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.

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.

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 [EC]) 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 observations, and UL56 codon 25 is a genetic locus for letermovir resistance distinct from loci previously described.

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. Brincidofovir (BCV or CMX001, Chimerix) is an experimental orally bioavailable hexadecyloxypropyl conjugate of the nucleotide analog cidofovir (CDV) (1). A phase 3 clinical trial of BCV for the prevention of human cytomegalovirus (CMV) infection in bone marrow transplant recipients (ClinicalTrials registration no. NCT01769170) was recently completed, and BCV unexpectedly failed to meet its primary efficacy endpoint despite showing a statistically significant antiviral effect during the ontreatment phase (2). These results and those of a prior successful phase 2 trial (3) showed that short-term prophylactic use of BCV did not result in the detection of CMV UL54 DNA polymerase gene mutations conferring drug resistance (4). The genetic pathways and biochemical mechanisms of resistance to BCV are expected to be the same as those of the parent compound CDV (5), into which BCV is metabolized prior to formation of the active antiviral CDP diphosphate (CDV-PP). Intracellular levels of CDV-PP differ markedly after exposure to CDV or BCV (6), as reflected in the much lower BCV concentrations (0.2 to 0.5 nM for BCV versus 200 nM for CDV) required to reduce CMV replication by 50% in cell culture (50% effective concentration [EC 50 ]). In vitro exposure to BCV may select for mutations different from those previously reported for CDV or ganciclovir (GCV), as higher intracellular active drug concentrations may select for mutants with higher associated levels of drug resistance at the cost of reduced growth fitness. In one report, the UL54 exonuclease domain III amino acid substitution D542E (7) was detected after many cell culture passages under BCV and shown to confer cross-resistance to CDV but not to GCV or foscarnet (FOS). Eight additional in vitro selection experiments under BCV are reported here with characterization of the resistance and relative growth phenotypes of the emergent mutations.The reference BCV compound was provided by Chimerix and added to the human fibroblast cultures starting with 0.2 nM BCV for all experiments after CMV inoculation at a low multiplicity of infection (MOI), followed by weekly propagation under increasing drug concentrations as permitted by interim viral growth, as previously described (8). We commonly use an error-prone UL54 exonuclease mutant (e.g., D413del, strain T4138) to accelerate the evolution of resistance mutations, but this mutant is already resistant to BCV, CDV, and GCV (Table 1), with EC 50 s ϳ5-fold increased over those f...