Dynamics of the Emergence of a Human Cytomegalovirus UL97 Mutant Strain Conferring Ganciclovir Resistance in a Pediatric Stem-Cell Transplant Recipient

Stem-cell transplant recipients are at risk of developing ganciclovir-resistant human cytomegalovirus (HCMV) infection caused by mutations in the viral UL97 gene. Knowledge of the relative proportions of coexisting HCMV wild-type and mutant strains may contribute to a better understanding of the dynamics of in vivo mutant strain selection under ganciclovir. Currently, genotype resistance screening for UL97 is routinely performed by restriction fragment length polymorphism detection and sequencing. We present here the longitudinal course of a pediatric recipient of an allogeneic stem-cell transplant infected with a ganciclovir-resistant HCMV strain. EDTA-treated blood samples were analyzed longitudinally. The patient acquired a primary HCMV infection shortly before transplantation and reactivated the virus following allogeneic hematopoietic stem cell transplantation, thus receiving an intensive antiviral treatment schedule. Three different methods for UL97 mutation analysis, restriction fragment length polymorphism detection, sequencing, and a new, real-time PCR approach were performed. In conclusion, for our pediatric patient, during peak viral load, the UL97 wild-type strain predominates, while during clinical deterioration with low viral load, the predominant mutant strain persists.The emergence of an human cytomegalovirus (HCMV) infection needing ganciclovir (GCV) therapy in stem-cell transplant recipients is a severe complication. The incidence of resistance to GCV in pediatric stem-cell transplant recipients was roughly estimated at 3.8%.¹ Early and rapid detection of GCV resistance in human pediatric and adult hematopoietic stem-cell transplantation (HSCT) as well as in solid organ-transplantation is very important for determining therapeutic course.^2,3,4GCV resistance is caused by mutations in the UL97 and the UL54 genes.² Most clinical GCV-resistant isolates have clustered mutations in the UL97 gene, especially in the region between codons 400 and 665.^5,6 The determination of relative proportions of wild-type and mutant strains out of mixed viral populations provides important information with regard to therapeutic options.^1,7 Phenotypic and genotypic assays are used for the diagnosis of GCV-resistant HCMV infections. For a culture-based phenotypic assay a viral isolate is necessary. Even rapid modifications using cell-associated virus strains in an HCMV immediate early plaque reduction assay⁸ are very time-consuming and laborious, as compared with genotypic methods. In contrast, both sequencing and PCR-based restriction fragment length polymorphism (RFLP) assays are suitable to detect mutations in the HCMV phosphotransferase gene (UL97) associated with GCV resistance. Mutations in the polymerase-gene (UL54), which contains no clustered regions for mutations involved in drug resistance for cidofovir and foscarnet, are detectable only by sequencing.Here, we present a case of an HCMV-infected infant after allogeneic stem cell-transplantation developing the UL97-mutation C603W, which conferred GCV resistance with fatal outcome. To screen the dynamics of the development of GCV resistance, three genotypic screening methods for the detection of this mutation were compared: RFLP assay, sequencing analysis, and our newly established LightCycler real-time PCR approach.^9,10     

Improved Detection of Mutated Human Cytomegalovirus UL97 by Pyrosequencing

Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.Human cytomegalovirus (HCMV) is an important opportunistic pathogen for humans of all ages with immature or compromised immune functions (17). Drugs commonly used for the treatment of HCMV infections are ganciclovir (GCV), cidofovir, and foscarnet, which all target the viral DNA polymerase. However, all these compounds cause considerable adverse effects. Their prolonged application frequently results in the selection of drug-resistant HCMV mutants with eventual failure of therapy (reviewed in reference 6).The first-line drug GCV is pharmacologically a prodrug that requires phosphorylation to the monophosphate by the HCMV UL97-encoded protein kinase to gain its antiviral activity. As a consequence, over 80% of GCV-resistant isolates carry mutations in the UL97 gene (4, 8). UL97 mutations known to confer GCV resistance occur predominantly at codons 460 and 520 and at several codons between 590 and 607, particularly at codon positions 592, 594, 595, and 603 (3). During the selection process of a spontaneously occurring UL97 mutation under drug pressure, mixed virus populations exist in the patient. At present, direct dideoxy chain termination sequencing (CTSQ) of PCR products spanning this part of the UL97 gene is the method of choice for the detection of mutant viruses (16). However, the sensitivity of the CTSQ method in detecting minor mutant fractions in samples with heterogeneous mutant-wild-type HCMV mixtures is only moderate. Approximately 20% of the total virus population has to be mutant virus in order to be solidly detected (2; B. Schindele and B. Ehlers, unpublished data). However, fast and early detection of emerging resistant virus is of particular importance, since mixed HCMV UL97 populations can influence GCV susceptibility, and detection may predict the outgrowth of resistant virus variants (5). Therefore, the early and sensitive detection of a resistance-associated HCMV mutant is highly desirable.Pyrosequencing (PSQ) (18) has been successfully used to detect lamivudine-resistant variants of hepatitis B virus in heterogeneous virus populations (12) and drug-resistant influenza virus quasispecies (11). In the present study, we evaluated whether PSQ is also suitable for the fast detection of GCV-resistant HCMV. The method was compared with CTSQ by analyzing a collection of samples containing mixed HCMV populations derived from patients with active HCMV infections. The results show that the PSQ method has considerable advantages over the currently used CTSQ method in detecting minor fractions of HCMV UL97 mutants in mixed virus populations.     

