Assessment of the Human Cytomegalovirus UL97 Gene for Identification of Resistance to Ganciclovir in Iranian Immunosuppressed Patients

authors:

avatar Hossein Keyvani 1 , 2 , avatar Sedigheh Taghinezhad Saroukalaei 3 , 2 , avatar Amir Hossein Mohseni 3 , 2 , *

School of Medicine, Iran University of Medical Sciences, Tehran, IR Iran
Research and Development Department, Keyvan Virology Specialty Laboratory (KVSL), Tehran, IR Iran
Department of Microbiology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, IR Iran

how to cite: Keyvani H, Taghinezhad Saroukalaei S, Mohseni A H. Assessment of the Human Cytomegalovirus UL97 Gene for Identification of Resistance to Ganciclovir in Iranian Immunosuppressed Patients. Jundishapur J Microbiol. 2016;9(5):e31733. https://doi.org/10.5812/jjm.31733.

Abstract

Background:

Human cytomegalovirus (HCMV) infections are a major cause of morbidity and mortality among immunocompromised patients. Prolonged antiviral therapy is a cause of mutation and drug resistance in the HCMV genome.

Objectives:

The aim of this study was to identify resistance to ganciclovir (GCV) in Iranian immunosuppressed patients at two different stages of the disease: early (before GCV is initiated) and late (after six months of GCV therapy).

Patients and Methods:

In this study, 87 specimens from Iranian patients were amplified using nested PCR amplification of the UL97 gene. Sequence analyses of products were performed for identifying the mutated codons.

Results:

The present study show that the most frequent GCV-resistant mutations occurred in codons A594V (26.43%), H520Q (18.39%), and M460V (13.79%), consequently occurring at a low frequency in the L595S (2.29%), E596G (1.14%), and Del 594 (1.14%) codons, and with intermediate frequency in the C592G (10.34%), M460I (9.19%), and C603W (6.89%) codons. We describe for the first time a new GCV-resistance mutation, the deletion of codon 594, in the UL97 gene of Iranian HCMV patients after GCV therapy, following renal transplantation.

Conclusions:

The findings of the present study can be utilized to detect GCV resistance patterns among Iranian immunocompromised patients and to treat HCMV infections accordingly.

1. Background

Human cytomegalovirus (HCMV) infections are a major cause of morbidity and mortality among immunocompromised patients. Susceptible groups include renal transplant recipients, bone marrow recipients, and AIDS patients (1, 2). Hence, early diagnosis for the accurate treatment of HCMV is especially necessary to avoid poor clinical outcomes (3-6). Antiviral agents are used in the management of HCMV infections. Currently, the most common antiviral agents include ganciclovir (GCV), foscarnet, and cidofovir. These agents inhibit HCMV DNA synthesis by suppressing the DNA polymerase of the causative virus. Human cytomegalovirus infections usually require long-term treatment with antiviral agents for efficacy against the infective viruses. This can give rise to resistant strains of HCMV (7-10). Resistance to GCV has been evaluated by many researchers because this agent is used as the first-line treatment in 90% of HCMV patients (11).

Ganciclovir, a nucleoside analogue, must be phosphorylated to exert antiviral activity as an inhibitor of viral DNA polymerase UL54. The UL97 kinase, a virus-encoded product, activates the drug by monophosphorylation (12-17). HCMV resistance to GCV is mainly associated with the presence of mutations within UL97, DNA polymerase, or both viral genes (12, 18-25). Although mutations conferring GCV resistance have been mapped in both genes, the majority of the mutations appear in the UL97 gene, rather than as UL54 gene mutations (26). In the last two decades, several methods based on phenotypic testing have been described for the detection of HCMV-resistant mutations, such as the plaque reduction assay, ELISA, and DNA hybridization assays (27-29). However, these tests are burdensome, time-consuming, and labor-intensive (30). Therefore, there is an increasing need for early detection of the emerging mutants (31, 32).

2. Objectives

The purpose of the present study was the examination of GCV-resistant HCMV in Iranian immunosuppressed patients, before and after treatment.

3. Patients and Methods

3.1. Patients

A total of 87 specimens from Iranian patients were targeted in the present study. Clinical isolates from 48 bone marrow transplant patients and 39 renal transplant patients with suspected HCMV infections were identified and sent to the Keyvan virology specialty laboratory (KVSL) for molecular examination of the UL97 gene. The specimens were collected at two different stages of the disease: early (before GCV was initiated) and late (after six months of GCV therapy). All of the selected patients completed questionnaires and informed consent forms for participation in this study.

3.2. Nucleic Acid Extraction

Plasma samples (200 µL) were used for DNA extraction using the QIAamp DNA blood mini kit (Qiagen, USA). The extractions were carried out according to the manufacturer’s instructions.

3.3. Detection of HCMV in Specimens

Extracted DNA of plasma samples was subjected to TaqMan® real-time PCR targeting the UL83 gene, with the primers and probe proposed by Khansarinejad et al. (33). The sequence of the forward primer was 5′-TGAACATCCCCAGCATCAACG-3′, of the reverse primer was 5′-CAGTCCCGAGACMGTGAGAC-3′, and of the probe was 5′-FAM TGCCACATCTGCTTGCCCGACGC-BHQ1-3´. These primers and probe amplified the 137 bp segment of the UL83 gene in HCMV. Internal control of DNA extraction and PCR amplification was conducted by amplification of the human β globin gene. The primers consisted of the following sequences: forward primer: 5′-CAGAGCCATCTATTGCTTAC-3′, reverse primer: 5′-CATGGTGTCTGTTTGAGGTT-3′, and probe: 5′-JOE-ACACAACTGTGTTCACTAGC-BHQ1-3′ (34). All primers and probes were purchased from Bioneer Co., Ltd. (Daejeon, Korea). Real-time PCR was performed with TaqMan universal master mix (Takara Bio, Shiga, Japan). DNA was added to a 25 PCR mixture containing 12.5 μL of TaqMan universal PCR master mix, 300 nM of each primer, and 200 nM of probe. The reaction consisted of 10 minutes at 95°C, followed by 40 cycles of amplification, including 15 s at 95°C and 1 minute at 60°C.

3.4. Analysis of the UL97 Gene

Specific regions of the UL97 gene of HCMV in the early and late samples were amplified using nested PCR, as defined by Benzi et al. (35). The first round was done with the following primers: UL97-F1 (5′-GTTTCATCACGACCAGTGGA-3′) and UL97-R1 (5′-GGTCCTCCTCGCAGATTATG-3′). The second round was carried out with UL97-F2 (5′-TCATCACGACCAGTGGAAGCT-3′) and UL97-R2 (5′-GCGACACGAGGACATCTTGG-3′). The UL97 fragment was amplified using 1.5 mM MgCl2, 0.2 mM dNTP, 1 U Taq DNA polymerase, 10X PCR buffer (Kowsar Biotech Co., Tehran, Iran), 300 nM of each primer (Bioneer Co., Daejeon, Korea), and 50 ng genomic DNA of HCMV. The thermal cycler was run at 94°C for 30 seconds, 52°C for 40 seconds, and 72°C for 2 minutes for 40 cycles, and 72°C for 7 minutes. All of the nested PCR products were subjected to direct Sanger dideoxy DNA sequencing using internal primers of the UL97 gene (UL97-F2 and UL97-R2) (Bioneer Co., Daejeon, Korea). The results of the sequencing were analyzed with the CLC sequence viewer, version 6.4 (CLC Bio Co., Aarhus, Denmark).

4. Results

The fluorescent signals were observed in the FAM and JOE channels related to the UL83 and β globin genes, respectively. These data showed specific amplification of UL83 and β globin genes by real-time PCR amplification of genomic DNA of all samples. To confirm the specificity of the PCR and the real-time PCR assay, genomic DNA from other human herpesviruses (herpes simplex virus types 1 and 2, varicella-zoster virus, and human herpesvirus 6) was amplified. The negative results after amplification of all samples proved the specificity, and there was no cross-reactivity of all assays (data not shown).

Specific nested PCR primers amplified the vulnerable region in the UL97 gene. The binding site of primers on the UL97 gene of HCMV (2124 bp) were nucleotide numbers 1254 - 1273, 2070 - 2089, 1257 - 1277, and 1916 - 1935 for UL97-F1, UL97-R1, UL97-F2, UL97-R2, respectively. Accordingly, in the first round of the assay, the expected 836-bp PCR products were exhibited. The presence of the expected 679-bp band in the second round of the assay proved the specific amplification of the UL97 gene during the nested PCR reaction (Figure 1).

Gel Electrophoresis of Nested PCR Products of the UL97 Gene
M, 100 bp marker; 1, HCMV positive control; 2, Iranian HCMV sample; 3, negative control.

Based on the previous report, the predominant codon numbers in the UL97 gene of HCMV corresponding to GCV resistance have been reported: L405P, M460I, M460T, M460V, V466G, H520Q, ACRA591-594Del4, 591-607Del, C592G, A594E, A594G, A594P, A594T, A594V, L595F, L595S, L595W, L595Del, 595-603Del, E596G, G598S, K599T, L600Del, T601Del, 601-603Del, C603R, C603S, C603W, C607F, and C607Y (36). In order to analyze the sequenced PCR product of the UL97 gene, a series accession number from the GenBank database was selected. Then, using the CLC Sequence Viewer software, the unknown sequences of the UL97 gene from Iranian immunosuppressed patients were aligned with wild-type and mutant nucleotide sequences.

The codon locations of the samples were evaluated with the reported mutant codon, which has been published previously (12, 31, 35, 36). Of the 87 samples, only one (1.14%) point mutation occurred in the early-phase analysis of the samples, but many mutations occurred after treatment with GCV: A594V (26.46%), H520Q (18.39%), M460V (13.79%), C592G (10.34%), M460I (9.19%), C603W (6.89%), L595S (2.29%), E596G (1.14%), and Del 594 (1.14%) (Figure 2). Comparison of the early and late sequences of the UL97 gene exhibited the presence of point mutations in the specific codon of the UL97 gene mainly after drug consumption in the late phase. In addition, for the first time among immunocompromised patients in Iran, a new mutation (Del codon 594) was discovered in the HCMV UL97 gene in a patient who had undergone renal transplantation. The frequencies of early and late mutations of the UL97 gene are listed in Table 1. The mutant sequence has been submitted to GenBank (KM081676).

Percentage of UL97 Gene Mutations in HCMV Infection Samples in Iran
Percentage of UL97 Gene Mutations in HCMV Infection Samples in Iran
Table 1.

List of Accession Numbers (Wild-Type and Mutant Sequences) and Frequency of Early and Late Mutations Related to the UL97 Gene

CNWTNMNWTANMANEAMLAM
H520QCAT, CACCAA, CAGAY642448AY642438016
L595SCTT, CTC, CTA, CTGTCT, TCC, TCA, TCGHQ158765AY64243702
M460VATGGTT, GTC, GTA, GTGAY642435HCU07356012
C603WTGT, TGCTGGAY642443AY64245106
E596GGAA, GAGGGT, GGC, GGA, GGGAF345351-01
A594VGCT, GCC, GCA, GCGGTT, GTC, GTA, GTGAY642439HM208324123
M460IATGATT, ATC, ATAHQ158775AY64244708
C592GTGT, TGCGGT, GGC, GGA, GGGHQ158791HM35264809
Del 594GCG-KC664775KM08167601
No mutation--BK000394-01

5. Discussion

Prolonged GCV therapy in transplant patients is the main cause of mutations occurring in the UL97 gene and the subsequent emergence of GCV resistance. One of the most difficult therapeutic challenges is to determine the wild-type or mutant viruses in transplant patients (37, 38). Mutations of M460V, M460I, H520Q, A594V, L595S, L595F, C603W, and C607Y for resistance of HCMV to GCV have already been reported. In addition, N510S, A590T, A591V, A591D, C592G, L595W, L595T, E596G, E596D, N597I, G598V, K599M, C603Y, A606D, and V665I have also been discovered among clinically GCV-resistant isolates (39). Our study results showed that the early-phase analysis of mutations identified only one renal transplant patient’s sample as positive for the presence of A594V. This suggests that the presence of resistant viruses is weak in the early stages of the disease, whereas a high frequency of the mutated viruses can be detected in the late analysis of mutations.

Erice et al. elucidated no evidence so far for GCV resistance of A591V, C592G, L595W, L595T, E596G, N597I, G598V, K599M, C603Y, A606D, and V665I. We found one point mutation of E596G among our clinical specimens with resistance to HCMV (39). Hantz et al. evaluated 18 infected patients, and discovered high-level resistance mutations of M460V (3/17), A594V (2/17), L595S (6/17), and L595F (2/17), and a 12-codon deletion (Del 591–603). They also found low-level resistance mutations of C592G, A594P, and C603W in the UL97 gene (11). Our data also support the fact that most of the mutations are seen in the UL97 gene, with a high frequency in the A594V (23/87), H520Q (16/87), and M460V (12/87) codons.

Some deletions have previously been explained in an immunocompromised patient in association with clinical resistance to GCV, in the codons of the UL97 gene. Hantz et al. discovered a novel GCV-resistance mutation (a deletion of codon 601 of the UL97 gene) in kidney transplant patients (12). Marfori et al. found a new viral UL97 mutation (deletion of codons 601 - 603) in stem cell transplant recipients (40). Gentry et al. discovered that a deletion in base pair 498 of UL97 is responsible for drug resistance in the HCMV virus (41). Chou et al. reported a deletion mutation (codons 595 to 603) in the UL97 gene (16). For the first time, our study presents a description of a UL97 codon 594 deletion in a GCV-resistant HCMV isolate among Iranian immunocompromised patients. This deletion emerged in a renal transplant recipient after six months of GCV treatment for HCMV infection.

5.1. Conclusion

The results of our study support the hypothesis of a high prevalence of A594V, M460V, and H520Q mutations, low rates of point mutations in the L595S and E596G codons, and an intermediate prevalence of the C603W, M460I, and C592G mutations. Furthermore, the presence of Del 594 in Iranian immunosuppressed patients was also observed. The present study demonstrates an effective method for the early detection of mutations that cause GCV resistance, thereby guiding the treatment of HCMV infections.

Acknowledgements

References

  • 1.

    Jacobson MA, Mills J. Serious cytomegalovirus disease in the acquired immunodeficiency syndrome (AIDS): clinical findings, diagnosis, and treatment. Ann Intern Med. 1988;108(4):585-94. [PubMed ID: 2831765].

  • 2.

    Miller W, Flynn P, McCullough J, Balfour HJ, Goldman A, Haake R, et al. Cytomegalovirus infection after bone marrow transplantation: an association with acute graft-v-host disease. Blood. 1986;67(4):1162-7. [PubMed ID: 3006831].

  • 3.

    Numata S, Nakamura Y, Imamura Y, Honda J, Momosaki S, Kojiro M. Rapid quantitative analysis of human cytomegalovirus DNA by the real-time polymerase chain reaction method. Arch Pathol Lab Med. 2005;129(2):200-4. [PubMed ID: 15679421]. https://doi.org/10.1043/1543-2165(2005)129<200:RQAOHC>2.0.CO;2.

  • 4.

    Boriskin YS, Fuller K, Powles RL, Vipond IB, Rice PS, Booth JC, et al. Early detection of cytomegalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL21. 5: a two site evaluation. J Clin Virol. 2002 Feb;24(1-2):13-23. [PubMed ID: 11744424].

  • 5.

    Yun Z, Lewensohn-Fuchs I, Ljungman P, Vahlne A. Real-time monitoring of cytomegalovirus infections after stem cell transplantation using the TaqMan polymerase chain reaction assays. Transplantation. 2000;69(8):1733-6. [PubMed ID: 10836392].

  • 6.

    Sanchez JL, Storch GA. Multiplex, quantitative, real-time PCR assay for cytomegalovirus and human DNA. J Clin Microbiol. 2002;40(7):2381-6. [PubMed ID: 12089251].

  • 7.

    Balfour HJ. Management of cytomegalovirus disease with antiviral drugs. Rev Infect Dis. 1990;12 Suppl 7:S849-60. [PubMed ID: 2173114].

  • 8.

    Lalezari JP, Drew WL, Glutzer E, James C, Miner D, Flaherty J, et al. (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine (cidofovir): results of a phase I/II study of a novel antiviral nucleotide analogue. J Infect Dis. 1995;171(4):788-96. [PubMed ID: 7706804].

  • 9.

    Lalezari JP, Stagg RJ, Kuppermann BD, Holland GN, Kramer F, Ives DV, et al. Intravenous cidofovir for peripheral cytomegalovirus retinitis in patients with AIDS. A randomized, controlled trial. Ann Intern Med. 1997;126(4):257-63. [PubMed ID: 9036797].

  • 10.

    Reid R, Mar EC, Huang ES, Topal MD. Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. J Biol Chem. 1988;263(8):3898-904. [PubMed ID: 2831212].

  • 11.

    Hantz S, Garnier-Geoffroy F, Mazeron MC, Garrigue I, Merville P, Mengelle C, et al. Drug-resistant cytomegalovirus in transplant recipients: a French cohort study. J Antimicrob Chemother. 2010;65(12):2628-40. [PubMed ID: 20961907]. https://doi.org/10.1093/jac/dkq368.

  • 12.

    Hantz S, Michel D, Fillet AM, Guigonis V, Champier G, Mazeron MC, et al. Early selection of a new UL97 mutant with a severe defect of ganciclovir phosphorylation after valaciclovir prophylaxis and short-term ganciclovir therapy in a renal transplant recipient. Antimicrob Agents Chemother. 2005;49(4):1580-3. [PubMed ID: 15793144]. https://doi.org/10.1128/AAC.49.4.1580-1583.2005.

  • 13.

    Baldanti F, Silini E, Sarasini A, Talarico CL, Stanat SC, Biron KK, et al. A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate. J Virol. 1995;69(2):796-800. [PubMed ID: 7815545].

  • 14.

    Baldanti F, Michel D, Simoncini L, Heuschmid M, Zimmermann A, Minisini R, et al. Mutations in the UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates differentially affect GCV phosphorylation as determined in a recombinant vaccinia virus system. Antiviral Res. 2002 Apr;54(1):59-67. [PubMed ID: 11888658].

  • 15.

    Baldanti F, Lurain N, Gerna G. Clinical and biologic aspects of human cytomegalovirus resistance to antiviral drugs. Hum Immunol. 2004;65(5):403-9. [PubMed ID: 15172438]. https://doi.org/10.1016/j.humimm.2004.02.007.

  • 16.

    Chou S, Waldemer RH, Senters AE, Michels KS, Kemble GW, Miner RC, et al. Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir. J Infect Dis. 2002;185(2):162-9. [PubMed ID: 11807689]. https://doi.org/10.1086/338362.

  • 17.

    Chou S, Meichsner CL. A nine-codon deletion mutation in the cytomegalovirus UL97 phosphotransferase gene confers resistance to ganciclovir. Antimicrob Agents Chemother. 2000;44(1):183-5. [PubMed ID: 10602745].

  • 18.

    Alain S, Honderlick P, Grenet D, Stern M, Vadam C, Sanson-Le Pors MJ, et al. Failure of ganciclovir treatment associated with selection of a ganciclovir-resistant cytomegalovirus strain in a lung transplant recipient. Transplantation. 1997;63(10):1533-6. [PubMed ID: 9175826].

  • 19.

    Chou S, Marousek G, Parenti DM, Gordon SM, LaVoy AG, Ross JG, et al. Mutation in region III of the DNA polymerase gene conferring foscarnet resistance in cytomegalovirus isolates from 3 subjects receiving prolonged antiviral therapy. J Infect Dis. 1998;178(2):526-30. [PubMed ID: 9697736].

  • 20.

    Hanson MN, Preheim LC, Chou S, Talarico CL, Biron KK, Erice A. Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir. Antimicrob Agents Chemother. 1995;39(5):1204-5. [PubMed ID: 7625819].

  • 21.

    Wolf DG, Yaniv I, Honigman A, Kassis I, Schonfeld T, Ashkenazi S. Early emergence of ganciclovir-resistant human cytomegalovirus strains in children with primary combined immunodeficiency. J Infect Dis. 1998;178(2):535-8. [PubMed ID: 9697738].

  • 22.

    Wolf DG, Smith IL, Lee DJ, Freeman WR, Flores-Aguilar M, Spector SA. Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma. J Clin Invest. 1995;95(1):257-63. [PubMed ID: 7814623]. https://doi.org/10.1172/JCI117648.

  • 23.

    Wolf DG, Lee DJ, Spector SA. Detection of human cytomegalovirus mutations associated with ganciclovir resistance in cerebrospinal fluid of AIDS patients with central nervous system disease. Antimicrob Agents Chemother. 1995;39(11):2552-4. [PubMed ID: 8585743].

  • 24.

    Sullivan V, Biron KK, Talarico C, Stanat SC, Davis M, Pozzi LM, et al. A point mutation in the human cytomegalovirus DNA polymerase gene confers resistance to ganciclovir and phosphonylmethoxyalkyl derivatives. Antimicrob Agents Chemother. 1993;37(1):19-25. [PubMed ID: 8381637].

  • 25.

    Sullivan V, Talarico CL, Stanat SC, Davis M, Coen DM, Biron KK. A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Nature. 1992;358(6382):162-4. [PubMed ID: 1319560]. https://doi.org/10.1038/358162a0.

  • 26.

    Smith IL, Cherrington JM, Jiles RE, Fuller MD, Freeman WR, Spector SA. High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J Infect Dis. 1997;176(1):69-77. [PubMed ID: 9207351].

  • 27.

