Amplification and Cloning of Full-length JEV cDNA and Preparation of Infectious RNA by in vitro Transcription

Preparation of infectious RNA by in vitro transcription from full length cDNA has led to advances in the study of many positive strand RNA viruses. There are two strategies of preparing full length cDNA of RNA viruses: (1)construction of infectious clone. (2)amplification of full length cDNA by long template RT PCR with the RNA promoter incorporated to 5 primer. In this study both ways were established to generate Japanese Encephalitis Virus (JEV) infectious RNA. Firstly JEV (SA14 strain) full length cDNA was amplified by long template RT PCR and then cloned into the cosmid vector. One clone in which JEV cDNA was downstream of T7 promoter was selected and the RNA derived from this clone showed infectious after transfection of BHK cell. In another way of preparing JEV full length cDNA by long template RT PCR, T7 promoter was introduced to 5 primer. RNA derived from this cDNA also showed infectious after transfection of BHK cell. The recovered viruses were further confirmed by intracerebral inocu