Cytomegalovirus Gene Research Articles

Cytomegalovirus Antiviral Resistance Among Kidney Transplant Recipients in a Phase 3 Trial of Letermovir vs Valganciclovir Prophylaxis

Abstract Background In a phase 3 trial, letermovir was noninferior to valganciclovir for cytomegalovirus (CMV) disease prophylaxis in kidney transplant recipients who were CMV-seronegative and received kidneys from donors who were CMV-seropositive. Genotypic antiviral resistance and CMV glycoprotein B (gB) genotype are reported. Methods Plasma samples with detectable CMV DNA were sequenced for the presence of known letermovir and valganciclovir resistance-associated amino acid substitutions (RASs) encoded by CMV gene regions (UL51, UL54, UL56, UL89, UL97) and prevalence of gB (UL55) genotypes (gB1–gB5). Results Among participants, 84 of 292 (letermovir) and 93 of 297 (valganciclovir) had evaluable data for ≥1 gene target. Letermovir RASs were not detected in participants who received letermovir prophylaxis; however, 3 had valganciclovir RASs (pUL97). Twelve participants who received valganciclovir prophylaxis had valganciclovir RASs (pUL54, pUL97), and 1 who did not receive letermovir during the trial had letermovir RASs (pUL56). All but 1 participant responded to valganciclovir treatment irrespective of breakthrough CMV DNAemia or frequency of RASs. gB1 was the most frequent genotype across all participants and subgroups. Conclusions Letermovir RASs were not detected with letermovir prophylaxis, supporting a low risk for development of resistance in kidney transplant recipients who were CMV-seronegative and received kidneys from donors who were CMV-seropositive. Clinical Trials Registration ClinicalTrials.gov, NCT03443869; EudraCT, 2017-001055-30.

Development and validation of a next-generation sequencing assay with open-access analysis software for detecting resistance-associated mutations in CMV.

Cytomegalovirus (CMV) resistance testing by targeted next-generation sequencing (NGS) allows for the simultaneous analysis of multiple genes. We developed and validated an amplicon-based Ion Torrent NGS assay to detect CMV resistance mutations in UL27, UL54, UL56, and UL97 and compared the results to standard Sanger sequencing. NGS primers were designed to generate 83 overlapping amplicons of four CMV genes (~10 kb encompassing 138 mutation sites). An open-access software plugin was developed to perform read alignment, call variants, and interpret drug resistance. Plasmids were tested to determine NGS error rate and minor variant limit of detection. NGS limit of detection was determined using the CMV WHO International Standard and quantified clinical specimens. Reproducibility was also assessed. After establishing quality control metrics, 185 patient specimens previously tested using Sanger were reanalyzed by NGS. The NGS assay had a low error rate (<0.05%) and high accuracy (95%) for detecting CMV-associated resistance mutations present at ≥5% in contrived mixed populations. Mutation sites were reproducibly sequenced with 40× coverage when plasma viral loads were ≥2.6 log IU/mL. NGS detected the same resistance-associated mutations identified by Sanger in 68/69 (98.6%) specimens. In 16 specimens, NGS detected 18 resistance mutations that Sanger failed to detect; 14 were low-frequency variants (<20%), and six would have changed the drug resistance interpretation. The NGS assay showed excellent agreement with Sanger and generated high-quality sequence from low viral load specimens. Additionally, the higher resolution and analytic sensitivity of NGS potentially enables earlier detection of antiviral resistance.

