Rescue of recombinant Thogoto virus from cloned cDNA

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome con sisting of six negative-stranded RNA segments. To rescue a recombinant THOV , the viral structural proteins were produced from expression plasmids by m eans of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RN As (vRNAs) were generated from plasmids under the control of the RNA polyme rase I promoter. Using this system, we could efficiently recover recombinan t THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introd uced into two vRNA segments. The availability of this efficient reverse gen etics system will allow us to address hitherto-unanswered questions regardi ng the biology of THOV by manipulating viral genes in the context of infect ious virus.