Acyclovir Is Phosphorylated by the Human Cytomegalovirus UL97 Protein

Acyclovir (ACV) has shown efficacy in the prophylactic suppression of human cytomegalovirus (HCMV) reactivation in immunocompromised renal transplant patients without the toxicity associated with ganciclovir (GCV). The HCMV UL97 gene product, a protein kinase, is responsible for the phosphorylation of GCV in HCMV-infected cells. This report provides evidence for the phosphorylation of ACV by UL97. Anabolism studies with the HCMV wild-type strain AD169 and with recombinant mutants derived from marker transfer experiments performed by using mutant UL97 DNA from both clinical isolates and a laboratory-derived strain resistant to GCV showed that mutations in the UL97 gene cripple the ability of recombinant virus-infected cells to anabolize both GCV and ACV. These mutant UL97 recombinant viruses were less susceptible to both GCV and ACV than was the wild-type strain. A recombinant herpes simplex virus type 1 strain, in which the thymidine kinase gene is deleted and the UL13 gene is replaced with the HCMV UL97 gene, was able to induce the phosphorylation of ACV in infected cells. Finally, purified UL97 phosphorylated both GCV and ACV to their monophosphates. Our results indicate that UL97 promotes the selective activity of ACV against HCMV.     

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.     

Cyclopropavir inhibits the normal function of the human cytomegalovirus UL97 kinase

Cyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.     

Detection of human cytomegalovirus mutations associated with ganciclovir resistance in cerebrospinal fluid of AIDS patients with central nervous system disease.

To examine the involvement of ganciclovir-resistant strains in the development of central nervous system (CNS) disease caused by human cytomegalovirus (HCMV), 14 AIDS patients with CNS disease caused by HCMV were studied for the presence of HCMV strains with UL97 gene mutations associated with ganciclovir resistance by using amplification and direct sequencing of HCMV DNA in cerebrospinal fluid (CSF). The CSF of all seven patients who had not received ganciclovir prior to the development of CNS disease and four patients who had been receiving the drug for 3 to 8 months contained wild-type UL97 sequences. The CSF of three patients who had received ganciclovir for 12 to 30 months contained HCMV strains with nucleotide changes leading to single-amino-acid substitutions within conserved UL97 sites implicated in nucleotide binding (position 460) and substrate recognition (position 591). Patients containing mutant and wild-type strains revealed a similar spectrum of clinical and histopathologic manifestations. These findings indicate that CNS disease in AIDS patients receiving prolonged ganciclovir therapy can be caused by ganciclovir-resistant HCMV strains. Direct genotypic analysis of HCMV DNA within CSF should help to identify ganciclovir-resistant virus and to guide anti-HCMV therapy.     

人巨细胞病毒对更昔洛韦耐受性的基因型鉴别方法的建立和应用

目的建立实验室检测人巨细胞病毒（HCMV）更昔洛韦（GCV）耐药的基因型分析的常规方法。方法采用巢式聚合酶链反应（PCR）直接扩增33例HCMV感染患者的外周血白细胞及前期实验同步获得的33例临床分离毒株的HCMVUL97基因片段,对扩增产物进行测序,与HCMV标准毒株AD169序列比对,参照国外报道的耐药相关的热点突变,判定各临床分离毒株的耐药基因型。结果除有1株为HCMVUL97呈M460V突变型外,余32株同标准株AD169的序列一致。同一患者的分离毒株与外周血白细胞中测得HCMVUL97基因序列完全一致。结论建立的实验室检测HCMV耐药的基因型分析完全可以替代传统的耐药表型分析,前者无需分离活病毒,不仅需时短,而且相对安全,其技术要点更适宜临床推广应用。     

