A robust in vitro hepatitis C virus (HCV) infection system has been established using the HCV JFH-1 molecular clone and Huh-7-derived cell lines, enabling the production of infectious virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for studying host-virus interactions and facilitates the discovery of antiviral drugs and vaccines. The system allows for the production of viral titers of 10^4-10^5 infectious units per ml of culture supernatant, with infection spreading throughout the culture within a few days after inoculation at low multiplicities of infection (MOI), and the virus can be serially passaged without loss in infectivity. The system was developed using in vitro transcribed genomic JFH-1 RNA delivered to cells by electroporation or liposome-mediated transfection. The system was tested with various cell lines, including Huh-7.5.1 and Huh-7 cells, and showed that HCV can infect these cells and produce infectious particles. The system was also used to study the effects of anti-E2 and anti-CD81 antibodies on HCV infection, showing that these antibodies can inhibit HCV infection. The system was also used to study the biophysical properties of infectious HCV JFH-1 particles, showing that they have a density similar to that of human isolates. The system was also used to study the in vitro tropism of JFH-1 HCV, showing that it is limited to Huh-7-derived cell lines. The system was also used to study the kinetics of HCV infection in Huh-7.5.1 and Huh-7 cells, showing that HCV can produce similar amounts of progeny virus as Huh-7.5.1 cells but with delayed kinetics. The system was also used to study the effects of interferon on HCV infection, showing that both IFN-α and -γ prevent JFH-1 virus infection of Huh-7.5.1 cells. The system provides a robust model for studying HCV infection and has the potential to accelerate the discovery of antiviral drugs and contribute to the development of an HCV vaccine.A robust in vitro hepatitis C virus (HCV) infection system has been established using the HCV JFH-1 molecular clone and Huh-7-derived cell lines, enabling the production of infectious virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for studying host-virus interactions and facilitates the discovery of antiviral drugs and vaccines. The system allows for the production of viral titers of 10^4-10^5 infectious units per ml of culture supernatant, with infection spreading throughout the culture within a few days after inoculation at low multiplicities of infection (MOI), and the virus can be serially passaged without loss in infectivity. The system was developed using in vitro transcribed genomic JFH-1 RNA delivered to cells by electroporation or liposome-mediated transfection. The system was tested with various cell lines, including Huh-7.5.1 and Huh-7 cells, and showed that HCV can infect these cells and produce infectious particles. The system was also used to study the effects of anti-E2 and anti-CD81 antibodies on HCV infection, showing that these antibodies can inhibit HCV infection. The system was also used to study the biophysical properties of infectious HCV JFH-1 particles, showing that they have a density similar to that of human isolates. The system was also used to study the in vitro tropism of JFH-1 HCV, showing that it is limited to Huh-7-derived cell lines. The system was also used to study the kinetics of HCV infection in Huh-7.5.1 and Huh-7 cells, showing that HCV can produce similar amounts of progeny virus as Huh-7.5.1 cells but with delayed kinetics. The system was also used to study the effects of interferon on HCV infection, showing that both IFN-α and -γ prevent JFH-1 virus infection of Huh-7.5.1 cells. The system provides a robust model for studying HCV infection and has the potential to accelerate the discovery of antiviral drugs and contribute to the development of an HCV vaccine.