Epstein-Barr virus (EBV) in infectious mononucleosis: detection of the virus in tonsillar B lymphocytes but not in desquamated oropharyngeal epithelial cells

Abstract

Aims: Despite its well established tropism for B cells, the nature of the cellular compartment(s) mediating primary and persistent Epstein-Barr virus (EBV) infection is still a matter of controversy. In view of the association of EBV with several lymphoid and epithelial malignancies, resolution of this issue is important.

Methods: Desquamated oropharyngeal epithelial cells from 10 patients with acute infectious mononucleosis and from seven chronic virus carriers were studied for evidence of EBV infection using in situ hybridisation for the detection of the small EBV encoded RNAs (EBERs) and of the viral genome. In addition, immunocytochemistry was used to detect the BZLF1 transactivator protein of EBV.

Results: There was no evidence of latent or replicative EBV infection in oropharyngeal epithelial cells in any of the samples. In contrast, EBV infected B cells were readily identified in a tonsil from a patient with infectious mononucleosis.

Conclusions: The results suggest that oropharyngeal epithelial cells are not a major site of EBV infection and provide further support for the notion that B cells mediate primary and persistent EBV infection.

Figure 1

Detection of Epstein-Barr virus (EBV) infected cells in a tonsil from a patient with infectious mononucleosis. (A) Double labelling using radioactive in situ hybridisation (black silver grains) and immunohistochemistry (red staining) reveals numerous EBV encoded small RNA (EBER) positive/CD20 positive B cells. (B) These cells are often admixed with crypt epithelial cells but appear to be cytokeratin negative. (C) Production of the BZLF1 protein (blue staining) of EBV, indicating a switch from latent to replicative infection is seen in CD79a positive (red staining) B cells morphologically resembling plasma cells, but not in cytokeratin positive (red staining) epithelial cells (D).

Figure 2

Validation of cytoblock techniques. (A) In situ hybridisation with ³⁵S labelled antisense probes reveals expression of Epstein-Barr virus (EBV) encoded small RNAs (EBERs) in most B95.8 cells. (B) A small proportion of B95.8 cells is also labelled with the sense control probes, indicating hybridisation to replicating viral DNA (see text). (C) In situ hybridisation with a probe specific for the BamHI W fragment of the EBV genome results in the labelling of a proportion of B95.8 cells, whereas the EBV negative Ramos cells (D) are unlabelled. (E) In situ hybridisation with a ³⁵S labelled U6 specific RNA probe results in the accumulation of silver grains over scattered small inflammatory cells, whereas nuclei of epithelial cells are negative. (F) In situ hybridisation with a digoxigenin labelled Cot1 specific DNA probe results in red nuclear staining of most epithelial and inflammatory cells. (G) EBER specific in situ hybridisation with ³⁵S labelled RNA probes shows no evidence of latent EBV infection in desquamated oropharyngeal epithelial or inflammatory cells from a patient with acute infectious mononucleosis. (H) Using a ³⁵S labelled probe specific for the BamHI W fragment of the EBV genome, viral DNA is not detected in desquamated oropharyngeal cells.