As we previously showed that HEK293 cells support replication of the DNA of an autonomous human parvovirus (B19V) in the presence of adenovirus helper genes or adenovirus [33] , we first investigated whether the adenovirus helper function is necessary for pIHBoV1 replication in HEK293 cells. Specifically, we transfected pIHBoV1 into HEK293 cells (untreated or infected with adenovirus), alone or with pHelper. Interestingly, we found that pIHBoV1 replicated well in the absence of helper virus. Indeed, all the three representative forms of replicated bocavirus DNA [27, 34] (DpnI digestion-resistant dRF DNA, mRF DNA and ssDNA) were detected in each test case, and at similar levels ( Figure 2A ). DpnI digestion-resistant DNA bands are newly replicated DNA in cells as DpnI digestion only cleaves plasmid DNA prepared from prokaryotic cells, which is methylated at the dam site [35] . In contrast, these DNA forms of the viral genome were absent in pIHBoV1-transfected primary airway epithelial cells (NHBE; Figure 2B , lanes 7&8) and present at very low levels (over 20 times lower than in pIHBoV1-transfected HEK293 cells) in pIHBoV1-transfected human airway epithelial cell lines BEAS-2B ( Figure 2B , lanes 5&6), A549 and 16HBE14o-( Figure 2C ), even in the presence of adenovirus. Thus, replication in these cells appears to be non-existent or poor in these contexts.