Fig 1.
The ability of various primary hepatocytes to facilitate viral entry.
(A) Schematic representation of the experimental setting. Multiple primary hepatocytes from different species were seeded in 12-well plates and infected with 200 HDV genomes per cell (MOI = 200) in the presence of 4% PEG8000 with or without 500 nM MyrB. One day post-infection, cells were washed three times with PBS and changed with fresh medium, the supernatants were collected at indicated time points. (B) Cells were fixed in 4% paraformaldehyde for the detection of HDAg by Immunofluorescence staining. HDAg positive cells were labeled with a white arrow. Scale bar = 50 µm. (C) Representative fields were randomly selected for imaging, and the proportion of positive cells was calculated. (D) Cells were harvested at 7 dpi and extracted RNA for the detection of HDV RNA. Dotted line represents assay limit of detection. Data was presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.MOI, multiplicity of infection. dpi, days post-infection.
Fig 2.
Alignment of NTCP sequences from different species.
NTCP amino acid sequences from human (Protein ID. NP_003040.1), tree shrew (Protein ID. AFK93890.1), cat (Protein ID. XP_003987831.1), rabbit (Protein ID. NP_001076237.1), Syrian hamster (Protein ID: XP_005072871.1), Siberian hamster (Protein ID: XWU18636.1), guinea pig (Protein ID. XP_005008790.2), bull (Protein ID. NP_001039804.1), goat (Protein ID. XP_005686078.2), dog (Protein ID. XP_537494.2), pig (Protein Protein ID. XP_001927730.1), cynomolgus macaque (Protein ID. ALX38777.1), and mouse (Protein ID. AAH94023.1) are aligned using the alignment function of MEGA11 (Mega Limited). HBV-susceptible hosts (human and tree shrew) and HBV-unsusceptible hosts (mouse) are used as positive and negative reference, respectively. Residues 84-87 and 157-165 are boxed with blue. Residues 87 and 158 are labeled in red.
Fig 3.
Cross-Species analysis of rcDNA repair and cccDNA formation in primary hepatocytes.
(A) Schematic representation of the experimental setting. Multiple primary hepatocytes from different species were seeded in 12-well plates and transfected with rcDNA-I, the supernatants were collected at 2-, 4-, and 6 days post-transfection (dpt). (B-C) Secreted HBeAg and HBsAg in the supernatants at indicated time points were determined by ELISA.
Fig 4.
Susceptibility of primary hepatocytes from various species to HBV infection.
(A) Schematic representation of the experimental setting. Multiple primary hepatocytes from different species were seeded in 12-well plates and infected with HBV at 800 genomes per cell (MOI = 800) in the presence of 4% PEG8000 with or without 500 nM MyrB. One day post-infection, cells were washed three times with PBS and changed with fresh medium, the supernatants were collected at 5 dpi and 7 dpi. (B-D) Secreted HBeAg and HBsAg in the supernatants at 5 dpi and 7 dpi were determined by ELISA. Data was presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. MOI, multiplicity of infection. dpi, days post-infection.
Fig 5.
Comprehensive analysis of the HBV life cycle in primary cat hepatocytes.
(A) Schematic representation of the experimental setting. Primary cat hepatocytes were seeded in 6-well plates and infected with 2000 HBV genomes per cell (MOI = 2000) in the presence of 4% PEG8000 with or without 500 nM MyrB. (B) Secreted HBeAg and HBsAg in the supernatants at 3 dpi, 5 dpi, and 7 dpi were determined by ELISA. (C) HBV DNA of the cell culture supernatants at 7 dpi was detected by qPCR using an HBV viral DNA quantitative fluorescence diagnostic kit. (D) The expression of HBc protein was detected by western blotting. β-actin was used as an internal control. (E) The expression and localization of HBsAg protein was detected by immunofluorescence staining. Scale bar = 100 µm. (F) Cellular DNA was Hirt extracted and denatured at 85°C, treated with EcoRI restriction nuclease, and subjected to Southern blotting using 32P labeled HBV probes. The Hirt DNA samples from HBV-infected HepG2-hNTCP cells served as the positive control. (G) Cellular total RNA was extracted and reverse-transcribed, and HBV pgRNA and total RNA were quantified by RT-qPCR using HBV-specific primers. (H) Cellular total RNA was extracted and then subjected to Northern blotting assay using 32P labeled HBV probes. Ribosomal RNA 18S and 28S served as the loading control. (I) Cellular HBV core-associated DNA was extracted and then subjected to Southern blotting assay using 32P labeled HBV probes. The DNA samples from HBV-infected HepG2-hNTCP cells served as the positive control. (J) Transmission electron microscopy (TEM) was used for cellular ultrastructural examination of MVBs containing HBV particles (white arrow). Dotted line represents assay limit of detection. Data was presented as mean±SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. MOI, multiplicity of infection. HBc, HBV core protein; pgRNA, pre-genomic RNA; rcDNA, relaxed circular DNA; dslDNA, double-strand linear DNA; ssDNA, single-strand DNA; PF-rcDNA, protein-free rcDNA; cccDNA, covalently closed circular DNA; dpi, days post-infection.
