We have previously reported that bovine papillomavirus type 1 (BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S.cerevisiae (budding yeast) cultures (Zhao and Frazer 2002,Journal of Virology,76:3359-64 and 76:12265-73).Here,we report the episomal replications of BPV-1 DNA in long term virion-infected S.cerevisiae culture up to 108 days.Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages,early active replication (day 3-16),middle weak replication (day 23-34/45) and late stable replication (day 45-82).Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form,stranded DNA,monomers and higher oligomers were detected in the virion-infected yeast cells over the time course.Higher oligomers shown as covalently closed circular DNAs (cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle.In this study,the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication,but they appeared to be suppressed at the middle weak replication.Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S.cerevisiae system.