Figure 4

Infectivity of virus particles produced from Huh-7.5 cells transfected with JFH1-GFP chimeric genome and JFH1-ΔE-E2-GFP deleted mutant clone. Huh-7.5 cells were transfected with 20 μg of in vitro transcribed HCV RNA. After 72 hours, cells along with supernatants were harvested. Four rounds of freezing and thawing using dry ice lysed the cells. Cell free supernatants were collected by centrifugation at 3500 rpm using a tabletop centrifuge. The titer of HCV in the supernatant was determined by real-time RT-PCR. The TCID50 of infectious supernatant was determined by using 10-serial dilution of the virus stock. (A) Intracellular GFP expression in the infected Huh-7.5 cells at 0, 24, 48, 72 and 96 h using MOI of 10 or TCID50 (i.e 10⁵ virus particle/ml). At different time intervals, cells were taken out from the culture, fixed and GFP examined under a fluorescence microscope. Increased expression of GFP in the infected culture was seen. (B) Intracellular GFP expression in Huh-7.5 cells infected using supernatants of E1-E2 deleted mutant construct. No GFP signal was seen in cells infected using culture supernatants of E1-E2 deleted clone. (C) Real-time RT-PCR was used to quantify the HCV RNA level in the infected cells using a primer targeted to the HCV 5'UTR region. HCV RNA titer in the infected cultures was increased with time suggesting that replication of HCV genome in the infected culture.