Investigation of Ganciclovir Resistance in Cytomegalovirus Isolates by Phenotypic and Genotypic Methods

Ganciclovir-resistant cytomegalovirus (CMV) strains are reported following long-term antiviral agent use, especially for immune-suppressive patients. In this study, it was aimed to investigate the mutations in the UL97 gene of CMV, which causes ganciclovir (GCV) resistance by genotypic and phenotypic methods in patients who developed CMV infection following hematopoietic cell (HCT) or solid organ transplantation (SOT). Thirty patients who had HCT or SOT in Mediterranean University Hospital and developed CMV infection during routine follow-up with a viral load of CMV over 1000 copies/mL were included in the study. CMV DNA was analyzed by an automated system (Cobas Ampliprep/COBAS TaqMan CMV Test, Roche Diagnostics, Germany) quantitatively. DNA sequence analysis of the regions including codons 420-664 in the UL97 gene region was done by the Sanger sequencing method to detect mutations causing antiviral resistance and compared with defined mutations. In order to investigate antiviral resistance by phenotypic methods, heparinized blood samples of the patients were collected, 'buffy coat (leukocyte layer)' was inoculated into MRC-5 cells by centrifugation method and CMV growth in these cells was controlled with monoclonal antibodies when growth was detected, virus titer was determined and plaque reduction test was applied as recommended. It was determined that 22 of the 30 patients were HCT recipients and eight were SOT (five kidney, three liver) recipients. When the CMV serology pattern of the patients was evaluated before transplantation, 29 (96.7%) patients were found to be seropositive and one (3.3%) patient was found to be seronegative. Totally, nine CMV UL97 mutations were detected in seven (23.3%) pediatric patients who had HCT, including six seropositive and one seronegative case. In addition, one mutation (D605E) not known to cause GCV resistance was detected in a seronegative recipient and three previously unidentified mutations were detected (1474T, F499S, V559A) in a seronegative recipient. Five of the mutations defined were UL97 mutations with a defined clinical resistance against GCV in each of the five recipients (C603W, C592G, H520Q, M460V, A594T). In the plaque reduction test using 3 µM, 12 µM, 48 µM and 96 µM concentrations of GCV in CMV strains, the IC50 value was determined to be ≥ 8 µM for the five CMV strains, and the phenotypic presence of GCV resistance was shown. Clinical resistance associated with CMV UL97 mutation was detected in five (22.7%) of 22 patients who had HCT. GCV resistance was also demonstrated in these patients by phenotypic methods. No UL97 mutation was detected in the patients who had SOT.

Fibroblasts are a site of murine cytomegalovirus lytic replication and Stat1-dependent latent persistence in vivo

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we show that murine cytomegalovirus, a β-herpesvirus, persists for the long term and across organs in PDGFRα-positive fibroblastic cells, with similar or higher genome loads than in the previously known sites of murine cytomegalovirus latency. Whereas murine cytomegalovirus gene transcription in PDGFRα-positive fibroblastic cells is almost completely silenced at 5 months post-infection, these cells give rise to reactivated virus ex vivo, arguing that they support latent murine cytomegalovirus infection. Notably, PDGFRα-positive fibroblastic cells also support productive virus replication during primary murine cytomegalovirus infection. Mechanistically, Stat1-deficiency promotes lytic infection but abolishes latent persistence of murine cytomegalovirus in PDGFRα-positive fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic murine cytomegalovirus replication and a reservoir of latent murine cytomegalovirus in vivo and STAT1 is required for murine cytomegalovirus latent persistence in vivo.

Decoding murine cytomegalovirus.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

A preliminary study on the UL97 gene of cytomegalovirus isolated from patients suspected of ganciclovir resistance in Sri Lanka

