Microscope (Fig. 1B). Results showed particles of 40?0 nm size of genotype 1b which was similar to the sizes described earlier [18] and 35?5 nm for genotype 3a. The size difference may be due to the difference in the amount of E1 and E2 proteins incorporated into each virus like particle. The purified HCV-LPs binding to Huh7 cells were analyzed by flow cytometry at 37uC. It was observed that with constant concentration of VLP (7 mg), at different time points, the intensity of fluorescence increased gradually upto 4 h which declined afterwards (Figure S1). Further, the binding efficiency of the HCV-LPs was compared at 4th hr time point. HCV-LP corresponding to genotype 3a showed marginally higher interaction (,80 ) with the Huh7 cells than the HCV-LP of genotype 1b (,70 ) (Figure S2).In vitro Transcription of Viral RNAThe pJFH1 construct (generous gift from Dr. Takaji Wakita, National Institute of Infectious Diseases, Tokyo, Japan) was linearized with XbaI. HCV RNA was synthesized from linearized pJFH1 template using Ribomax Large scale RNA production system-T7 according to manufacturer’s instructions (Promega).Transfection and Generation of JFH1 VirusHuh7.5 cells were transfected with in vitro synthesized JFH1 RNA transcript using Lipofectamine 2000 (Invitrogen) in OptiMEM (Invitrogen). Infectious JFH1 virus particles were generated as described previously [28]. Uninfected Huh7.5 cells were used as a mock control.Characterization of Monoclonal Antibodies Against 1b and 3a Genotype of HCV-LPBALB/c 1662274 mice were immunized with the HCV-LPs (both genotype 1 and genotype 3) and hybridoma were established by fusion of splenocytes with mouse myeloma cells. Approximately 200 hybridomas from two independent experiments were screened. A total of five mAbs were obtained out of which two (E8G9 and H1H10) were against genotype 3a and three (E1B11, D2H3 and G2C7) were against genotype 1b. The cross reactivity of the monoclonal antibodies was determined by ELISA employing HCV-LP of other genotype as coating antigen (500 ng). As seen in Table 23727046 1, mAbs E8G9 against 3a HCV-LP and G2C7 against 1b HCV-LP showed maximum reactivity and were also cross reactive with both HCV-LPs to the same extent. mAbs E8G9 and D2H3 reacted strongly with the envelope protein in Western blot analysis suggesting that they recognize linear epitopes. The other three mAbs (E1B11, G2C7 and H1H10) reacted well in ELISA and dot blot but not in Western blot indicating that they are generated against conformational epitopes. The characteristics of the monoclonal antibodies are summarized in Table 1.Virus Neutralization buy CX-5461 AssayAnti-E2 antibodies (E8G9 and H1H10) generated against genotype 3a VLP were tested for their ability to neutralize virus infectivity. Huh7.5 cells were seeded into 24 well plate 16 h prior to the day of infection. JFH1virus was incubated with serial CX-4945 site dilutions of E2 mAbs at 37uC for 1 hr. The antibody-virus mixture was then transferred on the cells. Infectivity was analyzed three days (for HCV negative sense strand detection) or three hours (for input HCV positive sense strand detection) post infection by realtime RT- PCR.Quantification of Viral RNAViral RNA was quantified by real-time RT-PCR analysis. Cells were harvested three hours (for HCV positive sense strand detection) or three days (for HCV negative sense strand detection) post infection and total RNA was isolated which was reverse transcribed with HCV 39 primer (for positive sense) or HCV 59 primer (for HCV negat.Microscope (Fig. 1B). Results showed particles of 40?0 nm size of genotype 1b which was similar to the sizes described earlier [18] and 35?5 nm for genotype 3a. The size difference may be due to the difference in the amount of E1 and E2 proteins incorporated into each virus like particle. The purified HCV-LPs binding to Huh7 cells were analyzed by flow cytometry at 37uC. It was observed that with constant concentration of VLP (7 mg), at different time points, the intensity of fluorescence increased gradually upto 4 h which declined afterwards (Figure S1). Further, the binding efficiency of the HCV-LPs was compared at 4th hr time point. HCV-LP corresponding to genotype 3a showed marginally higher interaction (,80 ) with the Huh7 cells than the HCV-LP of genotype 1b (,70 ) (Figure S2).In vitro Transcription of Viral RNAThe pJFH1 construct (generous gift from Dr. Takaji Wakita, National Institute of Infectious Diseases, Tokyo, Japan) was linearized with XbaI. HCV RNA was synthesized from linearized pJFH1 template using Ribomax Large scale RNA production system-T7 according to manufacturer’s instructions (Promega).Transfection and Generation of JFH1 VirusHuh7.5 cells were transfected with in vitro synthesized JFH1 RNA transcript using Lipofectamine 2000 (Invitrogen) in OptiMEM (Invitrogen). Infectious JFH1 virus particles were generated as described previously [28]. Uninfected Huh7.5 cells were used as a mock control.Characterization of Monoclonal Antibodies Against 1b and 3a Genotype of HCV-LPBALB/c 1662274 mice were immunized with the HCV-LPs (both genotype 1 and genotype 3) and hybridoma were established by fusion of splenocytes with mouse myeloma cells. Approximately 200 hybridomas from two independent experiments were screened. A total of five mAbs were obtained out of which two (E8G9 and H1H10) were against genotype 3a and three (E1B11, D2H3 and G2C7) were against genotype 1b. The cross reactivity of the monoclonal antibodies was determined by ELISA employing HCV-LP of other genotype as coating antigen (500 ng). As seen in Table 23727046 1, mAbs E8G9 against 3a HCV-LP and G2C7 against 1b HCV-LP showed maximum reactivity and were also cross reactive with both HCV-LPs to the same extent. mAbs E8G9 and D2H3 reacted strongly with the envelope protein in Western blot analysis suggesting that they recognize linear epitopes. The other three mAbs (E1B11, G2C7 and H1H10) reacted well in ELISA and dot blot but not in Western blot indicating that they are generated against conformational epitopes. The characteristics of the monoclonal antibodies are summarized in Table 1.Virus Neutralization AssayAnti-E2 antibodies (E8G9 and H1H10) generated against genotype 3a VLP were tested for their ability to neutralize virus infectivity. Huh7.5 cells were seeded into 24 well plate 16 h prior to the day of infection. JFH1virus was incubated with serial dilutions of E2 mAbs at 37uC for 1 hr. The antibody-virus mixture was then transferred on the cells. Infectivity was analyzed three days (for HCV negative sense strand detection) or three hours (for input HCV positive sense strand detection) post infection by realtime RT- PCR.Quantification of Viral RNAViral RNA was quantified by real-time RT-PCR analysis. Cells were harvested three hours (for HCV positive sense strand detection) or three days (for HCV negative sense strand detection) post infection and total RNA was isolated which was reverse transcribed with HCV 39 primer (for positive sense) or HCV 59 primer (for HCV negat.