The pH dependency for initiation of infection by the hepadnavirus duck hepatitis B virus (DHBV) was investigated in primary duck hepatocytes. First, an infection assay was developed using a radioimmunoblot to measure DHBV e antigen secreted into tissue culture fluid from infected hepatocytes. The quantity of this viral marker was proportional to the duration of inoculation and the amount of DHBV used as inoculum. The role of pH in initiation of DHBV infection was investigated by using this

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... , but no dependence on low pH was found. DHBV was able to infect hepatocytes in the presence of NH4C1 and monensin, agents that raise the pH in intracellular vesicles and prevent penetration of viruses dependent on low pH in endosomes. In control experiments, infection by Semliki Forest virus, which is low pH dependent, was inhibited, whereas herpes simplex virus type 1 infection, which is pH independent, occurred. Attempts to trigger DHBV-cell fusion by exposure of DHBV prebound to hepatocytes to mildly acidic pH were unsuccessful. In these experiments, it was also observed that internalization of DHBV occurred only between pH 6.8 and 8.0. Additionally, in the absence of cells, infectivity of DHBV was stable at pH 4.6 to 4.8, which is lower than the pH encountered in endosomes (pH 5 to 6.6). Thus, no evidence for a role for mildly acidic pH in the initiation of DHBV infection was found. Therefore, we propose that the infection route followed by DHBV resembles that of the group of enveloped viruses, including herpesviruses, that fuse with their host cells at neutral pH. To initiate infection, the genome and associated components of viral particles must gain entry to the cytoplasm. In enveloped viruses, this translocation is achieved when viral surface proteins mediate fusion between viral and cellular membranes. Two groups of enveloped viruses are distinguished by the pH at which fusion occurs (see references 5, 10, 25, and 29 for reviews). For one group, mildly acidic pH triggers rapid fusion by exposing a fusion peptide buried within the three-dimensional structure of a surface protein at neutral pH. The mechanism of this low-pH-dependent fusion is best understood for influenza virus (27) , and other representatives include the alphavirus Semliki Forest virus (SFV), the rhabdovirus vesicular stomatitis virus, and flaviviruses. Internalization of these viruses is usually rapid. Conversion to the fusogenic form during infection occurs within the mildly acidic pH (pH 5 to 6.6) environment of endosomes, and consequently, agents that raise the pH in these vesicles inhibit penetration. In contrast, viruses belonging to the second group undergo fusion at neutral pH with relatively slow kinetics, and entry of these viruses is not inhibited by raising the pH of intracellular vesicles. These viruses have been called pH-independent viruses and include coronaviruses, herpesviruses, paramyxoviruses, and the retroviruses human immunodeficiency virus type 1 and Rous sarcoma virus (see reviews in references 5, 10, 25, and 29 and also see references 1, 4, 13, and 26). The hepadnaviruses are a conserved group of animal viruses, for which human hepatitis B virus is the type member. Although the basic features of hepadnaviral replication are becoming understood in increasing detail, very little is known about the initiation of infection. This is in part because the full replication cycle occurs only in highly differentiated cells from specific hosts and no infectable cell * Corresponding author. lines have been established. Consequently, infection studies are only possible in primary hepatocytes. Duck hepatitis B virus (DHBV) provides the best model system, since primary duck hepatocytes are easily obtained and conditions for maintenance and infection have been described previously (2, 18, 22, 28) . Nevertheless, the mechanism of initiation of infection by DHBV is not yet understood, and the cell receptor unit(s) remains elusive. Additionally, little is known about the role played by hepadnaviral surface proteins in attachment and entry into hepatocytes. In DHBV, there are two forms of surface protein, DHBV surface antigen (DHBsAg) and a larger DHBpre-S/S protein, consisting of an N-terminal extension, the pre-S region, in addition to the DHBsAg sequence. It is believed that the pre-S region carries the viral attachment site, but there is only scant evidence (8). To begin to understand hepadnavirus infection processes, we determined the pH requirement for DHBV infection by performing several types of experiments. The results show that DHBV most likely initiates infection at neutral pH in a manner analogous to that of pH-independent viruses. (The major findings of this work were presented at the meeting Molecular Biology of Hepatitis B Viruses held in San Diego, Calif., 5 to 9 August 1990.) MATERIALS AND METHODS Primary duck hepatocytes and DHBV. Primary duck hepatocytes were prepared and cultured essentially as described by Galle et al. (2), but triiodothyronine was omitted from maintenance medium. To seed cells, this medium was supplemented with 5% fetal calf serum, and the standard maintenance medium (called MM) lacked fetal calf serum but contained dimethyl sulfoxide (1.5%). Hepatocytes were seeded in 6-, 12-, or 24-well plates (Costar) at a density of 2.5 X 104 to 10 x 104 cells per cm2, and the medium was changed