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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.

In lieu of a licensed vaccine, antivirals are being considered as an intervention to prevent congenital human cytomegalovirus (HCMV) infection. Ideally, antiviral therapies should undergo pre-clinical evaluation in an animal model prior to human use. Guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV. However, GPCMV is not susceptible to the most commonly used HCMV antiviral, ganciclovir (GCV), rendering in vivo study of this agent problematic in the guinea pig model. Human cytomegalovirus (HCMV) susceptibility to GCV is linked to the UL97 gene. We hypothesized that GPCMV susceptibility to GCV could be improved by inserting the HCMV (Towne) UL97 gene into the GPCMV genome in place of the homolog, GP97. A chimeric GPCMV (GPCMV::UL97) expressed UL97 protein, and replicated efficiently in cell culture, with kinetics similar to wild-type GPCMV. In contrast, deletion of GP97 resulted in a virus (GPCMVdGP97) that grew poorly in culture. GPCMV::UL97 had substantially improved susceptibility to the inhibitory effects of GCV in comparison to wild-type GPCMV. Additionally, GPCMV::UL97 exhibited improved susceptibility to another antiviral undergoing clinical trials, maribavir (MBV; benzimidazole riboside 1263W94), which also acts through UL97.

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.

The effect of renal function on the pharmacokinetics of maribavir, a novel anticytomegalovirus agent, was evaluated in 12 adults with normal renal function (creatinine clearance [CrCl] >80 mL/min) and 19 adults with renal impairment classified as mild (n = 5), moderate (n = 5), or severe (n = 9), as measured by CrCl 50-80, 30-49, and

Cytomegalovirus (CMV) infection is one of the most important infectious complications of solid-organ transplantation, a serious, life-threatining, opportunistic pathogen in HIV-infected patients, and may cause hearing defects and irreversible central nervous system disease in infants infected during gestation. Four drugs are currently licensed for prophylaxis, pre-emptive therapy, and treatment of CMV infection -- ganciclovir, and its oral prodrug valganciclovir, foscarnet, cidofovir, and fomivirsen. All four drugs are effective against CMV infection. Toxicities, drug-drug interactions, poor bioaailibility, and the development of drug resistance, however, are clinically relevant and common limitations of these drugs. Novel compounds are on the horizon that possibly will become useful alternatives to currently licensed drugs. Maribavir, a benzimidazol, is the most promising novel drug and closest to clinical application. Several phase II clinical trials proved its good tolerability and effectivity. Other compounds are currently evaluated in pre-clinical and phase I trials with promising preliminary data. In addition, analogs of cidofovir posess significantly improved pharmacological and virological characteristics allowing their oral administration. This review summarizes the current status in drug development and will introduce the most recent patents on this line of research.

Human cytomegalovirus (HCMV) is the leading cause of congenital infections. Symptomatic newborns present with a range of sequelae including disorders of the CNS such as visual impairment, microcephaly, mental retardation and hearing loss. HCMV congenital infection causes gross changes in brain morphology and disturbances in glial and neuronal distribution, number and migration. In these studies, we have evaluated the effectiveness of the antiviral maribavir in inhibiting HCMV infections of ES cell-derived neuronal progenitor cells (NPC). We used EZ-spheres generated from H9 ES cells which are pre-rosette NPCs that retain long-term potential to differentiate into diverse central and peripheral neural lineages following directed differentiation. Our results demonstrate that the maribavir disrupts HCMV replication and viral yield in undifferentiated EZ-sphere-derived NPCs. In addition, we observed that maribavir limits HCMV replication and reduces the percentage of infected cells during differentiation of NPCs. Finally, early steps in differentiation are maintained during infection by treating with maribavir, likely an indirect effect resulting from decreased viral spread. Future studies of NPC proliferation and differentiation during infection treated with maribavir could provide the impetus for studying maribavir as an antiviral agent for congenital HCMV disease.

Opinion statement In treating cytomegalovirus (CMV) infection, it is crucial to decide whether one is treating pre-emptively or if one is treating established disease. Disease may be further divided into viral syndrome and tissue-invasive disease. Generally, mild disease in immunosuppressed patients may be treated with oral valganciclovir. Treatment may also be started with valganciclovir for CMV retinitis in AIDS patients. In other tissue-invasive syndromes, starting with intravenous ganciclovir or foscarnet at full doses (adjusted for renal function) is preferred. Treatment at full doses should be continued until symptom resolution and until blood antigenemia (or DNAemia) is cleared. Patients receiving treatment must be closely monitored for side effects to the drugs, as well as for response. Drug-resistant CMV is a therapeutic challenge; combination therapy with both ganciclovir and foscarnet may be tried. In extreme cases, resorting to unconventional agents like leflunomide or maribavir may be necessary. Immune reconstitution, through reduction in immunosuppression, or the introduction of anti-retroviral therapy, should be attempted. CMX001 is a novel agent active against double-stranded viruses; thus far, resistance to CMX001 does not confer resistance to ganciclovir or foscarnet. Hence, prophylaxis or pre-emptive treatment with CMX001 may allow the use of ganciclovir or foscarnet for treatment.

Multidrug-resistant (MDR) cytomegalovirus (CMV) emerged after transient responses to ganciclovir, foscarnet, and cidofovir in a CMV-seropositive recipient who underwent allogeneic hematopoietic stem cell transplantation from a CMV-seronegative donor. Experimental treatments using leflunomide and artesunate failed. Re-transplantation from a CMV-seropositive donor supported by adoptive transfer of pp65-specific T cells and maribavir was followed by lasting suppression. This case illustrates that successful MDR CMV therapy may require individualized multidisciplinary approaches.

Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21Cip1. In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21Cip1. Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21Cip1in wild-type virus but not a pUL27-deficient virus. We manipulated the p21Cip1levels to evaluate the functional consequence to MBV. Overexpression of p21Cip1restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21Cip1in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21Cip1and support the idea that CDKs can complement some activities of pUL97.IMPORTANCEHCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21Cip1. In contrast, we provide evidence that p21Cip1functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21Cip1activators might be useful in combination with MBV to effectively inhibit HCMV infections.

