HCMV Infection Research Articles

Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels (154.32)

Abstract Human cytomegalovirus (HCMV) productively infects CD34+ progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. CIITA mRNA levels were significantly lower in HCMV-infected matLC than in mock-infected cells. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV were decreased by HCMV, a result not seen after incubation of matLC with LPS. Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. The stability of CIITA mRNA was not diminished by HCMV, arguing that diminished transcription is the mechanism for CIITA mRNA reduction. HCMV-infected matLC expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. Thus, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, resulting in reduced MHC II biosynthesis as a candidate immunoevasive mechanism.

Effects of congenital HCMV infection on synaptic plasticity in dentate gyrus (DG) of rat hippocampus

This study was carried out to investigate whether the congenital HCMV infection affect the induction and maintenance of LTP /DP. Rat models of Sprague–Dawley rats congenitally infected by HCMV were made. Field excitatory postsynaptic potentials (EPSPs) were recorded in the hippocampal slices of offspring rats (50–65 days) to study alterations of LTP /DP in area dentate gyrus (DG) of the hippocampus after congenital infection. The Ca 2+ and mRNA level of calmodulin (CaM) in the hippocampus neurons of the experiment group (congenital infected by HCMV) and the control group were measured;The input/output (I/O) curves of the EPSP slope PS amplitude in area DG in experiment group were significantly depressed when compared to control group ( P < 0.05). LTP of the EPSP slope and PS amplitude in area DG of the hippocampus was 137 ± 4% (EPSP) and 225 ± 11% (PS) in control rats and 115 ± 9% (EPSP) and 163 ± 7% (PS) in experiment rats (EPSP: F = 25.29, P < 0.05;PS: F =74.33 P < 0.05, two-way ANOVA with Tukey test); DP of the EPSP slope and PS amplitude was 86 ± 3% (EPSP) and 85 ± 2% (PS) in control rats and 94 ± 5% (EPSP) and 93 ± 4% (PS) in congenitally infected rats (EPSP: F = 5.62, P < 0.05;PS: F =4.22, P < 0.05, two-way ANOVA with Tukey test) . At the same time, intracellular [Ca 2+] and mRNA level of CaM in the hippocampus neurons of the experiment group were significantly increased than that of in the controls ([Ca 2+]: P < 0.01;CaM mRNA: P < 0.01) . The results demonstrate that congenital HCMV infection could reduce the range of synaptic plasticity in the Sprague–Dawley rats, which may trigger the dysfunction of learning and memory through disrupting the calcium balance.

Peptide inhibition of human cytomegalovirus infection

BackgroundHuman cytomegalovirus (HCMV) is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV)- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB), a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection.ResultsUsing the Wimley-White Interfacial Hydrophobicity Scale (WWIHS), several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF) were infected with the Towne-GFP strain of HCMV (0.5 MOI), preincubated with peptides at a range of concentrations (78 nm to 100 μM), and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100 μM and 50 μM, respectively, and 60% at a concentration of 2.5 μM. While peptides 264-291 and 297-315, individually failed to inhibit viral infection, when combined, they showed 67% inhibition of HCMV infection at a concentration of 0.125 μM each.ConclusionsPeptides designed to target putative fusogenic domains of gB provide a basis for the development of novel therapeutics that prevent HCMV infection.

Genotyping of Human Cytomegalovirus (Hcmv) Glycoprotein B (Gb) in Hematopoietic Stem Cell Transplant Recipients With Active HCMV infection: Impact of gB Genotypes on the Patient's Outcome

Genotyping of Human Cytomegalovirus (Hcmv) Glycoprotein B (Gb) in Hematopoietic Stem Cell Transplant Recipients With Active HCMV infection: Impact of gB Genotypes on the Patient's Outcome

Prognostic markers of symptomatic congenital human cytomegalovirus infection in fetal blood

