L'auteur n'a pas fourni de titre en anglais

L'auteur n'a pas fourni de résumé en françaisL'auteur n'a pas fourni de résumé en anglai

Exploration et fonctionnalité de particules virales authentiques en vue de l'étude de la réplication du virus de l'hépatite C

L'auteur n'a pas fourni de résumé en anglaisL'auteur n'a pas fourni de résumé en françai

Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody.

International audienceThe nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum-derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single-chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi-angle laser light scattering. The scFv mimicked the antibody in binding to the native serum-derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics

New insights into HCV replication in original cells from Aedes mosquitoes

International audienceAbstractBackgroundThe existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines.MethodsFirst, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments.ResultsOur binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism).ConclusionsThese results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development

Additional file 3: Figure S2. of New insights into HCV replication in original cells from Aedes mosquitoes

Fluorescence observation of adherent Ktmos1 cells. The Ktmos1 Aedes aegypti cells were grown on thin glass (0,17 mm), 2 chambers LabTek (Nunc). The cells were fixed after different periods of cultivation with 2% PFA for 20 min at 37 °C. After permeabilization by PBS containing 0,1% Triton X100 for 2 min, the nuclei were stained by Hoechst 33,258 (Sigma). Observation was performed on motorized inverted Olympus IE81 microscope using the DIC (Differential Interference Contrast) and the DAPI filter. The panel (A) shows a late metaphase stage of a dividing cell. The panel (B) shows Ktmos1 cells in monolayer. (TIFF 925 kb

Additional file 5: Figure S4. of New insights into HCV replication in original cells from Aedes mosquitoes

HCV Infection protocols. (A) for human HepaRG hepatocytes (“H”) and (B) for insect cells, Ktmos1 (“K”, Ae Aegypti) and C6/36 (“C”, Ae Albopictus). The infection was performed using HCVsp, LAT isolate, genotype 3. D, day; − before infection; D0, day of infection; D4, D7, D14, D21, D28, days post-infection and medium change. P17, P18, passages 17 and 18. HepaRG®, HepaRG cells from KIT902 (Biopredic International). Over the time, HepaRG and Ktmos1 cells in monolayer became more and more differentiated. (TIFF 300 kb

Additional file 2: Figure S1. of New insights into HCV replication in original cells from Aedes mosquitoes

Global process of the Ktmos1 cell generation from eggs hatching to the final supracellular structures. (A) Macroscopic picture showing the eggs of Aedes aegypti collected from insectary. (B) Large hollow vesicles developing at the cut ends of the larvae fragments. (C) Microscopic examination of the adherent cells and molecular identification of cells and larvae extracts by PCR targeting rDNA ITS: the upper panel shows cells in monolayer; the lower panel indicates species-diagnosis PCR of cellular samples with hollow vesicles (lane 1), adherent cells (lane 2) and “Dome-like” structures (lane 3). HEK 293 cells are the negative control, ground larvae extracts of Aedes aegypti bora bora strain are the positive control. The approximate size of the amplified product is 550 pb. (D) Microscopic examination of the hollow vesicles as supracellular structures (D1 and D2). (TIFF 751 kb

Additional file 7: Figure S6. of New insights into HCV replication in original cells from Aedes mosquitoes

Absence of HCV RNA detection in HEK 293 cells. Cells were collected at days 0 (D0), 4 (D4), 21 (D21) and 28 (D28) p.i. The inoculum HCVsp (LAT isolate, genotype 3) was used as positive control. Non-infected (mock) cells (−) and HCV-infected (+) HEK 293 cells. (TIFF 1236 kb

Epstein-Barr virus infection and clinical outcome in breast cancer patients correlate with immune cell TNF-α/IFN-γ response.

International audienceFor nearly two decades now, various studies have reported detecting the Epstein-Barr virus (EBV) in breast cancer (BC) cases. Yet the results are unconvincing, and their interpretation has remained a matter of debate. We have now presented prospective data on the effect of EBV infection combined with survival in patients enrolled in a prospective study. We assessed 85 BC patients over an 87-month follow-up period to determine whether EBV infection, evaluated by qPCR in both peripheral blood mononuclear cells (PBMCs) and tumor biopsies, interacted with host cell components that modulate the evolution parameters of BC. We also examined the EBV replicating form by the titration of serum anti-ZEBRA antibodies. Immunological studies were performed on a series of 35 patients randomly selected from the second half of the survey, involving IFN-γ and TNF-α intracellular immunostaining tests performed via flow cytometry analysis in peripheral NK and T cells, in parallel with EBV signature. The effect of the EBV load in the blood or tumor tissue on patient survival was analyzed using univariate and multivariate analyses, combined with an analysis of covariance. Our study represents the first ever report of the impact of EBV on the clinical outcome of BC patients, regardless of tumor histology or treatment regimen. No correlation was found between: (i) EBV detection in tumor or PBMCs and tumor characteristics; (ii) EBV and other prognostic factors. Notably, patients exhibiting anti-ZEBRA antibodies at high titers experienced poorer overall survival (p = 0.002). Those who recovered from their disease were found to have a measurable EBV DNA load, together with a high frequency of IFN-γ and TNF-α producing PBMCs (p = 0.04), which indicates the existence of a Th1-type polarized immune response in both the tumor and its surrounding tissue. The replicative form of EBV, as investigated using anti-ZEBRA titers, correlated with poorer outcomes, whereas the latent form of the virus that was measured and quantified using the EBV tumor DNA conferred a survival advantage to BC patients, which could occur through the activation of non-specific anti-tumoral immune responses