Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools

AbstractThe detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs) in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA. This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3–9.5% of the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a ‘hyper sensitive ELISA’ for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell transplant

Preliminary study comparing dogs’ responses to travel in electric cars

Many pet dogs (Canis lupus familiaris) will travel in a car at some point in their life, which can be stressful and lead to a range of potentially problematic responses. Electric vehicles are noticeably quieter than ones with internal combustion engines but there are no studies on their effect on dogs. Therefore, the aim of this study was to undertake a preliminary examination using a mixed methods approach to identify potential issues worthy of further investigation by comparing the dog’s behavioural and physiological (heart rate) responses when travelling in these two types of car. 20 dogs undertook the same ~10 minute journey twice, once in each of the vehicles. As a pilot study, we focus primarily on reporting qualitative evaluations of results and effect sizes, with statistical significance references given where apparent. The behaviour of dogs seemed to be affected by the type of vehicle that they travelled in, with dogs appearing to be behaviourally more restless in the diesel vehicle than the electric one, indicated by shorter bouts of lying relaxed and alert. In a clinical assessment of individual dogs, we noted that two dogs appearing to suffer from nausea seemed to show potential improvements associated with travel in the electric vehicle

Stimulation of the Human RAD51 Nucleofilament Restricts HIV-1 Integration In Vitro and in Infected Cells

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51