D−/R−; toxoplasma: D+/R−.
Antibodies against HSV-1/HSV-2 were not tested before trans-
plantation. The conditioning regimen consisted of Melphalan,
Fludarabine, antithymocyte globulin, and total body irradiation.
During the immediate posttransplant period, the patient
received ciprofloxacine and ACV prophylactically (3× 400 mg
by mouth or 3× intravenous 5 mg/kg daily). She did not
develop any manifestation of graft-versus-host disease (GVHD)
and left the hospital with a prophylactic treatment including
ACV (400 mg 3 times per day), fluconazole, and sulfamethox-
azol plus trimethoprim.
In December 2006, she received 2 doses of Rituximab (at
days 60 and 67 posttransplantation) because of Epstein-Barr
virus reactivation. In January 2007, she developed signs of
GVHD and received corticosteroids and cyclosporine A. In
March 2007, she presented with a herpetic gingivostomatitis
under ACV treatment and then received PFA (90 mg/kg 2
times per day intravenously). She also had pneumonia due to
Streptococcus pneumonia resistant to penicillin that responded
to moxifloxacin (400 mg once daily given intravenously for 3
days and then orally for 7 days). At 167 days posttransplanta-
tion, she was hospitalized because of complete blindness,
cough, and worsening of her general condition. The patient
was not neutropenic at that time. However, due to a microan-
giopathy, cyclosporine was replaced by mycophenolate mofetil
by mouth while corticosteroids were maintained. At the time
of hospitalization, no HSV lesions were observed in the
mouth, but ulcerative lesions developed quickly under prophy-
lactic ACV. Considering the patient’s blindness, ganciclovir
(GCV) was started to cover a possible cytomegalovirus (CMV)
retinitis. An invasive pulmonary aspergillosis was diagnosed,
and blindness was attributed to aspergillus endophtalmitis.
Antifungal treatment (Voriconazole) was initiated; GCV was
interrupted and replaced by PFA (90 mg/kg bid) at day 170
posttransplantation because of worsening of the mouth ulcera-
tions. A cerebral nuclear magnetic resonance image identified
several focal lesions, and treatment with pyrimethamine and
clindamycine was started. This treatment was rapidly stopped;
at day 178 posttransplantation, liposomal Amphotericine B
was added because of progression of pulmonary aspergillosis
noted in chest computed tomographic scan. Polymerase chain
reaction amplification for HSV and CMV was negative in the
cerebrospinal spinal fluid, and several mouth rinses were per-
formed because of the lack of improvement of the oral HSV
lesions despite PFA treatment. In the meantime, the patient
developed a new pneumococcal pneumonia complicated with
bacteremia and received piperacillin–
tazobactam (4 g 4 times per day). At day 200 posttransplantation,
PFA was stopped and the patient received intravenous acyclo-
vir (10 mg/kg 3 times per day). The HSV isolate recovered at
that time showed ACVrand PFAr, and antiviral treatment was
discontinued. The oral lesions remained stable, but mouth
rinses and swabs remained positive for HSV-1. In the
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