Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol

• A full-length TMUV cDNA is generated using a PCR-based protocol.
• Infectious viral particles are rescued from BHK-21 cells transfected with in vitro transcribed RNA.
• Genomic characterization reveals that the rescued virus is genetically indistinguishable from the parental virus except genetic markers.
• The rescued virus possesses similar growth properties in BHK-21 cells and virulence in mice with the parental virus.
• Overall the technical platform will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV.

Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV.