12 research outputs found
Détection de séquences d'ARN du VHC dans des extraits de moustiques du genre Aedes : Etude cinétique aprÚs infections expérimentales
Abstract: In order to explore the ability of mosquitoes to replicate HCV, we conducted a series of experimentalinfections (four investigations spread between 2006 and 2009) on two different types of mosquito; Aedes(Aedes (Ochlerotatus) caspius Pallas 1771; Aedes (Aedimorphus) vexans Meigen 1830) and Culex Culex(Culex) pipiens Linnaeus 1758 wild (Ae. vexans, Ae. caspius Cx. pipiens) and lab adapted (Ae. vexans). Aftercollecting mosquitoes, females aged between 5 and 10 days were fed by a viremic blood meal, a mixture ofsheep (or rabbit) red blood cells and serum of patients infected with HCV (genotype 1b). The mosquitoeswere then maintained in breeding for several days (15 to 30 days) by making sampling points to monitorthe infection. After development of all stages upstream of the detection conditions (milling and extraction,kits for useâŠ) and detection conditions (choice of method of amplification and detection, kits for use ...)We succeeded to provide evidence of HCV replication in the mosquito Aedes vexans. In the firstexperimental infection, which lasted three weeks and performed on Ae. vexans, we detected HCV RNA in~50% of mosquitoes of the 21st days post-infection (DPI), by endpoint PCR. The second infectionexperiment, conducted on two types of mosquitoes: Aedes sp. and Culex pipiens, has allowed us to: (i)confirmation â by qRT-PCR â the results of the first experiment (presence of HCV RNA in the mosquitoAedes sp. 21 DPI) with an infection rate of ~33%; (ii) demonstrate that only Aedes mosquitoes containedHCV RNA, the Culex mosquitoes were all negative, (iii) show that the RNA detected was issued fromreplication, since adaptive mutations were identified in the sequence of the RdRp (RNA dependent RNApolymerase), Val262 â Ala262, Pro265 â Ser265, and Asn316 â Asp316, with a specificity for the genus ofAedes, since â once again â all Culex mosquitoes at 21 DPI were HCV RNA-negative. Another way todistinguish replication originated RNA from residual RNA was to show dissemination of the virus indifferent mosquitoâ tissues. To do this, we conducted a third experimental infection (on Ae. caspius and Ae.vexans), over a period of 15 days and analyzed the presence of HCV RNA separately in the heads andbodies (previously separated by dissection). In conducting samples at different times after infection (0, 4,8 and 15 days), we succeeded to obtain a kinetics of infection separately in heads and bodies, detectionperformed by WTA (whole transcriptome amplification) followed by qPCR. On the day of infection (D0),the infection rate in the heads and bodies of mosquitoes was 9.1% and 100% respectively, the rate rose to57.1% and 85.7% respectively at 15 DPI, after it has been totally negative after eight days post-infection.On the other hand, we have identified adaptive mutations in the IRES sequence of mosquito at 15 DPIcompared to those of day 0. It is interesting to note that HCV RNA-positive mosquitoes all belonged to thevexans species, showing that replication is species specific. Different regions of the HCV genome werethen, amplified. Finally, we made use of lab adapted mosquitoes (lab strain Ae. vexans KK, Team ABFailloux, Bunyavirus Molecular Genetics, Institute Pasteur - Paris). Infection of this strain has also shown,using different approaches for detection (qRT-PCR, qPCR-WTA, HCV-GA namely), that HCV RNA waspresent at 30 DPI in ~33% of heads and ~66% of the mosquitoâ bodies, while infection rates were at~28% and ~ 71% in the head and body respectively at 0 DPI. These results were based on sequencingperformed on the amplified regions of the HCV genome. Taken together; these results show that HCV isable to replicate in Ae. vexans mosquitoes, suggesting that HCV, like other flaviviruses, can alternatebetween two hosts (primates and mosquitoes) and has lost its potential for replication in the mosquitoover time, the primate providing a more stable replication environment (longer lifetime).dans la sĂ©quence de la RdRp (ARN PolymĂ©rase ARN dĂ©pendante), Val262âAla262, Pro265âSer265, etAsn316â Asp316, avec une spĂ©cificitĂ© pour le genre Aedes, puisque tous les moustiques Culex Ă 21 JPIĂ©taient ARN VHC-nĂ©gatifs. Un autre moyen de distinguer lâARN issu de la rĂ©plication de lâARN rĂ©siduel aĂ©tĂ© de montrer lâexistence dâune dissĂ©mination au sein du moustique. Pour ce faire, nous avons rĂ©alisĂ© unetroisiĂšme expĂ©rience dâinfections (genres Ae. caspius et Ae. vexans), sur une durĂ©e de 15 jours et avonsanalysĂ© la prĂ©sence de lâARN du VHC distinctement