Background: Human cytomegalovirus (HCMV) is an opportunistic virus causing infections in immunocompromised patients. The widespread use of ganciclovir to treat CMV infections has led to the development of ganciclovir resistance. To date, no studies have been reported in Sri Lanka on mutations in viral genes detected from CMV identified by PCR. No Sri Lankan studies have been reported to date of ganciclovir resistance associated CMV gene mutations. Objective: To screen the CMV UL97 gene identified in a cohort of patients who did not respond to ganciclovir for resistance conferring mutations. Methodology: A retrospective study was carried out on suspected ganciclovir resistant patients who had static or increased levels of CMV viraemia treated with ganciclovir for 2-3 weeks to determine mutations of UL97 gene. DNA of CMV was extracted; Codons 439 – 645 of the UL97 gene was amplified by PCR only from patients who did not respond to ganciclovir and two fragments of 354bp (1317-1671) and 285bp (1650-1935) were sequenced. Results: Twelve of the 340 patients with CMV infection had static or increased levels of CMV viraemia despite being treated with ganciclovir for 2-3 weeks. No mutation was found in the UL97 gene detected from the 12 patients which could confer ganciclovir resistance. However, there was an incidental finding of a single nucleotide change from thymine to cytosine at the 1368 nucleotide position of the UL97 gene which was detected in all 12 patients. Conclusion: No mutation was found in the UL97 gene from CMV patients in this preliminary study which could confer ganciclovir resistance. UL54 gene of CMV should be considered in addition to UL97 gene in future studies as mutations reported in both genes are known to contribute to ganciclovir resistance.

Efficiency of Promoters of Human Genes FAP and CTGF at Organism Level in a Danio rerio Model.

The identification of tissue-specific promoters for gene therapeutic constructs is one of the aims of complex tumor therapy. The genes encoding the fibroblast activation protein (FAP) and the connective tissue growth factor (CTGF) can function in tumor-associated stromal cells but are practically inactive in normal adult cells. Accordingly, the promoters of these genes can be used to develop vectors targeted to the tumor microenvironment. However, the efficiency of these promoters within genetic constructs remains underexplored, particularly, at the organism level. Here, we used the model of Danio rerio embryos to study the efficiency of transient expression of marker genes under the control of promoters of the FAP, CTGF, and immediate early genes of Human cytomegalovirus (CMV). Within 96 h after the injection of vectors, the CTGF and CMV promoters provided similar equal efficiency of reporter protein accumulation. In the case of the FAP promoter, a high level of reporter protein accumulation was observed only in certain zebrafish individuals that were considered developmentally abnormal. Disturbed embryogenesis was the factor of changes in the exogenous FAP promoter function. The data obtained make a significant contribution to understanding the function of the human CTGF and FAP promoters within vectors to assess their potential in gene therapy.

S2722 Cytomegalovirus Colitis in an Immunocompetent Patient Presenting With New Onset Ulcerative Colitis: A Clinical Vignette

Introduction: Cytomegalovirus (CMV) colitis is a rare disease among immunocompetent patients presenting with inflammatory bowel disease (IBD). CMV can cause steroid-refractory IBD, which may require antiviral and infliximab (IFX) combination therapy. Case Description/Methods: A 23-year-old woman was admitted for dehydration and poor oral intake in the setting of eight bloody bowel movements per day, tenesmus, and weight loss. She presented to an outside hospital 5-weeks prior with abdominal cramps and bright red blood per rectum. The CT scan showed bowel wall thickening and mucosal enhancement in the rectosigmoid and descending colon. A colonoscopy 2-weeks prior to admission was suggestive of severe ulcerative colitis (UC) and she was started on mesalamine/budesonide. Due to poor clinical response, she was switched to prednisone 40 mg for 5 days. Outpatient medical management failed and, upon admission, she was converted to methylprednisolone 20 mg every eight hours. Serum CMV PCR was positive at 1,758 IU/mL and colonic biopsies showed scattered CMV inclusions by immunohistochemical staining. She was started on ganciclovir 5mg/kg/dose IV in addition to the steroid regimen and IFX infusions were deferred. By day 2 of ganciclovir IV and day 7 of methylprednisolone IV, IBD symptoms began to resolve. On discharge at hospital day 12, patient was transitioned to twice daily oral valganciclovir 900 mg for a 3-week course and an oral prednisone taper. CMV PCR was 1,185 IU/mL on discharge and < 100 IU/mL at one week follow up. IFX therapy was initiated 45 days post discharge with improved symptoms, as characterized by a 17-lb weight gain at 3-month follow-up. (Figure) Discussion: CMV colitis is a rare diagnosis in immunocompetent patients with early, steroid naïve IBD. Serum PCR positive infection has been reported more commonly after 2-3 weeks of steroid therapy. CMV infection is a recognized complication and marker of poor prognosis in moderate to severe UC, especially those who present with steroid refractory disease. It is believed that the inflammatory mediators associated with UC play a synergistic role in CMV reactivation and subsequent disease. In addition, local corticosteroid induced immunosuppression may further facilitate CMV gene transcription, resulting in disease progression and poor steroid response. There remains no consensus for management of UC with concomitant CMV colitis however, in our patient the use of antiviral and biologic therapy (IFX) promoted disease regression and clinical improvement.Figure 1.: H&E Stain (20x) demonstrating active chronic colitis (left). Immunohistochemical staining (40x) showing CMV immunoreactivity (right).

