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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 conferringgt;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.

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 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.

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 log(10) 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 log(10) copies/mL) for one week, followed by 10 weeks of rising viral loads reaching 4.3 log(10) 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.

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.

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.

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.

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.

Symptomatic cytomegalovirus (CMV) disease has been the standard endpoint for clinical trials in organ transplant recipients. Viral load may be a more relevant endpoint due to low frequency of disease. We performed a meta-analysis and systematic review of the literature. We found several lines of evidence to support the validity of viral load as an appropriate surrogate end-point, including the following: (1) viral loads in CMV disease are significantly greater than in asymptomatic viremia (odds ratio, 9.3 95% confidence interval, 4.6-19.3); (2) kinetics of viral replication are strongly associated with progression to disease; (3) pooled incidence of CMV viremia and disease is significantly lower during prophylaxis compared with the full patient follow-up period (viremia incidence: 3.2% vs 34.3%; P < .001) (disease incidence: 1.1% vs 13.0%; P < .001); (4) treatment of viremia prevented disease; and (5) viral load decline correlated with symptom resolution. Based on the analysis, we conclude that CMV load is an appropriate surrogate endpoint for CMV trials in organ transplant recipients.

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.

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.

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.

Antiviral therapy is required in the transplant setting for viral infections that arise from preexisting latent or persistent infection in the recipient and/or donor as well as from nosocomial or community sources. A preventive or prophylactic antiviral approach where feasible is preferable to the treatment of symptomatic viral disease. Acute infections need prompt diagnosis and antiviral treatment for the best outcome. Selection of antiviral therapy involves multiple considerations including potential benefit, toxicity, logistical complexity, and the possibility of drug resistance. This chapter reviews antiviral drug therapy for the most common viruses affecting transplant recipients: herpesviruses including herpes simplex virus, varicella-zoster virus, and cytomegalovirus, influenza, respiratory syncytial virus (RSV), and hepatitis viruses B and C. The mechanism of action, major pharmacologic features, therapeutic uses related to transplant recipients, and genetic pathways of antiviral drug resistance are described for the FDA-approved drugs and some experimental compounds in late stage clinical trial.

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 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(50)]) 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.

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 duringin vitroCMV 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

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.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.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.

International audience; The human cytomegalovirus (HCMV) terminase complex consists of several components acting together to cleave viral DNA into unit length genomes and translocate them into capsids, a critical process in the production of infectious virions subsequent to DNA replication. Previous studies suggest that the carboxyl-terminal portion of the pUL56 subunit interacts with the pUL89 subunit. However, the specific interacting residues of pUL56 remain unknown. We identified a conserved sequence in the C-terminal moiety of pUL56 (671 WMVVKYMGFF 680). Overrepresentation of conserved aromatic amino acids through 20 herpesviruses homologues of pUL56 suggests an involvement of this short peptide into the interaction between the larger pUL56 terminase subunit and the smaller pUL89 subunit. Use of Alpha technology highlighted an interaction between pUL56 and pUL89 driven through the peptide 671 WMVVKYMGFF 680. A deletion of these residues blocks viral replication. We hypothesize that it is the consequence of the disruption of the pUL56-pUL89 interaction. These results show that this motif is essential for HCMV replication and could be a target for development of new small antiviral drugs or peptidomimetics. Human cytomegalovirus (HCMV), a beta herpesvirus, can cause serious diseases in immunocompromised patients. Current antiviral inhibitors (ganciclovir, cidofovir and foscarnet) all target the viral DNA polymerase. They have adverse effects and prolonged treatment can select for drug resistance mutations either in the viral pol-ymerase pUL54, the kinase pUL97 or both of them 1, 2. Thus, we need new drugs targeting others stages of repli-cation. The terminase complex is highly specific for HCMV, has no counterpart in the human organism, and thus represents a target of choice for new antivirals development. This has been confirmed by the recent development of letermovir in the transplant setting 3, 4. DNA packaging process requires several proteins such as pUL56 and pUL89, the large and small termi-nase subunits, respectively. Recently, four additional proteins were shown to be also implicated in this process, namely, pUL51, pUL52, pUL77, pUL93 5-10. This process is driven by specific interactions of protein-DNA and protein-protein to cleave and package unit length genomic DNA into an empty capsid. Evidence suggests that the large subunit pUL56 has a crucial role in DNA cleavage/packaging, containing many of the functional sites required for this process like interaction with the portal protein pUL104, endonu-clease activity, and more interestingly an ATP-binding site (amino acids 709 to 723) 11. Although the association between pUL56 and pUL89 has already been reported, the residues of pUL56 involved in the terminase complex integrity are still unknown 12, 13. Nevertheless, co-immunoprecipitation experiments showing an interaction between the C-terminal half of pUL56 (pUL56-Cter) and pUL89 were confirmed by other results 12, 13. Because knowledge of terminase functional and interaction domains is important both for the development of drugs targeting the DNA packaging stage and for the improvement of existing ones such as letermovir, the aim of the current study is to identify a minimum peptide of pUL56 with a putative key role in its interaction with pUL89. Sequence alignments encouraged us to focus on the putative involvement of one part of the pUL56

