[Modulation of transcriptomic signature of the infected host: a new therapeutic strategy for the management of severe viral infections? Example of the flu]. / La modulation de la signature transcriptomique de l'hôte infecté : une nouvelle stratégie thérapeutique dans les viroses graves ? Exemple de la grippe.

During the last decades, emergence and reemergence of viruses were responsible for epidemic and pandemic infectious diseases, with variable degrees of severity. Current preventive strategies are not sufficient at all, and available therapeutic drugs are very limited. Indeed, genetic variations of viruses can impair the efficacy of antiviral compounds by the apparition of resistance. Moreover, current delay needed for de novo development of drugs does not allow a rapid response in case of important epidemic or pandemic events. In this context, new therapeutic approaches are necessary. An innovative concept is to repurpose already marketed compounds that can reverse the host cellular transcriptomic response to the infection. By targeting the host, these molecules exhibit a broad-spectrum activity and are potentially effective even against new emergent strains. This strategy implements the characterization of specific host gene expression profiles, the in silico screening of drugs, and their validation in in vitro and in vivo models, until their evaluation in clinical trials. Here, we will present this approach, with the example of the flu.

Modulation of p53ß and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response.

In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53ß and p53γ through alternative splicing of exons 9ß and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53ß and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9ß/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9ß/9γ, we demonstrate that cell growth can be driven by modulating p53ß and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53ß and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53ß enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53ß and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53ß and p53γ promote apoptosis in MCF7 cells, p53ß and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53ß and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53ß and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers.

The chicken chorioallantoic membrane tumor assay as model for qualitative testing of oncolytic adenoviruses.

Oncolytic adenoviruses are promising anticancer agents. To study and optimize their tumor-killing potency, genuine tumor models are required. Here we describe the use of the chicken chorioallantoic membrane (CAM) tumor model in studies on oncolytic adenoviral vectors. Suspensions of human melanoma, colorectal carcinoma and glioblastoma multiforme cell lines were grafted on the CAM of embryonated chicken eggs. All cell lines tested formed 5-10 mm size tumors, which recapitulated hallmarks of corresponding human specimens. Furthermore, melanoma tumors were injected with adenoviral vector-carrying gene encoding the fusion protein of parainfluenza virus type 5. This led to the induction of cell fusion and syncytia formation in the infected cells. At 6 days post-injection, histological and immunohistochemical analyses of tumor sections confirmed adenovirus replication and syncytia formation. These results demonstrate that the CAM model allows rapid assessment of oncolytic viruses in three-dimensional tumors. Hence, this model constitutes an easy and affordable system for preclinical characterization of viral oncolytic agents that may precede the mandatory process of animal testing. Application of this model will help reducing the use of human xenografts in mice for preclinical evaluation of oncolytic viruses and other anticancer agents.

In vitro characterization of naturally occurring influenza H3NA- viruses lacking the NA gene segment: toward a new mechanism of viral resistance?

Among a panel of 788 clinical influenza H3N2 isolates, two isolates were characterized by an oseltamivir-resistant phenotype linked to the absence of any detectable NA activity. Here, we established that the two H3NA- isolates lack any detectable full-length NA segment, and one of these could be rescued by reverse genetics in the absence of any NA segment sequence. We found that the absence of NA segment induced a moderate growth defect of the H3NA- viruses as on cultured cells. The glycoproteins density at the surface of H3NA- virions was unchanged as compared to H3N2 virions. The HA protein as well as residues 188 and 617 of the PB1 protein were shown to be strong determinants of the ability of H3NA- viruses to grow in the absence of the NA segment. The significance of these findings about naturally occurring seven-segment influenza A viruses is discussed.

Engineering of a parainfluenza virus type 5 fusion protein (PIV-5 F): development of an autonomous and hyperfusogenic protein by a combinational mutagenesis approach.

The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope with the target cell membrane. For the paramyxovirus parainfluenza virus type 5 (PIV-5), this fusion involves an attachment protein (HN) and a class I viral fusion protein (F). We investigated the effect of 20 different combinations of 12 amino-acid substitutions within functional domains of the PIV-5 F glycoprotein, by performing cell surface expression measurements, quantitative fusion and syncytia assays. We found that combinations of mutations conferring an autonomous phenotype with mutations leading to an increased fusion activity were compatible and generated functional PIV-5 F proteins. The addition of mutations in the heptad-repeat domains led to both autonomous and hyperfusogenic phenotypes, despite the low cell surface expression of the corresponding mutants. Such engineering approach may prove useful not only for deciphering the fundamental mechanism behind viral-mediated membrane fusion but also in the development of potential therapeutic applications.

Cold oxygen plasma technology efficiency against different airborne respiratory viruses.

BACKGROUND: Respiratory infections caused by viruses are major causes of upper and lower respiratory tract infections. They account for an important mortality and morbidity worldwide. Amongst these viruses, influenza viruses and paramyxoviruses are major pathogens. Their transmission is mainly airborne, by direct transmission through droplets from infected cases. OBJECTIVES: In the context of an influenza pandemic, as well as for the reduction of nosocomial infections, systems that can reduce or control virus transmission will reduce the burden of this disease. It may also be part of the strategy for pandemic mitigation. STUDY DESIGN: A new system based on physical decontamination of surface and air has been developed. This process generates cold oxygen plasma (COP) by subjecting air to high-energy deep-UV light. To test its efficiency, we have developed an experimental device to assess for the decontamination of nebulized respiratory viruses. High titer suspensions of influenza virus type A, human parainfluenza virus type 3 and RSV have been tested. RESULTS: Different experimental conditions have been evaluated against these viruses. The use of COP with an internal device allowed the best results against all viruses tested. We recorded a reduction of 6.5, 3.8 and 4 log(10) TCID50/mL of the titre of the hPIV-3, RSV and influenza virus A (H5N2) suspensions. CONCLUSIONS: The COP technology is an efficient and innovative strategy to control airborne virus dissemination. It could successfully control nosocomial diffusion of respiratory viruses in hospital setting, and could be useful for the reduction of influenza transmission in the various consultation settings implemented for the management of cases during a pandemic.

Characterization of naturally occurring parainfluenza virus type 2 (hPIV-2) variants.

BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.

[Reverse genetic in paramyxovirus]. / La génétique inverse chez les paramyxovirus : applications et perspectives.

Paramyxoviruses constitute a large family, including human animal pathogens with high prevalence and also major economic impact. During last fifteen years, development of reverse genetic systems, which allow generating viruses from cloned cDNA, revolutionized research on Paramyxoviruses. From the elaboration of minireplicons to the constitution of performing reverse genetic systems, this new strategy contributed to our understanding of viral cycle, pathogenicity or viral assembly, for example. This approach opened the way to the development of viral vectors and live-attenuated vaccines that could be useful tools in therapeutics and prophylaxis.

Modifications of the sex pheromonal communication of trichogramma brassicae by a sublethal dose of deltamethrin

Pyrethroid insecticides, by their intensive use and their persistence that can exceed 3 months, largely contribute to the environmental pollution. In this work, we determined the effects of a very low dose of deltamethrin on the sex pheromonal communication of Trichogramma brassicae. The dose used was a dose that would theoretically kill one insect over 1000 (an LD 0.1). We found that this dose slightly but very significantly increased the arrestment behavior of treated males responding to the female pheromone. On the other hand when females were exposed to the same dose of insecticide, the response of males to their pheromone was very significantly decreased. In Trichogramma, like in other insects, sex pheromonal communication probably involves nervous transmissions both for the reception and the emission of the pheromone. Then, the sublethal effects of deltamethrin, observed in this work, are certainly due to multiple actions of this insecticide on nervous transmissions. Trichogramma is a beneficial insect that contributes to the control of pest populations of moths. Actions of this insecticide at a dose that can correspond to environmental pollution could be a real threat to the equilibrium of these populations.

A chromosomal analysis of the phenotypic plasticity of some life history traits in relation to developmental temperature.

The capacity to delay egg deposition in D. melanogaster females in the absence of a sexual partner is genetically determined and opposite types can be artificially selected. In natural populations, the relative frequency of these genotypes varies geographically and seasonally, with temperature as a selective factor. However, the retention duration of these genotypes can be modified by developmental temperature change. To study the genetic control of this response, chromosome substitution between opposite types of line was carried out in order to produce every possible homozygous chromosomal combination of the three major chromosomes (X,2,3). Eggs of these eight constructed lines were developed at two different temperatures (25 degrees C and 14 degrees C). Low temperature development directly affected the number of ovarioles but also modified the subsequent expression of adult characteristics such as retention duration and fecundity. The comparison of the eight lines revealed that, although the 3 chromosomes were involved in the genetic determinism of each trait, only one or two of them were sensitive to temperature change, and these differed according to the trait. For retention duration and fecundity, the effect of chromosome 3 from the long retention strain was particularly affected by low temperature, showing antagonism between the selective effect detected in natural populations and the effect on phenotypic plasticity studied here.

Chromosomal analysis of initial retention capacity in virgin Drosophila melanogaster females.

In natural temperate populations, virgin Drosophila melanogaster females present highly variable periods of preoviposition duration (from 2 to 25 days). Strains were selected for long and short initial retention capacity. Chromosome substitution between two of these lines produced, by appropriate mating procedures, every possible homozygous chromosomal combinations of the X, II and III chromosomes. Analyses of these lines demonstrate that both X and III chromosomes are involved in this egg-laying control, and have complementary effects.