In vitro HBoV1 infection has been reported only once in welldifferentiated human airway epithelia (HAE) [31] . That study provided only minimal information on virus replication, and did not include observations of pathophysiology. Obviously, the lack of a sustainable and highly reproducible system that enables highyield virus production, as well as the ability to conduct reverse genetics is a significant barrier to further elucidation of HBoV1 replication and pathogenesis. In the current study, we have successfully sequenced the full-length HBoV1 genome and cloned it in a plasmid referred to as pIHBoV1. Furthermore, we have demonstrated that transfection of human embryonic kidney 293 (HEK293) cells with pIHBoV1 results in efficient production of HBoV1 virions at a high titer, and that these virions are able to productively infect both primary and conditionally transformed polarized HAE.