Cytomegalovirus (CMV) is a herpes virus that causes severe disease in the immunocompromised because virus replication is normally held in check by cell-mediated immune responses in patients with past infection. Patients receiving immunosuppressive drugs to allow allo-transplantation, patients with AIDS (acquired immunodeficiency syndrome) or foetuses with immature immune responses are therefore at risk of developing CMV end-organ disease. A high viral load is required for CMV end-organ disease to develop. Ganciclovir is a potent inhibitor of CMV replication and can be used either for prophylaxis or for pre-emptive therapy (administering the drug when CMV is detected in the blood). Both of these strategies effectively control CMV end-organ disease in transplant patients. Three new drugs have recently completed phase II clinical trials (maribavir, brincidofovir and letermovir). Phase II studies of prototype CMV vaccines have recently published encouraging results, suggesting that CMV infection may ultimately be preventable by means of universal immunisation. Key Concepts CMV viraemia precedes CMV end-organ disease. Prompt treatment of CMV viraemia prevents end-organ disease. The probability of end-organ disease is non-linear with respect to viral load. This threshold relationship is seen also in babies with congenital CMV infection and hearing loss. Prompt treatment of congenital CMV infection can reduce the risk of progressive hearing loss. Quantitative viraemia biomarkers can be used to assess the potency of antiviral drugs in humans. These same biomarkers can be used to determine if vaccine given pre-transplant can help control CMV replication post-transplant. Although clinically silent, CMV infection is associated with excess mortality in the general public. Universal immunisation has the potential to provide health benefits greater than appreciated at present. Keywords: cytomegalovirus; viral load; threshold; dynamics; indirect effects

Since human cytomegalovirus (CMV) is extremely species specific and does not replicate in experimental animal tissues, animal models for the evaluation of antiviral agents for these infections have utilized surrogate animal viruses including murine CMV, rat CMV and guinea pig CMV. Murine CMV and rat CMV infections in normal and immunocompromised animals provide models of disseminated infection and are ideal for screening of new agents. While guinea pig CMV infection in immunocompromised animals also provides a model for disseminated disease, the model for congenital CMV is unique among all the experimental models. While these models have played a major role in the development of ganciclovir, foscarnet and cidofovir, they do not provide information directly related to human CMV, nor are they useful for evaluation of agents that are active only against human CMV. The SCID-hu mouse models in which human tissue is infected with human CMV has been very useful in the development of new antiviral agents such as maribavir and cyclopropavir. Collectively these experimental CMV infections provide a variety of models representing various aspects of CMV infection in humans that are highly predictive for antiviral efficacy in humans.

Cytomegalovirus infections are associated with severe morbidity and mortality is patients at risk for disease because of immune system disabilities; in particular, recipients of stem cell (HSCT) or solid organ (SOT) transplants. There are three systemic drugs approved for CMV treatment: ganciclovir, or its prodrug valganciclovir, foscarnet, and cidofovir. An anti-sense therapeutic, ISIS 2922, is also approved specifically as in intravitreal treatment for CMV retinitis. Ganciclovir, and more recently, valganciclovir, have been useful in proactive approaches of CMV disease management; in both prophylactic and preemptive regimens in HSCT and SOT populations. The major anti-herpes agent valacyclovir has also been approved for prophylaxis of renal transplant recipients, or SOTs outside of the US. These drugs have provided major advances in CMV disease management, although they are limited by intolerable toxicities, oral bioavailability and efficacy, and risk of drug resistance with extended use. Several drugs are in early clinical development which may address these limitations; this review will provide an overview of our current arsenal of available drugs, and of those in the early clinical development pipeline.

The rapid in vivo degradation of the potent human cytomegalovirus inhibitor 2-bromo-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BDCRB) compared with a structural L-analog, maribavir (5,6-dichloro-2-(isopropylamino)-1-β- L-ribofuranosyl-1H-benzimidazole), has been attributed to selective glycosidic bond cleavage. An enzyme responsible for this selective BDCRB degradation, however, has not been identified. Here, we report the identification of two enzymes, 8-oxoguanine DNA glycosylase (OGG1) and N-methylpurine DNA glycosylase (MPG), that catalyze N-glycosidic bond cleavage of BDCRB and its 2-chloro homolog, 2,5,6-trichloro-1-(β-D-ribofuranosyl)benzimidazole, but not maribavir. To our knowledge, this is the first demonstration that free nucleosides are substrates of OGG1 and MPG. To understand how these enzymes might process BDCRB, docking and molecular dynamics simulations were performed with the native human OGG1 crystal coordinates. These studies showed that OGG1 was not able to bind a negative control, guanosine, yet BDCRB and maribavir were stabilized through interactions with various binding site residues, including Phe319, His270, Ser320, and Asn149. Only BDCRB, however, achieved orientations whereby its anomeric carbon, C1′, could undergo nucleophilic attack by the putative catalytic residue, Lys249. Thus, in silico observations were in perfect agreement with experimental observations. These findings implicate DNA glycosylases in drug metabolism.

Maribavir (1263W94, VP-41263) is an oral anticytomegalovirus agent under clinical development. The pharmacokinetics and safety of maribavir and the effects of maribavir on the activities of cytochrome P450 (CYP) 1A2, CYP 2C9, CYP 2C19, CYP 2D6, CYP 3A, N -acetyltransferase-2 (NAT-2), and xanthine oxidase (XO) were evaluated in a randomized, double-blind, placebo-controlled study. Twenty healthy subjects received a five-drug phenotyping cocktail of caffeine (CYP 1A2, NAT-2, XO), warfarin plus vitamin K (CYP 2C9), omeprazole (CYP 2C19), dextromethorphan (CYP 2D6), and midazolam (CYP 3A) 4 days before and after 7 days of treatment with maribavir at 400 mg twice daily (16 subjects) or placebo (4 subjects) for 10 days. Maribavir did not affect the CYP 1A2, CYP 2C9, CYP 3A, NAT-2, or XO activities. Bioequivalence was not demonstrated for CYP 2C19 and CYP 2D6, suggesting a decrease or inhibition of CYP 2C19 and CYP 2D6 activities. The pharmacokinetics of maribavir following a single dose and after 10 days of treatment were similar, with minimal accumulation at steady state. Maribavir was safe and well tolerated. Taste disturbance was the most frequently reported adverse event. These results will further guide evaluation of the drug interaction potential and clinical development of maribavir.