To identify fetal cord blood prognostic markers of symptomatic congenital human cytomegalovirus infection (HCMV). Retrospective observational study. Fetal medicine unit in Milan and Medical virology unit in Pavia, Italy. HCMV-infected and -uninfected fetuses of mothers with primary HCMV infection during the period 1995-2009. Overall, 94 blood samples from as many fetuses of 93 pregnant women experiencing primary HCMV infection were examined for multiple immunological, haematological and biochemical markers as well as virological markers. Congenital HCMV infection was diagnosed by detection of virus in amniotic fluid, and symptomatic/asymptomatic infections were determined by ultrasound scans, nuclear magnetic resonance imaging, histopathology or clinical examination at birth. Blood sample markers were retrospectively compared in symptomatic and asymptomatic fetuses with congenital infection. A statistical analysis was performed to determine the value of each parameter in predicting outcome. Univariate analysis showed that most nonviral and viral markers were significantly different in symptomatic (n = 16) compared with asymptomatic (n = 31) fetuses. Receiver operator characteristics analysis indicated that, with reference to an established cutoff for each marker, the best nonviral factors for differentiation of symptomatic from asymptomatic congenital infection were β(2) -microglobulin and platelet count, and the best virological markers were immunoglobulin M antibody and DNAaemia. β(2) -Microglobulin alone or the combination of these four markers reached the optimal diagnostic efficacy. The determination of multiple markers in fetal blood, following virus detection in amniotic fluid samples, is predictive of perinatal outcome in fetuses with HCMV infection.

Study of Viral Infections in Infants with Biliary Atresia

Viral infections may have an important role in the pathogenesis of biliary atresia, and related clinical outcomes. In this research for determination of the possible role of HBV, HCV, HCMV, adenovirus, and BK virus infections in biliary atresia related clinical complications, the molecular and antigenic prevalence of these viral agents were studied. In this retrospective study, 34 formalin fixed paraffin embedded (FFPE) biopsy and autopsy liver tissue samples of neonates with biliary atresia were evaluated. The molecular prevalence of these viral infections was assayed by different PCR and RT-PCR methods. The antigenic prevalence of HBV, HCV, and HCMV infections was also studied in these liver tissue samples by immunohistochemistry (IHC) method. HBV, HCV, and adenovirus genomes were detected in 9%, 6%, and 6% of liver autopsy and biopsy tissues of infants with biliary atresia, respectively. HBV and HCV co-infection was confirmed in 6% of FFPE samples. The genome of other investigated viruses was not detected in FFPE liver tissues. Detection of viral infection in FFPE liver tissue samples of newborns with biliary atresia, suggests the need for complete studies for the determination of accurate role of these viral infections in pathogenesis of biliary atresia.

Detection of human cytomegalovirus in normal and neoplastic breast epithelium

IntroductionHuman cytomegalovirus (HCMV) establishes a persistent life-long infection, and can cause severe pathology in the fetus and the immunocompromised host[1]. Breast milk is the primary route of transmission in humans worldwide, and breast epithelium is thus a likely site of persistent infection and/or reactivation, though this phenomenon has not previously been demonstrated. Increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. We hypothesized that persistent HCMV infection occurs in normal adult breast epithelium and that persistent viral expression might be associated with normal and neoplastic ductal epithelium.MethodsSurgical biopsy specimens of normal breast (n = 38) breast carcinoma (n = 39) and paired normal breast from breast cancer patients (n = 21) were obtained. Specimens were evaluated by immunohistochemistry, in situ hybridization, PCR and DNA sequencing for evidence of HCMV antigens and nucleic acids.ResultsWe detected HCMV expression specifically in glandular epithelium in 17/27 (63%) of normal adult breast cases evaluated. In contrast, HCMV expression was evident in the neoplastic epithelium of 31/32 (97%) patients with ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) cases evaluated (p = 0.0009).ConclusionsThese findings are the first to demonstrate that persistent HCMV infection occurs in breast epithelium in a significant percentage of normal adult females. HCMV expression was also evident in neoplastic breast epithelium in a high percentage of normal and neoplastic breast tissues obtained from breast cancer patients, raising the possibility that viral infection may be involved in the neoplastic process.

