Hepadnaviral lymphotropism and its role in virus persistence in the woodchuck model of hepatitis B

Hepatitis B virus (HBV) causes lifelong liver disease in up to 400 million persons worldwide. The true extent ofHBV exposure is unknown, but could be as high as 2 billion people. This is mainly due to the existence of occult infection and the inadequate sensitivity of HB V -specific serological assays. Fallowing our previous findings indicating that the lymphatic system is a site ofhepadnavirus replication, the aims of this study were to determine virological factors underlying hepadnavirallymphotropism as it pertains to virus persistence. Specifically, we focussed on the identification of how virus dose and the potential existence of variants may induce lymphatic system-restricted infection. We employed the woodchuck/woodchuck hepatitis virus (WHV) system, which represents the closest pathogenic model of human HBV infection and hepatitis B. Our results revealed that a significant portion of circulating lymphoid cells are infected with WHY, whether or not infection is serologically evident or occult. This was documented by applying an in situ PCR combined with flow cytometry technique, established during this study, that enumerated WHV -infected cells without the necessity of nucleic acid extraction. We identified that primary occult WHV infection, that is normally limited to the lymphatic system, is induced by exposure to low doses ( < 10³ vge) of wild-type virus and is unlikely due to infection with or the appearance of viral variants inclined to preferentially invade lymphoid cells. We also showed that exposure to small amounts ofWHV ( < 10³ vge) did not induce protective anti-viral immunity in that the infected host remained susceptible to infection with large WHY doses. In a subsequent in vitro study, we documented that WHY could be serially passaged in both lymphoid cells and hepatocytes, which does not lead to the emergence of cell typespecific virus variants. The passaged WHY maintained its infectivity and pathogenicity when administered to virus-naive woodchucks. This proved that lymphotropism is a natural propensity of wild-type WHY in both in vivo and in vitro conditions. By analysing animals intrahepatically transfected with recircularized, complete recombinant WHY DN~ we documented that recombinant WHY DNA initially establishes infection in lymphoid cells and, at this location, infectious virus is produced. The infection of the liver was always secondary. Since dendritic cells (DC) are known to be a reservoir of virus replication in many persistent viral infections, we aimed to recognize whether this lymphoid cell subset is also the site of WHY persistent replication. We prepared monocytic derived DC from animals with serologically evident and occult chronic WHY infections and demonstrated that DC are indeed one of the cell types where the virus persistently propagates. The new features of hepadnavirus infection uncovered in this study imply that when the host is exposed to a low hepadnavirus dose, the lymphatic system is a primary target of invading virus. Because of the similarities between WHY and HBY, it is reasonable to assume that infection of lymphoid cells also plays a major role in the initiation and long-term persistence of HB V in humans