UL49 is an essential subunit of the viral pre-initiation complex that regulates human cytomegalovirus gene transcription

SummaryMore than half the world’s population is infected with human cytomegalovirus (HCMV), causing congenital birth defects and impacting the immuno-compromised. Many of the >170 HCMV genes remain uncharacterized, and this gap in knowledge limits the development of novel antivirals. In this study, we investigated the essential viral protein UL49 and found it displayed leaky late expression kinetics, and localized to nuclear replication compartments. Cells infected with mutant UL49 virus were unable to produce infectious virions and phenocopied other beta-gamma viral pre-initiation complex (vPIC) subunit (UL79, UL87, UL91, UL92, and UL95) mutant infections. RNA-seq analysis of vPIC mutant infections revealed a consistent diminution of genes encoding capsid subunits, including TRX2/UL85 and MCP/UL86, envelope glycoproteins gM, gL and gO, and egress-associated tegument proteins UL99 and UL103. Therefore, as a member of the vPIC, UL49 serves as a fundamental HCMV effector that governs viral gene transcription required to complete the replication cycle.

What drives ‘Memory Inflation’ of CD8 T cells during Cytomegalovirus Latency?

Cytomegalovirus (CMV) infection is controlled by virus-specific CD8 T cells. After clearance of productive CMV infection viral latency will be established in multiple host tissues and CD8 T cells persist at extra-lymphoid sites. Although the overall absolute number of tissue-infiltrating CD8 T cells declines over time, CD8 T cells specific for certain viral epitopes accumulate over time also in absolute numbers and assume the phenotype CD44+CD62L-CD127-KLRG1+ characteristic of effector cells. This phenomenon has been coined with the popular term ‘memory inflation’ (MI). While the knowledge about the immunology of MI expanded in the last years, the ‘molecular drivers’ of MI and the epitope-selectivity are still not understood. Latency is a dynamic state in which not all viral genomic loci are silenced all the time. Instead, certain genes are transiently in an ‘on’ state, a phenomenon recently coined with the term ‘sleepless latency’. It has been proposed that the ‘inflationary’ CD8 T cells are involved in the immune surveillance of viral latency by sensing and terminating episodes of viral transcriptional reactivation. This implies transcription of epitope-encoding viral genes, translation to the respective antigenic protein, and efficient antigen processing and epitope presentation, all of which represent bottlenecks for MI to occur. To date, the IE1 gene of murine CMV is the only viral epitope-encoding gene for which transcripts were detected during latency, corresponding to an IE1 epitope-specific MI, but the amount of IE1 protein is below the limits of molecular detection. Furthermore, it remained debatable if the epitope is presented during latency to drive CD8 T-cell expansion or if MI is driven by a latency-associated MI-promoting altered homeostasis and cytokine-chemokine microenvironment. Here I will give evidence for further viral transcripts expressed in latency (TELs), the expression of a functional protein and the presentation of peptide to CD8 T cell in latency.

