TRAF6 and IRF7 Control HIV Replication in Macrophages

The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication

Serotyping of Streptococcus pneumoniae based on capsular genes polymorphisms.

Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. A novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Autolysin, pneumolysin and eight genes located in the capsular operon were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system to identify capsular serotypes. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. We show that typing only 12 polymorphisms located in the capsular operon allows the identification at the serotype level of 22 serotypes and the assignation of 24 other serotypes to a subgroup of serotypes. Overall, 126 samples (75.9%) were correctly serotyped, 14 were assigned to a member of the same serogroup, 8 rare serotypes were erroneously serotyped, and 18 gave negative serotyping results. Most of the discrepancies involved rare serotypes or serotypes that are difficult to discriminate using a DNA-based approach, for example 6A and 6B. The assay was also tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. Overall, 89% of specimens positive for pneumolysin were serotyped, demonstrating that this method does not require culture to serotype clinical specimens. The assay showed no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype prevalence changes after new conjugate vaccines introduction

Algorithm for results analysis.

<p>Samples were assessed for the presence of <i>S. pneumoniae</i> by detecting the pneumolysin and the autolysin genes. If <i>S. pneumoniae</i> was detected in the sample, the serotyping probes were analyzed in order to identify the serotype.</p

Schematic representation of experimental procedure.

<p>Multiplex PCR amplifies up to 12 genes and is performed outside of the INFINITI system. Amplification products are loaded in the INFINITI system at the primer extension step. Primer extension oligonucleotides include a tag sequence that will hybridize to the microarray and a specific detection sequence that allows for primer extension of <i>S. pneumoniae</i> genes. Fluorescent nucleotides are incorporated during primer extension. Once the reaction is complete, the INFINITI system automatically transfers the labeled products to the microarray for hybridization. Tag sequences hybridize to anti-tags located on the microarray. Microarrays are then washed, dried and loaded into the integrated confocal scanner where fluorescence is measured. The report generated by the instruments is analyzed off-line using the Pneumotyper software.</p

Capsular operon of <i>S. pneumoniae</i> serotype 19A.

<p>Expected amplicons are shown under the sequence, with genotyped positions marked with vertical lines. The expected genotype for serotype 19A is in large characters while the other possible genotypes are shown in smaller type. Positions are in nucleotides.</p

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach

International audienc

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach

International audienc