Translational Control of the Abundance of Cytoplasmic Poly(A) Binding Protein in Human Cytomegalovirus-Infected Cells

Irrespective of their effects on ongoing host protein synthesis, productive replication of the representative alphaherpesvirus herpes simplex virus type 1, the representative gammaherpesvirus Kaposi's sarcoma herpesvirus, and the representative betaherpesvirus human cytomegalovirus [HCMV] stimulates the assembly of the multisubunit, cap-binding translation factor eIF4F. However, only HCMV replication is associated with an increased abundance of eIF4F core components (eIF4E, eIF4G, eIF4A) and the eIF4F-associated factor poly(A) binding protein (PABP). Here, we demonstrate that the increase in translation factor concentration was readily detected in an asynchronous population of HCMV-infected primary human fibroblasts, abolished by prior UV inactivation of virus, and genetically dependent upon viral immediate-early genes. Strikingly, while increased mRNA steady-state levels accompanied the rise in eIF4E and eIF4G protein levels, the overall abundance of PABP mRNA, together with the half-life of the polypeptide it encodes, remained relatively unchanged by HCMV infection. Instead, HCMV-induced PABP accumulation resulted from new protein synthesis and was sensitive to the mTORC1-selective inhibitor rapamycin, which interferes with phosphorylation of the mTORC1 substrate p70 S6K and the translational repressor 4E-BP1. While virus-induced PABP accumulation did not require p70 S6K, it was inhibited by the expression of a dominant-acting 4E-BP1 variant unable to be inactivated by mTORC1. Finally, unlike the situation in alpha- or gammaherpesvirus-infected cells, where PABP is redistributed to nuclei, PABP accumulated in the cytoplasm of HCMV-infected cells. Thus, cytoplasmic PABP accumulation is translationally controlled in HCMV-infected cells via a mechanism requiring mTORC1-mediated inhibition of the cellular 4E-BP1 translational repressor.

Identification of Binary Interactions between Human Cytomegalovirus Virion Proteins

Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis.

Association Between Mannose-Binding Lectin Gene Polymorphism and Pediatric Cytomegalovirus Infection

Mannose-binding lectin (MBL) is an important constituent of the human innate immune system, and can bind to a wide range of pathogens, including viruses such as influenza A, HIV, herpes simplex 2, and SARS-CoV. MBL deficiency results from single nucleotide polymorphisms (SNPs) in exon 1, and the promoter region of the human MBL2 gene has been found to be associated with susceptibility to a number of infections. However, studies on the interactions between MBL and CMV infection are limited. In this study, we investigated 104 children suffering from HCMV infection, in an effort to find any association between MBL and HCMV infection of children in China. We analyzed the genotypes of 104 HCMV patients and 105 healthy controls, and investigated the distributions of polymorphisms at -550(H/L), -221(Y/X), and +4(P/Q), together with their structural variants. Although there was no significant difference in the distribution of B alleles between HCMV patients and healthy controls, the frequencies of the high-MBL-level related genotype of YA type in HCMV patients were significantly lower than those seen in healthy controls, while low-level related genotypes of XB type were more common in HCMV patients. In addition, CMV-DNA quantification revealed higher viral loads of the XB type in HCMV patients. Thus we can speculate that as an acute response protein and a pattern-recognition molecule of the innate immune system, MBL may play a role in protecting against HCMV infection in children, and MBL gene mutations may be a significant risk factor for the development of infantile HCMV infection.

Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Kinetic studies with CPV as the substrate for pUL97 demonstrated a Km of 1,750+/-210 microM. Introduction of 1.0 or 10 nM maribavir, a known pUL97 inhibitor, and subsequent Lineweaver-Burk analysis demonstrated competitive inhibition of CPV phosphorylation, with a Ki of 3.0+/-0.3 nM. Incubation of CPV with pUL97 combined with GMP kinase [known to preferentially phosphorylate the (+)-enantiomer of CPV-MP] established that pUL97 stereoselectively phosphorylates CPV to its (+)-monophosphate. These results elucidate the mechanism of CPV phosphorylation and help explain its selective antiviral action.

