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The preparation of infectious beak and feather disease circovirus virions (BFDV) has until now relied on the extraction of virus from whole tissue of deceased or euthanized parrots known to be infected with the virus. Extraction from diseased tissue is necessary, as the virus has yet to be grown in vitro using tissue-cultured cells from any source. While infectious DNA clones have been synthesized for porcine and duck circoviruses, and both replicate in host cells and result in active viral infection in animals, this has not been shown for BFDV. The aim of this study was to prepare an infectious BFDV genomic clone that could be used as challenge material in birds for vaccine testing. A putatively infectious BFDV genomic clone was designed and tested in mammalian cell culture, and in the plant Nicotiana benthamiana in the presence of plant-specific ssDNA geminivirus replication components. Replication was assessed using rolling-circle amplification, qPCR, replication-deficient clones and rescue plasmids. We showed that a synthetic partially dimeric BFDV genomic clone self-replicated when transfected into 293TT mammalian cells, and was also replicated in N. benthamiana in the presence of geminivirus replication elements. This is the first report of a BFDV genome replicating in any cell system, and the first report of a circovirus replicating with the aid of a geminivirus in a plant. Both of these developments could open up possibilities for making reagents and vaccines for BFDV, testing vaccine efficacy and investigating viral replication using rationally designed artificial genomes.

Xenogenic rolling-circle replication of a synthetic beak and feather disease virus genomic clone in 293TT mammalian cells and Nicotiana benthamiana