The evolution of in vitro culture systems for the study of hepatitis E virus infection.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis worldwide, is increasingly recognized as an important zoonotic pathogen capable of causing chronic infection and diverse extrahepatic manifestations. However, progress in understanding the biology and pathogenesis of HEV has historically been constrained by the limited availability of robust and reproducible in vitro culture systems. Early work relied heavily on hepatoma-derived cell lines, which, while experimentally tractable, often support inefficient viral replication and incompletely reflect hepatic physiology. Recently, substantial advances in cell culture methodologies have expanded the repertoire of available models for investigating HEV. Here, we critically review the spectrum of in vitro culture systems currently used to study HEV infection, focusing on how increasing physiological complexity has enabled deeper mechanistic insight into viral replication, host tropism, and pathogenesis. We discuss classical hepatoma cells-based systems alongside more advanced hepatic models, including stem cell-derived cultures and organoids. We further highlight the emergence of extrahepatic and physiological barrier models, such as in vitro placental and blood-brain barrier systems, which provide new opportunities to investigate HEV-associated extrahepatic disease manifestation. Throughout, we emphasize how differences in innate immune competence, cellular differentiation state, and infection modality in different culture systems shape experimental outcomes. Finally, we consider gaps and future directions in the field, including the integration of microfluidic multi-organ platforms, improved organoid co-culture systems, and multi-omics approaches. Together, these advances aim to bridge the gap between simple cell culture models and human disease, supporting the development of more analytical platforms for studying HEV infection dynamics.