p. either in the absence (Fig. 1A,a) or in the presence (Fig. 1A,b) of 1% NHS only during the infection process. Culture medium was subjected to ultracentrifugation for removing cellular material and proteins and pelleting

HCV RNA-associated particles. As illustrated in Fig. 1A, HCV RNA(+) was detected by RT-PCR at days 7 and 14 p.p. in the absence of NHS (a) or at days 4 and 7 p.p. in the presence of NHS (b), reflecting adsorption and/or partial penetration of the inoculum into the cells. No detection was observed at day 21 (−NHS) or 14 (+NHS) p.p. Thus, residual cell-surface bound HCV RNA was completely Selleckchem BAY 73-4506 eliminated (<100 copies/mL, which is the detection limit of the qPCR technique used). Thereafter, the extracellular HCV RNA progressively increased and reached 6log10

copies/mL at days 42-49 (+NHS) corresponding to production of newly synthesized HCV RNA-positive particles. The infection was efficient learn more because the HCV RNA increased by 4 logs from 14 to 49 days (+NHS). These results reflect that HCV actively replicated in the infected cells and spread into uninfected cells because the HepaRG cells did not proliferate during the differentiated phase of the culture. Interestingly, whereas a common cyclic pattern was observed when the infection was performed without NHS (a), a continuous pattern occurred in the presence of 1% NHS (b). More rapid penetration of HCVsp and best synchronization of infection resulted from the presence of NHS during the infection. This condition was therefore used in subsequent experiments. The HCV amplification was also assessed by determination of E1E2 antigenic activity (Fig. 1B) by indirect ELISA (a) and western blotting (b) using the D32.10 mAb. The increased HCV E1E2 in the medium from day 28 to day 49 p.p. correlated well with the HCV RNA peak detection and supported de novo synthesis and release of enveloped RNA-containing particles. The cutoff values were calculated by using three control samples from uninfected HepaRG cells (mean optical density values: 0.296 ± 0.124 for 1/10 dilution, 0.093 ± 0.025 this website for 1/50 dilution, and 0.071 ± 0.010 for 1/100 dilution).

A good correlation between ELISA and western blot techniques was observed. Examination of HCV core antigen with a commercial immunoassay confirmed the production of complete virions containing both HCV RNA and core antigen and expressing E1E2 envelope proteins. Next we asked whether the HCV particles produced by HCVsp-infected HepaRG cells were infectious. To this end, the ability of HCV particles released into the cell culture media 25 days after infection 1 (corresponding to D28* p.p., cf. Fig. 1A,b) to infect naive HepaRG cells at 3 days p.p. was tested (Fig. 1C). HCV RNA(+) was analyzed by RT-PCR in the supernatants collected each week and subjected to ultracentrifugation as described above. After early detection at day 1 p.i.