The viral etiology of an influenza-like illness during the 2009 pandemic.

Many viruses are known to cause influenza-like illness (ILI); however, in nearly 50% of patients, the etiologic agent remains unknown. The distribution of viruses in patients with ILI was investigated during the 2009 A/H1N1 influenza pandemic (A/H1N1p). From June 2009 to January 2010, 660 patients with suspected influenza were questioned and examined, and nasal swabs were collected. All patient samples were tested for influenza virus, and 286 negative nasal swabs were tested further for 18 other respiratory viruses using real-time RT-PCR. Two waves of ILI were observed in the epidemic curve (weeks 35-42 and 42-49). At least eight viruses co-circulated during this period: human rhinovirus (HRV) (58), parainfluenza 1-4 viruses (PIV) (9), human Coronavirus (hCoV) OC43 (9), enterovirus (5), adenovirus (AdV) (4), and human metapneumovirus (hMPV) (2); however, 204 samples remained negative for all viruses tested. ILI symptoms, according to the Centers for Disease Control and Prevention criteria for ILI definition, were reported in 75% of cases. These patients had positive swabs for A/H1N1p, HRV, hCoV-OC43, PIV, AdV, and hMPV without significant difference with non-ILI patients. This study found that many respiratory viruses circulated during this period and that the A/H1N1p did not impact on the kinetics of other respiratory viruses. The proportion of non-documented cases remains high. ILI could not distinguish A/H1N1p infection from that due to other respiratory viruses. However, in multivariate anlaysis, cough, chills, hyperemia, and dyspnea were associated significantly with influenza virus versus other respiratory viruses.

Evolutionary conservation of the product of human c-myc exon 1 and its inducible expression in a murine cell line.

The human c-myc proto-oncogene contains an open reading frame within its first exon which is translated into protein (MYCHEX1). While the murine c-myc exon 1 is obviously non coding, we show that in mouse cells there are polypeptides closely related to human MYCHEX1. These polypeptides share the same immunological reactivities with the human polypeptides. Furthermore, the 32 kDa polypeptide of murine cells has, like its human counterpart, the ability to dimerise in a 58 kDa form in denaturing and reducing SDS-PAGE. The human gene was introduced into a murine cell line by transfection. A cell line was studied, in which the inducible expression of the gene allows a substantial increase in the concentration of the corresponding protein. This inducible protein behaves in any respect like the murine one, either in SDS-PAGE or in a specific immunoassay. These shared properties constitute a further proof that the human and mouse MYCHEX1 proteins are encoded by the sequence overlapping the human myc exon 1 and a related murine sequence. The gene contained in the human c-myc exon 1 is not, therefore, a specific feature of human cells.

Evaluation of the Xpert Flu test and comparison with in-house real-time RT-PCR assays for detection of influenza virus from 2008 to 2011 in Marseille, France.

Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert(®) Flu assay (Cepheid) with three real-time RT-PCR assays using 127 nasopharyngeal samples, of which 75 were positive for influenza A (with 52 identified as A/H1N1-2009) and 52 were positive for influenza B. The Xpert(®) Flu assay presented a quasi-absence of non-interpretable tests, and showed sensitivity and specificity of 100% and 100% for Flu A, 98.4% and 100% for A/H1N1-2009, and 80.7% and 100% for Flu B.

Comparative detection of enterovirus RNA in cerebrospinal fluid: GeneXpert system vs. real-time RT-PCR assay.

Enteroviruses (EVs) constitute the most common cause of aseptic meningitis in both children and adults. Molecular techniques have now been recognized as the reference standard for the diagnosis of EV infections, and the rapidity of the molecular diagnosis of EV meningitis has been shown to be a determining factor in the management of patients. The rapid documentation of EV RNA in cerebrospinal fluid (CSF) is key to adapting patient management and the therapeutic regimen. To shorten the time needed for virological documentation, we implemented EV RNA detection in two point-of-care (POC) laboratories. Here, we present the results of the POC detection of EV RNA with the Xpert EV kit on the GeneXpert integrated system, and a comparison with the real-time RT-PCR (rtRT-PCR) assay routinely used in the core virology laboratory. From January to September 2009, a total of 310 CSF samples were tested. The rtRT-PCR gave 81 positive, 225 negative and four 'indeterminate' results. POC results were concordant in 81.6% (253/310). Most of the discrepancies consisted of 'indeterminate' results at the POC level (16%). Calculated performances (excluding the indeterminate results) of the Xpert EV kit on the GeneXpert system in POC settings were 100%, 98.9%, 97.6% and 100% for Sensibility, Specificity, positive predictive value and negative predictive value, respectively. Taken together, these results indicate that the implementation of POC detection of EV RNA can provide robust results in <4 h, and may have a significant impact on patient management, therapeutic attitude, and hospitalization costs.

Interim report on the A/H1N1 influenza virus pandemic in Marseille, France, April-November 2009.

