A newly developed eGFP-traceable recombinant rhesus lymphocryptovirus, a tool to study viral infection and replication in vitro and ex vivo, confirms gp350 as key for viral entry.

Epstein-Barr virus (EBV) is implicated in multiple epithelial and lymphoid malignancies worldwide; however, no licensed prophylactic vaccine exists, partly due to the lack of an adequate preclinical animal model to test candidate vaccines. Rhesus lymphocryptovirus (rhLCV) faithfully recapitulates EBV infection, persistence, and pathogenesis in rhesus macaques, offering an invaluable surrogate model. While recombinant EBV expressing enhanced green fluorescent protein (eGFP) enables swift identification of infected cells in vitro and ex vivo, an analogous tool for rhLCV has been lacking. Here, employing en passant recombination technology, we engineered a recombinant rhLCV expressing eGFP (rhLCV.eGFP) from an rhLCV bacterial artificial chromosome derived from the B-lymphoblastoid rhesus macaque cell line LCL 8664. Whole-genome sequencing confirmed sequence integrity, and functional assays demonstrated infectivity in peripheral blood mononuclear cells (PBMCs) from multiple primate species and EBV-susceptible human cell lines, and transformation competence in rhesus macaque PBMCs. To define the role of gp350/220 (gp350) in viral entry, we generated a glycoprotein gp350-deficient mutant (rhLCV.eGFPΔgp350), verified by Sanger sequencing and immunoblotting with a novel anti-gp350 monoclonal antibody (4A6). This mutant exhibited markedly reduced infectivity in rhLCV.eGFP-susceptible human B and epithelial cell lines, underscoring gp350 as a key determinant of B cell tropism, analogous to EBV gp350. Our successful engineering of rhLCV.eGFP provides a powerful platform for visualizing infection, dissecting viral entry mechanisms and cellular tropism, and advancing rhLCV as a surrogate EBV model for vaccine design, defining correlates of protection, studies of latency and reactivation, and preclinical testing of candidate antivirals and immunotherapies. Epstein-Barr virus (EBV) infection is associated with various epithelial and lymphoid diseases. The absence of a suitable animal study model for EBV infection has hindered mechanistic studies of pathogenesis and preclinical testing of vaccines and EBV‑targeted therapies. Rhesus lymphocryptovirus (rhLCV), an EBV homolog, accurately recapitulates EBV infection and pathogenesis in rhesus macaques, offering a promising EBV surrogate model. In this study, we engineered a stable recombinant rhLCV expressing enhanced green fluorescent protein (eGFP), rhLCV.eGFP, for easy identification of infected or transformed cells in vitro and ex vivo. Further illustrating the utility of this platform, we also generated a gp350-deficient mutant rhLCV.eGFP and demonstrated markedly reduced infectivity in otherwise susceptible B and epithelial cells, confirming the EBV gp350-homologous role of rhLCV gp350 in viral entry. Thus, rhLCV.eGFP will serve as an invaluable resource for studying EBV biology and advancing the development of EBV prophylactic and therapeutic strategies using the rhLCV surrogate model.