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1 Available online at Letter to Editor: Detection of a UL97 Gene Mutation Conferring Ganciclovir Resistance in Human Cytomegalovirus: Prevalence of the D605E Polymorphism in Korean Immunocompromised Patients Heungsup Sung 1, Dongheui An 1, Sang-Oh Lee 2, Sang-Ho Choi 2, Sung-Han Kim 2, Hyun-Sook Chi 1, and Mi-Na Kim 1 Departments of 1 Laboratory Medicine and 2 Infectious Diseases, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea. Abstract. The purpose of this study was to determine the prevalence of ganciclovir (GCV) resistanceconferring human cytomegalovirus (HCMV) UL97 gene mutations and UL97 polymorphisms in Korean immunocompromised patients. A partial sequence of the HCMV UL97 gene spanning codons 430 to 644 was amplified in 77 samples from 32 patients by nested polymerase chain reaction (PCR) and sequenced directly. A cysteine-to-glycine mutation at codon 592 (C592G) conferring GCV resistance was detected in a 2-year-old girl after a 40-day GCV treatment, but overall, UL97 gene mutations associated with GCV resistance were rare in GCV-treated patients. An aspartate-to-glutamate substitution at codon 605 (D605E) was detected in 29 of 32 patients (90.6%), but 17 of 19 (89.5%) GCV-naïve patients also possessed D605E. These findings indicate that the D605E polymorphism, which is frequent in Korean patients and thus may be a natural sequence variant, could be a genetic marker for HCMV in Asian countries. Key words: ganciclovir, human cytomegalovirus, UL97 mutation, UL97 polymorphism Introduction Human cytomegalovirus (HCMV) infections are common and usually asymptomatic in immunocompetent persons; however, HCMV infections are frequent, and occasionally severe, in immunocompromised hosts [1,2]. Tissue-invasive HCMV disease is a serious complication in immunosuppressed transplant recipients or patients with AIDS [3]. Ganciclovir (GCV) is the drug of choice for the treatment of HCMV disease, and is also used as a prophylaxis or preemptive therapy in groups at high risk for HCMV diseases [4,5]. Long-term treatment with GCV can lead to the production of drug-resistant mutants [3]. The mechanisms of resistance to GCV in HCMV include mutations in the catalytic domain of the UL97 phosphotransferase gene and alterations in the UL54 viral DNA Address correspondence to Heungsup Sung, M.D., Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, , South Korea; tel ; fax ; sung@amc.seoul.kr polymerase gene [6,7]. Several genotypic studies have identified UL97 mutations in about 90% of GCV-resistant HCMV clinical isolates [8]. Wellcharacterized GCV-resistance mutations at UL97 codons 460, 520 and impair the phosphorylation of GCV that is necessary for its antiviral activity, presumably by altering substrate recognition [7]. The D605E variant of UL97 was first described in one of eight HCMV isolates from an immunocompromised host in France [9]. Although the UL97 D605E variant has not been commonly observed in the HCMV strains circulating in Western countries, its frequency is high in Asian countries, estimated at 91.8% in Japanese infants and children, and 78% in Chinese transplant recipients [10,11]. In this study, we investigated the prevalence of GCV resistance-conferring HCMV UL97 gene mutations and UL97 polymorphisms in Korean immunocompromised patients /12/ by the Association of Clinical Scientists, Inc.

2 430 Table 1. Demographic characteristics of the 32 patients in the study population. Number % Patients/Specimens 32/77 Sex (male/female) 18/14 56/44 Median age: years (range) 39 (8 months 71 years) Patient group - Stem cell transplantation Liver transplantation Kidney transplantation Others (all were steroid users) 9 28 HCMV serologic status - Transplantation groups (donor/recipient) 23 Positive/Positive Positive/Negative Others group 9 Positive Materials and Methods Patients and specimens. One hundred and nine specimens were originally obtained from 43 patients enrolled between December 2008 and June All specimens had 200 HCMV DNA copies/ml measured with an Artus CMV PCR Test (QIAGEN Gaithersburg, Inc., Gaithersburg, MD, USA) using a LightCycler 2.0 system (Roche Ltd Basel, Switzerland). Seventy-seven specimens that had an amplifiable HCMV UL97 gene from 32 patients were ultimately analyzed in this study. All blood specimens were extracted using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). Patient demographic information, including clinical data, was obtained from the hospital s electronic database. Age, gender, underlying disease, immunosuppressive status, and anti-hcmv prophylaxis and treatment were reviewed. The study was approved by the hospital s Institutional Review Board (# ). UL97 gene PCR. Extracted whole, genomic DNA was used as the template for amplification of UL97 codons 430 to 644, where the known drug resistance mutations are located [3,7,12]. This region was amplified by nested polymerase chain reaction (PCR) using the primers HLF97-F (5 -CTG CTG CAC AAC GTC ACG GTA CAT C-3 ) and HLF97-R (5 -CTC CTC ATC GTC GTC GTA GTC C-3 ), previously described by Lurain et al. [12] for the first PCR, and the primers 460/520 (5 - GTG GAA GCT GGC GTG CAT-3 ) and (5 -CGA CAC GAG GAC ATC TTG-3 ) for the second PCR. Reactions were carried out with a Mastercycler ep thermal cycler (Eppendorf, Foster City, CA, USA) using 30 cycles of 94 C for 1 minute and 60 C for 10 minutes for the first PCR, and 35 cycles of 94 C for 30 seconds, 56 C for 30 seconds, and 72 C for 30 seconds for the second PCR. DNA sequencing and data analysis. PCR products were purified using a Power Gel Extraction kit (TaKaRa Bio Inc., Shiga, Japan). The purified templates were sequenced using an ABI Prism BigDye Terminator v3.1 Cycle Sequencing kit (Applied BioSystems, Foster City, CA, USA) and analyzed on an ABI 3730xl DNA Analyzer (Applied BioSystems). The quality control strain was HCMV AD169 (ATCC VR-538), and established sequences were compared to those from the AD169 strain as a wild-type GCV-sensitive reference. DNA sequences were analyzed using MEGA 4.1 software [13]. Drug resistance mutations were defined as changes in the UL97 coding region known to confer resistance to GCV [3,7].

3 HCMV UL97 mutations and polymorphisms 431 Figure 1. A C592G mutation (A) and a premature stop at codon 607 (B) in HCMV UL97. Results Patient demographic data. The demographics, underlying disease/condition, and clinical characteristics of the 32 patients are shown in Table 1. HCMV disease had developed in 12 patients: five (three pneumonia, one enterocolitis and retinitis, and one esophagitis) among the stem cell transplant recipients, one (hepatitis) among the liver transplant recipients, and six (three pneumonia, one esophagitis, one gastritis, and one colitis) among the others patient group. UL97 gene mutations. HCMV DNA load in the 77 specimens averaged 137,473 ± 520,364 copies/ ml (range, 980 3,800,000 copies/ml). The 77 specimens were divided into four groups according to GCV treatment history: 23 specimens from 19 patients were from GCV-naïve patients, 13 specimens from nine patients were from patients treated previously with GCV, 35 specimens from 19 patients were from patients currently undergoing GCV treatment, and six specimens from three patients previously treated with GCV and currently undergoing foscarnet (FOS) treatment (Table 2). Of the 23 specimens from GCV-naïve patients, 11 were from among the nine patients with acyclovir prophylaxis. A C592G mutant conferring GCV resistance was detected in a 2-year-old girl after a 40-day GCV treatment (Table 2, Figure 1). A premature stop at

4 432 Figure 2. A D605E-encoding mutation in HCMV UL97. (A) Forward and reverse electropherogram before GCV treatment shows an aspartic acid at codon 605. (B) Forward and reverse electropherogram after GCV treatment shows aspartic acid and glutamic acid at codon 605. codon 607 was also detected in the same specimen. Since the viral titer of HCMV was not decreased after 7 weeks GCV treatment, and the mutation in the UL97 gene conferring GCV resistance was detected, we started FOS monotherapy. The HCMV viral load was converted to negative after 7 days FOS therapy. FOS was discontinued after two consecutive negative results after a total 15-day FOS therapy. M550I, which is alleged to be a drug-sensitive mutation, was detected in a 4-year-old girl on FOS treatment for 20 days and previously treated for 110 days with GCV. The D605E variant was detected in 72 specimens (93.5%) from 29 patients (90.6%), but was also found in 17 of 19 (89.5%) GCV-naïve patients. One specimen from a kidney transplant recipient (18 months post-transplantation) who had received GCV for 3 days harbored UL97 genes with aspartate and glutamate at codon 605 (Figure 2). The frequencies of D605E among patients in GCV-naïve, previously GCV-treated, currently GCV-treated, and previously GCV-treated/currently FOS-treated groups were 89.5% (17/19), 88.9% (8/9), 100% (19/19), and 100% (3/3), respectively, and were not different among groups (Table 2). An L531M variant was detected in a patient treated previously for 21 days with GCV. The most common silent mutations detected were G503G (100%), G598G (100%), P525P (80.5%, 62/77), T504T (22.1%, 17/77), and H454H (19.5%, 15/77).

