FIG. 5.

Infection of ducklings with the I2 mutant. Virus particles containing the mutant I2 or wild-type genome were prepared by transfection of LMH cells and used to infect four 4-day-old ducklings at a dose of 5 × 10⁹ viral genomes per bird. (A) Virus present at the indicated times in 25 μl of serum was extracted and analyzed by agarose gel electrophoresis and blot hybridization. The top panel was exposed approximately four times as long as the bottom panel (compare hybridization marker signals). (B) Liver samples (equivalent to 5 mg) were analyzed for viral replicative intermediates present at day 6 postinfection by gel electrophoresis and blot hybridization. Each lane contained the DNA extracted from an equivalent of 5 mg of liver tissue. (C) rcDNA extracted from enveloped virus in the blood and cccDNA from the liver at day 6 postinfection was subjected to PCR amplification and direct sequencing of the PCR products through the r region. Primers corresponding to nucleotides 2492 to 2516 and 70 to 45 were used for amplification of the rcDNA. The sequencing gel shows the presence of the I2 mutation in the day 6 postinfection samples in comparison with the wild-type sequence.