dans les tĂȘtes et les corps (prĂ©alablement sĂ©parĂ©s pardissection). En effectuant des prĂ©lĂšvements Ă diffĂ©rentes dates aprĂšs infection (0, 4, 8 et 15 jours), nousavons rĂ©ussi Ă obtenir une cinĂ©tique dâinfection, sĂ©parĂ©ment, dans les tĂȘtes et dans les corps, dĂ©tectionrĂ©alisĂ©e par WTA (Whole Transcriptome Amplification) suivie de qPCR. Au jour de lâinfection (J0), le tauxdâinfection dans les tĂȘtes et les corps des moustiques Ă©tait de 9,1% et 100% respectivement, ce taux estpassĂ© Ă 57,1% et 85,7% respectivement Ă J15 aprĂšs une nĂ©gativation huit jours aprĂšs infection. Dâautrepart, nous avons identifiĂ© des mutations dâadaptation dans la sĂ©quence de lâIRES des moustiques Ă J15comparĂ©s Ă ceux Ă J0. Il est intĂ©ressant de noter que les moustiques ARN VHC-positifs appartenaient tous Ă lâespĂšce vexans, montrant ainsi que la rĂ©plication est spĂ©cifique dâespĂšce. DiffĂ©rentes rĂ©gions du gĂ©nomedu VHC ont, ensuite, Ă©taient amplifiĂ©es. Enfin, nous avons eu recours Ă des moustiques dâĂ©levage, adaptĂ©saux conditions de laboratoire (souche dâĂ©levage Ae. vexans KK, Ă©quipe A-B Failloux, GĂ©nĂ©tique MolĂ©culairedes Bunyavirus, Institut Pasteur - Paris). Lâinfection de cette souche a Ă©galement montrĂ©, en utilisantdiffĂ©rentes approches de dĂ©tection (qRT-PCR, WTA-qPCR, HCV-GA notamment), quâĂ 30 JPI, lâARN du VHCĂ©tait prĂ©sent dans ~33% des tĂȘtes et ~66% des corps, alors que les taux dâinfection Ă J0 Ă©taient de ~28%et ~71% dans les tĂȘtes et les corps respectivement. Les rĂ©sultats se sont appuyĂ©s sur des sĂ©quençageseffectuĂ©s sur les rĂ©gions amplifiĂ©es du gĂ©nome du VHC. Lâensemble de ces rĂ©sultats montrent que le VHCest capable de se rĂ©pliquer dans les moustiques du genre Ae. vexans, ce qui suggĂšre que le VHC, Ă lâimagedâautres Flavivirus, puisse alterner entre deux hĂŽtes (primates et moustiques) et quâil ait perdu sonpotentiel de rĂ©plication chez le moustique au fil du temps, le primate lui fournissant un environnement derĂ©plication plus stable (durĂ©e de vie plus longue)
AR collaborates with ERα in aromatase inhibitor-resistant breast cancer
Androgen receptor (AR) is an attractive target in breast cancer because of its frequent expression in all the molecular subtypes, especially in estrogen receptor (ER)-positive luminal breast cancers. We have previously shown a role for AR overexpression in tamoxifen resistance. We engineered ER-positive MCF-7 cells to overexpress aromatase and AR (MCF-7 AR Arom cells) to explore the role of AR in aromatase inhibitor (AI) resistance. Androstendione (AD) was used as a substrate for aromatization to estrogen. The nonsteroidal AI anastrazole (Ana) inhibited AD-stimulated growth and ER transcriptional activity in MCF-7 Arom cells, but not in MCF-7 AR Arom cells. Enhanced activation of pIGF-1R and pAKT was found in AR-overexpressing cells, and their inhibitors restored sensitivity to Ana, suggesting that these pathways represent escape survival mechanisms. Sensitivity to Ana was restored with AR antagonists, or the antiestrogen fulvestrant. These results suggest that both AR and ERα must be blocked to restore sensitivity to hormonal therapies in AR-overexpressing ERα-positive breast cancers. AR contributed to ERα transcriptional activity in MCF-7 AR Arom cells, and AR and ERα co-localized in AD + Ana-treated cells, suggesting cooperation between the two receptors. AR-mediated resistance was associated with a failure to block ER transcriptional activity and enhanced up-regulation of AR and ER-responsive gene expression. Clinically, it may be necessary to block both AR and ERα in patients whose tumors express elevated levels of AR. In addition, inhibitors to the AKT/IGF-1R signaling pathways may provide alternative approaches to block escape pathways and restore hormone sensitivity in resistant breast tumors
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 5: Figure S4. of New insights into HCV replication in original cells from Aedes mosquitoes
HCV Infection protocols. (A) for human HepaRG hepatocytes (âHâ) and (B) for insect cells, Ktmos1 (âKâ, Ae Aegypti) and C6/36 (âCâ, Ae Albopictus). The infection was performed using HCVsp, LAT isolate, genotype 3. D, day; â before infection; D0, day of infection; D4, D7, D14, D21, D28, days post-infection and medium change. P17, P18, passages 17 and 18. HepaRGÂź, 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 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 (ĂąÂÂ) and HCV-infected (+) HEK 293 cells. (TIFF 1236 kb
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 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 ĂąÂÂDome-likeù 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