Influence of human cytomegalovirus infection on the NK cell receptor repertoire in children

Human cytomegalovirus (hCMV) infection is usually asymptomatic but may cause disease in immunocompromised hosts. It has been reported that hCMV infection may shape the NK cell receptor (NKR) repertoire in adult individuals, promoting a variable expansion of the CD94/NKG2C+ NK cell subset. We explored the possible relationship between this viral infection and the expression pattern of different NKR including CD94/NKG2C, CD94/NKG2A, immunoglobulin-like transcript 2 (ILT2, CD85j), KIR2DL1/2DS1, KIR3DL1, and CD161 in peripheral blood lymphocytes from healthy children, seropositive (n=21) and seronegative (n=20) for hCMV. Consistent with previous observations in adults, a positive serology for hCMV was associated with increased numbers of NKG2C+ NK and T cells as well as with ILT2+ T lymphocytes. Moreover, the proportions of CD161+ and NKG2C+CD56-CD3- NK cells also tended to be increased in hCMV+ individuals. Excretion of the virus was associated with higher proportions of NKG2C+ NK cells. Altogether, these data reveal that hCMV may have a profound influence on the NKR repertoire in early childhood.

Monitoring human cytomegalovirus infection with nested PCR: comparison of positive rates in plasma and leukocytes and with quantitative PCR

BackgroundHuman cytomegalovirus (HCMV) infection poses a significant health threat to immunocompromised individuals. Here we performed this study to set up a highly sensitive nested PCR method applicable for detecting HCMV infection in high-risk individuals. In this work, 106 blood specimens from 66 patients with potential HCMV infection were obtained. Total DNA was extracted separately from plasma and peripheral blood leukocytes (PBL) of each sample. HCMV DNA was detected in parallel by nested PCR and quantitative real-time PCR (qRT-PCR), and the results were compared.ResultsSerial dilution test revealed that the detection limit of nested PCR was 180 copies/ml. The nested PCR showed a higher positive rate than qRT-PCR (34.9% vs. 12.3%, p < 0.001). The positive rate of nested PCR based on PBL DNA was significantly higher than that based on plasma DNA (34.9% vs. 18.9%, p = 0.002). Of the 14 patients with serial samples, 11 were positive for HCMV DNA in PBL while only 7 were positive in plasma. Moreover, for each patient, nested PCR using PBL DNA also detected more positive samples than that using plasma DNA.ConclusionCombined use of nested PCR with PBL DNA is highly sensitive in defining HCMV infection. This assay is particularly useful in the case of quantification not essential.

Role of caveolin ‐ 1 in the entry of HCMV into human extravillous trophoblast cells

ObjectiveTo establish the cell model of HCMV infect human extravillous trophoblast cells (EVTs)and use this valuable cell model to study the role of caveolin‐1 in the entry of HCMV into EVTs.MethodsEVTs separated from early gestation villous tissue (5–6 weeks gestation) was used to establish the cell model of HCMV infection. The dynamic change of caveolin‐1 in EVTs after infection of HCMV was characterized by using flow cytometry and immunocytochemistry and RT‐PCR.ResultsThe positive products of HCMV immediate early protein can be observed in the nucleuses of infected cells using immunocytochemistry method, mRNA from HCMV IE gene was detected in the EVT cells 24 and 36 hours after infection. The result of flow cytometry shows that the percentage of EVTs apoptosis does not increase at early period, but improve 48 hours later after infection; The expression of caveolin‐1 in uninfected EVT cells is higher, began to reduce at 12 hours after infection, the lowest expression of caveolin‐1 occurs at 24 hours after infection. The expression of caveolin‐1 began to increase at 48 hours, with peak expression occurring at 96 hours after infection.ConclusionEstablished a cell model of HCMV infect EVT cells successfully. The dynamic change of caveolin‐1 in EVTs imply that HCMV enters EVT cells via unique endocytic pathway that involves caveolae/caveolin‐1.

