We then tested HBV infection on tsNTCP knockdown PTHs. Infection with HBV can be assessed by measuring secreted viral antigens HBV S antigen (HBsAg) and HBV e antigen (HBeAg). HBV inocula may contain residual HBsAg that can release and interfere with the detection of newly synthesized HBsAg during the first few days of infection. To differentiate de novo HBsAg synthesis from the contaminating inoculum, we assayed HBsAg secretion over time from days 6 to 12 after infection with the culture medium changed every 2-3 days. In addition, the kinetics of production of HBeAg with minimal or no residuals in the inoculum was also examined in the same time course experiment. As shown in Figure 4A (lower-left), both HBsAg and HBeAg levels were markedly reduced by transfection of tsNTCP-specific but not a control siRNA at all three time points tested, demonstrating that tsNTCP expression is required for bona fide HBV infection. To confirm that tsNTCP functions at the viral entry level for HBV as it does for HDV, we tested AAV8-HBV virus infection on tsNTCP knockdown PTHs. AAV8-HBV is a recombinant adenovirus-associated virus containing a 1.05× overlength HBV genome, for which viral entry is mediated by AAV8 capsid instead of HBV envelope proteins. AAV8-HBV infection of PTHs can nevertheless transduce the HBV genome into cells and lead to subsequent HBV viral antigen expression. NTCP knockdown did not affect AAV8-HBV infection in PTHs, as shown by the kinetics of HBeAg ( Figure 4A , lower-right). This result shows that NTCP has no effect on post-entry steps of HBV infection.