Abstract: In order to explore the ability of mosquitoes to replicate HCV, we conducted a series of experimental infections (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). After collecting mosquitoes, females aged between 5 and 10 days were fed by a viremic blood meal, a mixture of sheep (or rabbit) red blood cells and serum of patients infected with HCV (genotype 1b). The mosquitoes were then maintained in breeding for several days (15 to 30 days) by making sampling points to monitor the infection. After development of all stages upstream of the detection conditions (milling and extraction, kits for use…) 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 first experimental 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 infection experiment, conducted on two types of mosquitoes: Aedes sp. and Culex pipiens, has allowed us to: (i) confirmation – by qRT-PCR – the results of the first experiment (presence of HCV RNA in the mosquito Aedes sp. 21 DPI) with an infection rate of ~33%; (ii) demonstrate that only Aedes mosquitoes contained HCV RNA, the Culex mosquitoes were all negative, (iii) show that the RNA detected was issued from replication, since adaptive mutations were identified in the sequence of the RdRp (RNA dependent RNA polymerase), Val262 → Ala262, Pro265 → Ser265, and Asn316 → Asp316, with a specificity for the genus of Aedes, since – once again – all Culex mosquitoes at 21 DPI were HCV RNA-negative. Another way to distinguish replication originated RNA from residual RNA was to show dissemination of the virus in different mosquito’ 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 and bodies (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, detection performed 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 to 57.1% 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 DPI compared to those of day 0. It is interesting to note that HCV RNA-positive mosquitoes all belonged to the vexans species, showing that replication is species specific. Different regions of the HCV genome were then, amplified. Finally, we made use of lab adapted mosquitoes (lab strain Ae. vexans KK, Team AB Failloux, 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 was present at 30 DPI in ~33% of heads and ~66% of the mosquito’ bodies, while infection rates were at ~28% and ~ 71% in the head and body respectively at 0 DPI. These results were based on sequencing performed on the amplified regions of the HCV genome. Taken together; these results show that HCV is able to replicate in Ae. vexans mosquitoes, suggesting that HCV, like other flaviviruses, can alternate between two hosts (primates and mosquitoes) and has lost its potential for replication in the mosquito over time, the primate providing a more stable replication environment (longer lifetime).