We have previously reported that primary hepatocytes from T. belangeri
can be infected with HBV (17
). However, the practical use of this system was limited by the low efficiency of infection. Here, we show that human serum interferes with viral attachment, thereby greatly reducing viral infection. On the other hand, purification of virions by gradient centrifugation strongly enhances both HBV binding and infection. Thus, newly synthesized cccDNA and ssDNA and viral RNA were unambiguously detectable by Southern and Northern blot analysis, respectively. Infection of PTH with Nycodenz gradient-purified HBV particles represents an authentic biological process, because the viral host tropism was preserved in cell culture and Nycodenz itself does not affect binding of HBV to PTH. This is in contrast to polyethylene glycol-based protocols (6
), because polyethylene glycol may induce nonphysiological membrane fusion.
The biochemical nature of the inhibitory serum component is unclear at present. Preliminary results indicate that the inhibitory factor cannot be removed by dialysis and that albumin and immunoglobulins, which are the most abundant serum proteins, do not account for the inhibitory effect (J. Köck and F. von Weizsäcker, unpublished data). Interestingly, attachment of duck hepatitis B virus to primary duck hepatocytes is not inhibited by duck serum (J. Köck and F. von Weizsäcker, unpublished). This may explain in part the high efficiency of duck hepatitis B virus infection in vitro (16
Compared to human hepatocytes, PTH have the principal advantage of being readily available from in-house-bred animals. PTH, therefore, allow the highly reproducible infection with HBV under the experimental conditions described, while the suitability of primary hepatocytes prepared from human liver for infection experiments is very variable (5
PTH were also infected by WMHBV but not by WHV. This finding substantiates PTH as a useful in vitro model system for studying hepadnavirus infection and rules out the possibility that viral uptake in PTH is promiscuous. The permissiveness of PTH to primate but not rodent hepadnaviruses is in line with the fact that tupaias are phylogenetically closely related to primates but not to rodents (12
It is important to note that the replication rate of HBV in PTH is low. Newly synthesized HBV ssDNA was not detected until about 2 weeks postinfection. It seems unlikely that the low replication rate of HBV in PTH is due to inadequate cell preparation or culture conditions, since WMHBV ssDNA was visible as early as 6 days postinfection (Fig. A). The precise viral and/or cellular factors determining the replication rate of HBV and WMHBV in PTH remain to be further elucidated. Initial experiments comparing HBV and WMHBV RNA synthesis in PTH revealed an overall low abundance of pgRNA, in HBV- as well as WMHBV-infected cells. The ratio of pgRNA to sgRNA, however, was slightly higher in WMHBV- than in HBV-infected PTH. Since pgRNA is pivotal for replication and capsid formation, subtle changes in pgRNA production might translate into substantial effects on viral replication rates. Compatible with this notion, there are significant differences in the core promoter regions of WMHBV and HBV. We are currently preparing a replication-competent WMHBV construct that would allow us to directly address this question in transfected cells.