Available treatments for hepatitis B can control the virus but are rarely curative. This led to a global initiative to design new curative therapies for the 257 million patients affected. Discovery and development of these new therapies is contingent upon functional in vitro and in vivo hepatitis B virus (HBV) infection models. However, low titer and impurity of conventional HBV stocks reduce significance of in vitro infections and moreover limit challenge doses in current in vivo models. Therefore, there is a critical need for a robust, simple and reproducible protocol to generate high-purity and high-titer infectious HBV stocks. Here, we outline a three-step protocol for continuous production of high-quality HBV stocks from supernatants of HBV-replicating cell lines. This purification process takes less than 6 h, yields to high-titer stocks (up to 1 × 10¹¹ enveloped, DNA-containing HBV particles/mL each week), and is with minimal equipment easily adaptable to most laboratory settings.

A single hepatitis B virus (HBV) particle is sufficient to establish chronic infection of the liver after intravenous injection, suggesting that the virus targets hepatocytes via a highly efficient transport pathway. We therefore investigated whether HBV utilizes a physiological liver-directed pathway that supports specific host-cell targeting in vivo. We established an ex vivo system of perfused human liver tissue that recapitulates the liver physiology to investigate HBV liver targeting. This model allowed us to investigate virus-host cell interactions in a cellular microenvironment mimicking the in vivo situation. HBV was rapidly sequestered by liver macrophages within one hour after a virus pulse, but was detected in hepatocytes only after 16 hours. We found that HBV associates with lipoproteins. Electron- and immunofluorescence microscopy corroborated a co-localization in recycling endosomes within peripheral and liver macrophages. Importantly, recycling endosomes accumulated HBV and cholesterol, followed by transport of HBV back to the cell surface along the cholesterol efflux pathway. To reach hepatocytes as final target cells, HBV was able to utilise the hepatocyte-directed cholesterol transport machinery of macrophages. Our results propose that HBV by binding to liver targeted lipoproteins and utilizing the reverse cholesterol transport pathway of macrophages hijacks the physiological lipid transport pathways to the liver to most efficiently reach its target organ. This may involve trans-infection of liver macrophages and result in deposition of HBV in the perisinusoidal space from where HBV can bind its receptor on hepatocytes.