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Human cytomegalovirus (HCMV) resistance to antiviral drugs is a major drawback of repeated or long-duration treatment in immunocompromised patients. Resistance testing is usually performed by genotypic assays. For accurate interpretation of these assays, the role of new mutations in HCMV resistance has to be assessed. Two previously unknown UL54 single point mutations (D515Y and V787A) were characterized for phenotypic drug-resistance by marker transfer analysis using bacterial artificial chromosome (BAC) mutagenesis. Increases in 50% inhibitory concentrations of ganciclovir and foscarnet were found for both mutated recombinant strains showing that mutations D515Y and V787A induce resistance to both antivirals. Importantly, none of those impacted the viral growth kinetics. For a better understanding of their molecular resistance mechanisms, a 3D homology model was used to localize the mutated amino-acids in functional domains of UL54 and predict their impact on UL54 function and resistance. However, 3D homology model analysis has limits and phenotypic characterization using BAC-HCMV is still essential to measure the role of unknown mutations., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pre-emptive antiviral treatment efficiently prevents occurrence of cytomegalovirus (CMV) disease in allogeneic stem cell transplant recipients. However, successive treatment courses can be necessary. The current study was aimed at determining factors that could influence the response to antiviral treatment, in particular the donor CMV serostatus. A total of 147 consecutive CMV-seropositive recipients (R+) were included and prospectively monitored for 6 months after transplantation. Reactivation of CMV occurred in 111 patients, 61 of 78 with a CMV-positive donor (D+) and in 50 of 69 with a CMV-negative donor (D-) (p 0.45). Baseline viral loads and initial viral doubling times did not differ between D+/R+ and D-/R+. Fifteen D+/R+ and four D-/R+ had self-resolving CMV infections. A total of 92 patients received antiviral treatment and 81 (88%) had a significant decrease in CMV load under therapy. Repeated CMV episodes were observed in 67% of those and were significantly more frequent in D-/R+ than in D+/R+ (p <0001). Half-life of CMV under treatment was significantly longer in D-/R+ than in D+/R+. Treatment failure observed in eight recipients was associated with low leucocyte count at reactivation onset, and was significantly more frequent in D-/R+ (six patients) than in D+/R+ (two patients) (p <0.0001). CMV strains resistant to antivirals were found in two D-/R+. Donor CMV serostatus influenced neither CMV reactivation occurrence nor the kinetics of CMV DNA load in the early phase of CMV replication but had a significant impact on response to antiviral therapy. Virological drug-resistance remained rare., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Monitoring cytomegalovirus circulating viral load is an important parameter of the follow-up in immunocompromised patients. It can be measured either by DNAemia or by pp65 antigenemia. The French national reference center for cytomegaloviruses organized an investigation of practice in 37 teacher hospital virology laboratories to assess the situation in France in 2010., Methods: A questionnaire was sent to collect following information: method used in routine for monitoring of circulating viral load of CMV, assay used, sample matrix and extraction method., Results: Thirty-six over thirty-seven laboratories filled the questionnaire. Among these, 67% used the quantitative PCR in routine, 11% antigenemia and 22% antigenemia or quantitative PCR; 87% of the laboratories use whole blood for quantitative PCR, whereas 10% and 3% use plasma and leukocytes respectively. Among the laboratories using DNAemia, 100% used real-time PCR assays, 91% use an automated extraction and 9% a manual extraction., Conclusion: Thus in France, measurement of DNAemia by real-time PCR is a tool, which gradually replaces the antigenemia for the monitoring of cytomegalovirus infection among immunocompromised patients. The very great diversity of the methods used justifies the installation of a national quality control on total blood, matrix used by 87% of the laboratories., (Copyright © 2011. Published by Elsevier SAS.)