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 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

Methylenecyclopropane nucleoside (MCPN) analogs are being investigated for treatment of human cytomegalovirus (HCMV) infection because of favorable preclinical data and limited ganciclovir cross-resistance. Monohydroxymethyl MCPNs bearing ether and thioether functionalities at the purine 6 position have antiviral activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) in addition to HCMV. The role of the HCMV UL97 kinase in the mechanism of action of these derivatives was examined. When tested against a kinase-inactive UL97 K355M virus, a moderate 5- to 7-fold increase in 50% effective concentration (EC 50 ) was observed, in comparison to a 13- to 25-fold increase for either cyclopropavir or ganciclovir. Serial propagation of HCMV under two of these compounds selected for three novel UL97 mutations encoding amino acid substitutions D456N, C480R,and Y617del. When transferred to baseline laboratory HCMV strains, these mutations individually conferred resistance to all of the tested MCPNs, ganciclovir, and maribavir. However, the engineered strains also demonstrated severe growth defects and abnormal cytopathic effects similar to the kinase-inactive mutant. Expressed and purified UL97 kinase showed in vitro phosphorylation of the newly tested MCPNs. Thus, HCMV UL97 kinase is involved in the antiviral action of these MCPNs, but the in vitro selection of UL97-defective viruses suggests that their activity against more typical ganciclovir-resistant growth-competent UL97 mutants may be relatively preserved.

The prevalent use of ganciclovir and its oral prodrug valganciclovir as prophylaxis and therapy for cytomegalovirus (CMV) infection has increased the awareness of drug resistance, the risk of which increases with prolonged drug exposure, impaired host defenses, or insufficient drug delivery [1]. In high-risk populations such as seronegative recipients of a CMV-seropositive donor organ, the incidence of ganciclovir resistance among treated individuals is estimated to be in the 5%–12% range. Drug resistance is suspected when rising plasma viral loads or progressive disease occur during prolonged antiviral treatment. Although ganciclovir resistance was originally documented by phenotypic testing of CMV culture isolates, this is a slow process, unsuitable in contemporary clinical practice where viral isolation is rarely performed. Instead, genotypic resistance testing is now standard, and is based on the detection of diagnostic mutations in the UL97 kinase gene involved in the initial phosphorylation of ganciclovir or in the UL54 DNA polymerase gene that encodes the antiviral drug target for ganciclovir. The accuracy of genotypic resistance testing depends on a comprehensive and validated database linking specific mutations with levels of drug resistance. Extensive clinical experience has defined a set of UL97 mutations that are the most frequent initial laboratory markers of ganciclovir resistance (M460V/I, H520Q, C592G, A594V, L595S, and C603W), accounting for about 80% of documented cases [1]. In the remaining cases, less common UL97 mutations (clustered at codons 590–607) or UL54 mutation may be the first genetic marker of ganciclovir resistance. UL54 mutations typically add to pre-existing UL97 mutation to increase the level of ganciclovir resistance and confer cross-resistance to other anti-CMV drugs. An ongoing difficulty with interpretation of UL54 mutations is the relatively frequent occurrence of natural sequence polymorphisms unrelated to drug resistance. This difficulty is compounded in prophylaxis studies, where no baseline comparator sequence is available. UL97 and UL54 sequence variants remain incompletely documented despite several surveys of clinical CMV sequences [1–6]. A randomized nonblinded treatment trial comparing 3 weeks of oral valganciclovir and intravenous ganciclovir, followed by 4 weeks of valganciclovir, in solid organ transplant recipients (VICTOR study, NCT00431353) involved 321 subjects [7], of which 275 were included in a published resistance substudy [8]. Study subjects included those with a prior antiviral treatment history. Based on genotypic testing of prospectively collected samples, 13 subjects (4.7% of those tested) were considered to have confirmed or probable ganciclovir resistance mutations during the treatment period (0–49 days). Among the 13 cases, 10 had UL97 mutations only, 1 had both UL97 and UL54 mutations, and 2 were considered to have a probable mixture of resistance-associated and wild-type UL54 sequences without UL97 change [8]. Left unresolved were 110 UL54 amino acid sequence variants that could not be classified as known resistance mutations or natural sequence polymorphism. This study aimed to resolve the status of these sequence variants in relation to ganciclovir resistance. The extensive recombinant phenotyping needed for this purpose relied on recent technical advances in the construction and testing of cloned CMV strains.

Human cytomegalovirus UL97 kinase mutations that commonly confer ganciclovir resistance cluster in different parts of the gene than those conferring resistance to maribavir, an experimental UL97 kinase inhibitor. The drug resistance, growth, and autophosphorylation phenotypes of several unusual UL97 mutations in the kinase catalytic domain were characterized. Mutations V466G and P521L, described in clinical specimens from ganciclovir-treated subjects, conferred a UL97 kinase knockout phenotype with no autophosphorylation, a severe growth defect, and high-level ganciclovir, cyclopropavir, and maribavir resistance, similar to mutations at the catalytic lysine residue K355. Mutations F342S and V356G, observed after propagation under cyclopropavir in vitro , showed much less growth attenuation and moderate- to high-level resistance to all three drugs while maintaining UL97 autophosphorylation competence and normal cytopathic effect in cell culture, a novel phenotype. F342S is located in the ATP-binding P-loop and is homologous to a c-Abl kinase mutation conferring resistance to imatinib. UL97 mutants with relatively preserved growth fitness and multidrug resistance are of greater concern in antiviral therapy than the severely growth-impaired UL97 knockout mutants. Current diagnostic genotyping assays are unlikely to detect F342S and V356G, and the frequency of their appearance in clinical specimens remains undefined.

HCMV is a member of the family Herpesviridae and represents a worldwide distributed pathogen with seropositivity rates in the adult population ranging between 40% and 90%. Notably, HCMV infection is a serious, sometimes life-threatening medical problem for newborns and immunosuppressed individuals, including transplant recipients and patients under antitumoral chemotherapy. Current standard therapy with valganciclovir has the disadvantage of inducing drug-resistant virus mutants and toxicity-related side effects. Our analysis stresses the earlier finding that kinase inhibitors of the quinazoline class exert an antiviral response by targeting the viral protein kinase pUL97 without inducing resistance. Therefore, quinazolines have been used as a core structure to gain insight in the mode of inhibitor-kinase interaction. Here, we demonstrate that (i) the novel quinazolines Vi7392 and Vi7453 are highly active against HCMV laboratory and clinically relevant strains including maribavir- and ganciclovir-resistant variants, (ii) antiviral activity is not cell-type specific and was also detected in a placental explant tissue model using a genetically intact HCMV strain (iii) the viral kinase pUL97 represents a target of the anticytomegaloviral activity of these compounds, (iv) induction of pUL97-conferring drug resistance was not detectable under single-step selection, thus differed from the induction of ganciclovir resistance, and (v) pUL97 drug docking simulations enabled detailed insights into specific drug-target binding properties providing a promising basis for the design of optimized kinase inhibitors. These novel findings may open new prospects for the future medical use of quinazoline drug candidates and the use of drug-target dynamic simulations for rational design of antivirals. (C) 2016 Elsevier B.V. All rights reserved.

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.

Resistance to the experimental human cytomegalovirus (CMV) UL97 kinase inhibitor maribavir has been mapped to UL97 mutations at codons 353, 397, 409 and 411, in the kinase ATP-binding region, and to mutations in the UL27 gene. We studied the maribavir susceptibility phenotypes of additional UL97 mutations observed in vitro and in clinical trials, and the effect of simultaneous mutation in both UL97 and UL27. In vitro selection under maribavir identified a new locus of UL97 mutation within the conserved kinase p-loop (L337M), which conferred low grade maribavir resistance (3.5-fold increased EC50) without ganciclovir cross-resistance. During maribavir Phase III CMV prevention clinical trials, three previously unknown UL97 sequence variants were detected in plasma samples after 27-98 days of drug exposure (I324V, S334G and S386L). These variants did not confer any drug resistance despite proximity to mutations that confer maribavir resistance. The UL27 resistance mutation R233S, when added to strains containing UL97 mutations L337M or V353A, doubled their maribavir EC50s. These results expand the range of UL97 maribavir-resistance mutations into another part of the kinase ATP-binding region, but offer no genotypic evidence that development of drug resistance affected the outcomes of Phase III maribavir clinical trials after drug exposure of up to 14 weeks. There is a potential for increased maribavir resistance in UL27-UL97 double mutants.