Sequencing of cytomegalovirus UL97 gene for genotypic antiviral resistance testing

The widespread use of ganciclovir (GCV) to treat cytomegalovirus (CMV) infections in immunosuppressed patients has led to the development of drug resistance. Phenotypic assays for CMV drug resistance are presently too time-consuming to be therapeutically useful. To support the development of genotypic assays for GCV resistance, the complete sequences of the UL97 phosphotransferase genes in 28 phenotypically GCV-sensitive CMV clinical isolates were determined. The gene was found to be highly conserved, with nucleotide sequence identity among strains ranging from 98.6 to 100% and amino acid sequence identity of >99%. Primers for a genotypic assay were designed to amplify codons 400 to 707, because all known UL97 mutations conferring drug resistance occur at three sites within this region. This part of the UL97 gene was amplified from over 50 clinical isolates, and two sequencing reactions for the coding strand were successfully used to identify GCV resistance mutations. This genotypic assay can be performed in 48 h using genomic DNA extracted from cell monolayers at very low levels of virus infectivity, thus rapidly providing therapeutically useful results.     

Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma.

Specific mutations in the UL97 region of human cytomegalovirus (HCMV) have been found to confer resistance to laboratory-adapted strains subjected to ganciclovir selection. In this study, mutations in the UL97 region of HCMV isolates obtained from patients receiving ganciclovir therapy were examined to determine whether they would confer ganciclovir resistance, and if these mutations could be detected directly in the plasma of AIDS patients with progressive HCMV disease despite ganciclovir treatment. A single nucleotide change within a conserved region of UL97 was found in five resistant isolates, resulting in an amino acid substitution in residue 595: from leucine to phenylalanine in one, and from leucine to serine in four resistant isolates. A sixth resistant isolate demonstrated a single nucleotide change, leading to a threonine to isoleucine substitution in residue 659. The role of the 595 amino acid substitution in conferring ganciclovir resistance was confirmed by marker transfer experiments. In further studies, direct sequencing of HCMV DNA present in plasma obtained from persons with resistant viruses revealed the identical amino acid substitutions in plasma as those present in the cultured viruses. These findings indicate that clinical resistance to ganciclovir can result from specific point mutations in the UL97 gene, and that the emergence of the resistant genotype can be detected directly in patient plasma.     

Alkoxyalkyl esters of cidofovir and cyclic cidofovir exhibit multiple-log enhancement of antiviral activity against cytomegalovirus and herpesvirus replication in vitro

The incidence of cytomegalovirus (CMV) retinitis is declining in AIDS patients but remains a significant clinical problem in patients with organ transplants and bone marrow transplants. Prophylaxis with ganciclovir (GCV) or valganciclovir reduces the incidence of CMV disease but may lead to the emergence of drug-resistant virus with mutations in the UL97 or UL54 gene. It would be useful to have other types of oral therapy for CMV disease. We synthesized hexadecyloxypropyl and octadecyloxyethyl derivatives of cyclic cidofovir (cCDV) and cidofovir (CDV) and found that these novel analogs had 2.5- to 4-log increases in antiviral activity against CMV compared to the activities of unmodified CDV and cCDV. Multiple-log increases in activity were noted against laboratory CMV strains and various CMV clinical isolates including GCV-resistant strains with mutations in the UL97 and UL54 genes. Preliminary cell studies suggest that the increase in antiviral activity may be partially explained by a much greater cell penetration of the novel analogs. 1-O-Hexadecyloxypropyl-CDV, 1-O-octadecyloxyethyl-CDV, and their corresponding cCDV analogs are worthy of further preclinical evaluation for treatment and prevention of CMV and herpes simplex virus infections in humans.     

Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations

Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol (EMB)-resistant Mycobacterium tuberculosis strains worldwide. The identification of these mutations in resistant strains has been achieved by labor-intensive DNA sequencing or by tedious amplification protocols followed by restriction endonuclease digestion. In this report, we describe PCR-restriction fragment length polymorphism (RFLP)-based methods for determining substitutions at embB codons 306 and 497 and iniA codon 501 directly in BACTEC cultures of M. tuberculosis isolates. The wild-type and mutant alleles are revealed by easily interpretable and different RFLP patterns. The methods optimized initially on reference strains were tested directly on BACTEC cultures of 25 randomly selected clinical M. tuberculosis isolates, seven of which were determined to contain EMB-resistant strains by phenotypic drug susceptibility testing. The PCR-RFLP methods identified mutations in four of seven EMB-resistant strains with three isolates containing mutated embB codon 306 and one isolate containing mutated embB codon 497. The results of PCR-RFLP were confirmed by DNA sequencing. The worldwide prevalence figures for mutations at embB codons 306 and 497 and iniA codon 501 suggest that nearly half of EMB-resistant M. tuberculosis strains could be identified within one working day even in developing countries equipped with simple PCR technology instead of weeks required for phenotypic drug susceptibility testing. Further, since EMB resistance is also associated with multiple-drug resistance from some geographical locations, detection of EMB resistance may also lead to rapid identification of multidrug-resistant strains of M. tuberculosis.     

Recombinant Phenotyping of Cytomegalovirus UL97 Kinase Sequence Variants for Ganciclovir Resistance

A strain of human cytomegalovirus, T2211, modified from standard laboratory strain AD169 to contain a secreted alkaline phosphatase reporter gene for rapid viral quantitation, was cloned as a bacterial artificial chromosome, BA1, and then mutagenized to create recombinant viruses containing viral UL97 kinase sequence variants found in clinical specimens after ganciclovir treatment, but with no phenotypic data to determine their role in drug resistance. Seven control strains and 14 other recombinant strains were phenotyped for ganciclovir resistance and compared with similar strains created using prior technology to show a good concordance of findings. Sequence changes V466M, H469Y, A478V, N510S, A588V, K599R, L600I, G623S, T659I, and V665I were found to confer no significant ganciclovir resistance, while mutations L405P, M460T, A594E, and C603R conferred 3- to 9-fold increases in ganciclovir 50% inhibitory concentrations. Different mutations at codons 594 (A594V, A594E) and 603 (C603W, C603S) conferred varied amounts of ganciclovir resistance. Advances in recombinant phenotyping make it easier to show that many uncharacterized UL97 sequence variants do not confer ganciclovir resistance, but some are newly confirmed as resistance associated, including one (L405P) which is outside the codon range where such mutations are usually found. This information should improve the interpretation of genotypic data generated by diagnostic laboratories.Prolonged use of ganciclovir (GCV) or its prodrug valganciclovir, often for several months in transplant recipients undergoing primary cytomegalovirus (CMV) infection, may result in the development of drug resistance, which is suspected when circulating viral loads increase during ongoing therapy (17). In current clinical practice, GCV resistance is usually confirmed by analysis of CMV DNA sequences amplified from posttreatment clinical specimens, since viral culture isolates or pretreatment baseline CMV sequences are not commonly available. GCV resistance typically results from mutations in the viral UL97 kinase gene responsible for the initial phosphorylation of GCV, but sometimes mutations in the viral UL54 DNA polymerase (pol) gene emerge to confer added GCV resistance and cross-resistance to cidofovir and/or foscarnet. With both genes, an extensive and evolving database of mutations related to drug resistance has been documented and used for interpretation of genotypic test results.In the UL97 gene, a number of well-defined (“canonical”) mutations at codons 460, 520, and 590 to 607 are found in GCV-resistant clinical strains, with mutations M460V/I, H520Q, C592G, A594V, L595S, and C603W occurring most frequently (6). Point and deletion mutations in the codon range 590 to 607 confer various degrees of GCV resistance, while other changes in this region confer no significant resistance (6). Noncanonical UL97 sequence changes have repeatedly been found in patients who have received GCV. Sequences of pretreatment and/or drug-susceptible CMV isolates are available to indicate that certain sequence variants are probably normal interstrain polymorphisms unrelated to drug resistance (26), but in many cases, insufficient data exist to determine if the sequence variants represent polymorphisms, resistance-related mutations, or reporting errors (6, 16, 26, 32). Speculation about the significance of uncharacterized UL97 sequence variants has resulted in some misleading attributions of GCV resistance (5, 31), which can be confusing to clinicians and diagnostic laboratories.The role of viral gene mutations in drug resistance is determined by recombinant phenotyping (marker transfer), whereby a specific sequence change is transferred to a reference laboratory strain of CMV and the resulting effect on drug susceptibility is assessed by the drug concentration required to reduce viral growth by 50% (EC₅₀). Confirmed UL97 GCV resistance mutations usually confer 3- to 15-fold increases in the GCV EC₅₀ (6). Many UL97 sequence changes have not been phenotyped, mainly because the size of the CMV genome makes its site-specific mutagenesis technically nontrivial. In recent years, however, the work has benefited from use of standard laboratory CMV strains modified with reporter genes to facilitate viral quantitation (10) and mutagenesis of the CMV genome as a bacterial artificial chromosome (BAC) (4, 27). Here, we combine these advances to determine the significance of many UL97 sequence changes described in the existing literature or encountered more recently in diagnostic laboratories.     

