UL97 Gene Research Articles

Human Cytomegalovirus (CMV) UL97 D605E Mutation Has a Higher Prevalence in Infants With Primary CMV Infection Compared With Transplant Recipients With CMV Recurrence

Mutations in the UL97 gene are a major mechanism of human cytomegalovirus (CMV) resistance to gancyclovir (GCV). Some mutations may show different regional distributions. To analyze UL97 mutations in Chinese people, we scanned the UL97 gene fragment among virus isolates from 27 infants as well as blood samples from 28 solid organ transplant (SOT) and 42 bone marrow transplant (BMT) recipients with active CMV infections as defined by DNAemia or PP65 antigenemia. Only a known GCV-resistant mutation M406V was found in a BMT recipient. However, the D605E mutation was identified in 18 of 27 (66.7%) infants as well as 11 of 28 (39.3%) SOT and 17 of 42 (40.5%) BMT recipients. It was significantly different between the infants and transplant recipients (P < .05). So far, the influence of D605E mutation on GCV-resistance is controversial. In this study, 18 D605E mutants, 9 wild type (WT) isolates, and AD169 controls cultured in fibroblasts were tested for phenotypic drug resistance using a plaque reduction assay. The dose of GCV required for 50% inhibition of plaque formation (IC50) was 1.20 ± 0.67 μmol/L (D605E), 1.71 ± 0.64 μmol/L (WT), and 1.43 ± 0.70 μmol/L (AD169), respectively. This small difference could be caused by analytical error. We concluded that the UL97 D605E mutation showed a different prevalence between infants with primary CMV infection and transplant recipients with CMV recurrence. However, it was not related to a resistant phenotype to GCV.

MBV Use in Practice for Cytomegalovirus (CMV) Infection at Six French Transplant Centres

Introduction: MBV is a UL97 inhibitor with good oral bioavailability, low host cell toxicity and has theoretical benefits for inhibiting cross resistant viruses. We examined clinical and virological outcome of 9 patients treated with maribavir (MBV) in day to day use in France during 2011-2012. Methods: The CNR receives clinical information and resistance-suspected isolates from all transplant centers in France. During 2011-2012, 9 patients from 6 centers developed clinical (in 9/9) and/or genetic (in 6/9) resistance to CMV infection and were treated with maribavir (MBV). Data from these patients were collected including CMV viral load, resistance mutations in UL97 and UL54 genes, and clinical and virological responses to MBV therapy. Viral load was determined in each center using in house (n=3) or commercial assays (Argene n=2, Abbott n=1). Antiviral resistance (AVR) was determined by full-length sequencing of UL97 and UL54 genes using our published sequence-based methods, and GCV levels were measured when relevant. MBV response was considered adequate if viral load reduction was at least two logs or became undetectable. Results: Patients received bone marrow (n=3) and solid organ transplants (n=6 D+/R−) in 6 different centres, using similar standard immunosuppression regimens with CNI, mycophenolic acid, corticosteroids, with or without sirolimus. They received either ValGCV prophylaxis (n=5) or pre-emptive therapy (n=3), or ValACV prophylaxis (n=1). CMV UL97 or UL54 resistance mutations were present at the time of MBV treatment in 6 and 4 patients, respectively, without any MBV resistance mutation. Patients were given MBV in three cases following foscarnet (FOS) nephrotoxicity, in 1 case following UL54 mutation A809V which confers resistance to GCV/FOS, and in other cases for viral load plateauing on treatment. Two patients received MBV in association to FOS. An adequate MBV response was observed in 4/9 patients, with reduction in previously stable or increasing CMV viral load, in 5/9 patients there was either no response or uncertain response (slow decrease or efficient second prophylaxis) (see table) though clinical improvement in one patient. Conclusion: Our results showed good clinical and virological response to MBV in approximately half MBV-treated patients following CMV-resistant infection. There was variable response, although no clear parameter emerged as a clinical surrogate for non-response to MBV. These results are of great interest given the lack of efficacy in phase III trials of MBV prophylaxis in HSCT recipients, possibly due to low dosing regimens and drug initiation timing strategies used in the study. Additional clinical and surrogate laboratory markers are needed to determine antiviral response in order to guide MBV use, and dosage ranging studies would inform further MBV use.Table: [Summary table of MBV treated patients at six Frenc]

