A High throughput Reverse Genetics System for Hepatitis C Virus

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 DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is one of medically important RNA viruses and causes a wide spectrum of liver diseases, which are also a leading indication for liver transplantation in the United States. Given its high priority in public health, an in vito model for HCV life cycle is highly demanded for both basic and translational HCV research, however, current in vitro HCV models could only mimic some steps of viral life cycle. There is so far only one particular HCV genotype 2a strain, named JFH1, is able to be propagated in vitro. The JFH1-based cell culture has been established through the rescue of infectious viruses from cloned viral genomes, so called reverse genetics approach. A well-known constraint in such an approach is the unpredictability of viral mutations introduced internally or externally during the preparation of full-length viral clones. Some of these mutations may be lethal or detrimental for successful rescue of infectious viruses, in particular in the setting of HCV due to its coordinated genome network and remarkable genetic diversity. When the number of strains/clones tested is large enough to overcome the barrier of mutations from either heterogeneous viral population or experimental introduction, HCV cell culture could be established with strains other than JFH1. However, this goal is hard to be achieved by conventional reverse genetics that is notable for its tedious process. Based on our previous work, we thus propose to develop a rapid reverse genetics platform capable of screening clinical HCV isolates in a high-throughput manner. We hypothesize that the establishment of such a platform is feasible by the integration of our long RT-PCR technique (US patent No: 7,786,294 B2) and a seamless assembly method. The proposed platform is a plasmid-based reverse genetics in which long RT-PCR and 3'UTR amplicons of HCV will be fused into the target plasmid using the Gibson assembly method, followed by direct transfection into Huh-7 cells and subsequent measurement of potential infectious viruses. We will screen a total of 61 patient-derived HCV 1a strains that have already been characterized for viral mutations at the full-length genome level. The method developed with the proposed project has general applicability in RNA viruses other than HCV.