5 HCMV UL97 mutations and polymorphisms 433 Table 2. UL97 mutations detected in 77 specimens from 32 Korean immunocompromised patients. GCV Previously Currently Currently -naïve GCV-treated GCV-treated FOS-treated Specimens/Patients 23/19 13/9 35/19 6/3 Mutations - Drug-resistance mutation C592G - - 1/1 - - Confirmed drug-sensitive mutation M550I /1 D605E 21/17 10/8 35*/19 6/3 - Miscellaneous L531M - 1/1 - - Premature stop at codon /1 - *One specimen harbored UL97 605D and 605E mixed forms (Figure 2). Discussion In this study, we observed only one GCV-resistant mutation, C592G. The UL97 C592G mutation is one of the seven most common GCV resistanceconferring UL97 mutations and is related to lowgrade GCV resistance [14,15]. The frequency of GCV-resistant HCMV strains among clinical isolates appeared to vary considerably. Among transplant recipients, the risk of resistance is highest in HCMV-seronegative recipients of seropositive organs [3]. The low frequency of GCV-resistant HCMV in this study might be related to the fact that only one transplant recipient was an HCMVseronegative recipient of seropositive organ. The D605E change is the only reported sequence polymorphism found in baseline sensitive HCMV isolates in the UL97 codon range 590 to 607, where GCV resistance mutations are clustered [12]. The transfer of this amino acid change to a control strain does not confer a change in GCV sensitivity, alone or in combination with common UL97 resistance mutations [15]. According to the limited available data, the D605E variant is rare in HCMV isolates from immunocompromised patients from Western countries [16,17]. In the present study, we found a high frequency of the D605E variant in HCMV isolated from immunocompromised patients in Korea. Because of its high prevalence in the strains of HCMV circulating in Japan and China, this polymorphism could be a genetic marker for HCMV in Asian countries [10,11]. On the other hand, a mixed population of HCMV strains harboring aspartate and glutamate at codon 605 was found in a GCV-treated patient. Population-based sequencing could only detect the major variants; that is, those present at more than 10% of the total virus population [18]. Minor variants could be lost due to the relatively low signalto-noise ratio in PCR sequencing strategies. The mixed population observed in this study might be due to either treatment-related mutations or a change in the population ratio among quasispecies between recipient and donor after treatment. An M550I and an L531M variant were observed in this study. The recombinant virus containing UL97 mutations encoding M551I had GCV IC 50 values equal to or less than that of the control strains, implying that UL97 M551I polymorphic variants of HCMV are susceptible to GCV [19]. L531M, a novel mutation with an unknown phenotype, was observed in a patient treated previously with GCV. The exact role of this mutation natural polymorphism or GCV-resistance mutation remains unclear and will require further investigation.

