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.

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.

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.

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.

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.

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.

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.

Analysis of Mutations in the Gene Encoding Cytomegalovirus DNA Polymerase in a Phase 2 Clinical Trial of Brincidofovir Prophylaxis.

Brincidofovir is an oral antiviral in development for prevention of cytomegalovirus disease. Cytomegalovirus genotyping results from a phase 2 trial comparing brincidofovir to placebo for prophylaxis against cytomegalovirus infection in hematopoietic cell transplant recipients provided initial data on the clinical resistance profile for brincidofovir. In this study, no known resistance-associated mutations were detected in brincidofovir-treated subjects; identified genotypic substitutions did not confer resistance to cytomegalovirus antivirals in vitro, suggesting that these changes represent polymorphisms unrelated to brincidofovir resistance. Lack of evidence for genotypic resistance during prophylaxis suggests that first-line use of brincidofovir for prevention of cytomegalovirus infection may preserve downstream options for patients.

Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97.

The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.

Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance.

Eight in vitro selection experiments under brincidofovir pressure elicited the known cytomegalovirus DNA polymerase amino acid substitutions N408K and V812L and the novel exonuclease domain substitutions D413Y, E303D, and E303G, which conferred ganciclovir and cidofovir resistance with 6- to 11-fold resistance to brincidofovir or 17-fold when E303G was combined with V812L. The new exonuclease domain I resistance mutations selected under brincidofovir pressure add to the single instance previously reported and show the expected patterns of cross-resistance.

Rapid In Vitro Evolution of Human Cytomegalovirus UL56 Mutations That Confer Letermovir Resistance.

Letermovir (LMV) is an experimental cytomegalovirus terminase inhibitor undergoing phase 3 clinical trials. Viral mutations have been described at UL56 codons 231 to 369 that confer widely variable levels of LMV resistance. In this study, 15 independent experiments propagating an exonuclease mutant viral strain in escalating LMV concentrations replicated 6 of the 7 published UL56 mutations and commonly elicited additional resistance-conferring mutations at UL56 codons 231, 236, 237, 244, 257, 261, 325, and 329. Mutations were first detected earlier in LMV (median, 3 passages) than in 8 parallel experiments with foscarnet (median, 15 passages). As LMV concentrations increased, the typical initial UL56 change F261L, which confers low-grade resistance, combined or was replaced with mutations conferring higher-grade resistance, eventually enabling normal viral growth in 30 µM LMV (>5,000-fold the 50% effective concentration [EC50] for the wild type). At high LMV concentrations, the UL56 changes C325F/R were commonly detected, as well as a combination of changes at codons 236, 257, 329, and/or 369. Recombinant viruses containing individual UL56 mutations and combinations were constructed to confirm their resistance phenotypes and normal growth in cell culture. Several double and triple mutants showed much higher LMV resistance than the respective single mutants, particularly those including changes at both codons 236 and 257. The multiplicity of pathways to high-grade LMV resistance with minimal viral growth impact suggests a low viral genetic barrier and the need for close monitoring during treatment of active infection.

Approach to drug-resistant cytomegalovirus in transplant recipients.

PURPOSE OF REVIEW: The purpose of this study is to provide updated information on diagnosis of cytomegalovirus (CMV) drug resistance, treatments for drug-resistant infection and potential uses of experimental antiviral compounds. RECENT FINDINGS: For established CMV antivirals, uncommon viral UL97 kinase and UL54 DNA polymerase drug resistance mutations are sporadically described that expand an extensive existing database. Some novel mutations reported from treated patients have no drug-resistant phenotype and may be genotyping artefacts. Next-generation sequencing technology may enable earlier detection of emerging resistance mutations in treated patients. Management options for drug-resistant infection include optimization of host defenses, antiviral dose escalation, substitutions or combinations of standard or experimental antivirals. Maribavir and letermovir have antiviral targets distinct from the classic DNA polymerase. UL97 mutations elicited by ganciclovir and maribavir are different, although a single p-loop mutation can confer significant cross-resistance. High-grade resistance mutations in the UL56 terminase gene are readily selected in vitro under letermovir and await clinical correlation. SUMMARY: Technical advancements can enhance the accurate and timely genotypic detection of drug resistance. Antivirals undergoing clinical trial offer the prospect of new viral targets and drug combinations, but unresolved issues exist with regard to their therapeutic potential for drug-resistant CMV and their genetic barriers to resistance.

Differential properties of cytomegalovirus pUL97 kinase isoforms affect viral replication and maribavir susceptibility.

UNLABELLED: The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE: The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo.

A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion.

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.

Phenotypic evaluation of previously uncharacterized cytomegalovirus DNA polymerase sequence variants detected in a valganciclovir treatment trial.

BACKGROUND: In a large randomized trial comparing oral valganciclovir and intravenous ganciclovir for treatment of cytomegalovirus disease in solid organ transplantation, confirmed genotypic drug resistance was uncommon (<5%), but definitive interpretation was limited by the detection of 110 uncharacterized UL54 viral DNA polymerase sequence variants. METHODS: Based on treatment history and genetic locus of the sequence changes, 39 of the sequence variants were prioritized for recombinant phenotyping by construction of cloned viral mutants and drug susceptibility testing in cell culture. RESULTS: Four amino acid substitutions were newly confirmed to alter ganciclovir susceptibility: A505V and I726T conferred a borderline decrease in ganciclovir and cidofovir susceptibility, while Q578L and G841S conferred slightly decreased ganciclovir and foscarnet susceptibility. A nonviable phenotype was found for 8 mutations distributed among amino terminal, exonuclease and catalytic domains. Retesting of stored study specimens could not confirm the original detection of >20 sequence variants, including the nonviable mutations and several resistance mutations. CONCLUSIONS: Newly phenotyped UL54 sequence variants did not significantly change the reported incidence of drug resistance in the clinical trial. Unrecognized sequence variants in diagnostic genotyping reports should be confirmed by additional testing in order to improve clinical decision making.

Cytomegalovirus mutants resistant to ganciclovir and cidofovir differ in susceptibilities to synguanol and its 6-ether and 6-thioether derivatives.

The methylenecyclopropane nucleoside (MCPN) analogs synguanol and its 6-alkoxy (MBX2168) and 6-alkylthio (MBX1616) derivatives retained good in vitro activities against several common ganciclovir-resistant UL97 kinase variants of human cytomegalovirus. Foscarnet-MCPN cross-resistance was observed among UL54 polymerase variants. UL54 exonuclease domain ganciclovir-cidofovir dual-resistant variants were remarkably more hypersensitive to these MCPNs than to cyclopropavir, with some 50% effective concentration ratios that were <0.1× the wild type. Different categories of MCPNs may have therapeutically exploitable mechanistic differences in viral DNA polymerase inhibition.

Improved detection of emerging drug-resistant mutant cytomegalovirus subpopulations by deep sequencing.

In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.