Duck hepatitis B virus replication in primary bile duct epithelial cells

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 immunohistoch emistry using CAM 5.2, a cytokeratin marker which was shown to react specif ically 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 appea r 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 cult ures of duck IBDE cells were acutely infected with DHBV, dual-labeled confo cal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 o r a combination of anti-pre-SI proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells s howed that the DHBV pre-S1 and core proteins were similar to their counterp arts in infected primary duck hepatocyte cultures. Southern blot analysis o f infected IBDE preparations using a digoxigenin-labeled positive-sense DHB V riboprobe revealed the presence of hepadnavirus covalently closed circula r (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear D NA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acu tely infected IBDE cultures is indicative of DHBV reverse transcriptase act ivity, while the establishment of a pool of viral CCC DNA reveals the abili ty of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supporte d hepadnavirus replication as shown by the de novo synthesis of DHBV protei ns and DNA replicative intermediates.