A new mutation in the human cytomegalovirus UL97 gene may confer ganciclovir resistance in Chinese kidney transplant recipients

Mutations in the UL97 gene are the most common mechanism of human cytomegalovirus resistance to ganciclovir in transplant recipients. In this study, UL97 fragments were amplified and sequenced in 70 Chinese kidney transplant recipients who were diagnosed as having an active cytomegalovirus infection. A new mutation, C518Y, was identified in two kidney recipients, and this strain showed high-grade ganciclovir resistance by plaque reduction assay. The known mutations L595 W and C607F were detected in one recipient, but the D605E mutation was found in 42.9 % (30/70) of kidney recipients. The prevalence of this mutation was higher than that in Europe and may be associated with different regions or races.

Lack of resistance-associated mutations in UL54 and UL97 genes of circulating Cytomegalovirus strains isolated in a medical center in Taiwan

Human cytomegalovirus (HCMV) is a large DNA virus and a member of the betaherpesvirus family. HCMV infection is extremely common in human populations and can cause severe diseases in immunocompromised hosts. Ganciclovir is the most widely used antiviral drug for cytomegalovirus infection and works by blocking the amplification of HCMV. HCMV strains resistant to ganciclovir have been detected in recent decades and mainly result from mutations in UL97 (protein kinase) and UL54 (DNA polymerase) genes. In order to understand the prevalence of resistance of HCMV in Taiwan, we studied 40 clinical isolates to detect the mutations of UL97 and UL54 that might be related to resistance. The results showed that no mutation known to cause ganciclovir resistance was detected in any strain, but some polymorphisms (N685S, A688V, A885T, N898D in UL54; D605E in UL97) were frequently observed. Our results suggest that resistant HCMV strains are not prevalent in Taiwan.

Prolonged KI Polyomavirus Infection in Immunodeficient Child

Prolonged KI Polyomavirus Infection in Immunodeficient Child

Detection of ganciclovir resistance mutations by pyrosequencing in HCMV-infected pediatric patients

BackgroundHuman cytomegalovirus (HCMV) is an opportunistic pathogen especially for immuno-suppressed subjects that might develop pharmacological resistance in patients undergoing prolonged antiviral treatment. Ganciclovir (GCV) is the drug used as first choice therapy in affected children and a GCV-resistant phenotype is mainly linked to mutations of the viral protein kinase UL97. ObjectivesHere a new quantitative pyrosequence (PSQ) method is presented that allows detection and quantification of the viral species carrying the more frequent UL97 mutations responsible for GCV resistance in clinical samples (>80% of known cases). Study designThe system has been validated using two independent approaches (cloning and sequencing of UL-97 gene fragments and real-time PCR) and clinical samples derived from 3 pediatric patients. ResultsThe UL97 pyrosequencing analysis has indicated a significant increase of mutant viruses carrying the H520Q and C592G mutations.In particular, the H520Q viral mutation, known to increase GCV resistance (IC50=10) increased around 5 times during hospitalization. In addition, C592G (known to have IC50=2.9) also increased 3 times. ConclusionsPSQ is a quick, cheap, high throughput and sensitive analysis method to detect GCV-associated resistance mutation useful to follow antiviral therapy in perinatal CMV-infection as well as in immune-suppressed patients.

Incidence of cytomegalovirus UL97 and UL54 amino acid substitutions detected after 100 or 200 days of valganciclovir prophylaxis