The Association of Prothrombin Gene Mutations and Cytomegalovirus Infection with Abortion Among Iraqi Women

Abortion is categorized as the termination of conception caused by the failure or removal of the embryo from the uterus before the conclusion of pregnancy. Microorganisms and genetic factors are two of the many factors associated with abortion. Cytomegalovirus is a widespread congenital virus infection pathogen that affects a wide variety of people. The prothrombin gene is one of the essential causes that trigger blood clotting and the function of abortion women, therefore the aim of the study is to detect and associate Cytomegalovirus and prothrombin gene mutation (Gene ID: 14061 in NCBI) with abortion through genetic and immunological methods. Five ml of whole blood was collected from an intravenous puncture and divided into two tubes, one with EDTA and one without (plain tube) from 74 women with an abortion history as a patient group and 74 women without an abortion record who had at least one successful fertility as a control group. Allele-specific PCRs are used to amplify gene regions with genetic primers containing prothrombin gene polymorphisms. Current results have shown the greatest risk of abortion was observed in women patients with IgG seropositivity in 65 women with frequency (87.8%) and the lowest rate of abortion was in IgM seropositivity in 3 women with frequency (4.1%) and 6 (8.1%) were positive for IgM-and IgG indicating they have both an old and recent infections. Furthermore, allele-specific PCRs are used to amplify prothrombin G20201A polymorphism. The result of this study demonstrated there is no association between prothrombin genotype level frequency and abortion in CMV-infected women. While, there is a highly significant association between A and G Alleles combinations and abortion in CMV-infected women.

P1617: CLINICAL SIGNIFICANCE OF CYTOMEGALOVIRUS DRUG RESISTANCE GENE MUTATIONS IN CYTOMEGALOVIRUS INFECTION AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background: Cytomegalovirus (CMV) infection is the most important complication after allo-HSCT and threatens the prognosis of patients seriously. Currently, there are limited data on CMV drug-resistant gene mutations in CMV infection after transplantation in China. Aims: In order to further understand the expression of CMV drug-resistant gene mutations in patients with poor efficacy of CMV infection in single center tertiary hematology hospital in china. Methods: The clinical data of 41 patients with CMV infection after allo-HSCT were retrospectively analyzed from January 2019 to July 2021 in transplantation Center of Hematology Hospital, Chinese Academy of Medical Sciences, due to refractory and probable refractory CMV infection, who were tested for CMV resistance gene mutation. The current recommended definitions are based on Guidelines for the management of cytomegalovirus infection from ECIL 7. A total of 21 patients with AML, 8 patients with ALL, 6 patients with AA, 5 patients with MDS and 1 patients with Fanconi anemia are included. Donors were HLA haploid-identical related (N = 36), HLA-identical siblings and un-related (N = 5). Informed consent was obtained. Results: ① Of All 41 patients included, 1 patient (2.4%) had UL97 mutation, 19 patients (46.3%) had UL54 mutation, no patients occur simultaneously with UL54 and UL97 mutation. The overall mutation frequency was 48.8% (20/41), and all mutation types were missense mutation. ② UL54 gene were detected 8 cases of T691S missense mutation (8/41, 19.5%), 3 cases of M827I missense mutation (3/41, 7.3%) and 1 case of A786V missense mutation (1/41, 2.4%). All above these 3 mutation sites had been reported, but the drug resistance significance was unknown. There were 1 site mutation (A543S) of UL97 gene and 19 sites mutation of UL54 gene that had not been reported. The most common mutation site is the T691S mutation in UL54 gene. In all the 41 patients, only one patient exhibited a mutation with definite anti-CMV drug resistance significance: Q578H mutation of UL54 gene, and finally was successfully treated with twice CMV-CTL infusions (figure). ③ Compared with patients without UL54 mutation, patients with UL54 mutation strains had a higher proportion of CMV-DNA re positive rate in 10 days after remission of DNAemia (42.1% vs.9.5%, χ2=5.647, P=0.028), and hadthe significantly longer complete clearance time of CMV DNAemia (40 days vs. 24 days, Z=2.198, P=0.027). ④ Compared with those patients without CMV gene mutation, the patients with T691S mutation had a higher incidence in terms of CMV-DNA turned re positive in 10 days and 14 days after remission of DNAemia (71.4% vs. 19.0%, P=0.008; 71.4% vs. 9.5%, P=0.001; 71.4% vs. 9.5%, P=0.001, respectively). The median time of CMV-DNA reaching the peak was significantly later (14 days vs. 10 days, P=0.016). The median clearance time of CMV DNAemia was significantly longer (56 days vs. 24 days, P=0.002), and the differences were statistically significant. Compared with the non-T691s UL54 mutation, the patients with T691S mutation also had similar results. Image:Summary/Conclusion: CMV gene missense mutations were detected in 48.8% of patients infected with CMV after transplantation, most of which occurred in UL54 gene and T691S mutation is a common missense mutation. The incidence of missense mutations with clear drug resistance was low (2.4%) and might be induced by antiviral therapy. The median clearance time of CMV DNAemia in UL54 mutated patients was significantly longer, and the proportion of CMV-DNA turned re positive was increased, which might be caused by T691S mutation in UL54 gene.

