WT1 quantification is an informative molecular marker for MRD in pediatric AML and is now performed as prospective analysis in ELAM02 protocol.

A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).Human cytomegalovirus (HCMV) infection is characterized by a primary infection leading to a lifelong persistence of the viral genome. Periodically, the virus reactivates from latency and recovers its ability to multiply. HCMV is a major cause of morbidity and mortality in bone marrow or solid-organ transplant recipients and in AIDS patients. Early diagnosis of HCMV infection in high-risk patients is essential in order to start preemptive treatments (3,13,15). The detection of the pp65 antigen in leukocytes is a sensitive method widely used for the early diagnosis of HCMV infection, but it is laborintensive, requires immediate processing, and relies on a subjective interpretation of the slides (1, 7). Qualitative PCR detection of HCMV DNA in leukocytes or plasma is considered the most sensitive method, but it lacks specificity for the diagnosis of HCMV disease (2, 6, 16). Quantification of HCMV DNA has been proposed to be more specifically associated with the disease (1, 18). Real-time PCR based on the TaqMan technology (4, 5) provides an accurate means to quantify viral DNA with the major advantage of avoiding post-PCR handling that can be the source of DNA carryover. Recent studies report the utility of real-time PCR for the quantification of HCMV DNA (9,11,12,14,17). In these studies, the primers used for PCR were located in the major immediate-early gene (12,14,17), the US17 gene (9), or the glycoprotein B gene (17). As suggested by Yun et al., the sensitivity of quantitative PCR may be dependent on the viral target gene (17); however, the most appropriate region for amplification has not been established, as the sequence diversity of clinical isolates remains to be characterized. In the present study, a real-time PCR assay was developed to quantify HCMV DNA in peripheral blood leukocytes (PBLs) using a target sequence located in the UL83 gene which codes for the lower matrix protein detected in the pp65 antigenemia test. The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin) and the results were compared to those of the pp65 antigenemia assay.DNA extractions were performed in all experiments using the QIAamp blood kit (QIAGEN S. A., Courtaboeuf, France) according to the manufacturer's recommendations, except that DNA was eluted in 200 l of distilled water. To amplify HCMV DNA, primers and probe were defined in the UL83 region as follows: pp549s (direct primer), 5Ј-GTCAGCGTTC GTGTTTCCCA-3Ј; pp812as (reverse primer), 5Ј-GGGACAC AACACCGTAAAGC-3Ј; and pp770s (fluorogenic probe), 5ЈFAM-CCCGCAACCCGCAACCCTTCATG-3ЈTAMRA. No cross-reactivity was observed when the specificity of the primers and probe was tested for other human herp...

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We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.

Summary. Post-transplant lymphoproliferative disorder (PTLD) after haemopoietic stem cell transplantation is a serious complication that occurs in 8±22% of patients with high-risk factors. We retrospectively investigated tolerance and efficacy of humanized anti-CD20 monoclonal antibody (rituximab) as first-line treatment in 12 children with B-cell PTLD. At diagnosis, eight patients had tumoral involvement. The other four patients had fever, associated with raised Epstein±Barr virus (EBV) viral load and monoclonal gammopathy. Rituximab was given at the dose of 375 mg/ m 2 once a week by intravenous infusion (1±9 infusions). Only 1/48 infusions was associated with a grade 2 clinical adverse event. Eight out of 12 (66%) patients responded to the treatment and were in complete remission. All patients without tumoral involvement responded to the treatment. A rapid decrease in fever within 1 week was observed in all responders. Non-responders did not show any clinical response during the first week. Tumoral involvement and immunodepression seemed to be more marked in nonresponders. Rituximab was an effective and well-tolerated treatment of B-cell PTLD. Early treatment before tumoral involvement seemed to be the most effective approach. Lack of rapid response should lead to intensification of PTLD treatment. Pre-emptive treatment should be considered and evaluated in further longitudinal multicentre studies.

Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log 10 . Reproducibility was evaluated in intra-and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10 6 PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10 6 PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (P ‫؍‬ 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5 end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4 ؉ cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.

Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.

The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.