BackgroundThe IMPACT study was a randomized, double-blind study comparing 100 to 200 days of VGCV prophylaxis (900mg once daily) in D+/R− kidney transplant recipients. Although extending the duration of prophylaxis resulted in a significant reduction in confirmed cytomegalovirus (CMV) disease (100-day: 36.8% vs 200-day: 16.1%1), the consequence of extending the duration of prophylaxis on the development of viral resistance remains unknown. ObjectiveTo determine whether extending valganciclovir prophylaxis from 100 days to 200 days increased the incidence of ganciclovir resistance. Study designGenotypic analysis of CMV UL97 and UL54 was conducted on virus isolated from patients meeting the predefined resistance analysis criteria (RAC). ResultsA greater number of patients met the RAC in the 100 day prophylaxis arm (50/163; 31%) compared to the 200 day prophylaxis arm (22/155; 14%). Sequence data were successfully generated for all 200-day patients and 48/50 100-day patients. Three patients in each treatment arm (100 day: 3/163 (1.8%) vs 200 day: 3/155 (1.9%)) had a single known valganciclovir resistance mutation detected (100 day: UL97 gene: M460V, C592G twice; 200 day: UL97 gene: C603W, M460V and UL54 gene: P522S). Overall, a resistance mutation was more likely to be detected if the patient met the RAC during prophylaxis (5/12 (42%)) compared to post-prophylaxis (1/58 (2%)). All six patients with known ganciclovir resistance mutations cleared the virus; three cleared virus without treatment and three cleared virus following treatment. ConclusionsExtending valganciclovir prophylaxis from 100 days to 200 days did not significantly affect the incidence of ganciclovir resistance.

Failure of Pre-Emptive Treatment of Cytomegalovirus Infections and Antiviral Resistance in Stem Cell Transplant Recipients

Treatment of cytomegalovirus (CMV) infections after stem cell transplantation (SCT) does not always lead to a rapid viral response. The causes of treatment failure may be either viral resistance or immunological failure to control viral replication. This study investigated the response to pre-emptive treatment in CMV infections in order to define risk factors for treatment failure, including the role of antiviral resistance. Adult recipients of allogeneic T-cell depleted SCT were studied retrospectively (n=92). CMV infections were treated with (val)ganciclovir according to a CMV DNA-load-based pre-emptive strategy. Treatment failure was defined as a CMV DNA load of 1,000 copies/ml or more after at least 2 weeks of treatment. Resistance was analysed by nucleotide sequence analysis of the UL97 and UL54 genes in the first CMV DNA-positive sample and in samples during treatment failure. Treatment failure occurred in 26 of the 47 pre-emptively treated patients (55%) and in 39 of 86 (45%) treatment episodes. The risk of treatment failure was increased during first treatment episodes (P=0.01) and during the use of immunosuppressive medication (P=0.02). Antiviral resistance was found in only 1 patient (4%) with treatment failure. A slow response to pre-emptive antiviral treatment occurred frequently in CMV infections in SCT recipients. Antiviral resistance was observed but played a minor role in treatment failure.

Genotyping cytomegalovirus UL97 mutations by high-resolution melting analysis with unlabeled probe

Human cytomegalovirus (CMV) is an opportunistic pathogen, and infections with this virus can be treated with ganciclovir (GCV). Most GCV-resistant clinical CMV isolates contain a mutation in the UL97 gene. Genotypic assays for diagnostic screening of GCV-resistant CMV have been developed. High-resolution melting analysis (HRMA) with unlabeled probe is considered a perfect tool for this purpose. In this study, we have developed an HRMA-based genotypic test for the detection of UL97 mutations. Wild type and M460V/I mutants of UL97 were constructed. HRMA with unlabeled probe was used as a genotyping method for the detection of M460V/I mutations. The melting peaks obtained directly from PCR products did not enable us to distinguish the wild type from M460 mutants. The sensitivity and accuracy of HRMA were dramatically improved by using unlabeled probe. HRMA with unlabeled probe successfully distinguished M460V from M460I and served well for the detection of M460V/I mutations in clinical samples. HRMA with unlabeled probe proves to be a sensitive and cost-effective genotyping method for the detection of M460 mutations.

Estudios de resistencia. ¿Cuándo están indicados?

