Advances and challenges in experimental models for Epstein–Barr virus research

Epstein–Barr virus (EBV), the first identified human oncovirus, is a ubiquitous γ‐herpesvirus etiologically linked to diverse malignancies, lymphoproliferative disorders, and autoimmune diseases. Experimental models are pivotal for unraveling the infection and pathogenesis mechanisms, life cycle regulation, as well as development and evaluation of antiviral vaccines and therapeutics. Traditional two‐dimensional (2D) cell lines (e.g., lymphoblastoid cell lines [LCLs] and NPC43) have provided foundational insights into viral latency and oncogenesis, but lack physiological complexity. Emerging three‐dimensional (3D) models, such as air–liquid interface (ALI) cultures, spheroids, and patient‐derived organoids (PDOs), better mimic tissue architecture and tumor microenvironments and yet face challenges in scalability, dynamic microenvironments, and immune component integration. Animal models, such as humanized mice, rabbits, and tree shrews, have been developed that over-come in vitro limitations by recapitulating systemic interactions, immune responses, and disease progression. Despite these advances, persistent challenges remain, including species‐specific constraints, incomplete modeling of the tumor microenvironment, partial immune system representation, and limited disease heterogeneity. This review systematically summarizes the technical features, applicability, and limitations of current models, provides guidance for selecting experimental models in further studies, and emphasizes the need for innovative, patient‐tailored platforms to unravel EBV's lifelong pathogenesis and facilitate the translation of EBV vaccines and therapeutics.