Spread of Hepatitis B Viruses In Vitro Requires Extracellular Progeny and May Be Codetermined by Polarized Egress

ABSTRACT Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.