We report here the results of a 7-month survey of the influenza A/H1N1 pandemic in the Virology laboratory of the public hospitals of Marseille (April-November 2009). In total, 8 587 samples were analysed during this period, of which 1 974 (23%) were positive for the novel influenza variant. The analysis of results obtained using rapid influenza diagnostic tests (RIDTs) revealed a global sensitivity of 49.4% (vs. molecular qRT-PCR detection), strongly correlated with age groups (varying from 30% to 58% for patients >40 age and <10, respectively), indicating that RIDTs can be helpful in accelerating the management of suspected cases. Epidemiological analysis showed that the winter influenza wave began in October in Marseille (i.e. 2 to 3 months earlier than usual seasonal influenza outbreaks) and that the majority of autochthonous cases were observed in patients younger than 20 years old, with a low number of cases in patients over 60 years old. In November 2009, 22.2% (167/754) of patients with a laboratory diagnosis of influenza A/H1N1 infection were hospitalized, of which 9% (15/167) were admitted to an intensive care unit (ICU). Patients in the extreme age groups (>40 years old and <1) were significantly more often hospitalized than others, and 2.4% of hospitalized patients died. During the last 3 weeks of the period, the average number of bed-days attributable to H1N1sw-positive patients was 31.4, of which 5.9 were in ICUs.

ZFX transactivation of the HIV-1 LTR is cell specific and depends on core enhancer and TATA box sequences.

The ZFX gene is ubiquitously transcribed and highly conserved among vertebrates. The integrity of Zfx, its murine homologue, has been shown to be important for growth during embryogenesis and sustained gamete production. Alternative splicing was shown to result in production of mRNAs coding for either ZFX804or a shorter isoform initiated downstream, ZFX575. ZFX575was previously shown to be a potent transactivator of the HLA-A11 promoter. Here, the HIV-1 LTR is also shown to be potently transactivated by ZFX575in several cell types, while ZFX804activity is found to be similar to that of ZFX575, null or intermediary according to the cell type. In all cell types, the HIV-1 TATA box sequence is a key element of transactivation, while the Sp1 or NFkappaB sites are variably required, according to the cell type. Overall, the results suggest that ZFX575and ZFX804could play a role in HIV-1 LTR induction as co-activators enhancing productive interactions between upstream transactivators and the basal transcription complexes recruited by the TATA box.

Immunochemical detection of proteins related to the human c-myc exon 1.

Published sequence data of the human c-myc gene indicate the presence of a coding capacity for a polypeptide of 188 residues within the first exon. Using antibodies raised against five synthetic peptides corresponding to different non-over-lapping parts of this polypeptide, two proteins of 32 kd and 58 kd antigenically related to the synthetic peptides have been detected in extracts of human cells. The confidence of this detection has been reinforced by showing that epitopes corresponding to different peptides were indeed located on the same molecule and that the 58 kd protein appears to be a dimeric form of the 32 kd protein. That these proteins originate from the first exon was indicated by: hybrid-arrested translation experiments followed by immunodetection of the translation products; in vitro translation of messenger RNA derived from cloned exon 1 by SP6 polymerase transcription.

Nucleotide sequence of the human c-myc locus: provocative open reading frame within the first exon.

The nucleotide sequence of a HindIII-EcoRI DNA fragment, 8 kbp long, of a lambda recombinant containing the whole human c-myc gene has been deduced by the method of Maxam and Gilbert. This fragment encodes the complex c-myc locus and the sequence provides information relative to the 2.7 kb long c-myc transcript. It appears that although exons 2 and 3 would code for a 48-K protein homologous to the myc domain of the viral p110 gag-myc protein, the first exon, which has a large open reading frame ending with a stop codon just upstream from the donor splice site, could code on its own for a 20-K protein. Speculations about the role of that putative protein on the regulation of the expression of exons 2 and 3 are made.

Multiple nuclear factors bind to novel positive and negative regulatory elements upstream of the human MHC class I gene HLA-A11.

Quantitative expression of class I genes varies widely in different tissues, during development and in some neoplastic cells. Regulatory DNA sequences controlling levels of transcription of class I genes have been characterized in the murine and swine promoters. Although some regulatory features appear to be retained in humans, most of them are not evolutionary conserved. In this study, we identify novel DNA sequences and factors which contribute to the regulation of the human HLA-A11 gene. Two activator elements (-155 to -91 and -335 to -206) and one negative element (-172 to -156), distinct from those previously described are mapped within 335 bp upstream of exon 1 of the human HLA-A11 gene. Various nuclear factors bind to these regulatory elements, some of which are cell restricted or interact with evolutionary divergent regulatory sequences of the human class I gene promoter. Two of the five DNA-binding sites characterized in this work bind at least two proteins which compete for the occupancy of their site. The identification of these new regulatory elements provides a more comprehensive basis for the understanding of physiological or pathological modulation of MHC class I transcription in humans.

Transcriptional regulation of the MHC class I HLA-A11 promoter by the zinc finger protein ZFX.

Regulation of the human MHC class I HLA-A11 promoter is governed by a complex array of regulatory elements. One of these elements, shown here to be critical for the transcriptional activity of the promoter, was used to screen a lambda gt11 library and allowed the identification of a cDNA which coded for the zinc finger protein ZFX. ZFX was shown to bind the sequences AGGGCCCCA and AGGCCCCGA, located respectively at positions -271 to -263 and -242 to -234 of the HLA-A11 promoter, with similar affinities through its three C-terminal zinc fingers. ZFX575, a short isoform of ZFX, activates transcription from the HLA-All promoter in a Leydig cell line.

In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.