Humanized Mouse Models of Epstein Barr Virus Infection

Abstract The oncogenic Epstein Barr virus (EBV) is an exclusively human pathogen with related lymphocryptoviruses (γ1‐herpesviruses) only present in monkeys. Therefore, experimentation with EBV infection in a small animal model requires reconstitution or adoptive transfer of human lymphocyte populations, primarily EBV's main host cell, the human B cell. In this protocol we describe human immune system reconstitution after neonatal transfer of CD34 + hematopoietic progenitor cells in lymphodeplete immune compromised mouse strains, using NOD‐ scid γ c –/– (NSG) mice as a commonly used example. Such reconstituted humanized mice allow intraperitoneal and intranasal infection with EBV and we describe injection of 10 5 infectious particles of the prototypic EBV strain B95‐8 that can be produced from a recombinant bacmid (p2089) in HEK293 cells. Infection with this dose mimics symptomatic primary EBV infection, infectious mononucleosis (IM), with high viral loads plateauing 4 weeks after infection, with CD8 + T‐cell lymphocytosis at week 5 and 6 after infection. This IM‐like primary EBV infection in humanized mice leads to clonal EBV‐induced B‐cell lymphoproliferations that resemble large B‐cell lymphomas with the latency III program of EBV infection. We describe Basic Protocols to monitor viral loads, immunohistochemistry of infected tissues and spectral flow cytometry to characterize protective T‐cell expansion. The described mouse model has been used by us and others to characterize mutant EBV infections, cell‐mediated immune control of EBV, modulation of EBV pathogenesis by co‐infections with human immunodeficiency virus (HIV) and Kaposi sarcoma associated herpesvirus (KSHV), as well as passive transfer of vaccine elicited antibodies to test their protection against EBV infection. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : CD34 + human hematopoietic progenitor cell isolation and characterization Basic Protocol 2 : Human immune system reconstitution and characterization Basic Protocol 3 : Recombinant EBV production and humanized mouse infection Basic Protocol 4 : Viral load quantification, lymphoma assessment, and immunohistochemistry after EBV infection of humanized mice Basic Protocol 5 : Human T‐cell response analysis after EBV infection of humanized mice