Efficient HBV infection of PHHs or HepaRG cells in vitro normally requires high dose of virus inoculums, and only limited progeny viruses are produced after infection (Gripon et al., 1988; Gripon et al., 2002; Boehm et al., 2005) . To assess viral particles released from HBV-infected HepG2-NTCP cells, we first quantified viral DNA in the medium collected at different time points after the infection. As indicated by drastic decline of viral DNA level on day 4 post-infection, the majority of residual viruses from inocula were removed by changing the medium and washing during the first few days of infection. The levels of viral DNA in the media resulted from the ongoing infection during days 4-13 post-infection were low (equivalent to ∼1% of input viral DNA copies) despite significant amount of HBeAg secretion during this period. Similarly, only low levels of viral DNA were detected in the medium from HBV-infected PHHs ( Figure 6-figure supplement 4, right) . It is reasonable to speculate that some host factors that are lacking in cell cultures might be needed for efficient viral particles formation or releasing; or some cellular factors in cultures may hinder these processes during infection. The culture medium collected from infected HepG2-NTCP cells was subsequently tested for infection of PHHs. In line with the low HBV viral DNA level in the medium inoculum, very low number of intracellular HBV total RNA copies were detected in PHHs on day 13 post-infection ( Figure 6-figure  supplement 5) , indicating that only very limited HBV infection might have occurred, which may be attributed to the low multiplicity of infection.