Four siRNAs (tsNTCP-si1: 5′-CUAUGUAGGCAUUGUGAUAdTdT-3′, tsNTCP-si2: 5′-GUGUUAUCCU GGUGGUUAUdTdT-3′, tsNTCP-si3: 5′-GGACAUGAAUCUCAGCAUUdTdT-3′, tsNTCP-si4: 5′-GGGCAAGAGCAUCAUGUUUdTdT-3′) specifically targeting Tupaia slc10a1 were designed through siDESIGN Center (www.thermo.com/sidesign). The specificities of these siRNAs were examined by searching against NCBI cDNA databases and the in-house Tupaia hepatocyte transcriptome to ensure they are free of off-target. siRNA with sequence Ctrl-si: 5′-UUCUCCGAACGUGUCACGUdTdT-3′ that is a scramble sequence with no known mammalian target sequence was used as a negative control. 20 nM siRNA were transfected into PTHs 24 h after initial seeding with lipofectamine 2000 (Invitrogen). tsNTCP mRNA level in the siRNA-transfected cells was quantified by real-time RT-PCR 3 days after siRNA transfection. Transfected PTH cells were infected on day 4 after siRNA transfection with HBV, HDV, AAV8-HBV (carrying 1.05 copies of HBV genome), or Lenti-VSV-G-Luc viruses. The secreted viral antigen HBsAg or HBeAg from HBV-and AAV8-HBV-infected cells were examined as indicated. For Lenti-VSV-G-Luc virus-infected cells, luciferase activity was determined on 6 days post-infection.