The drugs currently available for treatment of severe human cytomegalovirus (HCMV) infections suffer from many drawbacks, particularly toxicity, and potential teratogenicity contraindicating their use in target populations such as pregnant women. The emergence of drug-resistant strains is still a problem for disease management, particularly in immunosuppressed populations where antivirals are used for extended periods of time. The flavonoid family of drugs contains promising candidates as they have low toxicity and inhibit different targets to currently available antivirals. We report here that, unlike their chalcon homologs, four flavonoids (baicalein, quercetin, quercetagetin and naringenin) inhibit various stages of HCMV replication, the most active anti-HCMV compound being baicalein and the less active and less selective being quercetagetin. These drugs could provide potential inhibitors of virus replication alone or in combination, without increased toxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antimalarial drug artesunate inhibits cytomegalovirus (HCMV) replication in human fibroblasts. Astrocytes, the major cell type of the brain, support cytomegalovirus (HCMV) replication. The aim of the study was to assess the antiviral activity of artesunate in astrocytoma cell line U373MG in comparison with ganciclovir. The antiviral concentration inhibiting by 50% (IC(50)) the synthesis of viral DNA was measured by real-time PCR in parallel in U373MG and MRC-5 cells. Reference HCMV strains susceptible and resistant to ganciclovir, and clinical isolates were tested. Ganciclovir and artesunate had similar activity in U373MG cells and MRC-5 fibroblasts. The artesunate IC(50)s in U373MG cells (1.5-2.25 μM) were at least 36-fold lower than the 50% cytotoxicity concentrations. Then, the anti-HCMV activity of artesunate was demonstrated in a cancer cell line., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Unlabelled: Occult hepatitis B is defined as the presence of hepatitis B virus (HBV) DNA in the absence of detectable HBs antigen. The prevalence of occult hepatitis B among patients HIV-infected is uncertain, varying between 0% and 89%, and the clinical consequences of the coinfection are poorly known. The aim of this study was to evaluate the frequency of occult hepatitis B among HIV-infected patients and determine risk factors., Methods: This retrospective study was conducted with plasma samples from 31HIV-infected patients untreated for HBV infection and for whom at least one sample was available. All patients were found to be carriers of isolated anti-HBc antibodies between 2000 and 2008, and HBV DNA was quantified in 51 samples (one to three per patient) by real-time PCR using the Qiagen HBV PCR kit., Results: HBV DNA was found in samples from seven patients (22%). Occult hepatitis B seemed to be more frequent among patients coinfected with HCV (p=0.047). The number of CD4 cells was significantly less in samples containing detectable HBV DNA than in those with no detectable HBV DNA., Conclusion: The prevalence of occult hepatitis B seemed high, and HBV DNA titers were weak (< 20UI/mL), among patients infected with HIV and carrying isolated anti-HBc antibodies. These results would support screening HIV-infected patients for the presence of HBV DNA if confirmed with a larger patient population., (Copyright 2009 Elsevier Masson SAS. All rights reserved.)

A quantitative real-time PCR-based assay was developed for determination of cytomegalovirus (HCMV) susceptibility to antiviral drugs. After HCMV isolate-growth for 4 days, antiviral drug susceptibility was determined by measuring the reduction of intracellular HCMV DNA in the presence of increasing concentrations of either ganciclovir, or foscarnet or cidofovir. The 50% inhibitory concentration (IC(50)) was the drug concentration that reduced the number of HCMV genome copies by 50%. The IC(50) values were measured for seven HCMV reference strains sensitive or resistant to one or more antiviral drugs. The antiviral susceptibility of 21 HCMV isolates was then tested and the results were consistent with prior determination of their phenotype and/or genotype by plaque reduction assay and sequencing. The real-time PCR susceptibility assay reported here was found to be highly reproducible, simpler to perform than the plaque reduction assay, and amenable to use in the routine diagnostic virology laboratory.

We described a colorimetric method to determine the biochemical phenotype of wild-type and mutated cytomegalovirus (HCMV) DNA polymerases by measuring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. Mutations V715M and E756K, which are known to confer foscarnet-resistance, were used as controls. Mutation N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, were studied. The mutations were introduced by site-directed mutagenesis into wild-type gene UL54 cloned in an expression vector and then polymerases were synthesised by using a commercially available coupled transcription-translation system. The polymerase activity was measured with and without foscarnet. The activity of polymerases containing the V715M or E756K mutations was inhibited by foscarnet at concentrations 70- and 30-fold higher than that of wild-type polymerase, respectively. Change N495K and combination of K415R and S291P, induced a five- and ten-fold decrease in susceptibility to foscarnet, respectively. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. Therefore, this novel phenotypic method could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.

Cidofovir (CDF) or Vistid is a monophosphate nucleoside analogue that inhibits the DNA polymerase of herpes viruses including the cytomegalovirus (CMV). CDF is active on GCV-resistant strains with a mutation on the phosphotransferase gene (UL97). However, DNA polymerase gene mutations that induce resistance to GCV are responsible for cross-resistance to CDF. Resistance phenotypes to GCV and CDF were determined for 57 CMV strains isolated from blood and urine samples. Sixteen strains were recovered after CDF therapy. Of the remaining 41 CDF-naive strains, 34 were susceptible and seven resistant to GCV. Fifty percent inhibitory concentrations (IC50) for CDF were in the 0.2-2.6 microM range for CDF-naive strains susceptible to GCV. For GCV-resistant strains, IC50 values for CDF were < or = 3 microM for strains with a low level of resistance to GCV (GCV IC50 < 30 microM) and > or = 6 microM for three of the five strains with a high level of resistance to GCV (GCV IC50 > or = 30 microM).