In analogy with maribavir [1‐(β‐L‐ribofuranosyl)‐isopropylamino‐5,6‐dichlorobenzimidazole], a nucleoside analogue that acts against human cytomegalovirus (HCMV) by a non‐nucleoside mechanism, here I present three other examples of classes of nucleoside analogues (i.e. bicyclic furo[2,3‐d]pyrimidine as well as HEPT and TSAO derivatives) that act against either HCMV or human immunodeficiency virus (HIV) through a non‐nucleoside mode of action. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8) are responsible for a number of clinical manifestations in both normal and immunocompromised individuals. The parent benzimidazole ribonucleosides evaluated in this series, 2-bromo-5,6-dichloro-1-(β- d -ribofuranosyl)benzimidazole (BDCRB) and maribavir (1263W94), are potent and selective inhibitors of human CMV replication. These nucleosides act by two different mechanisms. BDCRB blocks the processing and maturation of viral DNA, whereas 1263W94 inhibits the viral enzyme pUL97 and interferes with DNA synthesis. In the present study, we have evaluated the in vitro antiviral activity of BDCRB, an analog, GW275175X (175X), and 1263W94 against the replication of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6, and HHV-8. By using various methodologies, significant activity was observed against human CMV and EBV but not against HSV-1, HSV-2, VZV, HHV-6, or HHV-8. Plaque reduction assays performed on a variety of laboratory and clinical isolates of human CMV indicated that all strains, including those resistant to ganciclovir (GCV) and foscarnet, were sensitive to all three benzimidazole ribonucleosides, with mean 50% effective concentration values of about 1 to 5 μM compared to that of GCV at 6 μM. The toxicity of these compounds in tissue culture cells appeared to be similar to that observed with GCV. These results demonstrate that the benzimidazole ribonucleosides are active against human CMV and EBV and suggest that they may be useful for the treatment of infections caused by these herpesviruses.

1263W94 [maribavir; 5,6-dichloro-2-(isopropylamino)-1, β- l -ribofuranosyl-1- H -benzimidazole], a novel benzimidazole compound, has been demonstrated to potently and selectively inhibit human cytomegalovirus replication in vitro and to have favorable safety profiles in animal species. Two phase I trials evaluated the safety and pharmacokinetics of escalating single doses of 1263W94 in 13 healthy subjects (dose, 50 to 1,600 mg) and 17 human immunodeficiency virus (HIV)-infected subjects (dose, 100 to 1,600 mg). No severe safety concerns were observed in the evaluation of adverse events, vital signs, electrocardiograms, and clinical laboratory tests following administration of a single dose of 1263W94. The most frequently reported adverse events in both populations were taste disturbance (80%) and headache (53%). 1263W94 was rapidly absorbed following oral administration, with peak concentrations in plasma ( C max ) occurring 1 to 3 h after dosing. The increases in the C max of 1263W94 and the area under the concentration-time curve from time zero to infinity (AUC 0-∞ ) for 1263W94 were dose dependent; C max increased slightly less than proportionally to the dose, and AUC 0-∞ increased slightly more than proportionally to the dose. 1263W94 was rapidly eliminated, with a mean half-life in plasma of 3 to 5 h; the half-life was independent of the dose level. Less than 2% of the 1263W94 dose administered was eliminated unchanged in urine. The principal metabolite of 1263W94 was 4469W94 (which is derived by N-dealkylation of 1263W94 via CYP3A4), which accounted for 30 to 40% of the dose in urine. Greater than 98% of the 1263W94 in plasma is bound to proteins, and the extent of binding appears to be constant over the dose range of 200 to 1,600 mg. In the trial with HIV-infected subjects, consumption of a high-fat meal decreased the 1263W94 AUC 0-∞ and C max in plasma by ∼30%.

Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.

This study describes the extent of cross-resistance and interactions for selected inhibitors of human cytomegalovirus (HCMV) DNA synthesis and DNA processing. HCMV isolates resistant to the benzimidazole D-ribonucleoside viral DNA processing inhibitors TCRB and BDCRB were sensitive to BAY 38-4766, a non-nucleoside inhibitor of viral DNA processing. This indicates that these two drug types have distinct interactions with the products of HCMV genes UL56 and UL89 required for viral DNA cleavage and packaging. These virus isolates also were sensitive to ganciclovir (GCV) but slightly resistant to the L-benzimidazole ribonucleoside viral DNA synthesis inhibitor 1263W94. Virus resistant to 1263W94 remained sensitive to BDCRB, GCV, and BAY 38-4766. Examination of drug-drug interactions in cell culture assays measuring inhibition of HCMV replication revealed strong synergism for the combination of BDCRB with 1263W94, and for combinations of 1263W94 with cidofovir (CDV) and foscarnet (PFA), but not with GCV. Combinations of GCV with CDV and PFA were synergistic as well. The combination of GCV with 1263W94 showed additive antiviral interactions, whereas, a combination of BAY 38-4766 with GCV showed antagonism. Interaction of BDCRB with BAY 38-4766 showed a mixed pattern of synergy and antagonism. The antiviral synergy observed between GCV and PFA or CDV serves to validate clinical combination therapies for these drugs. Antagonism seen for BAY 38-4766 with GCV indicates that these two drugs are unlikely to be useful for combination therapies. Notably, 1263W94 demonstrated greater synergy in combination with PFA or CDV than did GCV, suggesting some promise for this benzimidazole L-riboside in such combination therapies.

1263W94 [maribavir; 5,6-dichloro-2-(isopropylamino)-1,β- l -ribofuranosyl-1-H-benzimidazole] is a novel benzimidazole compound for treatment of human cytomegalovirus (HCMV) infection and disease, with potent in vitro activity against HCMV and good oral bioavailability. A phase I study was conducted to determine the pharmacokinetics (PK), anti-HCMV activity, and safety of 1263W94 administered as multiple oral doses to human immunodeficiency virus type 1-infected adult male subjects with asymptomatic HCMV shedding. Subjects received one of six dosage regimens (100, 200, or 400 mg three times a day, or 600, 900, or 1,200 mg twice a day) or a placebo for 28 days. 1263W94 demonstrated linear PK, with steady-state plasma 1263W94 profiles predictable based on single-dose data. 1263W94 was rapidly absorbed following oral dosing, and values for the maximum concentration of the drug in plasma and the area under the concentration-time curve increased in proportion to the dose. 1263W94 demonstrated in vivo anti-HCMV activity in semen at all of the dosage regimens tested, with mean reductions in semen HCMV titers of 2.9 to 3.7 log 10 PFU/ml among the four regimens evaluated for anti-HCMV activity. 1263W94 was generally well tolerated; taste disturbance was the most frequently reported adverse event over the 28-day dosing period.

Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathioneS-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified32P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.

The limitations of current therapies for human cytomegalovirus (HCMV) coupled with the continued impact of HCMV disease in the immunocompromised host are the driving force for the development of new drugs against HCMV. This review predominantly focuses on new non-DNA polymerase inhibitors of HCMV replication. Drugs such as tomeglovir (BAY-384766), 2-bromo-5,6-dichloro-1beta-D-ribofuranosyl benzimidazole (BDCRB) and GW-275175X act as inhibitors of the terminase complex that is involved in cleavage and packaging of the unit length DNA into the capsids. Although the viral protein kinase UL97 has been exploited as an activator of ganciclovir and its prodrug valganciclovir, a new inhibitor maribavir (benzamidavir) has been shown to be a highly potent inhibitor of this enzyme. Many of these compounds have undergone successful phase I clinical trials. There are other compounds which have been identified through drug-screening but are at the earlier stages of development.

Antiviral drugs and antiviral drug resistance can be matters of life and death for patients suffering from viral infections. They also can be valuable tools for understanding virus biology and biochemistry. Triumphs of antiviral therapy include acyclovir to treat genital herpes [1–4], triple combination chemotherapy for AIDS [5,6], and, quite recently, telaprevir or boceprivir in combination with interferon α and ribavirin for chronic hepatitis caused by hepatitis C virus (HCV) [7–11]. Triumphs of the use of antiviral drugs and drug resistance for laboratory investigation include the discovery of the M2 ion channel of influenza A virus (amantadine) [12–15] and the UL97 protein kinase of human cytomegalovirus (HCMV) (ganciclovir) [16–19], and the first crystal structure of human immunodeficiency virus (HIV) reverse transcriptase (nevirapine) [20]. Most antiviral drugs inhibit viral functions such as viral polymerases, but increasingly host functions are being targeted for antiviral development, as exemplified by the anti-HIV drug maraviroc [21]. Thus, although antiviral drugs and resistance are relatively mature fields, there are still breakthroughs emerging in the clinic and in the laboratory. The first highly successful antiviral drugs to be developed act against herpesviruses. Although these drugs, such as acyclovir for herpes simplex virus (HSV) and varicella zoster virus (VZV) infections, and ganciclovir for HCMV infections, are widely used, they suffer a number of drawbacks. Mark Prichard and Nathan Price review these drawbacks and ongoing efforts to develop new antiherpesvirus drugs and their mechanisms of action and resistance. These newer drugs include variations on old standbys — nucleoside analogs that target viral polymerases — as well as compounds that inhibit newer targets such as a viral helicase-primase involved in HSV DNA replication, a viral terminase involved in HCMV DNA packaging, and the viral UL97 protein kinase involved in multiple stages of HCMV infection. Some of these compounds have completed phase 2 or phase 3 trials. Prichard and Price argues that the future of antiherpesvirus therapy should include drug combinations, which would likely require drugs that act against multiple targets. Toward the end of better understanding the value of antiviral therapy for HCMV infections, Francisco Marty and Michael Boeckh detail the clinical trial of maribavir, which inhibits the UL97 protein kinase, for the management of infection in stem cell transplant populations. The importance of carefully defining primary endpoints is emphasized. Antiviral drugs are important components for the control of influenza. Influenza causes annual epidemics of respiratory viral infections that result in significant morbidity and mortality. Influenza vaccines have been shown to reduce the risk of infection and mitigate against the virus’ sequelae. Currently, there are two approved classes of antiviral drugs in the United States for the treatment of influenza: adamantanes (amantadine and rimantadine) and the neuraminidase (NA) inhibitors (NAIs; oseltamivir, and zanamivir). Several other classes of antiviral agents and immune modulators are also currently under investigation. Michael Ison teviews the currently approved and investigational antiviral agents and the mechanisms of resistance that impact their activity. Certainly, one of the greatest challenges to our armamentarium of antiviral drugs is the emergence of resistant mutants. The key question is whether antiviral use or natural virus evolution will lead to the emergence of drugresistant virus with comparable or superior fitness to their drug-susceptible counterparts. Currently, NAIs are the first choice for influenza prevention and treatment. Tatiana Baranovicha, Robert Webster and Elena Govorkova review the complex process of risk assessment for the fitness of NAI-resistant seasonal H1N1 and H3N2, pandemic 2009 H1N1, and highly pathogenic H5N1 influenza A viruses: identification of antiviral susceptibility, degree of functional NA loss, molecular markers of resistance, and evaluation of explicative ability for in vivo virulence and transmissibility in animal studies (mouse, ferret and guinea pig models). There are more drugs against HIV than against any other virus, and also more targets against which antiviral drugs have been developed. This plethora of drugs and targets has permitted combination chemotherapy, which is necessitated by the ease with which HIV develops drug resistance. Even with combination chemotherapy, resistance has continued to be a problem, although as reviewed by Daniel Kuritzkes, the patterns of resistance have changed over the past decade. He further reviews recent developments in resistance to several newer anti-HIV drugs, including the newer non-nucleoside reverse transcriptase inhibitors, the integrase inhibitors, and the CCR5 antagonists (e.g. maraviroc), which block a host co-receptor. Of note are the detailed molecular differences in resistance mechanisms even with drugs of the same class. Even as progress in HIV therapy continues, the virus will evolve to spur the development of still more anti-HIV drugs. The era of direct acting antiviral (DAA) therapy for HCV infection has dawned. The development of DAA therapy is an exciting advance for clinicians and patients, but it will also bring new challenges. Until 2011, the standard of care for chronic HCV was the administration of pegylated interferon α in combination with oral ribavirin, a nucleoside analogue. In 2011, the standard of care became these two agents combined with either telaprevir or boceprivir, which are inhibitors of the HCV NS3/4A protease. Although this three-drug combination is more efficacious than the previous standard of care, patients must still receive intravenous infusions of interferon, and take a drug, ribavirin, whose mechanism of action remains mysterious. Knowing more about how ribavirin abets therapy of HCV could open opportunities for more rational approaches. Jan Paeshuyse, Kai Dallmeier, and Johan Neyts review the large, perplexing, and often contradictory literature on ribavirin and HCV. The field is further complicated by the difficulty in knowing whether the drug’s direct in vitro action against HCV is relevant to its ability to augment interferon therapy, as the drug has little if any antiviral effect by itself in patients at clinically achievable concentrations. The authors identify at least five potential mechanisms by which ribavirin can act, some of which entail direct antiviral action and others of which entail effects on acquired or innate immunity. Most of these mechanisms are not mutually exclusive. Sorting out the ribavirin puzzle should shed light on HCV biology and the HCV-host interaction. For the first time, drug resistance has become an issue to consider in the management of HCV. Alex Thompson, Stephen Locarnini, and Michael Beard summarize the current literature concerning resistance to the HCV NS3/4A protease inhibitors, both investigational and clinical, and identify the key questions facing the field. In the meantime, there are a variety of new drugs on the horizon for HCV. Priscilla Yang, Min Gao, Kai Lin, Qingsong Liu, and Valerie Villareal discuss these. Of interest, some of these drugs, such as R7128 and BMS-790052, directly act against viral proteins to block HCV replication, while others such as Debio-025, act against host factors such as cyclophilins that are important for HCV. Several other viral and host targets have been identified, and therapeutic strategies exploiting these targets are in development. Both classes of drugs and targets have provided new insights into HCV biology, particularly regarding the enigmatic NS5A protein, and both types of drugs have considerable promise for improving therapy of HCV infections, especially as resistance appears to arise much more slowly for some of these drugs than for protease inhibitors. There is considerable optimism that combinations of newer drugs could lead to cures of HCV infections without resorting to interferon or ribavirin. The past twenty years have demonstrated the incredible abilities of viruses to evolve to become resistant to antiviral drugs, and how important it is to the effectiveness of antiviral therapy whether resistant variants are fit. Thus, drug resistance is both a spur to efforts to develop new drugs, and a key consideration in conceiving and executing drug development. It will be interesting to see how viruses and scientists adapt during the next two decades.