Human cytomegalovirus (CMV) UL54 DNA polymerase ( pol ) mutants with known patterns of resistance to current antivirals ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) were tested for cyclopropavir (CPV) susceptibility by a standardized reporter-based yield reduction assay. Exonuclease and A987G (region V) mutations at codons commonly associated with dual GCV-CDV resistance in clinical isolates paradoxically conferred increased CPV susceptibility. Various polymerase catalytic region mutations conferring FOS resistance with variable low-grade GCV and CDV cross-resistance also conferred CPV resistance, with 50% effective concentration (EC 50 ) increases of 3- to 13-fold. CPV EC 50 values against several pol mutants were increased about 2-fold by adding UL97 mutation C592G. Propagation of a CMV exonuclease mutant under CPV selected for pol mutations less often than UL97 mutations. In 21 experiments, one instance each of mutations E756D and M844V, which were shown individually to confer 3- to 4-fold increases in CPV EC 50 , was detected. Unlike GCV and CDV, exonuclease mutations are not a preferred mechanism of CPV resistance, but mutations in and near pol region III may confer CPV resistance by affecting its recognition as an incoming base for DNA polymerization.

This review assesses recently published data on cytomegalovirus (CMV) antiviral drug resistance.Resistance is typically encountered after prolonged ganciclovir treatment for posttransplant primary CMV infection and is diagnosed by the detection of characteristic mutations in the viral UL97 kinase and UL54 DNA polymerase genes in clinical specimens. One of seven canonical UL97 mutations is detected in most cases of ganciclovir resistance, but many viral sequence variants of unknown relevance are being reported after drug exposure in vitro and in vivo. Rapid technical advances in recombinant phenotyping have shown that many of these variants confer no detectable drug resistance, whereas some unusual resistance mutations are newly confirmed. All currently marketed CMV antiviral drugs, including foscarnet and cidofovir, target the viral DNA polymerase, and cross-resistance may result from some UL54 mutations. To decrease cross-resistance and toxicity, there is an ongoing effort to develop anti-CMV drugs with different resistance pathways and alternative targets, such as the UL97 kinase or UL56-UL89 terminase enzymes.An increasing volume of information correlating CMV genotypes and drug susceptibility phenotypes is becoming available. This will improve the interpretation of sequence-based assays currently used for clinical diagnosis and guide the development of new antiviral drugs.

Current therapy options to treat infections with human cytomegalovirus face severe limitations leading to a continued search for novel drug candidates. Here, we describe novel characteristics of the strong antiviral potency of the drug artesunate. In vitro virus replication systems were applied to analyze a number of laboratory and clinically relevant strains of human cytomegalovirus. An inhibitory block at a very early stage of infection was demonstrated. Time-of-addition experiments indicated that the antiviral efficacy could be optimized when artesunate was applied as fractional doses consecutively added post-infection. Artesunate showed a clearly higher anti-cytomegaloviral activity than its parental drug artemisinin (approximately 10-fold) or other artesunate-related compounds. Mean IC(50) values of artesunate for a variety of standard therapy-resistant virus mutants were within a 2-fold range compared to wild-type virus. Furthermore, a synergistic effect was identified when artesunate was combined with the mechanistically distinct antiviral compound maribavir. These findings point to unique antiviral properties of artesunate which may offer an advantage over standard antiviral therapy particularly in cases of drug resistance.

Select mutations in the human cytomegalovirus (HCMV) gene UL27 confer low-grade resistance to the HCMV UL97 kinase inhibitor maribavir (MBV). It has been reported that the 608-amino acid UL27 gene product (pUL27) normally localizes to cell nuclei and nucleoli, whereas its truncation at codon 415, as found in a MBV-resistant mutant, results in cytoplasmic localization. We now show that in the context of full-length pUL27, diverse single amino acid substitutions associated with MBV resistance result in loss of its nucleolar localization when visualized after transient transfection, whereas substitutions representing normal interstrain polymorphism had no such effect. The same differences in localization were observed during a complete infection cycle with recombinant HCMV strains over-expressing full-length fluorescent pUL27 variants. Nested UL27 C-terminal truncation expression plasmids showed that amino acids 596-599 were required for the nucleolar localization of pUL27. These results indicate that the loss of a nucleolar function of pUL27 may contribute to MBV resistance, and that the nucleolar localization of pUL27 during HCMV infection depends not only on a carboxy-terminal domain but also on a property of pUL27 that is affected by MBV-resistant mutations, such as an interaction with component(s) of the nucleolus.

The anti-malaria drug artesunate has been shown to be an effective inhibitor of cytomegalovirus (CMV) in vitro , in an experimental animal model, and in a recent single-case clinical use. In this first case-series of 6 stem cell transplant recipients who received preemptive artesunate treatment for CMV infection, we have examined the viral kinetics following institution of artesunate, and employed first-phase viral kinetics studies to calculate its antiviral effectiveness. Two patients demonstrated a rapid 0.8–2.1 log viral load decline by 7 days, with a viral decay half-live of 0.9–1.9 days. Four patients demonstrated a continued yet stalled viral growth slope during treatment. No adverse events were noted in treatment courses of up to 28 days. Overall, a divergent antiviral efficacy was revealed, ranging from 43% to 90%, which appeared to be primarily dependent on the virus baseline growth dynamics. Further dose escalation studies are needed to examine the role of artesunate in the treatment of CMV infection in the transplantation setting.

