Cell-based platforms for antiviral screening against hepatitis B virus: advantages, limitations, and future perspectives.

Hepatitis B virus (HBV) infection affects over 250 million people worldwide, resulting in 1.3 million deaths annually. While most healthy adults clear acute HBV infection, many children and immunocompromised adults develop chronic hepatitis B, leading to liver-related complications such as cirrhosis and hepatocellular carcinoma. Current treatments, including nucleos(t)ide analogs and pegylated interferon alpha, suppress viral replication but rarely achieve a functional cure. The development and optimization of robust in vitro systems is central to the discovery of effective antiviral compounds against HBV. In this review, we examine the main cell-based models used for HBV antiviral screening, highlighting their strengths and limitations in modeling viral replication and as platforms for antiviral compound testing. We discuss DNA- and RNA-based transfection systems as controllable tools to study specific stages of the HBV replication cycle, as well as stably transfected HBV-producing cell lines that enable reproducible compound screening. Infection-based models, including HepG2-NTCP and HepaRG cells, are evaluated for their ability to support viral entry and cccDNA formation, key features for assessing entry inhibitors and post-entry antivirals. We highlight primary human hepatocytes (PHH) and mouse-passaged PHH (mpPHH), which more closely recapitulate physiological hepatocyte biology but present challenges in scalability and variability. Finally, we explore emerging three-dimensional systems, such as spheroids and organoids, which aim to mimic the complex liver architecture and microenvironment. By comparing these platforms, this review provides a framework for selecting appropriate in vitro models for HBV antiviral compound discovery. Such a framework aims to accelerate progress towards a functional cure.