Reactivation of CMV and HSV-1 Among Severe COVID-19 Patients May be Triggered by Increased Level of Pulmonary Dioxin

PurposeReactivation of cytomegalovirus (CMV) infection was recently found to influence worse clinical outcome following SARS-CoV-2 infection (doi: 10.1186/s12979-020-00185-x). Other findings showed that CMV and herpes simplex virus 1 (HSV-1) reactivations are observed in patients with COVID-19 acute respiratory distress syndrome (ARDS) (HYPERLINK "https://doi.org/10.1186/s13054-020-03252-3" doi.org/10.1186/s13054-020-03252-3). Addressing occurrence of Herpesviridae reactivation in immunocompetent patients and still unspecified triggers of reactivation, we tested potent xenobiotic 2,3,7,8-tetrachrolodibenzo-p-dioxin (TCDD), which current body burden (DBB) ranges from 20 pg/g (TEQ in fat) in general population to 100 pg/g in older people.Methods & MaterialsIn silico quantitation of active dioxin response enhancers (DRE) in promoters of viral genes. Plaque assay of viral titer. Virus DNA hybridization assay. Clinico-epidemiological analysis.ResultsIn silico analysis revealed in regulatory region of CMV IE genes from 5 to 10 DRE, and from 6 to 8 DRE in regulatory region of HSV-1 IE genes. We established that a picomolar TCDD can trigger up-regulation of CMV and HSV-1 genes via AhR:Arnt transcription factor in macrophage (doi.org/10.1016/j.ijid.2012.05.265) and glial human cell lines (doi.org/10.1016/j.jalz.2016.06.1268), respectively. In fact, viral reactivation may be triggered in COVID-19 ARDS patients by higher pulmonary TCDD concentrations, as lipid storm within lungs of severe COVID-19 patients has been recently reported (doi.org/10.1101/2020.12.04.20242115). Due to hydrophobic character (Log PO/W: 7.05), TCDD partitions into inflammatory lipids in lung tissue thus augmenting its local concentration. Population-based epidemiological data on SARS-CoV-2 first wave of pandemic showed that picomolar TCDD can reactivate CMV (measured by high CMV seropositivity), as cumulative mortality rate 4.5 times was revealed in Lombardi region of Italy where after Seveso industrial accident TCDD plasma level in pre-exposed subjects still is 15 times the level in rest of Italy (doi.org/10.3389/fpubh.2020.620416). Also, Arctic Native (AN) peoples have TCDD body burden 7 times that in general population, as they consume dioxin-contaminated fat in seafood, and their COVID-19 mortality is 2.2 times of that among non-AN Alaskans (doi: 10.15585/mmwr.mm6949a3).ConclusionTCDD in picomolar range may trigger CMV expression in lung cells and commit virus to the lytic cycle, which can be applied to reactivation of Herpesviridae infection in immunocompetent patients with COVID-19 ARDS syndrome.