Cytomegalovirus (CMV) resistance to antiviral drugs is an emerging problem and is due to selection of mutations in the viral genome. Although ganciclovir resistance is the most common and widely studied, there is resistance to all antiviral agents. Risk factors for the development of resistance are the absence of preexisting immunity to CMV, lung and pancreas transplantation, high viral loads, intense concomitant immunosuppressive therapy and prolonged exposure to ganciclovir or suboptimal levels of this drug. Antiviral resistance should be suspected when, despite adequate treatment exposure for 2 weeks, an increase in viral load, or persistence or clinical progression of CMV disease are detected. However, failure to respond cannot always be attributed to antiviral resistance nor does resistance always lead to poor clinical outcome. When resistance is suspected, phenotypic and genotypic confirmation is required. The most common mutations are those in the UL97 gene, which confers ganciclovir resistance. However, foscarnet and cidofovir can be used. The UL54 mutation is not uncommon, whether alone or in combination with UL97 mutations. The combination of UL54 and UL97 mutations is associated with high-grade and multiple resistance. Early detection of resistance is essential to prevent unfavorable outcome and the development of multi-drug resistance. In patients with a slow response to treatment and without mutations associated with resistance, plasma ganciclovir levels and specific CMV immunity should be investigated.

The Genetic Basis of Human Cytomegalovirus Resistance and Current Trends in Antiviral Resistance Analysis

Infections due to resistant human cytomegalovirus (CMV) are an emerging problem, particularly in immunocompromised hosts. When managing such patients, clinicians should be aware of the possibility of developing CMV antiviral resistance, especially while on prolonged therapy or if severe immunosuppression is present. CMV resistance to current antiviral agents is mediated by alterations in either the UL97 kinase or DNA polymerase, encoded by the UL97 and UL54 genes, respectively. UL97 mutations are capable of conferring resistance to ganciclovir, while UL54 mutations can impart resistance to ganciclovir, cidofovir, and foscarnet. If treatment failure is suspected to be due to antiviral resistance, CMV resistance analysis should be obtained. Phenotypic resistance assays performed on clinical isolates measure antiviral susceptibilities directly, but are laborious and time-consuming. Therefore, genotypic resistance analysis has become the more common means of diagnosing CMV resistance. Mutations in UL97 or UL54 may be clinically associated with resistance, but their effect on antiviral susceptibility must be confirmed by marker transfer techniques such as recombinant phenotyping.

Incidence and Outcomes of Ganciclovir-Resistant Cytomegalovirus Infections in 1244 Kidney Transplant Recipients

Cytomegalovirus (CMV) infections in kidney transplant recipients are in most cases successfully treated with oral valganciclovir (VGCV). However, in a few percent of patients, mutations in the UL 97 or UL 54 gene lead to drug resistance. We investigated the incidence and outcomes of ganciclovir-resistant CMV viremia in all 1244 kidney recipients transplanted at our center from 2004 through 2008. CMV DNAemia was monitored in all patients at least weekly, and patients who were positive were treated preemptively with VGCV (900 mg once daily). Ganciclovir-resistant mutations were detected in 27 patients (2.2%), of which 26 occurred in the 209 CMV IgG-negative recipients receiving a CMV-positive kidney (12.5%). All had UL97 gene mutations, and none had UL54 gene mutations. Mean DNAemia half-life for the first (nonresistance) episode of CMV viremia was 3.8 ± 1.2 days. After established resistance, 25 of 27 patients had their mycophenolate mofetil dose reduced by approximately 50%, and 10 of these were also treated with intravenous foscarnet. The DNAemia half-life was 3.7 ± 1.4 days in the foscarnet-treated patients, significantly shorter than in the other 17 patients, 10.8 ± 6.7 days (P = 0.001). Time to DNAemia eradication was 30 ± 16 and 81 ± 51 days in the two groups, respectively (P = 0.001). Use of 900 mg VGCV once daily for preemptive CMV treatment is associated with a high incidence of CMV UL97-resistance gene mutations in D+/R- patients. Foscarnet treatment rapidly and safely eradicated CMV DNAemia, and also patients who only reduced the immunosuppression and continued on VGCV treatment eventually cleared the virus.

Characterization of the transcripts of human cytomegalovirus UL144

BackgroundThe genome of human cytomegalovirus (HCMV) has been studied extensively, particularly in the UL/b' region. In this study, transcripts of one of the UL/b' genes, UL144, were identified in 3 HCMV isolates obtained from urine samples of congenitally infected infants.MethodsNorthern blot hybridization, cDNA library screening, and RACE-PCR were used.ResultsWe identified at least 4 differentially regulated 3'-coterminal transcripts of UL144 in infected cells of 1,300, 1,600, 1,700, and 3,500 nucleotides (nt). The 1600 nt transcript was the major form of UL144 mRNA. The largest transcript initiated from the region within the UL141 open reading frame (ORF) and included UL141, UL142, UL143, UL144, and UL145 ORFs.ConclusionsThese findings reveal the complex nature of the transcription of the UL144 gene in clinical isolates.