Cytomegalovirus UL54 DNA polymerase mutations observed in clinical specimens are of diagnostic significance if confirmed to affect antiviral drug susceptibility.Validate an updated recombinant phenotyping method to determine the degree of drug resistance conferred by previously uncharacterized UL54 sequence variants, in comparison with known resistance-related mutations.Bacterial artificial chromosome clones of viral DNA were mutagenized by recombination, transfected to produce live virus and phenotyped by standardized reporter-based yield reduction assays.Sixteen recombinant viruses were constructed, representing baseline sequences, known resistance-related mutations and amino acid changes of unproven significance from clinical specimens. Phenotypes of baseline strains and known mutants were comparable to results from prior methods and helped to resolve some published inconsistencies. Mutations F412L, F412S, L545W were newly confirmed to confer ganciclovir and cidofovir resistance, while Q578H conferred ganciclovir and foscarnet resistance with borderline cidofovir resistance. Some foscarnet-resistant mutants were appreciably growth-retarded.Results add to known exonuclease domain mutations that confer ganciclovir-cidofovir cross-resistance, polymerase domain mutations that confer foscarnet resistance with variably decreased ganciclovir/cidofovir susceptibility, and increase the list of sequence variants with no measurable impact on drug susceptibility. The phenotypic diversity of similar UL54 genotypic variants complicates the interpretation of genotypic resistance testing. Technical improvements are facilitating the phenotyping of remaining unknown sequence variants.

Background. In a phase III controlled trial IMproved Protection Against Cytomegalovirus in Transplantation (IMPACT) comparing 200 with 100 days of valganciclovir prophylaxis in 318 cytomegalovirus D+/R— kidney transplant recipients, an equal number of patients (n=3 per arm) had known ganciclovir resistance mutations detected during viral breakthrough. In addition, many other viral sequence variants were observed that were of unknown significance for ganciclovir resistance. Recombinant phenotyping was performed to determine whether the previously uncharacterized genotypic changes affected ganciclovir susceptibility, especially in those receiving the longer duration of prophylaxis. Methods. Sequences encoding individual amino acid substitutions in the UL97 kinase or UL54 DNA polymerase gene were transferred by recombination into a cloned cytomegalovirus laboratory strain, followed by reporter-based yield reduction phenotypic assay of the resulting virus for ganciclovir susceptibility. Results. Twenty-six uncharacterized amino acid substitutions were detected, 2 in UL97 and 24 in UL54. All 10 substitutions in the 200-day arm and 9 of 17 substitutions in the 100-day arm (prioritized based on location and conservation) were selected for phenotyping; one substitution was detected in both subsets. Results were generated for nine of ten 200-day and eight of nine 100-day substitutions, with no substitution demonstrating a significant reduction in ganciclovir susceptibility. The two remaining amino acid substitutions, both in UL54, were not evaluated because of poor viral viability. Conclusion. Phenotypic evaluation of previously uncharacterized viral genotypes in the 200-day valganciclovir prophylaxis group showed no evidence of an increased incidence of genotypic ganciclovir resistance when compared with those in the 100-day prophylaxis group.

The experimental cytomegalovirus UL97 kinase inhibitor maribavir was used to treat 2 cases of infection in which viral mutations that conferred ganciclovir and foscarnet resistance had evolved sequentially. In one case, viral shedding was cleared without evidence of maribavir resistance in an isolate obtained after therapy. In the other case, a high-grade viremia was initially reduced 50-fold but rebounded 2 months later, coincident with the emergence of viral UL97 mutations T409M and H411Y, which confer maribavir resistance. The relatively rapid onset of maribavir resistance probably resulted from incomplete viral suppression in an immunosuppressed host with a high viral load.

Mutations in the human cytomegalovirus (CMV) UL97 protein kinase are the most common mechanism of ganciclovir (GCV) resistance in the clinical setting. A CMV strain with a previously unrecognized UL97 mutation N597D was identified in the blood of a heart transplant recipient who experienced a persistent CMV infection with high viral loads accompanying pain and fever while receiving valganciclovir (valGCV) therapy. The N597D mutation was transferred by mutagenesis to an antiviral sensitive CMV strain for analysis of antiviral susceptibility by standardized phenotypic assay. Recombinant phenotyping showed N597D conferred a less than twofold increase in GCV IC50 compared to the sensitive control strain. Despite the presence of this mutation, valGCV eventually resolved the infection after 6 weeks of therapy. A subsequent CMV reactivation was also responsive to valganciclovir. This case illustrates the diversity of UL97 mutations in the codon segment 590–607 usually associated with GCV resistance, with some mutations producing minimal levels of resistance that do not preclude a therapeutic response to the drug. Accurate interpretation of genotypic test results ultimately requires experimental determination of the level of resistance conferred by newly discovered UL97 mutations. J. Med. Virol. 81:507–510, 2009. © 2009 Wiley-Liss, Inc.

