FIG. 2.

In vivo analyses of rCVB3.6: growth kinetics and insert stability. Mice were inoculated with 10⁷ PFU of a P2 stock of rCVB3.6. (A) The viral titers in the spleen, pancreas, and heart were determined at the indicated time points p.i. (three mice at days 2, 6, 7, and 8 and five mice at day 4). For the day 6-, 7-, and 8-tested mice that did not harbor infectious virus, the success of the original infection was confirmed by titration of feces that had been collected at 2 days p.i.; all mice showed a high fecal titer (∼10⁶ PFU/g) (data not shown). (B) A portion of the rCVB3.6 sequence encoding the GP₃₃ and GP₆₁ epitopes was amplified by RT-PCR from RNA isolated from the virus stock used to inoculate mice (P2) or from the spleen, heart, and pancreas at the indicated time points p.i., and PCR products were analyzed by gel electrophoresis. Each lane on the gel represents an individual mouse. The expected PCR product from intact rCVB3.6 is 466 bp; the dashed white line shows the expected location of a wt RNA product (310 bp). All RNA samples also were incubated in the absence of reverse transcriptase, and in all cases PCR products were undetectable (not shown). To more precisely analyze the sequence stability, samples were cloned and sequenced as described in the text. (C) To ensure that the P2 stock that was inoculated into the mice did not contain any residue of the plasmid DNA from which infectious RNA was prepared, the PCR was carried out with or without reverse transcriptase (+RT or −RT, respectively).