The substitution of methionine by either isoleucine or valine at residue 460 in the UL97 phosphotransferase has been shown to be responsible for resistance to ganciclovir (GCV) in 30% of resistant cytomegalovirus (CMV) isolates [4]. These substitutions require one nucleotide change in the gene (G- > T 1 380 and A- > G 1378 respectively). The aim of this study was to develop a discriminative PCR assay for rapid detection of these DNA changes. A PCR assay was duplicated in parallel for each mutation; to detect G- > T 1380 each reaction mixture contained primer VSUL14 and either primer LNW to distinguish wild type residues or LNM to distinguish mutant residues, and for A- > G 1378 primers were VSUL8 and either MCMW to detect wild type sequences or MCMM to detect mutated residues. For optimal discrimination, primers MCMW and MCMM were designed with a mismatch at position 3'-1. The reference strains AD169, Davis and Towne, a laboratory GCV-resistant mutant RCL1.7, and 33 CMV isolates (10 resistant, 2 indetermined and 21 sensitive) were tested by PCR. AD169, Davis and Towne, and 30 isolates were amplified only with non modified primers, and the absence of 460 mutations was confirmed by sequencing. Two isolates P1 and P2, from a transplanted patient were amplified with both MCMM and MCMW: sequencing analysis shown the presence of a mixture of strain, one of them harbouring A- > G 1378 mutation. One resistant strain was amplified neither with MCMM nor with MCMW: a C- > T silent mutation at nt 1368 was present. As sequencing analysis confirmed PCR results, discriminative PCR enables isolates to be rapidly assessed for the presence or absence of 460 mutations. Moreover, it can distinguish Met to Val from Met to Ile mutations, and allows the analysis of mixtures of sensitive ad resistant strains.

The Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). Serological studies have shown that the clinical onset of NPC is preceded by the appearance of a high antibody titer of IgA to viral capsid antigens and early antigens. The titers increase with the total tumor burden and the antibodies decline with the response to therapy. In patients with confirmed clinical remission elevation of IgA serological titers is highly significant for prediction of relapse. This demonstrates the usefulness of EBV serology for the clinical management of NPC patients.

The prevalence of HIV infection was assessed among 15,611 consecutive patients attending a Parisian anonymous testing center from April 1988 to June 1993. Sera (17,910) were tested for the presence of anti-HIV antibodies using two different enzyme-linked immunosorbent assays. Seropositivity was verified by Western blotting. The sera were also assayed for HIV antigenemia detection in 2,493 cases. Six hundred and seventy-seven patients were found to be anti-HIV antibody positive: among them 666 were infected by HIV-1 and only 11 by HIV-2. Antigenemia was detected in 108 samples (4.3%). In all cases but 5, antigenemia was associated with the presence of specific antibodies. Risk factors for HIV infection could be determined for 5,735 patients. The HIV prevalence rates were 5.2% in 1988-89, 4.9% in 1990, 3.4% in 1991, 2.8% in 1992 and 1.8% for the 6 first months of 1993 (p < 0.01). Only one patient was coinfected with HTLV-1. This study shows a trend of decreasing seropositivity rates among the patients attending the anonymous testing center since 1990. By contrast, the percentage of seropositive patients with antigenemia was stable between 1988 and 1993.

Recently developed methods have greatly increased the sensitivity and speed of virological diagnosis of cytomegalovirus infection. Virus can be detected in infected cell cultures within 24 or 48 hours of specimen inoculation by using monoclonal antibodies to immediate-early antigens in immunocytochemistry procedures or DNA sequences in hybridisation in situ assays. CMV antigens can also be detected directly in infected cells within clinical specimens. An early antigen can be visualized in nuclei of circulating leukocytes from viremic patients. DNA hybridization is used for CMV analysis in Dot-blot, Southern-blot and in situ hybridization assays. DNA amplification, by polymerase chain reaction (PCR), has proven to be a very sensitive method for diagnosis of CMV infection and should be useful for investigation of CMV pathogenesis and latency. Serologic assays such as ELISA and latex agglutination assays are accurate for screening donors and recipients of blood and organ or marrow graft. Studies of viral protein epitopes recognized by human sera are in progress.

Rapid detection of human cytomegalovirus (HCMV) from blood was performed in parallel using inoculation into human fibroblastic cells of both 500 microliters of whole blood and of buffy coat derived from 9.5 ml of blood. Of the 46 samples tested, 20 were positive for HCMV, 18 when the buffy coat was inoculated (90%) and 17 (85%) when whole blood was used. Fifteen samples were positive by both techniques. Four samples gave discordant results: in three cases, viraemia was detected only by buffy coat assay and in one case only by whole blood assay. These results suggest that HCMV isolation from blood should be performed by inoculating whole blood into cell cultures if only small blood volumes can be collected.

In a double-blind controlled study, oral acyclovir was compared with placebo in 39 consecutive patients undergoing bone-marrow transplantation. Acyclovir was given at a dose of 200 mg every 6 h from 8 days before to 35 days after bone-marrow transplantation. Pharmacokinetic studies showed good absorption of the drug, despite intestinal damage related to chemoradiotherapy or gut graft-versus-host disease. There was no sign of toxicity. The protection against herpes simplex virus (HSV) infection was complete in the treated group compared with the placebo group even in patients with high anti-HSV antibody titres before transplantation. The same protection was observed against cytomegalovirus (CMV) infection. The frequencies of HSV and CMV infections were the same in both groups after the cessation of treatment.