Monitoring of resistanceassociated mutations in UL97 gene of cytomegalovirus in children after allogeneic hematopoietic stem cell transplantation

Objective. To perform genotyping of cytomegalovirus (CMV) phosphotransferase UL97 and investigate mutations associated with ganciclovir/valganciclovir (GCV/VGCV) resistance in children after allogeneic hematopoietic stem cell transplantation (HSCT) with confirmed CMV infection. Materials and Methods. This single-center prospective study was conducted from January 2020 to December 2021 and enrolled allogeneic HSCT recipients under 18 years of age with confirmed CMV infection. In case of possible GCV resistance, molecular genotyping with Sanger sequencing was performed. The search for mutations in the UL97 gene was carried out in the range of 425–670 codons. Results. Out of 168 patients with confirmed CMV infection, 72 patients met the criteria for possible resistance to GCV/VGCV. As a result of nucleotide sequencing of the CMV UL97 gene in 19 patients (11.3% of a total number of patients and 26.4% of patients meeting the resistance criteria) 11 genotypes of the following mutations were identified: H520Q, C592G, A594V, L595S, D605E, C603W, C607Y, C607F, M615V, M460V and E655K. The following mutations associated with resistance to HCV/VHCV: H520Q, C592G, A594V, L595S, C603W, C607Y, C607F, M460V were found in 12 patients (7% of a total number of patients and 9.7% of patients meeting the resistance criteria). Conclusions. Due to a high frequency of detection of the mutant form of phosphotransferase UL97 in allogeneic HSCT recipients with confirmed CMV infection, it is important to implement monitoring of resistant-associated mutations in order to administer appropriate antiviral therapy.

NanoString evaluation of murine Cytomegalovirus transcription in vivo and in vitro

BackgroundNext Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to measure gene expression levels, however the information gathered is quite small in comparison to NGS. A newer method called nanoString allows for highly multiplexed gene expression analysis by detecting mRNAs without the use of enzymes for reverse transcription or amplification even for single cells or low input material. The method can be done in 1.5 days and data are quickly analyzed by the accompanied user friendly software.Our aim was to investigate this new method and compare it to the existing alternatives, while investigating murine Cytomegalovirus (mCMV) infection and latency. MethodsmCMV infected murine embryonic fibroblasts (MEF), lung and salivary glands from BALB/c mice were evaluated at different stages of infection. A set of 30 custom designed nanoString probes were tested, 20 probes specific for mCMV genes, 6 probes for host genes known to be influenced by viral infection and 4 reference gene specific probes. nanoString counts were compared to published RNA-Seq RPKM. ResultsWe found that nanoString can be used for analysis of cytomegalovirus gene expression during acute infection in vitro and in vivo, both for virus specific and host genes. Although some transcripts show different expression rates in comparison to NGS data, the most abundant transcripts are comparable. When tissues are infected, there are significantly fewer transcripts than in MEFs, and consistent with previous work there are significant differences in relevant abundance between MEF and tissues. We were unable to detect our viral transcripts of interest in latently infected tissue. ConclusionsFor viruses with annotated transcriptomes, nanoString allows simultaneous quantitation of multiple virus and host genes. One huge advantage of the platform is rapid turnaround and simplicity of analysis. It should prove to be very useful to explore host virus interactions during acute infection, but it is unclear if it has adequate sensitivity for analysis during latency in immunocompetent mice.

