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

A novel and rapid full-length long RT-PCR technique was established to prod uce genome-length cDNA from Japanese encephalitis virus. In vitro positive strand RNA transcripts from the full-length RT-PCR amplicon including T7 pr omoter sequences at the 5 ' end were proved to be infectious upon transfect ion. The full-length amplicon without the T7 promoter was cloned into a cos mid 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 inse rt was not infectious, but transcripts of the full-length PCR amplicon from pJEV-1 produced infectious virus upon transfection. A transcript with an e ngineered Xho I site from two ligated PCR fragments amplified from pJEV-1 w as also infectious. Furthermore, the coding region for premembrane and enve lope proteins (preM-E) from pJEV-1 was subcloned and expressed in the Droso phila Expression System. The expressed protein showed correct molecular siz e and was immunoreactive with a Japanese encephalitis virus E protein-speci fic 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. (C) 2001 Elsevier Science B.V. All rights reserved.