Genotypic characterization of human cytomegalovirus UL97 phosphotransferase natural polymorphism in the era of ganciclovir and maribavir

The molecular mechanisms of human cytomegalovirus (CMV) resistance to both ganciclovir and maribavir reported so far rely predominantly on the presence of mutations within UL97 phosphotransferase. The accurate interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objective of this work was to extend the catalog of CMV UL97 phosphotransferase natural polymorphisms. The full-length UL97 gene sequence analysis from 4 laboratory strains and 35 clinical samples from patients who had not received any previous anti-CMV treatment was performed. At the nucleotide level, the interstrain identity was >98%. At the amino acid level, ten natural polymorphisms never previously described were identified. Together with all previous results reported in the literature, a new map of UL97 phosphotransferase natural polymorphism could be settled in the era of ganciclovir and maribavir.

Mass spectrometry-based comparative sequencing to detect ganciclovir resistance in the UL97 gene of human cytomegalovirus

Background Persistent infections with herpesviruses such as human cytomegalovirus (HCMV) frequently occur after solid organ or stem cell transplantation, and are due to either failure of the host to immunologically control the virus or emerging resistance of the virus to the antiviral drug(s) used. Antiviral therapy can be guided by viral drug susceptibility testing based on screening for known resistance-inducing mutations in the viral genome. Mass spectrometry-based comparative sequence analysis (MSCSA) might be advantageous for this purpose because of its suitability for semi-automation. Objectives The applicability of MSCSA to detect sequence polymorphisms and drug resistance-inducing mutations in the HCMV genome was investigated. Study design We analyzed the 3′ part of the HCMV UL97 gene, which encodes the kinase that is activated by the commonly used anti-HCMV drug ganciclovir. Sequences obtained by MSCSA of material from HCMV-infected patients (43 samples) and the HCMV type strain were compared to conventional cycle sequencing results. Results In 94.1% of all samples the results obtained by MSCSA of the UL97 gene were identical to those from conventional cycle sequencing. The threshold to detect mutant sequences in a mixture with wild-type material was 20% using either technique. Furthermore, MSCSA was successfully applied to study the development of drug resistance in a patient who developed encephalitis due to ganciclovir-resistant HCMV. Conclusions MSCSA was found to be equally accurate compared to conventional cycle sequencing in the analysis of UL97 of HCMV.

Characterization of 3′ termini of human cytomegalovirus UL138-UL145 transcripts in a clinical strain

The functions of some proteins encoded by human cytomegalovirus (HCMV) UL/b' genes have been studied; however, systematic analysis of the transcripts for this region is still insufficient. The results of both rapid amplification of cDNA ends (RACE) and cDNA library screening in this study proved that 3' termini of all transcripts in the UL138-UL145 region were located approximately 20 bp downstream from each potential poly (A) signal, which were at the positions of nucleotides 7184, 9954 and 12848 in the UL/b' sequence of the H strain, respectively. Thus, there were at least two large families of polycistronic transcripts in this gene region. The first family of 3'-coterminal transcripts contained UL139, UL140 and UL141 genes, and the second one consisted of UL142, UL143, UL144 and UL145 genes. The 3'-coterminal characterization further confirmed that multiple uses of polyadenylation signals were commonly used by HCMV to utilize genetic information.

Does Cytomegalovirus Develop Resistance following Antiviral Prophylaxis and Treatment in Renal Transplant Patients in Kuwait?