Detection of cytomegalovirus in plasma of patient’s HIV by duplex real time PCR

Cytomegalovirus (CMV) is able to cause serious and even deadly diseases in immunocompromised patients. It is important to have a sensitive, specific and molecular viral tests for its detection, using as targets, key genes for viral replication. The following genes have been used in the molecular detection of CMV: UL122 (replication) and UL83 (most abundant protein of the tegument). Detect and quantify CMV, by real-time duplex PCR, from a minimum amount of plasma. The UL122 and UL83 genes were amplified with different fluorophores, by real-time duplex PCR. To quantify CMV, curves were generated, starting with DNA-CMV (1.0-0.0000001 ng). The dynamic range of “master” duplex straight had a pendent (m) −3.0, the amplification efficiency was 115.44% plasmas from patients with HIV viral load ≥ 100,000 copies/mL, 11.36% were true positive for CMV and 88.64% had no amplifications or they were outside of the linear range of molecular detection. This test identified two important CMV genes (UL122 and UL83) in a single reaction (FAM:VIC), viral detection was confirmed from a minimum amount of plasma. This mean a smaller amount of biological sample required and would add a tool to the clinical area, as well as a lower consumption of reagents and materials.

Construction of Human Cytomegalovirus Mutants with Markerless BAC Mutagenesis.

To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they are studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of these loss-of-function viral mutants to the wild-type virus allow for the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 20years. The cloning of CMV genomes into Escherichia coli as bacterial artificial chromosomes (BAC) allows for not only quick and efficient deletion of viral genomic regions, individual genes, or single-nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain-of-function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined parental viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, providing selective growth advantage to the recombined genomes and thus clonal selection of viruses with even extremely poor fitness. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.

Functional Profile of Human Cytomegalovirus Genes and Their Associated Diseases: A Review.

The human cytomegalovirus (HCMV), whose genome is 235 ± 1.9 kbp long, is a common herpesvirus. However, the functions of many of its genes are still unknown. HCMV is closely associated with various human diseases and infects 60–90% of the global population. It can infect various human cells, including fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, and monocytes. Although HCMV infection is generally asymptomatic and causes subtle clinical symptoms, it can generate a robust immune response and establish a latent infection in immunocompromised individuals, including those with AIDS, transplant recipients, and developing fetuses. Currently available antivirals approved for the treatment of HCMV-associated diseases are limited by dose-limiting toxicity and the emergence of resistance; however, vaccines and immunoglobulins are unavailable. In this review, we have summarized the recent literature on 43 newly identified HCMV genes. We have described their novel functions on the viral replication cycle, latency, and host immune evasion. Further, we have discussed HCMV-associated diseases and current therapeutic targets. Our review may provide a foundational basis for studies aiming to prevent and develop targeted therapies for HCMV-associated diseases.

One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus.

The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5′ terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3′ terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.

New Insights Into the Molecular Mechanisms and Immune Control of Cytomegalovirus Reactivation.

Cytomegalovirus (CMV) is a β-herpesvirus that establishes lifelong latency in infected hosts. Following transplantation of a latently infected organ, reactivation can occur and consists of a spectrum of clinically apparent syndromes from mild symptoms to tissue-invasive, resulting in both direct and indirect sequelae. Before the advent of effective antiviral agents, the primary treatment was reduction in immunosuppression (IS). While antiviral agents provide effective prophylaxis, there are several important caveats associated with their use, including drug toxicity and resistance. The traditional view attributes CMV reactivation and the ensuing clinical disease primarily to IS, either intrinsic to disease-related immune compromise or from the extrinsic administration of IS agents. However, previous data from both animal models and human subjects showed that inflammatory signals could induce upregulation of latent viral gene expression. New data demonstrate that ischemia/reperfusion is necessary and sufficient to induce CMV reactivation following murine transplantation of a latently infected graft. In this article, we review a growing body of evidence that suggests that reactivation of both human CMV and murine CMV is first triggered by molecular events that activate CMV gene expression and lytic infection and viral dissemination are then facilitated by IS. The initial activation of viral gene expression may be mediated by oxidative stress, DNA damage, or inflammatory cytokines, and these factors may act synergistically. New therapeutic approaches are needed to capture this complex array of targets.