Fig 6.
Primary human hepatocytes support higher HBV infection and replication.
(A) Schematic representation of the experimental setting. Primary hepatocytes were seeded in 48-well plates and infected with HBV (MOI = 250, 500, 1000, and 2000) in the presence of 4% PEG8000 with or without 500 nM MyrB. (B) Secreted HBeAg and HBsAg in the supernatants at 3 dpi, 5 dpi, and 7 dpi were determined by ELISA. (C) Intracellular DNA was extracted at 7 dpi and detected by qPCR using an HBV viral DNA quantitative fluorescence diagnostic kit. (D) Cellular total RNA was extracted at 7 dpi and reverse-transcribed, HBV pgRNA and total RNA were quantified by RT-qPCR using HBV-specific primers. (E-G) Secreted HBeAg and HBsAg (MOI = 1000) in the supernatants at indicated time points were determined by ELISA. Intracellular HBV DNA and RNA were detected by qPCR at 11 dpi. Dotted line represents assay limit of detection. Data was presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. MOI, multiplicity of infection. pgRNA, pre-genomic RNA; dpi, days post-infection.
Fig 7.
Enhanced HBV susceptibility in primary cat hepatocytes overexpressing hNTCP.
(A) Schematic representation of the experimental setting. Primary cat hepatocytes were seeded in 6-well plates and infected with HBV at a multiplicity of infection of 2000 (MOI = 2000), or transduced with AAV-TTR-hNTCP (60000 vg/cell) in the presence of 4% PEG8000. 24 hours post-HBV infection or AAV transduction, cells were washed thrice with PBS and changed with fresh PMM. 72 hours post-AAV transduction, cells were infected with HBV (MOI = 2000). (B) Secreted HBeAg and HBsAg in the supernatants at indicated time points were determined by ELISA. (C) HBV DNA of the cell culture supernatants at 7 dpi was detected by qPCR using an HBV viral DNA quantitative fluorescence diagnostic kit. (D) The expression of hNTCP protein in primary cat hepatocytes overexpressing hNTCP or not was detected by western blotting at 7 dpi. β-actin was used as an internal control. (E) Cellular total RNA was extracted and reverse-transcribed, HBV pgRNA and total RNA were quantified by RT-qPCR using HBV-specific primers. (F) The Hirt DNA samples from 7 dpi were extracted and subjected to Southern blotting using 32P labeled HBV probes. Dotted line represents assay limit of detection. Data was presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. MOI, multiplicity of infection. pgRNA, pre-genomic RNA; PF-rcDNA, protein-free rcDNA; cccDNA, covalently closed circular DNA; dpi, days post-infection; vg: viral genomes.
Fig 8.
Evaluation of primary cat hepatocytes as a platform for testing anti-HBV drugs.
(A) Schematic representation of the experimental setting. Primary cat hepatocytes were seeded in 6-well plates and infected with 2000 HBV genomes per cell (MOI = 2000) in the presence of 4% PEG8000 with or without 500 nM MyrB. One day post-infection, cells were treated with or without ETV (500 nM) and Poly (I:C) (1 μg/ml), and the supernatants and cells were collected at 3 dpi, 5 dpi, and 7 dpi. (B) Secreted HBeAg and HBsAg in the supernatants at indicated time points were determined by ELISA. (C) HBV DNA of the cell cultural supernatants at 7 dpi was detected by qPCR using an HBV viral DNA quantitative fluorescence diagnostic kit. (D) The expression of HBc protein in primary cat hepatocytes was detected by western blotting. β-actin was used as an internal control. (E) The expression of HBsAg protein in primary cat hepatocytes was detected by immunofluorescence staining. Scale bar = 100 µm. (F) Cellular DNA from different time points was Hirt extracted and then subjected to Southern blotting assay using 32P labeled HBV probes. The Hirt DNA samples from HBV-infected HepG2-hNTCP cells served as the positive control. (G) The Hirt DNA samples from seven days post-infection was denatured at 85°C, treated with EcoRI restriction nuclease, and subjected to Southern blotting using 32P labeled HBV probes. The Hirt DNA samples from HBV-infected HepG2-hNTCP cells served as the positive control. (H) Cellular total RNA was extracted and reverse-transcribed, and HBV pgRNA and total RNA were quantified by RT-qPCR using HBV-specific primers. (I) Cellular total RNA was extracted and then subjected to Northern blotting assay using 32P labeled HBV probes. Ribosomal RNA 18S and 28S served as the loading control. (J) Intracellular DNA was extracted and detected by qPCR using an HBV viral DNA quantitative fluorescence diagnostic kit. Dotted line represents assay limit of detection. Data was presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. MOI, multiplicity of infection. HBc, HBV core protein; pgRNA, pre-genomic RNA; rcDNA, relaxed circular DNA; dslDNA, double-strand linear DNA; ssDNA, single-strand DNA; PF-rcDNA, protein-free rcDNA; cccDNA, covalently closed circular DNA; dpi, days post-infection.