International audience

Cytomegalovirus (CMV) remains a significant cause of morbidity and mortality in immunocompromised patients, particularly following allogeneic haematopoietic transplantation. One of the principal factors leading to this increased risk is the loss of T-cell immunity.In a recent review, we assessed the treatment strategies for prophylaxis and pre-emptive treatment of CMV, particularly where relevant to the high-risk patient populations following allogeneic haematopoietic transplantation. This review is a focused update to our previous article and presents a more detailed analysis of the developments in drugs, vaccines and adoptive T-cell therapies since that time. Relevant studies were selected from PubMed and clinicaltrials.gov. The search terms include allogeneic transplant, cytomegalovirus, multidrug-resistant virus and adoptive T-cell therapy.The current randomised controlled studies evaluating pharmacological agents for CMV should inform as to whether these provide significant clinical benefits. Adoptive cell therapy provides a more physiological approach to the problem of lack of CMV-specific immunity. Recent reports add to the evidence that culture-based techniques can create cellular products that are safe and efficacious, although without Phase III data to definitively support their routine application and the difficulty of satisfying GMP standards.

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 .

Summary Background Concerns have been raised about emergence of ganciclovir resistance as a result of the advent of both routine oral ganciclovir prophylaxis and highly potent immuno-suppression. We retrospectively assessed the occurrence of ganciclovir-resistant cytomegalovirus disease among transplant recipients who had received oral ganciclovir prophylaxis and highly potent immunosuppression. Methods We studied 240 recipients of liver, kidney, or pancreas transplants. Antiviral susceptibility testing of blood cytomegaloviral isolates was done when patients failed to respond to intravenous ganciclovir treatment for symptomatic cytomegalovirus infection. Portions of the UL97 gene associated with ganciclovir resistance were sequenced in cytomegalovirus isolates with phenotypic resistance to ganciclovir. Findings Ganciclovir-resistant cytomegalovirus disease developed in five (7%) of 67 seronegative recipients of cytomegalovirus-seropositive organs (D+/R−) compared with none of 173 seropositive recipients (p=0·002). Among the 25 (10·4%) patients who developed cytomegalovirus disease within 1 year after transplantation, five had ganciclovir-resistant cytomegalovirus disease. Among D+/R− transplant recipients, ganciclovir-resistant cytomegalovirus disease was more common among the group receiving the most potent immunosuppression—ie, recipients of kidney and pancreas or pancreas alone (four of 19) compared with all other transplant recipients (one of 48, p=0·02). Ganciclovir-resistant cytomegalovirus disease was diagnosed at a median of 10 months after transplantation (range 7–12) after lengthened exposure to ganciclovir, was associated with previously described mutations of the UL97 gene, and led to serious clinical complications. Interpretation Ganciclovir-resistant cytomegalovirus is an important cause of late morbidity among D+/R− transplant recipients who have had lengthened exposure to ganciclovir and have received highly potent immunosuppression. Strategies to reduce this complication, especially among D+/R− patients, are warranted.

The cytomegalovirus UL97 kinase inhibitor maribavir suppressed viral growth more effectively in lung fibroblasts than in skin fibroblasts, and some cellular kinase inhibitors enhanced its antiviral activity. These effects influence the phenotypic assay of drug susceptibility and suggest the possibility of therapeutically useful combinations of maribavir and cellular kinase inhibitors.

