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The factors involved in the establishment of persistent hepadnavirus infection are poorly understood. Recent findings demonstrate that the sequence of the genome of hepatitis B virus (HBV) is variable in infected individuals and that, in some cases, virus mutants predominate. Our objectives in the present study were to analyze the variability of woodchuck hepatitis virus (WHV) genomes in an infected animal and to determine whether sequence heterogeneity played a critical role in the ability of WHV to induce chronic infection. We cloned and determined the complete nucleotide sequence of three supercoiled genomes from an animal that became infected after inoculation with a standardized WHV serum pool (i.e., the WHV7 virus pool). We found that there were four nucleotide substitutions among the three genome sequences as well as a 73-nucleotide deletion in one of the recombinants. DNA transfection experiments revealed that only one of the three recombinants was capable of independent replication. These data suggest that a significant proportion of replicative templates in woodchucks that are infected with WHV are defective virus genomes. Next, we compared the outcome of acute infection after inoculation with a serum pool containing a uniform population of replication competent virus (i.e., the WHV7R pool) with a serum pool composed of WHV genomes of variable sequence. The WHV7R serum pool originated from a woodchuck that became a chronic carrier after in vivo transfection of the liver with the infectious WHV7 recombinant. Neonatal woodchucks were inoculated with 5 x 10(6) WHV genome equivalents of either the WHV7 pool or the WHV7R pool. All animals in the study became acutely infected with WHV. Of the animals infected with the WHV7 serum pool, 65% became chronic carriers, while 80% of the animals infected with the WHV7R serum pool developed chronic infection. Thus, infection of woodchucks with a serum pool containing defective virus resulted in a rate of chronic WHV infection that was similar to, or even lower than, a rate from a pool containing only wild-type virus. This suggests that the presence of defective virus in the inoculum is not a prerequisite for the establishment of persistent hepadnavirus infections.

Woodchuck hepatitis virus (WHV) DNA was cloned from viral particles obtained from the serum of a woodchuck with a naturally acquired infection. The complete nucleotide sequence of the virus genome was determined and found to be 3323 base pairs long. Transfection experiments demonstrated that the recombinant WHV DNA was infectious in each of 18 woodchucks tested and established a chronic carrier state in 1 of 13 neonates and 3 of 5 adult animals. WHV DNA from serum particles from the chronically infected neonate was cloned and the nucleotide sequence of three independent recombinants was compared directly with that of the input recombinant DNA. The consensus sequence of the three progeny genomes was identical to that of the parental DNA sequence. Therefore, transfection of woodchuck livers with recombinant WHV DNA induces active virus replication and gene expression and yields progeny genomes that are faithful copies of the input virus genome.