Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector

Abstract

A novel and rapid full-length long RT-PCR technique was established to produce genome-length cDNA from Japanese encephalitis virus. In vitro positive strand RNA transcripts from the full-length RT-PCR amplicon including T7 promoter sequences at the 5' end were proved to be infectious upon transfection. The full-length amplicon without the T7 promoter was cloned into a cosmid vector under the SP6 promoter. This stable clone, designated as pJEV-1, was characterised further and used as a genetic resource for generation of infectious RNA transcripts, gene manipulation and expression. The 'run-off' transcript from pJEV-1 with vector sequences at the either end of the insert was not infectious, but transcripts of the full-length PCR amplicon from pJEV-1 produced infectious virus upon transfection. A transcript with an engineered Xho I site from two ligated PCR fragments amplified from pJEV-1 was also infectious. Furthermore, the coding region for premembrane and envelope proteins (preM-E) from pJEV-1 was subcloned and expressed in the Drosophila Expression System. The expressed protein showed correct molecular size and was immunoreactive with a Japanese encephalitis virus E protein-specific antibody. The derivation of genome-size cDNA from Japanese encephalitis virus and the stable clone will facilitate investigation of this virus and elucidation of its pathogenesis at the molecular level.