Evidence for lytic infection by Epstein-Barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals

Abstract

Normal nasopharyngeal tissues from 23 individuals who died of causes unrelated to the upper respiratory system and had no evidence of Epstein-Barr virus (EBV)-related diseases were studied using in situ hybridisation (ISH) and immunohistochemistry for the detection of EBV RNA and expression of EBV proteins, respectively. ISH using 35S-labelled riboprobe for EBV EBER RNA showed occasional to a few EBER+ lymphocytes in the stroma of nasopharyngeal mucosa in 14/16 cases with available paraffin-embedded tissues. In addition, very rare intraepithelial EBER+ lymphocytes were also detected in 3/16 cases. However, in none of these cases was EBER detected in the epithelial cells. Similar results were obtained using a nonradioactive ISH method for EBER (Dako). In 3/23 cases, immunostaining using monoclonal antibodies for EBV proteins on cryostat sections showed occasional cells in the stroma expressing EBV nuclear antigen 2 (EBNA2), latent membrane protein-1 (LMP), and switch protein encoded by BZLF1 gene (ZEBRA) in two cases and only very rare LMP+ and ZEBRA+ cells in one other case. Double immunostaining combining alkaline phosphatase anti-alkaline phosphatase (APAAP) for CD markers and indirect immunofluorescence for LMP showed that the LMP+ cells were either CD19+ or less frequently CD3+, but none were CD68+. These results show that both B and T lymphocytes harbouring EBV can be found in the normal nasopharyngeal tissues. Interestingly, EBV proteins associated with lytic viral replication - diffuse early antigen (EA-D), viral capsid antigen (VCA), or membrane antigen (MA) - were also detected in rare cells in the stroma in one case, and in another case only one MA+ cell was detected. These results provide evidence for a lytic type of infection by EBV in the normal nasopharynx involving a very small proportion of stromal lymphocytes and support the view that nasopharyngeal lymphocytes can act as a reservoir for EBV in normal individuals.