FIG. 2.

recTHOV carries genetic markers. vRNA was isolated from virus particles obtained from supernatants of recTHOV-infected (rec) or wild-type THOV-infected (wt) cells. As a control, supernatants of Vero cells treated with the supernatants of 293T cells which had been transfected with plasmids for all vRNA segments except segment 4 (-S4) were used for RNA isolation. Segment 4 (A and B) and segment 5 (C and D) genomic vRNAs were detected by RT-PCR with primers that allow the amplification of a 1,115-bp segment 4 fragment (position 481 to 1574) and a 974-bp segment 5 fragment (position 467 to 1418). RT-PCRs without RT enzyme (−RT) were used as controls. The presence of the newly created PstI site in the cDNA of segment 4 (B) and of the additional ClaI restriction site in the cDNA of segment 5 (D) of the recTHOV was determined by restriction analysis of the RT-PCR products. The products were analyzed by agarose gel electrophoresis in the presence of ethidium bromide. The molecular sizes of the fragments are indicated at the right.