Indeterminate/moderate IgG avidity during HCMV infection: comparison of methods

Background. The IgG avidity test is usually used for differentiating between primary and non-primary HCMV infection within 3 months. Weak avidity is highly suggestive of a primary infection, while high avidity tends to exclude it.An indeterminate or moderate avidity, however, does not allow a clear dating. Since there are several avidity tests with different performances, those tests that are able to minimize the results with indeterminate/moderate avidity are particularly useful. Objectives. The aim of our work was to evaluate the results obtained with two IgG avidity tests in IgG and IgM anti-HCMV positive patients. Study Design. 113 anti-HCMV IgG and IgM positive samples were tested with Enzime Linked Fluorescent Assay (ELFA) and Chemiluminescence Immuno Assay (CLIA) IgG avidity test. Results. 21 samples (18.6%), 50 (44.2%) and 42 (37.2%) with ELFA and 53 samples (46.9%), 10 (8.8%) and 50 (44.2%) with CLIA were found to have respectively low, indeterminate/moderate and high avidity. Of the 50 ELFA indeterminate avidity samples, 32 (64%), 10 (20%) and 8 (16%) were found to have respectively low, moderate and high CLIA.avidity. For 11 cases of the 32 ELFA indeterminate avidity and CLIA low avidity, there were previous data showing a seroconversion within three months. In a case of the 8 ELFA indeterminate avidity and CLIA high avidity, there were, instead, previous data of IgG and IgM positivity already four months earlier. Conclusions. It appears that the CLIA test for IgG avidity is more effective than ELFA for dating HCMV infection.

Active human Cytomegalovirus infection and Glycoprotein B genotypes in Brazilian pediatric renal or hematopoietic stem cell transplantation patients

A prospective analysis of active Human Cytomegalovirus infection (HCMV) was conducted on 33 pediatric renal or hematopoietic stem cell post-transplant patients. The HCMV-DNA positive samples were evaluated for the prevalence of different gB subtypes and their subsequent correlation with clinical signs. The surveillance of HCMV active infection was based on the monitoring of antigenemia (AGM) and on a nested polymerase chain reaction (N-PCR) for the detection of HCMV in the patients studied. Using restriction analysis of the gB gene sequence by PCR-RFLP (Restriction Fragment Length Polymorphism), different HCMV strains could be detected and classified in at least four HCMV genotypes. Thirty-three pediatric recipients of renal or bone marrow transplantation were monitored. Twenty out of thirty-three (60.6%) patients demonstrated active HCMV infection. gB1 and gB2 genotypes were more frequent in this population. In this study, we observed that gB2 had correlation with reactivation of HCMV infection and that patients with mixture of genotypes did not show any symptoms of HCMV disease. Future studies has been made to confirm this.

The correlation of HBV infection and HCMV reactive infection after liver transplantation

Objective To study the correlation of HBV infection pretransplantation and posttrans-plantation and HCMV recurrence after liver transplantation (LT). Methods We reviewed historical patient medical records of LT patients in recent two years in our hospital. All the patients were divided into HBV in-fection group and a control group based on a peripheral blood HB antigen assay before LT. The HBV infec-tion group was divided into HBV reactive infection group and HBV non-relapse group. HCMV antigen pp~65 was detected by immunohistochemical methods. HB antigens and antibodies were detected by time-resolved fluorescence immunoassay, and liver enzyme levels were detected by conventional methods. Results Com-paring two groups of patients, pp65-positive rates of LT patients with HBV infection and control group pa-tients were 84.3% and 57.9% respectively (P=0.024). While in HBV recurrence infection group and non-recurrence infection group, the incidences of HCMV recurrence were 90.9% and 83.3% (P=0.843). The changes in the liver transaminases level in both groups have no statistical significance (P>0.05). Conclusion Pretransplantation HBV infection may increase the incidence of HCMV recurrence. Posttrans-plantation HBV reactive infection, however, may not increase the incidence of HCMV reactive infection. Meanwhile, compare with either HBV infection or HCMV infection alone, co-infection may not serious in liv-er enzymes levels. Key words: Hepatitis B virus; Liver transplantation; Cytomegalovirus; Transaminase