The use of available antiviral agents for the prevention of cytomegalovirus (CMV) disease is limited by frequent toxic effects and the emergence of resistance. CMX001 has potent in vitro activity against CMV and other double-stranded DNA viruses. We evaluated the safety and anti-CMV activity of CMX001 in patients who had undergone allogeneic hematopoietic-cell transplantation.From December 2009 through June 2011, a total of 230 patients with data that could be evaluated were enrolled in the study. We randomly assigned these adult CMV-seropositive transplant recipients from 27 centers to oral administration of CMX001 or placebo. Patients were assigned in a 3:1 ratio to five sequential study cohorts according to a dose-escalating, double-blind design. Randomization was stratified according to the presence or absence of acute graft-versus-host disease and CMV DNA in plasma. Patients received the study drug after engraftment for 9 to 11 weeks, until week 13 after transplantation. Polymerase-chain-reaction analysis of CMV DNA in plasma was performed weekly. Patients in whom CMV DNA was detected at a level that required treatment discontinued the study drug and received preemptive treatment against CMV infection. The primary end point was a CMV event, defined as CMV disease or a plasma CMV DNA level greater than 200 copies per milliliter when the study drug was discontinued. The analysis was conducted in the intention-to-treat population.The incidence of CMV events was significantly lower among patients who received CMX001 at a dose of 100 mg twice weekly than among patients who received placebo (10% vs. 37%; risk difference, -27 percentage points; 95% confidence interval, -42 to -12; P=0.002). Diarrhea was the most common adverse event in patients receiving CMX001 at doses of 200 mg weekly or higher and was dose-limiting at 200 mg twice weekly. Myelosuppression and nephrotoxicity were not observed.Treatment with oral CMX001 at a dose of 100 mg twice weekly significantly reduced the incidence of CMV events in recipients of hematopoietic-cell transplants. Diarrhea was dose-limiting in this population at a dose of 200 mg twice weekly. (Funded by Chimerix; CMX001-201 ClinicalTrials.gov number, NCT00942305.).

Background Human cytomegalovirus infections are still significant causes of morbidity and mortality in transplant recipients. The use of antiviral agents is limited by toxicity and evolving resistance in immunocompromised patients with ongoing viral replication during therapy. Here, we present the first documented case of genotypic resistance against maribavir in a bone marrow transplant (BMT) recipient. Case presentation The female 13-year-old patient was suffering from a refractory cytopenia. Ganciclovir, foscarnet, cidofovir, leflunomide and maribavir, an inhibitor of the cytomegalovirus UL97 protein, were administered to treat a therapy-resistant cytomegalovirus infection. Viral mutations conferring resistance against nucleotide and pyrophosphate analogs as well as maribavir (MBV) have evolved sequentially. Particularly, impressive was the fast emergence of multiple mutations T409M, H411Y and H411N conferring maribavir resistance after less than 6 weeks. Conclusion We describe the fast emergence of cytomegalovirus variants with different maribavir resistance associated mutations in a bone marrow transplant recipient treated with MBV 400 mg p.o. twice per day. The results suggest that a high virus load permitted a selection of several but distinct therapy-resistant HCMV mutants. Since a phase II study with MBV is intended for the treatment of resistant or refractory HCMV infections in transplant recipients this has to be kept in mind in patients with high viral loads during therapy (NCT01611974).

International audience; Maribavir (MBV), a UL97 inhibitor, shows good oral bioavailability, low host cell toxicity, and theoretical benefits to inhibit cross-resistant viruses. We herein examined clinical and virological outcomes of 12 patients, including 3 bone marrow recipients and 9 organ recipients infected with resistant cytomegalovirus (CMV) and treated with MBV during 2011-2012. All received at least 800-mg daily doses. They had developed clinical (12/12) and/or virological (11/12) resistance to CMV infection. Based on a decrease of viral load in blood >1.5 log copies/mL half of them responded to MBV treatment. The individual changes varied from a rapid decrease in viral load (n = 4) to no response (n = 3) with some late response slowly decreasing viremia (n = 3). In 2 cases MBV was used as secondary prophylaxis. No clear parameter emerged as a clinical surrogate for nonresponse to MBV. These results contrast with the lack of efficacy in phase III trials of MBV prophylaxis among stem cell recipients, which were possibly due to low doses or inadequate timing of drug initiation in the study. Additional clinical and surrogate laboratory markers are needed to determine antiviral responses to guide MBV use. Dosage ranging studies might benefit future MBV use.

We report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb′ region of the viral genome. A UL97-null (Δ97) mutant of strain TB40/E, which encodes a full-length ULb′ region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a Δ97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb′ region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(β- l -ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb′ regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ∼100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb′ regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb′ region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb′ region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.

Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus.

Ganciclovir-resistant cytomegalovirus (CMV) infection is an emerging problem in solid organ transplant (SOT) recipients. Treatments such as foscarnet and cidofovir are fraught with serious side effects that may limit their use in this condition. The aim of this paper is to elucidate the mechanisms on how resistance occurs, when to suspect it clinically and what special tests are necessary to diagnose it. Based on recent literature, the paper also brings to light two medications, maribavir and leflunomide, which have been described to have anti-CMV activity.

A new series of 2-substituted 5,6-dichlorobenzimidazole ribonucleosides has been synthesized and tested for activity against two human herpes viruses and for cytotoxicity. 2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) was prepared by ribosylation of the heterocycle 2,5,6-trichlorobenzimidazole followed by a removal of the protecting groups. The 2-bromo derivative (BDCRB) was made in a similar fashion from 2-bromo-5,6-dichlorobenzimidazole. In contrast, the 2-iodo derivative presented a more difficult problem since the appropriate heterocycle was unavailable. This prompted us to prepare the 2-amino derivative followed by nonaqueous diazotization and removal of the blocking groups. Biological evaluation revealed marked differences in the activities of these compounds and the closely related known compound 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB). DRB was weakly active against both human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1), (IC50's = 42 and 30 microM, respectively) but was cytotoxic to uninfected human foreskin fibroblasts and KB cells in the same dose range. Similar results were obtained with the heterocycle 2,5,6-trichlorobenzimidazole. In marked contrast, the ribonucleoside of 2,5,6-trichlorobenzimidazole (TCRB) was active against HCMV (IC50 = 2.9 microM, plaque assay; IC90 = 1.4 microM, yield assay) but only weakly active against HSV-1 (IC50 = 102 microM, plaque assay). Little to no cytotoxicity was observed in HFF and KB cells at concentrations up to 100 microM. By changing the substituent at the 2-position from chlorine to bromine (BDCRB), a 4-fold increase in activity against HCMV was observed without any significant increase in cytotoxicity. In contrast, the 2-I and 2-NH2 derivatives were only weakly active against HCMV and HSV-1 with activity not well-separated from cytotoxicity. These data establish that for maximum activity against HCMV with separation from cytotoxicity, ribose is preferred at the 1-position and that Cl or Br is apparently preferred at the 2-position. The activity and selectivity of both TCRB and BDCRB were better than that observed with either ganciclovir or foscarnet.

This editorial summarizes recent developments in the management of ganciclovir-resistant human cytomegalovirus (HCMV) infections. All current drugs available for systemic treatment, including ganciclovir (GCV), valganciclovir, foscarnet and cidofovir, target the viral polymerase. However, all such compounds are hampered by dose-related toxicities and the emergence of resistance. Different approaches (e.g., PCR-based direct sequencing, pyrosequencing, mass spectrometry-based comparative sequencing) allow the fast detection of resistant HCMV and are well suited to therapy monitoring. However, more studies are required on the dynamic of mixed HCMV populations under drug pressure. Alternate antiviral compounds with new mechanisms of action, such as artesunate, leflunomid, letermovir and maribavir, are now being investigated in clinical studies. An advantage of some of the new substances is lesser toxicity issues, which might lead to new prophylactic and treatment strategies.

Resurgent interest in antiviral drugs for the treatment of herpesvirus has led to the development of new compounds that are progressing through clinical trials. This is important because there are few therapeutic options for resistant infections and some viruses such as human cytomegalovirus remain underserved. New compounds include conventional DNA polymerase inhibitors such as valomaciclovir and cyclopropavir, as well as CMX001 that has a broad spectrum of antiviral activity that includes all the herpesviruses. It also includes compounds with new molecular targets such as maribavir (MBV), FV-100, AIC361, and AIC246. Recent advances with each of these compounds will be reviewed including their virus specificity, mechanism of action, and stage of development. The potential of these new compounds to improve clinical outcome will also be discussed.

Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.

Cytomegalovirus (CMV) is a ubiquitous pathogen that establishes a lifelong asymptomatic infection in healthy individuals. Infection of immunesuppressed individuals causes serious illness. Transplant and AIDS patients are highly susceptible to CMV leading to life-threatening end-organ disease. Another vulnerable population is the developing fetus in utero, where congenital infection can result in surviving newborns with long-term developmental problems. There is no vaccine licensed for CMV and current antivirals suffer from complications associated with prolonged treatment. These include drug toxicity and emergence of resistant strains. There is an obvious need for new antivirals. Candidate intervention strategies are tested in controlled preclinical animal models but species specificity of human CMV precludes the direct study of the virus in an animal model.This review explores the current status of CMV antivirals and development of new drugs. This includes the use of animal models and the development of new improved models such as humanized animal CMV and bioluminescent imaging of virus in animals in real time.Various new CMV antivirals are in development, some with greater spectrum of activity against other viruses. Although the greatest need is in the setting of transplant patients, there remains an unmet need for a safe antiviral strategy against congenital CMV. This is especially important as an effective CMV vaccine remains an elusive goal. In this regard, greater emphasis should be placed on suitable preclinical animal models and greater collaboration between industry and academia.

Summary Background Available drugs against cytomegalovirus have adverse effects that compromise their prophylactic use in recipients of allogeneic stem-cell transplants. We assessed the safety, tolerability, and antiviral activity of oral maribavir in such patients. Methods In this placebo-controlled, randomised, double-blind, multicentre phase 3 study, we enrolled adult patients recipient-seropositive or donor-seropositive for cytomegalovirus who had undergone allogeneic stem-cell transplantation. Patients were recruited from 90 centres in Canada, Europe, and the USA. After engraftment, patients were stratified by recipient cytomegalovirus serostatus and conditioning regimen (myeloablative or reduced-intensity) and assigned (2:1) by masked computer-generated randomisation sequence to receive maribavir 100 mg twice daily or placebo for up to 12 weeks, with weekly blood cytomegalovirus surveillance. If the virus was detected, administration of study drug was stopped and pre-emptive anticytomegalovirus treatment started. The primary endpoint was cytomegalovirus disease within 6 months of transplantation. Analysis was by intention-to-treat. This study is registered with ClinicalTrials.gov, NCT00411645. Findings Between December, 2006, and May, 2008, 681 patients were enrolled and assigned to receive maribavir (454) or placebo (227). The incidence of cytomegalovirus disease within 6 months was 20 of 454 (4%) for the maribavir group and 11 of 227 (5%) for the placebo group (OR 0·90; 95% CI 0·42–1·92). During the 100 days following transplantation, cytomegalovirus infection rates as measured by pp65 antigenaemia were lower in the maribavir group (26·4%) than in the placebo group (34·8%; OR 0·67; 0·47–0·95), but not when measured by plasma cytomegalovirus DNA PCR (27·8% vs 30·4%; OR 0·88; 0·62–1·25), nor by initiation of treatment against cytomegalovirus (30·6% vs 37·4%; OR 0·73, 0·52–1·03). Maribavir was well tolerated: most adverse events, including incident acute graft-versus-host disease and neutropenia, affected both groups equally, except for taste disturbance (15% maribavir, 6% placebo). Interpretation Compared with placebo, maribavir prophylaxis did not prevent cytomegalovirus disease when started after engraftment. Cytomegalovirus disease as a primary endpoint might not be sufficient to show improvements in cytomegalovirus prevention in recipients of allogeneic stem-cell transplants in the setting of pre-emptive antiviral treatment. Clinical and virological composite endpoints should be used in future trials. Funding ViroPharma Incorporated.

The average 50% inhibitory concentration (IC 50 ) values for AD169 were 0.22 ± 0.09 μM 1263W94 and 5.36 ± 0.12 μM ganciclovir. For 35 human cytomegalovirus (HCMV) clinical isolates the average IC 50 was 0.42 ± 0.09 μM 1263W94, and for 26 ganciclovir-susceptible HCMV clinical isolates the average IC 50 was 3.78 ± 1.62 μM ganciclovir. Nine HCMV clinical isolates that were resistant to ganciclovir were completely susceptible to 1263W94.

Despite remarkable advances in the diagnostic and therapeutic modalities for its management, cytomegalovirus (CMV) remains one of the most important pathogens impacting on the outcome of transplantation. Not only does CMV directly cause morbidity and occasional mortality, it also influences many short-term and long-term indirect effects that collectively contribute to reduced allograft and patient survival. Prevention of CMV infection and disease is therefore key in ensuring the successful outcome of solid organ transplantation (SOT). In this regard, antiviral prophylaxis and pre-emptive therapy are similarly effective in preventing CMV disease after transplantation. However, current guidelines prefer antiviral prophylaxis over pre-emptive therapy in preventing CMV disease in high-risk SOT recipients, such as CMV-seronegative recipients of organs from CMV-seropositive donors (CMV D+/R-), and lung, intestinal and pancreas transplant recipients. Antiviral prophylaxis has the benefits of reducing not only the incidence of CMV disease, but also the indirect effects of CMV on allograft and patient survival. The major drawback of antiviral prophylaxis is delayed-onset CMV disease, which occurs in 15-38% of CMV D+/R- SOT recipients who received 3 months of prophylaxis. Allograft rejection, over-immunosuppression and lack of CMV-specific immunity are factors that predispose patients to delayed-onset CMV disease. A recent randomized trial in CMV D+/R- kidney recipients demonstrates a significant reduction in the incidence of CMV disease when valganciclovir prophylaxis is extended to 200 days (compared with the standard 100 days) after transplantation; however, the safety and cost of this prolonged approach has yet to be assessed. In some studies, delayed-onset CMV disease has been significantly associated with allograft loss and mortality. In the vast majority of patients, CMV disease responds to treatment with intravenous ganciclovir. Recently, oral valganciclovir was demonstrated to have an efficacy that is comparable to intravenous ganciclovir in treating mild to moderate cases of CMV disease in SOT recipients. Reduction in the degree of immunosuppression should complement antiviral treatment of CMV disease. Although it remains rare, ganciclovir-resistant CMV disease is increasingly seen in clinical practice, potentially fostered by the prolonged use of antivirals in high-risk over-immunosuppressed transplant recipients. Treatment of drug-resistant CMV is currently non-standardized and may include foscarnet, cidofovir, CMV hyperimmune globulins or leflunomide. The investigational drug marivabir had the potential to treat ganciclovir-resistant CMV disease as it acts through a different mechanism. However, the recent phase III clinical trial in allogeneic bone marrow transplant recipients showed that maribavir was not significantly better than placebo for the prevention of CMV disease. Similarly, the preliminary data in a liver transplant population suggests that maribavir was inferior to oral ganciclovir for the prevention of CMV disease. This article reviews the recent data and other developments in the management of CMV infection after SOT.

Cytomegalovirus (CMV) infection is a prominent infection in transplant recipients. The immunosuppressive drug mizoribine was shown to have anti-CMV activity in vitro and was reported to have an anti-CMV effect in renal transplantation. This study characterized the anti-CMV activity of mizoribine in vitro and its synergistic activity with ganciclovir. Mizoribine suppressed replication and at the EC(50) for plaque inhibition of 12.0 microg/ml. Mizoribine and ganciclovir exerted a strong synergism in anti-CMV activity. Mizoribine depletes guanosine nucleotides by inhibiting inosine monophosphate dehydrogenase and may increase the ratio of ganciclovir to guanosine in treated cells, resulting in a strong synergistic augmentation of the anti-CMV activity of ganciclovir. Two clinical isolates with UL97 mutations were less susceptible to mizoribine than the Towne strain but were equally susceptible in the presence of guanine. Two mizoribine-resistant strains were isolated after culture for 3 months with 100 microg/ml mizoribine, but they were as sensitive to ganciclovir as the parent Towne strain. The anti-CMV activity of mizoribine was antagonized by 2'-deoxyguanosine. Mizoribine inhibited CMV replication directly, and the sequence of mizoribine-resistant mutants of UL97 and UL54 was identical to that of the parent Towne strain, indicating the different anti-CMV action from ganciclovir, foscarnet, and maribavir. Mizoribine as an immunosuppressive and anti-CMV drug in the clinical regimen was suggested to suppress replication of CMV in vivo and control CMV infection in transplant recipients in combination with ganciclovir.

Although many drugs inhibit the replication of Epstein-Barr virus (EBV) in cell culture systems, there is still no drug that is effective and approved for use in primary EBV infection. More recently, maribavir (MBV), an l -ribofuranoside benzimidazole, has been shown to be a potent and nontoxic inhibitor of EBV replication and to have a mode of action quite distinct from that of acyclic nucleoside analogs such as acyclovir (ACV) that is based primarily on MBV's ability to block the phosphorylation of target proteins by EBV and human cytomegalovirus protein kinases. However, since the antiviral mechanisms of the drug are complex, we have carried out a comprehensive analysis of the effects of MBV on the RNA expression levels of all EBV genes with a quantitative real-time reverse transcription-PCR-based array. We show that in comparisons with ACV, the RNA expression profiles produced by the two drugs are entirely different, with MBV causing a pronounced inhibition of multiple viral mRNAs and with ACV causing virtually none. The results emphasize the different modes of action of the two drugs and suggest that the action of MBV may be linked to indirect effects on the transcription of EBV genes through the interaction of BGLF4 with multiple viral proteins.

This review discusses the management of resistant cytomegalovirus and prevention strategies for fatal therapy failures. Five drugs, ganciclovir/valganciclovir, cidofovir, foscarnet and fomivirsen, have been approved so far for the treatment of human cytomegalovirus (HCMV) diseases. Except for fomivirsen, all of the approved drugs share the same target molecule, the viral DNA polymerase. The emergence of drug-resistant HCMV has also been reported for all of them. For optimal care of patients, the clinical virologist has to provide the most meaningful assays for monitoring of therapy and early detection of emerging drug-resistant HCMV. Additionally, a quantitative drug monitoring would be helpful. New antiviral agents are urgently needed with less adverse effects, good oral bioavailability and possibly novel targets or mechanisms of action to avoid cross-resistance and to improve the ability to suppress the selection of resistant virus strains by combination therapy. Compounds like maribavir, leflunomide and artesunate, which exhibit anti-HCMV activity in vitro and in patients need to be evaluated in clinical studies. Besides these, new therapy approaches like immunotherapy or new diagnostic techniques like pyrosequencing have to be considered in the future.

The UL97 kinase has been shown to phosphorylate and inactivate the retinoblastoma protein (Rb) and has three consensus Rb-binding motifs that might contribute to this activity. Recombinant viruses containing mutations in the Rb-binding motifs generally replicated well in human foreskin fibroblasts with only a slight delay in replication kinetics. Their susceptibility to the specific UL97 kinase inhibitor, maribavir, was also examined. Mutation of the amino terminal motif, which is involved in the inactivation of Rb, also renders the virus hypersensitive to the drug and suggests that the motif may play a role in its mechanism of action.