Mutations in the human CMV UL97 kinase gene are a major mechanism of viral resistance to two anti-CMV drugs, ganciclovir (GCV) and maribavir (MBV). GCV, the most widely used and established therapy for CMV, is a substrate for the UL97 kinase. Well-characterised GCV-resistance mutations at UL97 codons 460, 520 and 590-607 impair the phosphorylation of GCV that is necessary for its antiviral activity, presumably by altering substrate recognition. In contrast, MBV is an inhibitor of the UL97 kinase and is the first new CMV therapy to reach later stage clinical trials in many years. No MBV-resistant CMV isolates have yet been detected in clinical trials, but after culture propagation under drug, UL97 mutations that confer moderate to high-level MBV resistance have been identified at codons 353, 397, 409 and 411. These mutations are located upstream of the GCV-resistance mutations and are close to the ATP-binding and catalytic domains common to all kinases, consistent with MBV acting as a small molecule ATP-competitive kinase inhibitor. So far, no UL97 mutations are known to confer resistance to both GCV and MBV.

A human cytomegalovirus (CMV) UL54 pol exonuclease domain II mutation, D413A, found in a clinical specimen, conferred ganciclovir (GCV) and cidofovir resistance but not foscarnet resistance when incorporated into laboratory strain T2294. After several passages without drug, mutation was observed in five of eight plaques of T2294, and its plating efficiency under foscarnet was increased ∼30-fold over that of a control strain. When T2294 was serially passed under maribavir (MBV), phenotypic changes and viral UL97 mutations were detected by passage 5, much earlier than previously reported for other CMV strains. By passage 15, mutations included two cases of H411Y, one each of H411L and H411N, three of T409M, five of V353A, and one of L397R. Five instances of codon 409 or 411 mutations evolved into double mutations including V353A. Marker transfer experiments showed H411N/Y/L to confer 9- to 70-fold-increased MBV resistance and combinations of H411L/Y and V353A to confer >150-fold-increased MBV resistance, but no GCV resistance. These findings are consistent with defective exonuclease activity of the pol D413A mutant T2294, leading to the accelerated evolution of UL97 mutations under MBV. This recapitulated the known resistance mutations V353A, L397R, and T409M; suggested their relative frequency; and identified new ones at codon 411. These UL97 mutations predict an MBV binding region overlapping the kinase ATP binding site and located upstream of known GCV resistance mutations. The existence of viable pol D413A mutants may facilitate the selection of additional drug resistance mutations in vivo and the study of these mutations in vitro.

Background: We report on two allogeneic stem cell transplant recipients who developed cytomegalovirus disease associated with new viral mutations that conferred antiviral drug resistance. Methods: Blood specimens obtained during symptomatic disease were analyzed for mutations in the CMV UL97 and DNA polymerase genes and new mutations were assessed by recombinant phenotyping. Results: Rising cytomegalovirus (CMV) antigenemia occurred after 4–5 months of preemptive valganciclovir therapy, followed by symptomatic CMV disease including fatal pneumonia in one case. In one case, a new viral UL97 mutation (deletion of codons 601–603) was found which conferred 15-fold increased ganciclovir resistance. In the other case, a known UL97 resistance mutation M460V and a new DNA polymerase (pol) mutation D413A were found. D413A conferred ganciclovir and cidofovir resistance. Conclusions: Known and newly discovered drug resistance mutations arising during preemptive therapy may complicate post-transplant CMV disease in stem cell recipients. Improved recombinant phenotyping methods enable the rapid quantitation of the resistance conferred by newly identified UL97 and pol mutations.

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.

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.

Understanding the mechanisms of viral drug resistance is critical to the clinical management of individuals receiving antiviral therapy, the development of new antiviral drugs, and the surveillance of drug resistance. This chapter reviews the mechanisms of resistance to antiviral agents used to treat seven common viral infections, i.e., infections with herpes simplex virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), influenza A and B viruses, hepatitis B virus (HBV), and hepatitis C virus (HCV). Antiviral drug resistance is usually mediated by mutations in the molecular targets of drug therapy, and the development of drug resistance is the most compelling evidence that an antiviral drug acts by specifically inhibiting a virus rather than its cellular host. Drug-resistant viruses are identified by in vitro passage experiments with increasing concentrations of an inhibitory drug and by ex vivo analysis of virus isolates obtained from individuals receiving antiviral therapy. Antiviral agents, mechanisms of resistance, and mutations involved in drug resistance in herpesviruses are summarized in a tabular form. Most TK mutants are classified as TK negative, usually arising from frameshift or stop mutations that delete important functional domains, or TK partial, when a mutation reduces the phosphorylation of both natural nucleosides and antiviral drugs. Nucleoside analog RdRp inhibitors appear to be equally active against the different HCV genotypes. The genetic barrier to nucleoside analog resistance also appears to be higher than that for the other two main classes of HCV inhibitors.

Treatment of cytomegalovirus (CMV) infections with current antiviral drugs ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) may select for viral drug resistance mutations. CMV UL97 phosphotransferase gene mutations at codons 460, 520, and 590 to 607 have been shown to confer ganciclovir resistance (1, 4), while an increasingly diverse set of viral UL54 DNA polymerase (pol) mutations confer varying degrees of resistance and cross-resistance to the three drugs (2, 3). Resistance of CMV to antiviral drugs may be diagnosed genotypically, by looking for known viral resistance mutations in UL97 or pol, or phenotypically, by applying various drug concentrations to a growing virus in cell culture. Genotypic analysis is rapid and can be performed without the need for viral isolation, but mutations of indeterminate significance for resistance continue to be reported without phenotypic validation (8) and the degree of resistance conferred by combinations of mutations may be difficult to deduce by genotypic testing alone. Phenotypic analysis detects drug resistance without the need for genetic information, but even if a CMV isolate is available for testing, the standard plaque reduction assay is slow, technically demanding, and shows a high degree of interlaboratory variability (6). For diagnosis of CMV drug resistance in a clinical setting, genotypic methods are more practical, but this requires the continuing study of phenotypes corresponding to new mutations encountered in clinical isolates. The drug resistance phenotype corresponding to specific viral mutations can be determined by transferring them to a known drug-sensitive reference strain and testing for the resulting change in drug sensitivity. In the past few years this process, known as marker transfer or recombinant phenotyping, has been facilitated by the development of CMV strains containing unique restriction sites in UL97 or pol that permit the rapid generation of recombinant viruses containing desired mutations (2, 4). Phenotyping of the resulting recombinant viruses then becomes the rate-limiting step, especially when performed by the standard plaque reduction assay. Here, we report the construction of CMV strains containing unique restriction sites in UL97 and pol, along with a secreted alkaline phosphatase (SEAP) reporter gene inserted at the nonessential gene region US6. This enabled the generation of recombinant viruses containing desired mutations in either or both UL97 and pol, which could be quantitated by simply measuring the SEAP activity in the culture supernatant. Reduction in SEAP activity under drug was used to determine drug sensitivity. Results of transferring specific mutations were compared with results previously obtained using traditional phenotyping assays, and the effects of combinations of mutations were studied for the first time. (Presented in part at the Infectious Diseases Society of America Annual Meeting, Boston, Mass., October 2004 [abstract 641] and the 44th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, October 2004 [abstract 615].)

Hepatitis C virus (HCV)-related liver failure is the leading indication for liver transplantation worldwide. After transplantation, virological recurrence is the rule, but the spectrum of histological injury is wide, ranging from the development of allograft cirrhosis within a few years to minimal hepatitis despite long-term follow-up. The immunological correlates of this variable natural history are poorly understood. Here, we studied the kinetics of the cellular immune responses, viral replication, and allograft histology in 24 patients who had undergone liver transplantation for HCV-related liver failure. Using direct ex vivo methodologies (i.e., interferon-gamma ELISPOT and major histocompatibility complex class I–peptide tetrameric complexes), we found that patients who experienced viral eradication after antiviral therapy showed restoration of HCV-specific T-cell responses, whereas patients with progressive HCV recurrence that failed to respond to therapy showed declining frequencies of these viral-specific effector cells. The cytotoxic T lymphocytes that peripherally reconstituted after transplantation were clonotypically identical to those present within the recipient explant liver, defined at the level of the T-cell receptor beta chain (one epitope/one clone). Moreover, the subset of patients who spontaneously demonstrated minimal histologic recurrence had more vigorous CD4+ T-cell responses in the first 3 months, particularly targeting nonstructural proteins. We provide evidence that T-cell responses emerge after liver transplantation, and their presence correlates with improved histological and clinical outcomes. In conclusion, these results may help identify patients more likely to develop severe HCV recurrence and therefore benefit from current antiviral therapy, as well as provide a rationale for the future use of novel immunotherapeutic approaches. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;41:72–81.)

