Hepatitis B Virion and cccDNA Formation in Primary Tupaia Hepatocytes and Human Hepatoma Cell Lines upon HBV Genome Transduction with Replication-defective Adenovirus Vectors and in vivo Infection of Tupaias
Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is a hepatotropic DNA-containing virus that replicates via reverse transcription. Because of its narrow host range there is as yet no feasible small animal system for HBV infection. Some evidence suggests that tree shrews (tupaias) may be susceptible to infection with human HBV, albeit with low efficiency. Infection efficiency depends on interactions of the virus with factors on the surface and inside the host cell. To by-pass restrictions during the initial entry phase, recombinant replication-defective adenovirus vectors, either without or with a green fluorescent protein marker gene, were constructed and used to deliver complete HBV genomes into primary tupaia hepatocytes. It was shown that these cells, like the human hepatoma cell lines HepG2 and HuH7, are efficiently transduced and produce all HBV gene products required to generate the secretory antigens HBsAg and HBeAg, replication-competent nucleocapsids, and enveloped virions. It was further demonstrated that covalently closed circular HBV DNA is formed. Therefore, primary tupaia hepatocytes support all steps of HBV replication following deposition of the genome in the nucleus, including the intracellular amplification cycle. In preliminary in vivo experiments, newborn and adult tupaias were infected with recombinant adenoviruses by intravenous injection. In two of the newborn animals, HBV antigens and HBV virions were detectable for more than 4 months. As in transduced PTHs, in vivo infection was dose dependent and subject to dynamic changes. It seems that newborn tupaias are much more sensitive to these recombinant adenoviruses than adults. The reasons for these differences are not yet completely clear, but may be related to the induced, or preexisting neutralizing antibodies. Together, these data provide a rational basis for in vivo experiments aimed at developing tupaias into a useful experimental animal system for HBV infection.
Hepatitis B Virus, transduction, vector, adenovirus, tupaia
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