FIG. 9.

Tissue-specific loss of the LCMV epitope during rCVB3 infection. Two naive C57BL/6 mice were infected with 2 × 10⁶ PFU of rCVB3.2; 4 days later, their hearts or pancreata were harvested and RNA was prepared. Alternatively, RNA was prepared from tissue culture cells after four or five viral passages (P4 and P5, respectively). The RNA was used as a template for RT-PCR with primers flanking the SfiI cloning site (see Materials and Methods). (A) PCR products were subjected to agarose gel electrophoresis, and bands were stained with ethidium bromide. Arrows indicate the expected band sizes for products from rCVB3.2 (containing the GP33 epitope), rCVB3.1 (lacking the epitope but containing the SfiI cloning site), and wtCVB (lacking both the epitope and the SfiI site). (B) PCR products were cloned by the T-A method. The resulting bacterial plates were duplicated, and the bacterial colonies were analyzed by hybridization with radiolabeled probes specific for CVB or for the GP33 sequence. Following autoradiography, positive colonies were counted; the data from two independent experiments are shown. ND, not done.