巨细胞病毒UL97基因突变类型的分析

目的通过基因测序分析巨细胞病毒(CMV)的UL97基因突变情况,指导临床用药。方法回顾性分析2018年6-12月进行CMV检测的患者情况,分析CMV阳性率在不同标本的分布,并选取CMV拷贝数>1 000copy/mL的标本30例,进行UL97基因片段PCR扩增,对扩增产物进行测序,与CMV标准毒株AD169序列比对,参照国内外报道的耐药相关的热点突变,判定耐药基因突变类型。结果痰标本CMV阳性率检出率最高,达43.57%(210/482),器官移植后患者CMV阳性率最高,为25.00%(22/88);序列分析发现8例移植患者标本发生了UL97基因氨基酸的替代,其中有5例为3个月持续在其血中发现高载量的CMV。结论CMV感染患者UL97基因的检测有助于发现耐药突变,从而有效指导临床用药。     

Rapid detection of K-ras mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: comparison with restriction fragment length polymorphism analysis

BACKGROUND: Current methods for detection of K-ras gene mutations are time-consuming. We aimed to develop a one-step PCR technique using fluorescent hybridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis. METHODS: Bile samples were obtained from 116 patients with biliary obstruction, including gallstones (n = 64), benign biliary strictures (n = 6), pancreatic cancer (n = 20), and cholangiocarcinoma (n = 26). The DNA was extracted and subjected to K-ras mutation analysis by real-time PCR and RFLP analysis. Mutations were confirmed by direct sequencing. The sensitivity and specificity were calculated according to the clinical results. RESULTS: The analysis time for real-time PCR was <1 h, whereas RFLP analysis took more than 2 days. With the sensor probe designed for the GAT (G12D) mutant in codon 12 of the K-ras gene, the real-time PCR method also detected the GTT (G12V) mutant. In contrast, a specific sensor probe for the TGT (G12C) mutant detected GAT (G12D), AGT (G12S), and GTT (G12V) mutants in addition to the TGT mutant. The real-time PCR assay allowed the detection of mutation in a 3000-fold excess of wild-type bile DNA. In bile, K-ras codon 12 mutations were detected in 16 of 46 malignant cases by real-time PCR with the TGT probe and 15 by RFLP analysis. All benign cases were wild type. CONCLUSION: Real-time PCR with a cysteine-specific (TGT) sensor probe can rapidly detect K-ras gene mutations in bile and diagnose malignant biliary obstruction with high specificity.     

Cyclopropavir susceptibility of cytomegalovirus DNA polymerase mutants selected after antiviral drug exposure

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.     

Human Cytomegalovirus UL97 Kinase Is Involved in the Mechanism of Action of Methylenecyclopropane Analogs with 6-Ether and -Thioether Substitutions

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 (EC₅₀) 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.     

Diverse cytomegalovirus UL27 mutations adapt to loss of viral UL97 kinase activity under maribavir

In vitro resistance to maribavir (MBV), a cytomegalovirus UL97 kinase inhibitor currently in clinical trials, is known to result from viral UL97 mutations that confer moderate to high-level resistance and UL27 mutations that confer low-level resistance. To add to the four reported UL27 mutations, cytomegalovirus isolates or strains were propagated under MBV. Four clinical isolates evolved UL27 mutations, which were first detected after 8 to 30 passages under drug selection. In three separate experiments, laboratory strain T2294, which contained an exonuclease mutation, developed UL27 mutations at 10 to 12 passages under MBV. Most of these isolates and strains also developed a UL97 mutation, commonly T409M, before or after the appearance of the UL27 mutation. The passage of two laboratory strains genetically defective in UL97, in the absence of MBV, likewise resulted in UL27 mutations. The nine UL27 mutations observed included multiple instances of point, stop, and frameshift mutations, which were individually transferred to a reference CMV strain and which were shown to confer two- to threefold increases in MBV inhibitory concentrations. In contrast, seven common UL27 amino acid changes found in baseline clinical isolates conferred no MBV resistance. The mutants with UL27 mutations had slightly attenuated growth. The frequent mutation of UL27 suggests that its normal expression is mildly disadvantageous to the virus in the absence of UL97 kinase activity, whether the latter results from MBV inhibition or a genetic defect. Although the function of UL27 is unknown, it does not appear to be a direct antiviral target for MBV.     