6 434 A high prevalence of silent mutations at G503G, P525P, and G598G was observed in this study; mutations that are not commonly observed in Western HCMV strains [20]. Because the prevalence of these mutations in Japan HCMV strains is also high (98.4%, 70.5%, and 98.4% for G503G, P525P, and G598G, respectively), these polymorphisms could also be used as markers to distinguish Asian HCMV strains from those in Western countries [10]. Although this is a small study of HCMV UL97 gene mutations and polymorphisms in Korean immunocompromised patients, our study could show that GCV resistance related to UL97 gene mutations was rare in GCV-treated patients. The D605E polymorphism was frequently identified in Korean immunocompromised patients, whether GCVtreated or GCV-naïve. As such, it may be a natural sequence variant that could be used as a genetic marker for HCMV in Asian countries. Acknowledgement This work was supported by a grant from the National Research Foundation of Korea funded by the Korean Government (KRF-E00314). References 1. Tuthill M, Chen F, Paston S, De La Peña H, Rusakiewicz S, Madrigal A. The prevention and treatment of cytomegalovirus infection in haematopoietic stem cell transplantation. Cancer Immunol Immunother 2009;58: Razonable RR. Strategies for managing cytomegalovirus in transplant recipients. Expert Opin Pharmacother 2010;11: Lurain NS, Chou S. Antiviral drug resistance of human cytomegalovirus. Clin Microbiol Rev 2010;23: Ijichi O, Michel D, Mertens T, Miyata K, Eizuru Y. GCV resistance due to the mutation A594P in the cytomegalovirus protein UL97 is partially reconstituted by a second mutation at D605E. Antiviral Res 2002;53: McGregor A. Current and new cytomegalovirus antivirals and novel animal model strategies. Inflamm Allergy Drug Targets 2010;9: Erice A. Resistance of human cytomegalovirus to antiviral drugs. Clin Microbiol Rev 1999;12: Chou S. Cytomegalovirus UL97 mutations in the era of ganciclovir and maribavir. Rev Med Virol 2008;18: Gilbert C, Boivin G. Human cytomegalovirus resistance to antiviral drugs. Antimicrob Agents Chemother 2005;49: Alain S, Mazeron MC, Pépin JM, Bergmann JF, Narwa R, Raskine L, Sanson Le Pors MJ. Value of a new rapid non-radioactive sequencing method for analysis of the cytomegalovirus UL97 gene in ganciclovir-resistant strains. J Virol Methods 1995;51: Tanaka K, Hori T, Yoto Y, Hatakeyama N, Yamamoto M, Suzuki N, Tsutsumi H. Human cytomegalovirus UL97 D605E polymorphism has a high prevalence in immunocompetent Japanese infants and children. Microbiol Immunol 2011;55: Zhou L, Fan J, Zheng SS, Ma WH. Prevalence of human cytomegalovirus UL97 D605E mutation in transplant recipients in China. Transplant Proc 2006;38: Lurain NS, Weinberg A, Crumpacker CS, Chou S; Adult AIDS Clinical Trials Group-CMV Laboratories. Sequencing of cytomegalovirus UL97 gene for genotypic antiviral resistance testing. Antimicrob Agents Chemother 2001;45: Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24: Chou S, Waldemer RH, Senters AE, Michels KS, Kemble GW, Miner RC, Drew WL. Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir. J Infect Dis 2002;185: Chou S, Van Wechel LC, Lichy HM, Marousek GI. Phenotyping of cytomegalovirus drug resistance mutations by using recombinant viruses incorporating a reporter gene. Antimicrob Agents Chemother 2005;49: Sánchez Puch S, Ochoa C, Carballal G, Zala C, Cahn P, Brunet R, Salomón H, Videla C. Cytomegalovirus UL97 mutations associated with ganciclovir resistance in immunocompromised patients from Argentina. J Clin Virol 2004;30: Hu H, Jabs DA, Forman MS, Martin BK, Dunn JP, Weinberg DV, Davis JL; Cytomegalovirus Retinitis and Viral Resistance Study Group. Comparison of cytomegalovirus (CMV) UL97 gene sequences in the blood and vitreous of patients with acquired immunodeficiency syndrome and CMV retinitis. J Infect Dis 2002;185: Leitner T, Halapi E, Scarlatti G, Rossi P, Albert J, Fenyö EM, Uhlén M. Analysis of heterogeneous viral populations by direct DNA sequencing. Biotechniques 1993;15: Martin M, Gilbert C, Covington E, Boivin G. Characterization of human cytomegalovirus (HCMV) UL97 mutations found in a valganciclovir/oral ganciclovir prophylactic trial by use of a bacterial artificial chromosome containing the HCMV genome. J Infect Dis 2006;194: Castor J, Cook L, Corey L, Jerome KR. Rapid detection directly from patient serum samples of human cytomegalovirus UL97 mutations conferring ganciclovir resistance. J Clin Microbiol 2007;45: