Ganciclovir and maribavir cross-resistance revisited: Relative drug susceptibilities of canonical cytomegalovirus mutants.

Therapeutic use of maribavir for human cytomegalovirus infection has renewed attention to the extent of cross-resistance with ganciclovir as the existing standard therapy. Each drug selects in vivo for a characteristic set of resistance mutations in the viral UL97 kinase gene. To improve the calibration of relative susceptibilities to each drug, genetic variants at relevant UL97 codons were extensively phenotyped using the same baseline viral clone, cell culture conditions and growth readout. Ganciclovir-selected mutations at codons 460, 520, 592, 594, 595 and 603 conferred 2.8-fold (C603Y) to 12-fold (M460I) increases in ganciclovir 50% inhibitory concentrations (EC50) over wild type baseline, while conferring maribavir EC50 fold changes ranging from 0.21-fold (M460I) to 1.9-fold (A594V). Maribavir-selected mutations at codons 409, 411 and 480 conferred maribavir EC50 fold changes ranging from 17 (H411Y) to 210 (C480F), while conferring ganciclovir EC50 fold changes ranging from 0.7 (H411Y) to 2.3 (C480F). The P-loop substitution F342Y, selected by either drug, is confirmed to confer 4.7-fold and 6-fold increases in maribavir and ganciclovir EC50s respectively, and suggests this part of the ATP-binding domain of UL97 to be involved in moderate resistance to both drugs. The maribavir hypersensitivity of M460I and M460V may be advantageous.

Drug Resistance Assessed in a Phase 3 Clinical Trial of Maribavir Therapy for Refractory or Resistant Cytomegalovirus Infection in Transplant Recipients.

BACKGROUND: This drug resistance analysis of a randomized trial includes 234 patients receiving maribavir and 116 receiving investigator-assigned standard therapy (IAT), where 56% and 24%, respectively, cleared cytomegalovirus DNA at week 8 (treatment responders). METHODS: Baseline and posttreatment plasma samples were tested for mutations conferring drug resistance in viral genes UL97, UL54, and UL27. RESULTS: At baseline, genotypic testing revealed resistance to ganciclovir, foscarnet, or cidofovir in 56% of patients receiving maribavir and 68% receiving IAT, including 9 newly phenotyped mutations. Among them, 63% (maribavir) and 21% (IAT) were treatment responders. Detected baseline maribavir resistance mutations were UL27 L193F (n = 1) and UL97 F342Y (n = 3). Posttreatment, emergent maribavir resistance mutations were detected in 60 (26%) of those randomized to maribavir, including 49 (48%) of 103 nonresponders and 25 (86%) of the 29 nonresponders where viral DNA initially cleared then rebounded while on maribavir. The most common maribavir resistance mutations were UL97 T409M (n = 34), H411Y (n = 26), and C480F (n = 21), first detected 26 to 130 (median 56) days after starting maribavir. CONCLUSIONS: Baseline maribavir resistance was rare. Drug resistance to standard cytomegalovirus antivirals did not preclude treatment response to maribavir. Rebound in plasma cytomegalovirus DNA while on maribavir strongly suggests emerging drug resistance. CLINICAL TRIALS REGISTRATION: NCT02931539.

Use of next-generation sequencing to detect mutations associated with antiviral drug resistance in cytomegalovirus.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality among immunocompromised hosts, including transplant recipients. Antiviral prophylaxis or treatment is used to reduce the incidence of CMV disease in this patient population; however, there is concern about increasing antiviral resistance. Detection of antiviral resistance in CMV was traditionally accomplished using Sanger sequencing of UL54 and UL97 genes, in which specific mutations may result in reduced antiviral activity. In this study, a novel next-generation sequencing (NGS) method was developed and validated to detect mutations in UL54/UL97 associated with antiviral resistance. Plasma samples (n = 27) submitted for antiviral resistance testing by Sanger sequencing were also analyzed using the NGS method. When compared to Sanger sequencing, the NGS assay demonstrated 100% (27/27) overall agreement for determining antiviral resistance/susceptibility and 88% (22/25) agreement at the level of resistance-associated mutations. The limit of detection of the NGS method was determined to be 500 IU/mL, and the lower threshold for detecting mutations associated with resistance was established at 15%. The NGS assay represents a novel laboratory tool that assists healthcare providers in treating patients who are infected with CMV harboring resistance-associated mutations and who may benefit from tailored antiviral therapy.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.

Relative frequency of cytomegalovirus UL56 gene mutations detected in genotypic letermovir resistance testing.

Genotypic testing for letermovir (LMV) resistance was performed by Sanger sequencing of cytomegalovirus terminase gene UL56 (codons 202-412) in 1165 diagnostic specimens, disclosing 36 sequence variants among 173 (14.8%) of the specimens, including one or more LMV resistance mutations in 134 specimens. Codon 325 mutations (C325Y/F/W/R) were the most common (108 specimens), followed by those at codon 369 (R369 S/G/T/K, 13 specimens) and V236M (11 specimens). Mutations V231L, N232Y, Q234R, L257F and V363I were detected in 1-3 specimens each. Combinations of codon 325 mutation and those at codons 236 or 369 were found in 6 specimens. Eleven novel sequence variants were phenotyped, validating Q234R, V363I and R369K as conferring 2- to 5-fold increased LMV 50% inhibitory concentrations (EC50). These findings indicate that UL56 codon 325 mutations conferring >3000-fold LMV EC50 are detected much more frequently in clinical practice than those conferring lower grade resistance, and suggest that a single step mutation to absolute LMV resistance is an ongoing concern in its therapeutic use.

Drug Resistance Mutations and Associated Phenotypes Detected in Clinical Trials of Maribavir for Treatment of Cytomegalovirus Infection.

BACKGROUND: In separate phase 2 trials, 120 patients received maribavir for cytomegalovirus (CMV) infection failing conventional therapy (trial 202) and 119 received maribavir for asymptomatic infection (trial 203). Overall, 172 cleared their CMV infection (CMV DNA <200 copies/mL) within 6 weeks. METHODS: Baseline and posttreatment plasma samples were tested for mutations in viral genes UL97, UL54, and/or UL27. Selected viral mutants were phenotyped for drug susceptibility. RESULTS: Baseline samples revealed UL54 mutations newly phenotyped as conferring resistance to standard DNA polymerase inhibitor(s), including K493N, P497S, K513T, L565V, V823A, A987V, and E989D. Of 29 patients (including 25 from trial 202) who cleared but later experienced recurrent CMV infection while on maribavir, 23 had available UL97 genotyping data; 17 had known resistance mutations (T409M or H411Y) and 5 additional had UL97 C480F alone. The newly phenotyped mutation C480F conferred high-grade maribavir resistance and low-grade ganciclovir resistance. Among 25 who did not respond to >14 days of therapy, 9 showed T409M or H411Y and 4 others showed C480F alone. CONCLUSIONS: After maribavir therapy (400-1200 mg twice daily), UL97 mutations T409M, H411Y, or C480F emerge to confer maribavir resistance in patients with recurrent CMV infection while on therapy or no response to therapy. CLINICAL TRIALS REGISTRATION: NCT01611974 and EudraCT 2010-024247-32.

Opposite effects of cytomegalovirus UL54 exonuclease domain mutations on acyclovir and cidofovir susceptibility.

Acyclovir has weak activity against human cytomegalovirus (CMV). Despite some efficacy as prophylaxis, more potent anti-CMV drugs are preferred. Acyclovir resistance of CMV has been little studied. The viral UL97 kinase phosphorylates acyclovir, and cross-resistance of ganciclovir-resistant mutants is documented. However, UL54 exonuclease domain mutants may confer ganciclovir and cidofovir resistance by a mechanism that does not apply to acyclovir as an obligate chain terminator. To test for differential susceptibilities, 11 exonuclease domain mutants were tested for their 50% inhibitory concentrations (EC50s) of acyclovir in comparison with cidofovir. The 5 mutants with the highest cidofovir EC50s (>10-fold increased over wild type) all had acyclovir EC50s less than 20% of wild type. The relatively common N408K mutant had an acyclovir EC50 of 6 µM, comparable to that reported for wild type varicella-zoster virus. Several foscarnet-resistant UL54 mutants outside the exonuclease domains, some with low-grade ganciclovir/cidofovir cross-resistance, showed various degrees of acyclovir resistance. Based on these in vitro data, acyclovir may become a therapeutic option when a highly cidofovir-resistant exonuclease mutation is present without a simultaneous mutation in UL97.

Ganciclovir and maribavir susceptibility phenotypes of cytomegalovirus UL97 ATP binding region mutations detected by expanded genotypic testing.

Because ganciclovir resistance mutations in the cytomegalovirus UL97 gene most commonly occur at codons 460, 520 and 590-607, diagnostic genotyping for drug resistance has often omitted the analysis of codons below 440. However, the UL97 kinase inhibitor maribavir selects for distinctive resistance mutations at codons 409 and 411, and ganciclovir/maribavir resistance mutations have also been described in the ATP binding region starting at codon 335. Expanded genotypic testing of UL97 codons 335-440 in 1535 clinical specimens disclosed 10 uncharacterized sequence variants that were phenotyped for ganciclovir and maribavir susceptibility. Notable findings included low-grade ganciclovir resistance conferred by amino acid substitutions K359N and E362D, decreased maribavir susceptibility of L348V, and maribavir hypersensitivity of V345I and E362D. Recently published substitutions F342Y and K359E/Q were also confirmed. The data indicate that mutations in the UL97 ATP binding region may arise in clinical specimens to affect the interpretation of ganciclovir and maribavir resistance. This region should now be included in the standard diagnostic genotyping of UL97, especially with the introduction of maribavir into therapeutic use.

Advances in the genotypic diagnosis of cytomegalovirus antiviral drug resistance.

Cytomegalovirus (CMV) drug resistance mutation maps are updated with recent information for polymerase inhibitors, the terminase inhibitor letermovir and the UL97 kinase inhibitor maribavir. Newly mapped mutations and their phenotypes provide more detail on cross-resistance properties and suggest the need to expand the CMV gene regions covered in diagnostic testing. Next-generation deep sequencing technology offers a more sensitive, higher resolution view of emerging antiviral resistance and is recommended for use in clinical trials. Issues of standardization and diagnostic utility in comparison with traditional Sanger sequencing remain unresolved. Quality control is important for the accurate and reproducible detection of mutant viral populations in clinical specimens.

Letermovir Resistance Analysis in a Clinical Trial of Cytomegalovirus Prophylaxis for Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: Letermovir (LET), a cytomegalovirus (CMV) deoxyribonucleic acid (DNA) terminase inhibitor, was recently approved for prophylaxis of CMV infection in adult CMV-seropositive recipients of allogeneic hematopoietic stem cell transplantation. Cytomegalovirus genotyping was performed to identify LET-resistance-associated variants (RAVs) among subjects in a Phase 3 trial. METHODS: The CMV UL56 and UL89 genes, encoding subunits of CMV DNA terminase, were sequenced from plasma collected from subjects with clinically significant CMV infection (CS-CMVi). Novel variants were evaluated by recombinant phenotyping to assess their potential to confer resistance to LET. RESULTS: Genotyping was successful for 50 of 79 LET subjects with CS-CMVi. Resistance-associated variants (encoding pUL56 V236M and C325W) were detected independently in subjects 1 and 3 who experienced CS-CMVi while receiving LET prophylaxis, and 2 other variants (encoding pUL56 E237G and R369T) were detected >3 weeks after subjects 2 and 3, respectively, had discontinued LET prophylaxis and received preemptive therapy with ganciclovir. CONCLUSIONS: The detected incidence of CMV resistance among subjects who received LET as prophylaxis in this Phase 3 trial was low. The LET RAVs that were detected mapped to the CMV UL56 gene at positions associated with reduced susceptibility to LET based on resistance selections in cell culture.

Novel UL97 drug resistance mutations identified at baseline in a clinical trial of maribavir for resistant or refractory cytomegalovirus infection.

In a Phase 2 clinical trial, 120 subjects with cytomegalovirus (CMV) infection refractory or resistant to standard therapy were randomized equally to 3 doses of oral maribavir treatment, and 70% achieved undetectable plasma CMV DNA within 12 weeks. At study entry, standard diagnostic UL97 genotyping was available for 71 subjects, with 60 (85%) revealing well-characterized ganciclovir resistance mutations that did not preclude a therapeutic response to maribavir. Central laboratory testing of a range of UL97 codons (288-468) not fully covered by standard genotyping was done on 93 subjects at baseline. This detected no previously known maribavir resistance mutations, but identified atypical mutations in 3 subjects, including a P-loop substitution F342Y, and ATP-binding region substitutions K359E/Q. By recombinant phenotyping, K359E and K359Q each conferred a nearly 4-fold increased ganciclovir 50% inhibitory concentration (EC50) without maribavir resistance, whereas F342Y conferred a 6-fold increased ganciclovir EC50 and a 4.5-fold increased maribavir EC50. The subject with F342Y detected at baseline did not achieve plasma CMV DNA clearance after 12 weeks of maribavir therapy and later developed an additional UL97 substitution H411Y known to confer 12- to 20-fold increased MBV EC50 by itself. The combination of F342Y and H411Y was shown to increase the maribavir EC50 by 56-fold. Diagnostic genotyping of UL97 should be expanded to cover the ATP-binding region beginning at codon 335 to enable the detection of atypical resistance mutations and further correlation of their clinical significance.

Definitions of Resistant and Refractory Cytomegalovirus Infection and Disease in Transplant Recipients for Use in Clinical Trials.

Despite advances in preventive strategies, cytomegalovirus (CMV) infection remains a major complication in solid organ and hematopoietic cell transplant recipients. CMV infection may fail to respond to commercially available antiviral therapies, with or without demonstrating genotypic mutation(s) known to be associated with resistance to these therapies. This lack of response has been termed "resistant/refractory CMV" and is a key focus of clinical trials of some investigational antiviral agents. To provide consistent criteria for future clinical trials and outcomes research, the CMV Resistance Working Group of the CMV Drug Development Forum (consisting of scientists, clinicians, regulatory officials, and industry representatives from the United States, Canada, and Europe) has undertaken establishing standardized consensus definitions of "resistant" and "refractory" CMV. These definitions have emerged from the Working Group's review of the available virologic and clinical literature and will be subject to reassessment and modification based on results of future studies.

Emergence of letermovir resistance in a lung transplant recipient with ganciclovir-resistant cytomegalovirus infection.

Following a year of valganciclovir prophylaxis, a lung transplant recipient developed cytomegalovirus (CMV) infection that became resistant to ganciclovir, as confirmed by detection of UL97 kinase mutation M460V and a previously uncharacterized UL54 DNA polymerase mutation L516P. The latter mutation is now shown to confer ganciclovir and cidofovir resistance. As predicted from the viral genotype, foscarnet therapy was effective, but resumption of valganciclovir as secondary prophylaxis resulted in a plasma viral load rebound to 3.6 log10 copies/mL several weeks later. Valganciclovir was then replaced by letermovir, resulting in gradual viral load reduction in the first 5 weeks to below the quantitation limit (2.7 log10 copies/mL) for 1 week, followed by 10 weeks of rising viral loads reaching 4.3 log10 copies/mL while on letermovir. At this point, CMV genotypic testing revealed UL56 mutation C325Y, which confers absolute resistance to letermovir. Retreatment with foscarnet was successful. This case adds to the considerable list of proven ganciclovir resistance mutations, and provides an early experience with letermovir resistance after off-label therapeutic use. This experience is consistent with in vitro observations of rapid emergence of letermovir-resistant CMV after drug exposure.

Antiviral activity of maribavir in combination with other drugs active against human cytomegalovirus.

The human cytomegalovirus (CMV) UL97 kinase inhibitor maribavir is in Phase III clinical trials as antiviral therapy, including use for infections refractory or resistant to standard therapy. To assess its activity in combination with approved and experimental CMV antivirals, and with the mTor inhibitor rapamycin (sirolimus), drug effects were tested by in vitro checkerboard assays and the data were analyzed using a three dimensional model based on an independent effects definition of additive interactions. Baseline virus and representative drug-resistant mutants were tested. According to the volume of synergy at 95% confidence, maribavir showed additive interactions with foscarnet, cidofovir, letermovir and GW275175X when tested against wild type and mutant viruses, strong antagonism with ganciclovir, and strong synergy with rapamycin, the latter suggesting a potentially useful therapeutic combination.

New Locus of Drug Resistance in the Human Cytomegalovirus UL56 Gene Revealed by In Vitro Exposure to Letermovir and Ganciclovir.

Letermovir is a human cytomegalovirus (CMV) terminase inhibitor recently approved as prophylaxis in stem cell transplant recipients. In further studies of emerging drug resistance, a baseline laboratory CMV strain was serially propagated in cell culture under a combination of letermovir and ganciclovir. In eight experiments, UL56 terminase gene mutations were detected beginning at 10 passages and included novel amino acid substitutions V236A, L328V, and A365S in a region previously associated with letermovir resistance. Outside this region, the UL56 substitution C25F was detected at moderate drug concentrations in two experiments as either the first detected mutation or an addition to a preexisting V231L substitution. In all cases, mutation at UL56 codon 325 conferring absolute letermovir resistance eventually developed at a median of 20 passages. No UL97 kinase or UL54 DNA polymerase mutations relevant to ganciclovir resistance were detected until many passages after the first detection of the UL56 mutations. UL56 substitutions V236A, L328V, and A365S were shown to confer borderline or low-grade letermovir resistance, while C25F conferred a 5.4-fold increase in letermovir resistance (50% effective concentration [EC50]) by itself and a 46-fold increase in combination with V231L. The evolution of resistance mutations sooner in UL56 than in UL54 or UL97 is consistent with prior in vitro observations, and UL56 codon 25 is a genetic locus for letermovir resistance distinct from loci previously described.

The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation.

Despite recent advances, cytomegalovirus (CMV) infections remain one of the most common complications affecting solid organ transplant recipients, conveying higher risks of complications, graft loss, morbidity, and mortality. Research in the field and development of prior consensus guidelines supported by The Transplantation Society has allowed a more standardized approach to CMV management. An international multidisciplinary panel of experts was convened to expand and revise evidence and expert opinion-based consensus guidelines on CMV management including prevention, treatment, diagnostics, immunology, drug resistance, and pediatric issues. Highlights include advances in molecular and immunologic diagnostics, improved understanding of diagnostic thresholds, optimized methods of prevention, advances in the use of novel antiviral therapies and certain immunosuppressive agents, and more savvy approaches to treatment resistant/refractory disease. The following report summarizes the updated recommendations.

A third component of the human cytomegalovirus terminase complex is involved in letermovir resistance.

Letermovir is a human cytomegalovirus (CMV) terminase inhibitor that was clinically effective in a Phase III prevention trial. In vitro studies have shown that viral mutations conferring letermovir resistance map primarily to the UL56 component of the terminase complex and uncommonly to UL89. After serial culture of a baseline CMV laboratory strain under letermovir, mutation was observed in a third terminase component in 2 experiments, both resulting in amino acid substitution P91S in gene UL51 and adding to a pre-existing UL56 mutation. Recombinant phenotyping indicated that P91S alone conferred 2.1-fold increased letermovir resistance (EC50) over baseline, and when combined with UL56 mutation S229F or R369M, multiplied the level of resistance conferred by those mutations by 3.5-7.7-fold. Similarly a combination of UL56 mutations S229F, L254F and L257I selected in the same experiment conferred 54-fold increased letermovir EC50 over baseline, but 290-fold when combined with UL51 P91S. The P91S mutant was not perceptibly growth impaired. Although pUL51 is essential for normal function of the terminase complex, its biological significance is not well understood. Letermovir resistance mutations mapping to 3 separate genes, and their multiplier effect on the level of resistance, suggest that the terminase components interactively contribute to the structure of a letermovir antiviral target. The diagnostic importance of the UL51 P91S mutation arises from its potential to augment the letermovir resistance of some UL56 mutations at low fitness cost.

Comparison of Cytomegalovirus Terminase Gene Mutations Selected after Exposure to Three Distinct Inhibitor Compounds.

Letermovir, GW275175X (a benzimidazole), and tomeglovir (Bay38-4766) are chemically unrelated human cytomegalovirus (CMV) terminase complex inhibitors that have been tested in human subjects. UL56 gene mutations are the dominant pathway of letermovir resistance, while UL89 and UL56 mutations are known to confer benzimidazole resistance. This study compares the mutations elicited by the three inhibitors during in vitro CMV propagation. GW275175X consistently selected for UL89 D344E and sometimes selected for UL89 C347S, UL89 R351H, or UL56 Q204R. Tomeglovir consistently selected for UL89 V362M and sometimes selected for UL89 N329S, T350M, H389N, or N405D or UL56 L208M, E407D, H637Q, or V639M. Adding to known and novel UL56 mutations, letermovir occasionally selected for UL89 N320H, D344E, or M359I. Recombinant phenotyping confirmed that UL89 D344E conferred 9-fold resistance (an increased 50% effective concentration [EC50]) for GW275175X and increased the letermovir and tomeglovir EC50s by 1.7- to 2.1-fold for the baseline virus and the UL56 Q204R, E237D, F261L, and M329T mutants. UL89 N320H and M359I conferred <2-fold letermovir resistance but 7-fold tomeglovir resistance; the N320H mutant was also 4-fold resistant to GW275175X. UL89 N329S conferred tomeglovir and letermovir cross-resistance. UL89 T350M conferred resistance to all three inhibitors. UL89 C347S conferred 27-fold GW275175X resistance. UL89 V362M and H389N conferred 98-fold and 29-fold tomeglovir resistance, respectively, without conferring cross-resistance. Thus, characteristic UL89 mutations confer substantial resistance to GW275175X and tomeglovir and are an uncommon accessory pathway of letermovir resistance. Instances of moderate cross-resistance and the proximity of the selected UL89 and UL56 mutations suggest targeting of a similar terminase functional locus involving UL56 and UL89 interaction.

Differentiated Levels of Ganciclovir Resistance Conferred by Mutations at Codons 591 to 603 of the Cytomegalovirus UL97 Kinase Gene.

Diagnostic mutations in the cytomegalovirus UL97 kinase gene are used to assess the level of associated ganciclovir resistance and therapeutic options. The best-known mutations at codons 460, 520, or 591 to 607 individually confer 5- to 10-fold-decreased ganciclovir susceptibility, except that a 3-fold decrease occurs in the case of the amino acid substitution C592G. Less common point and in-frame deletion mutations at codons 591 to 603 remain incompletely characterized. The ganciclovir susceptibilities of 17 mutants in this codon range were evaluated by use of the same recombinant phenotyping system and extensive assay replicates in two types of cell cultures. Amino acid substitutions K599E and T601M conferred no ganciclovir resistance, while A591V conferred 3.8-fold-decreased susceptibility. In-frame deletions of three or more codons conferred at least 8-fold-increased ganciclovir resistance, while the level of resistance conferred by one- or two-codon deletions varied from 4- to 10-fold, depending on their location. Measured levels of ganciclovir resistance were closely comparable when assays were performed in either fibroblasts or modified retinal epithelial cells. The significant revision of a few previously published resistance phenotypes and the new data strengthen the interpretation of genotypic testing for cytomegalovirus drug resistance.

Foscarnet resistance mutations mapping to atypical domains of the cytomegalovirus DNA polymerase gene.

Human cytomegalovirus UL54 DNA polymerase gene mutations that confer foscarnet resistance in clinical practice typically cluster in the amino terminal 2, palm and finger domains. Exposure to foscarnet in cell culture selected for mutations elsewhere in UL54, including amino acid substitutions S290R in the amino terminal 1 domain and E951D in the palm 2 domain. These are newly confirmed to confer foscarnet resistance and slightly decreased ganciclovir susceptibility. Other emergent substitutions N495K, T552N and T838A are known to confer foscarnet resistance, while additional ones Q783R and V798A only slightly affected susceptibility. An expanded set of domains is involved in foscarnet resistance and its genotypic diagnosis.