Recurrent and persistent cytomegalovirus infection in a kidney recipient caused by the L595S mutation in UL97 phosphotransferase gene

Ganciclovir (GCV) is the first therapeutic choice for prevention and treatment of active cytomegalovirus (CMV) infection in solid organ transplant recipients in Bahia state, Brazil. Prolonged and repeated GCV therapy may result in drug-resistant virus, associated with progressive and disseminated disease. We present a case report of a young male kidney recipient, who was CMV-seronegative with a CMV-seropositive donor (D(+)/R(-)), and who developed clinical GCV resistance, confirmed by mutation in viral UL97 phosphotransferase responsible for GCV activation. Under prophylactic therapy with intravenous GCV for 6 weeks post-transplantation, he developed severe anaemia and hepatic enzyme increases, probably due to drug side effects. At this moment, the drug was discontinued and he started to be monitored by pp65 antigen test. At week 10 post-transplantation, he presented fever, myalgia, thrombocytopenia and neutropaenia, with a positive CMV antigen test. During treatment with intravenous GCV, antigenaemia assay demonstrated a higher number of positive cells, requiring GCV at higher doses. Pre-emptive therapy lasted for 31 days and he started the maintenance therapy with oral GCV. However, antigenaemia assay demonstrated an extremely high number of positive cells, and he was rehospitalized and prescribed intravenous GCV. Severe leukopaenia led to GCV interruption, but immunosuppressive dose reduction helped to control the active CMV infection. GCV-resistant CMV infection resulted in increased morbidity, rehospitalization episodes and increased costs; therefore, implementation of resistance diagnostic tests in the transplantation routine is of great importance. We documented the first case of GCV-resistant CMV infection due to the L595S mutation in UL97 phosphotransferase gene in a kidney recipient from Bahia state, Brazil.     

The Cys607→Tyr Change in the UL97 Phosphotransferase Confers Ganciclovir Resistance to Two Human Cytomegalovirus Strains Recovered from Two Immunocompromised Patients

Two ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) strains recovered from an AIDS patient (strain VR4990) and a heart transplant recipient (strain VR5474) showed a Cys607→Tyr change in the UL97-encoded phosphotransferase. No amino acid substitutions were observed in the viral DNA polymerase. Marker transfer experiments showed marked reduction in GCV phosphorylation and drug susceptibility of the recombinant HCMV strain VR4990rec2-1-1. These results further extend the region of the carboxy-terminal domain of the UL97 phosphotransferase involved in GCV substrate recognition.     

Human cytomegalovirus double resistance in a donor-positive/recipient-negative lung transplant patient with an impaired CD4-mediated specific immune response

BACKGROUND: Emergence of human cytomegalovirus (HCMV) resistance to ganciclovir in solid-organ transplant recipients has been found to be mostly associated with primary HCMV infection. MATERIALS AND METHODS: The case of a donor-positive/recipient-negative (D(+)/R(-)) lung transplant patient developing ganciclovir and cidofovir resistance is described. HCMV infection was monitored by weekly determination of antigenaemia, viraemia and DNAaemia. HCMV-specific CD4 cell immunity was determined by cytokine flow cytometry. The emergence of drug-resistant HCMV strains was documented by sequencing of UL97 and UL54 genes of HCMV directly in blood samples. RESULTS: Following primary HCMV infection, the patient showed repeated reactivations for over a year, eventually resulting in the selection of a ganciclovir-resistant HCMV strain with a mutation in the UL97 gene product (A594V). Determination of HCMV-specific CD4 cell immunity showed a persistently impaired immune response. Subsequent foscarnet treatment allowed only transitory virus clearance from blood owing to renal toxicity. Further ganciclovir treatment induced a new mutation in both UL97 (H520Q) and UL54 (P522S) with final emergence of double resistance to both ganciclovir and cidofovir. The patient eventually died of lung failure. DISCUSSION: Determination of HCMV-specific CD4 cell immunity could be of help in predicting the emergence of drug-resistant strains in D(+)/R(-) transplant recipients.