The resistance of cytomegalovirus (CMV) to ganciclovir or valganciclovir is a factor in therapeutic failure and disease progression. CMV strains resistant to ganciclovir or valganciclovir have been associated with specific mutations in the UL97 and UL54 genes. Sequencing of both CMV UL97 and UL54 genes was performed to detect the presence of CMV antiviral resistance in six patients who received ganciclovir (and/or valganciclovir) and had prolonged detectable CMV DNA in their blood during antiviral treatment. Sequencing results showed no specific mutations in either UL97 or UL54 gene of CMV and therefore the CMV strains in kidney transplant patients who received ganciclovir either prophylactically or therapeutically were from the wild type. Our results suggest that CMV management and immunosuppression protocols for kidney transplant patients followed in the Organ Transplant Centre, Kuwait, is very effective in reducing the opportunity of developing CMV antiviral resistance.

The role of cytomegalovirus infection and disease in pediatric bone marrow transplant recipients in Mexico City in the context of viral drug resistance

We aimed to identify those pediatric patients undergoing ABMT with CMV EOD who developed GCV resistance. Forty-seven patients were analyzed following ABMT. Prospective post-transplant CMV monitoring was performed weekly for the detection of viral leukocyte DNAaemia, viral plasma DNAaemia, and viral DNAuria by PCR. Plasma DNAaemia was confirmed from whole blood by the detection of CMV pp67 late mRNA using NASBA technology. In the cases of persistence of viral DNA in plasma, and positive viral RNA detection in blood, CMV drug resistance screening by comprehensive PCR-based RFLP and sequencing of the viral UL97 gene were performed retrospectively. Thirty of the 47 (63.82%) patients showed active CMV infection with 27/30 (74.4%) patients belonging to the D+R+ group and 25/30 with proven viral replication. In total, 2/30 (6.6%) children developed CMV pneumonia proven by immunohistochemistry. Screening of the viral UL97 gene revealed in one of these two cases (1/30, 3.3%) the simultaneous presence of two point mutations in codon 460 (M460V, M460I) conferring GCV resistance. The CMV seroprevalence (81%) and the incidence of active infection (63.8%) in Mexican children undergoing ABMT are very high.

Pyrosequencing allows the detection of emergent ganciclovir resistance mutations after HCMV infection

Prenatally acquired human cytomegalovirus (HCMV) infection is the most frequent viral infection of newborns in developed countries. Virostatic therapy is accompanied by side effects and stepwise emergence of resistant virus variants. Different genotypic approaches show limited sensitivity in detecting on-growing minor resistant virus populations. Here, we demonstrate the superiority of pyrosequencing for the monitoring of mutant emergence. In a preterm baby born after 28 weeks of gestation and suffering from disseminated congenital HCMV infection, long-term control could not be achieved under ganciclovir/valganciclovir therapy and the infant died on the 113th day of life. Resistance-associated mutations in the HCMV UL97 gene were not detected by conventional DNA sequencing but postmortem pyrosequencing. Four different CMV variants carrying resistance-associated mutations each representing 11-17% of the total CMV population were found.

Drug-resistant cytomegalovirus in transplant recipients: a French cohort study

Cytomegalovirus (CMV) drug resistance is a therapeutic challenge in the transplant setting. No longitudinal cohort studies of CMV resistance in a real-life setting have been published in the valganciclovir era. We report findings for a French multicentre prospective cohort of 346 patients enrolled at initial diagnosis of CMV infection (clinical trial registered at clinicaltrials.gov: NCT01008540). Patients were monitored for detection of CMV infection for ≥2 years. Real-time detection of resistance by UL97 and UL54 gene sequencing and antiviral phenotyping was performed if viral replication persisted for >21 days of appropriate antiviral treatment. Plasma ganciclovir assays were performed when resistance was suspected. Resistance was suspected in 37 (10.7%) patients; 18/37 (5.2% of the cohort) had virological resistance, associated with poorer outcome. Most cases involved single UL97 mutations, but four cases of multidrug resistance were due to UL54 mutations. In solid organ transplant recipients, resistance occurred mainly during primary CMV infection (odds ratio 8.78), but also in two CMV-seropositive kidney recipients. Neither CMV prophylaxis nor antilymphocyte antibody administration was associated with virological resistance. These data show the feasibility of surveying resistance. Virological resistance was frequent in patients failing antiviral therapy. More than 1/5 resistant isolates harboured UL54 mutations alone or combined with UL97 mutations, which conferred a high level of resistance and sometimes were responsible for cross-resistance, leading to therapeutic failure.