Duck hepatitis B virus replication in primary bile duct epithelial cells

Abstract

Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV). The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells. Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes. A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells. Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures. When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures. Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear DNA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates.

FIG. 1

Schematic diagram of the method used for the isolation of IBDE cells from duckling liver.

FIG. 2

Reactivity of CAM 5.2 to duck IBDE cells. (A) Duck liver tissue sections were processed for immunoalkaline phosphatase staining as described in Materials and Methods. After reactivity with the substrate, Fast Red, the tissues were counterstained with Mayer's hematoxylin. Pink precipitates were detected in the cytoplasm of bile duct cells (arrows); no precipitates were detected in hepatocytes. (B) Primary cultures of total liver cells at day 2 of culture were fixed in 100% methanol for 5 min and processed for IFA using CAM 5.2. Fluorescent filaments were detected in approximately 10% of the total liver cells (solid arrows); these fluorescent cells were epithelium-like in morphology. Fluorescent staining was not observed in hepatocytes (open arrow) which were identified by the characteristic polygonal morphology; hepatocytes appeared dull red because of the Evans Blue stain.

FIG. 3

Characterization of primary cultures of duck IBDE cells and PDHs by IFA using polyclonal antibodies to duck albumin. Coverslip cultures of primary duck IBDE (A) or PDH cultures (B) were fixed with cold ethanol:acetic acid (3:1) for 5 min at day 2 of culture and processed for IFA using a 1/200 dilution of goat anti-albumin. After reaction with FITC-conjugated anti-goat immunoglobulin G containing Evans Blue, the cells were mounted and viewed. Cytoplasmic fluorescent staining was not detected in the primary IBDE cultures (A, arrow) but was detected in parallel cultures of PDHs (B, arrow). IBDE cells in panel A appeared red because of the Evans Blue counterstain.

FIG. 4

Phenotypic characterization of the primary duck IBDE cultures. At various days of culture, coverslips of primary IBDE cultures were fixed in 100% methanol for 5 min and processed for immunoperoxidase staining using CAM 5.2. After reactivity with the substrate, DAB, the cells were counterstained with Mayer's hematoxylin. Cytokeratin staining was detected as dark brown precipitates in the cytoplasm of cells at 2 (A and B) and 5 (C) days of culture. (A and B) Early in culture, IBDE cells grew as cell clusters which often contained a lumen (arrow). (C) By day 5 of culture, duct-like structures (arrow) were observed in IBDE colonies.

FIG. 5

Detection of DHBV pre-S1 in PDHs or primary cultures of IBDE cells acutely infected with DHBV. Coverslip PDH or IBDE cultures were infected with DHBV-positive duckling sera (A and B) or mock-infected as described in the Materials and Methods (C and D). At 11 days p.i. the cells were fixed in 100% methanol and processed for immunoperoxidase staining using anti-pre-S1. Cells were counterstained with Mayer's hematoxylin. Pre-S1 proteins were found localized in the cytoplasm of DHBV-infected PDHs (A) or in the cytoplasm of DHBV-infected IBDE cell colonies (B, arrow) as seen by the brown cytoplasmic staining. No pre-S1 staining was observed in mock-infected PDHs (C) or mock-infected IBDE cells (D).

FIG. 6

Colocalization of DHBV proteins and CAM 5.2-specific proteins in acutely infected or mock-infected primary cultures of IBDE cells. DHBV-infected (A and B) or mock-infected primary cultures of IBDE cells (C and D) were dual labeled with anti-DHBV core proteins (A and C) and CAM 5.2 (B and D) followed by staining with the appropriate TRITC-conjugated (A and D) and FITC-conjugated (B and C) secondary antibodies. DHBV-infected IBDE cell cultures were also dual labeled with anti-pre-S1/Texas red-conjugated secondary antibody (E) and FITC-conjugated CAM 5.2 (F). (A and B) Clusters of cells (arrows) emitting both TRITC and FITC signals can been seen in the IBDE colonies. Bars, 50 μm.

FIG. 7

Immunoblot analysis of primary cultures of IBDE cells infected with DHBV. Parallel studies were performed with DHBV-infected PDH cultures. Duck IBDE cells or PDH cultures were infected with DHBV-positive duckling sera, and at various times p.i. the cells were harvested and processed for immunoblot analysis using anti-core proteins (A) and anti-pre-S1 (B).

FIG. 8

Detection of DHBV DNA replicative intermediates in infected duck IBDE cells or culture medium. Parallel studies were performed in DHBV-infected PDH cultures. At various days p.i., cells or culture media were harvested and processed for total DNA or CCC DNA analysis. Ten micrograms of DNA was loaded on each lane. A minus-strand DIG-labeled DHBV riboprobe was used. (A) Detection of RC, DSL, and SS DNA in all preparations of DHBV-infected IBDE or PDH cells processed for total DNA analysis. (B) Detection of RC, DSL, and CCC DNA in preparations of DHBV-infected IBDE or PDH cells processed for CCC DNA analysis. (C) DHBV CCC DNA in infected IBDE or PDH preparations remained as a supercoiled species after the respective DNA preparations were boiled for 1 min and quenched on ice prior to gel analysis. (D) Detection of RC DNA extracted from culture medium of DHBV-infected IBDE or PDH cultures. For panels A through C, M represents DIG-labeled DNA molecular size markers; for panel D, M represents a full-length cDNA of the Australian strain of DHBV removed from the plasmid pT3T7 by digestion with EcoRI.