Abstract
Lifting adherent cells to the air-liquid interface (ALI) is a method conventionally used to culture airway epithelial cells into polarized apical and basolateral surfaces. Reactivation of Epstein-Barr virus (EBV) from monolayer epithelial cultures is sometimes abortive, which may be attributed to the lack of authentic reactivation triggers that occur in stratified epithelium in vivo In the present work, the ALI culture method was applied to study EBV reactivation in nasopharyngeal epithelial cells. The ALI culture of an EBV-infected cell line yielded high titers and can be dissected by a variety of molecular virology assays that measure induction of the EBV lytic cascade and EBV genome replication and assembly. EBV infection of polarized cultures of primary epithelial cells can be challenging and can have variable efficiencies. However, the use of the ALI method with established EBV-infected cell lines offers a readily available and reproducible approach for the study of EBV permissive replication in polarized epithelia.
Highlights
Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes a latent reservoir in peripheral B-lymphocytes with sporadic reactivation
The organotypic raft culture model established for studies in human papillomavirus (HPV) replication was recently applied to trigger EBV reactivation, resulting in the efficient production of infectious progeny virus that spreads in stratified primary keratinocytes [20]
The results of this study demonstrate that the air-liquid interface (ALI) method is proficient at reactivating EBV from the established HK1-EBV cell line, yielding high titers (~106 packaged genome equivalents per ALI culture [1.12 cm2]) that are secreted and infectious, providing an alternative method to interrogate EBV permissive replication from polarized epithelia in an established nasopharyngeal carcinoma (NPC)-derived cell line and allowing the elucidation of EBV pathogenesis in nasal epithelia
Summary
Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes a latent reservoir in peripheral B-lymphocytes with sporadic reactivation. The organotypic raft culture model established for studies in human papillomavirus (HPV) replication was recently applied to trigger EBV reactivation, resulting in the efficient production of infectious progeny virus that spreads in stratified primary keratinocytes [20].
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