A staining control for the HCMV pp65 antigen test

The HCMV phosphoprotein 65 (pp65) antigen test represents fast, cheap and sensitive method for the routine diagnosis of an ctive HCMV infection.1 However, the lack of economic and easy ccessible (immuno-)staining control slides for the validation of his test has been considered as one problem for its standardization nd quality assurance. Approaches to manufacture suitable stainng control slides weremanifold, including their preparation out of p65 positive patient peripheral blood leukocyte (PBL) samples or heir in vitro-generationby co-cultivationof donorPBLswithHCMV nfected endothelial cells.2 Commercially available HCMV pp65 ntigen test providers supply staining control slides that are based n HCMV infected and uninfected fibroblasts (ARGENE, France), or ased on donor PBLs preparations mixed with defined ratios of ecombinant, pp65 expressing SF9 insect cells (CMV BriteTM Kit, Q Products, The Netherlands). The disadvantages of these existing ather sophisticated, labor intensive and thus expensive methods re the lack of unlimited amounts of biologicalmaterial, the depenency on infectious HCMV and differences in staining intensity of ecombinant pp65, respectively.3

In Vitro and In Vivo Activities of the Novel Anticytomegalovirus Compound AIC246

Human cytomegalovirus (HCMV) remains a serious threat for immunocompromised individuals, including transplant recipients and newborns. To date, all drugs licensed for the treatment of HCMV infection and disease target the viral DNA polymerase. Although these drugs are effective, several drawbacks are associated with their use, including toxicity and emergence of drug resistance. Hence, new and improved antivirals with novel molecular targets are urgently needed. Here we report on the antiviral properties of AIC246, a representative of a novel class of low-molecular-weight compounds that is currently undergoing clinical phase II studies. The anti-HCMV activity of AIC246 was evaluated in vitro and in vivo using various cell culture assays and an engineered mouse xenograft model. In addition, antiviral properties of the drug were characterized in comparison to the current gold standard ganciclovir. We demonstrate that AIC246 exhibits excellent in vitro inhibitory activity against HCMV laboratory strains and clinical isolates, retains activity against ganciclovir-resistant viruses, is well tolerated in different cell types (median selectivity index, 18,000), and exerts a potent in vivo efficacy in a mouse xenograft model. Moreover, we show that the antiviral block induced by AIC246 is reversible and the efficacy of the drug is not significantly affected by cell culture variations such as cell type or multiplicity of infection. Finally, initial mode-of-action analyses reveal that AIC246 targets a process in the viral replication cycle that occurs later than DNA synthesis. Thus, AIC246 acts via a mode of action that differs from that of polymerase inhibitors like ganciclovir.

Active human cytomegalovirus infection and glycoprotein b genotypes in brazilian pediatric renal or hematopoietic stem cell transplantation patients

A prospective analysis of active Human Cytomegalovirus infection (HCMV) was conducted on 33 pediatric renal or hematopoietic stem cell post-transplant patients. The HCMV-DNA positive samples were evaluated for the prevalence of different gB subtypes and their subsequent correlation with clinical signs. The surveillance of HCMV active infection was based on the monitoring of antigenemia (AGM) and on a nested polymerase chain reaction (N-PCR) for the detection of HCMV in the patients studied. Using restriction analysis of the gB gene sequence by PCR-RFLP (Restriction Fragment Length Polymorphism), different HCMV strains could be detected and classified in at least four HCMV genotypes. Thirty-three pediatric recipients of renal or bone marrow transplantation were monitored. Twenty out of thirty-three (60.6%) patients demonstrated active HCMV infection. gB1 and gB2 genotypes were more frequent in this population. In this study, we observed that gB2 had correlation with reactivation of HCMV infection and that patients with mixture of genotypes did not show any symptoms of HCMV disease. Future studies has been made to confirm this.