The goal of this study was to develop a new surrogate challenge model for use in evaluating protective cell-mediated immune responses against hepatitis C virus (HCV) antigens. The use of recombinant Listeria monocytogenes organisms which express HCV antigens provides novel tools with which to assay such in vivo protection, as expression of immunity against this hepatotropic bacterial pathogen is dependent on antigen-specific CD8 + T lymphocytes. A plasmid DNA vaccine encoding a ubiquitin-NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma interferon-secreting T cells following vaccination of BALB/c mice. These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant L. monocytogenes strain (TC-LNS3) expressing the NS3 protein. Notably, sublethal infection of naive mice with strain TC-LNS3 induced similar NS3-specific T-cell responses. These findings suggest that recombinant strains of L. monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens.

BACKGROUND: The Viral Activation Transfusion Study (VATS) afforded an opportunity to determine whether blood transfusions, and in particular exogenous WBCs, “activate” CMV replication in HIV-infected, CMV-seropositive patients, and whether such patients can be superinfected by additional strains of CMV transmitted via blood transfusions. STUDY DESIGN AND METHODS: A total of 531 patients were randomized to receive either WBC-reduced (WBCR) or non-WBC-reduced (NWBCR) RBC units. Plasma CMV PCR assays were performed before transfusion and weekly after transfusion for 4 weeks. NWBCR cases with evidence of possible reactivation and/or superinfection were further studied for donor viremia by DNA PCR of frozen retention segments and new genotype acquisition using gB envelope sequence analysis of pre- and posttransfusion recipient specimens. RESULTS: VATS patients received a median of two RBC units during their initial transfusion. Whether positive or negative for CMV DNA at baseline, there were no significant treatment-arm differences in the percentage of patients who had positive qualitative CMV PCR or increases in CMV viral load at follow-up. Of 50 recipients randomized to NWBCR RBC and meeting criteria for possible CMV superinfection, 25 had sufficient CMV DNA load in a baseline and one or more viremic follow-up sample to permit comparison of gB genotypes. Only two recipients showed genotype shifts. Of 125 WBC pellets prepared from the seropositive units transfused into these 50 cases, only 1 tested weakly PCR positive for CMV DNA (insufficient copy number for genotyping). CONCLUSION: There was no evidence of “activation” of CMV by blood transfusion. Among the NWBCR RBC recipients, there was little evidence of possible transmission of new CMV strains. Hence, the current policy for transfusion support of HIV-infected patients, which allows transfusion of CMV-antibody-positive blood to CMV-seropositive patients, is appropriate.

We describe a 49-year-old man with human immunodeficiency virus infection and stable chronic renal insufficiency who developed acute oliguric renal failure and severe lactic acidosis and who died several weeks after tenofovir was added to an antiretroviral regimen that included didanosine. Although the role of tenofovir in precipitating acute renal failure is unclear, progressive accumulation of the drug and pharmacologic interaction that caused increased levels of didanosine were the likely antecedents of increased mitochondrial toxicity that led to lactic acidosis.

Objective: To characterize reverse transcriptase (RT) mutations by their association with extent of nucleoside RT inhibitor (NRTI) therapy. To identify mutational clusters in RT sequences from persons receiving multiple NRTI. Design: Atotal of 1210 RT sequences from persons with known antiretroviral therapy were analyzed: 641 new sequences were performed at Stanford University Hospital; 569 were previously published. Methods: Chi-square tests and logistic regression were done to identify associations between mutations and NRTI therapy. Correlation studies were done to identify mutational clusters. The Benjamini–Hochberg procedure was used to correct for multiple comparisons. Results: Mutations at 26 positions were significantly associated with NRTI including 17 known resistance mutations (positions 41, 44, 62, 65, 67, 69, 70, 74, 75, 77, 116, 118, 151, 184, 210, 215, 219) and nine previously unreported mutations (positions 20, 39, 43, 203, 208, 218, 221, 223, 228). The nine new mutations correlated linearly with number of NRTI; 777 out of 817 (95%) instances occurred with known drug resistance mutations. Positions 203, 208, 218, 221, 223, and 228 were conserved in untreated persons; positions 20, 39, and 43 were polymorphic. Most NRTI-associated mutations clustered into three groups: (i) 62, 65, 75, 77, 115, 116, 151; (ii) 41, 43, 44, 118, 208, 210, 215, 223; (iii) 67, 69, 70, 218, 219, 228. Conclusions: Mutations at nine previously unreported positions are associated with NRTI therapy. These mutations are probably accessory because they occur almost exclusively with known drug resistance mutations. Most NRTI mutations group into one of three clusters, although several (e.g., M184V) occur in multiple mutational contexts.