    Prix L, Maierl J, Jahn G, Hamprecht K. A simplified assay for screening of drug resistance of cell-associated cytomegalovirus strains. J Clin Virol. 1998;11(1):29-37. [PubMed ID: 9784141].

  • 28.

    Pepin JM, Simon F, Dussault A, Collin G, Dazza MC, Brun-Vezinet F. Rapid determination of human cytomegalovirus susceptibility to ganciclovir directly from clinical specimen primocultures. J Clin Microbiol. 1992;30(11):2917-20. [PubMed ID: 1333484].

  • 29.

    Gerna G, Sarasini A, Percivalle E, Zavattoni M, Baldanti F, Revello MG. Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture-positive blood samples. J Clin Microbiol. 1995;33(3):738-41. [PubMed ID: 7751388].

  • 30.

    Landry ML, Stanat S, Biron K, Brambilla D, Britt W, Jokela J, et al. A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Antimicrob Agents Chemother. 2000;44(3):688-92. [PubMed ID: 10681339].

  • 31.

    Liu JB, Zhang Z. Development of SYBR Green I-based real-time PCR assay for detection of drug resistance mutations in cytomegalovirus. J Virol Methods. 2008;149(1):129-35. [PubMed ID: 18280587]. https://doi.org/10.1016/j.jviromet.2007.12.011.

  • 32.

    Chakravarti A, Kashyap B, Matlani M. Cytomegalovirus infection: an Indian perspective. Indian J Med Microbiol. 2009;27(1):3-11. [PubMed ID: 19172051].

  • 33.

    Khansarinejad B, Soleimanjahi H, Mirab Samiee S, Hamidieh AA, Paryan M, Sanahmadi Y. Quantitation of human cytomegalovirus DNA in plasma using an affordable in-house qPCR assay. J Virol Methods. 2012;183(2):170-5. [PubMed ID: 22564892]. https://doi.org/10.1016/j.jviromet.2012.04.010.

  • 34.

    Kanaan A, Cour I, Alvarez-Lafuente R, Benedicto M, Culebras E, Prats D, et al. Significance of nested PCR and quantitative real time PCR for cytomegalovirus detection in renal transplant recipients. Int J Antimicrob Agents. 2004;24(5):455-62. [PubMed ID: 15519477]. https://doi.org/10.1016/j.ijantimicag.2004.06.012.

  • 35.

    Benzi F, Vanni I, Cassina G, Ugolotti E, Di Marco E, Cirillo C, et al. Detection of ganciclovir resistance mutations by pyrosequencing in HCMV-infected pediatric patients. J Clin Virol. 2012;54(1):48-55. [PubMed ID: 22300656]. https://doi.org/10.1016/j.jcv.2012.01.006.

  • 36.

    Lurain NS, Chou S. Antiviral drug resistance of human cytomegalovirus. Clin Microbiol Rev. 2010;23(4):689-712. [PubMed ID: 20930070]. https://doi.org/10.1128/CMR.00009-10.

  • 37.

    Gilbert C, Boivin G. Human cytomegalovirus resistance to antiviral drugs. Antimicrob Agents Chemother. 2005;49(3):873-83. [PubMed ID: 15728878]. https://doi.org/10.1128/AAC.49.3.873-883.2005.

  • 38.

    Baldanti F, Gerna G. Human cytomegalovirus resistance to antiviral drugs: diagnosis, monitoring and clinical impact. J Antimicrob Chemother. 2003;52(3):324-30. [PubMed ID: 12888590]. https://doi.org/10.1093/jac/dkg354.

  • 39.

    Erice A. Resistance of human cytomegalovirus to antiviral drugs. Clin Microbiol Rev. 1999;12(2):286-97. [PubMed ID: 10194460].

  • 40.

    Marfori JE, Exner MM, Marousek GI, Chou S, Drew WL. Development of new cytomegalovirus UL97 and DNA polymerase mutations conferring drug resistance after valganciclovir therapy in allogeneic stem cell recipients. J Clin Virol. 2007;38(2):120-5. [PubMed ID: 17157554]. https://doi.org/10.1016/j.jcv.2006.11.005.

  • 41.

    Gentry BG, Vollmer LE, Hall ED, Borysko KZ, Zemlicka J, Kamil JP, et al. Resistance of human cytomegalovirus to cyclopropavir maps to a base pair deletion in the open reading frame of UL97. Antimicrob Agents Chemother. 2013;57(9):4343-8. [PubMed ID: 23817384]. https://doi.org/10.1128/AAC.00214-13.