Detection of Epstein-Barr Virus in Periodontitis: A Review of Methodological Approaches

Abstract

Periodontitis, an inflammatory condition that affects the structures surrounding the tooth eventually leading to tooth loss, is one of the two biggest threats to oral health. Beyond oral health, it is associated with systemic diseases and even with cancer risk. Obviously, periodontitis represents a major global health problem with significant social and economic impact. Recently, a new paradigm was proposed in the etiopathogenesis of periodontitis involving a herpesviral-bacterial combination to promote long-term chronic inflammatory disease. Periodontitis as a risk factor for other systemic diseases can also be better explained based on viral-bacterial etiology. Significant efforts have brought numerous advances in revealing the links between periodontitis and Epstein-Barr virus (EBV), a gamma herpesvirus ubiquitous in the adult human population. The strong evidence from these studies may contribute to the advancement of periodontitis research and the ultimate control of the disease. Advancing the periodontitis research will require implementing suitable methods to establish EBV involvement in periodontitis. This review evaluates and summarizes the existing methods that allow the detection and diagnosis of EBV in periodontitis (also applicable in a more general way to other EBV-related diseases), and discusses the feasibility of the application of innovative emerging technologies.

Keywords: EBER-ISH; Epstein–Barr virus; PCR-based methods; detection methods; immunohistochemistry; immunophenotyping; periodontitis.

Figure 1

Outline of the methodological approaches for Epstein–Barr virus (EBV) detection in periodontitis. Actual and prospective methods are listed. Abbreviations: CIHC, chromogenic immunohistochemistry; EBER—EBV-encoded RNA; FACS—fluorescence-activated cell sorting; FCM—flow cytometry; FRET—fluorescence resonance energy transfer; IF—immunofluorescent detection; IHC—immunohistochemistry; ISH—in situ hybridization; LAMP—loop-mediated isothermal amplification; MACS—magnetic-activated cell sorting; PCR—polymerase chain reaction; qPCR—real-time quantitative PCR; RT-qPCR—reverse transcription qPCR.

Figure 2

Immunofluorescent (IF) costaining of CK19 (junctional epithelial cell marker cytokeratin 19) and LMP2 (EBV latent membrane protein 2) to detect EBV-infected epithelial cells in samples taken from a periodontitis patient. The cell nuclei are counterstained with DAPI. Reprinted from [27].

Figure 3

(a) EBV-encoded RNA in situ hybridization (EBER-ISH) of periodontal epithelial cells (pECs) and (b) chromogenic immunohistochemistry (CIHC) for CK19 (cytokeratin 19; junctional epithelial cell marker) immunostaining of periodontitis samples to reveal EBV-infected ECs. The size bar represents 15 µm. (c) EBER-ISH coupled with CIHC of CCL20 (chemokine ligand 20) to show the production of the inflammatory chemokine CCL20 by EBV-infected pECs in periodontitis patients. EBV-infected (solid arrows) and EBV^- pECs (dotted arrows) are presented. Reprinted from [27].

Figure 4

Multiplex chromogenic immunohistochemistry (CIHC) for the detection of T cells (CD3 (cluster of differentiation) marker), B cells (CD20), plasma cells (PCs; CD138) and antibody-producing cells (cytoplasmic kappa light chain) coupled with EBV-encoded RNA in situ hybridization (EBER-ISH) in the gingival tissue specimens of periodontitis patients. Panel (a) shows hematoxylin and eosin (H&E) overlapping between CD3, CD20, CD138, EBER and Kappa stainings on a specimen from a periodontitis patient (magnification ×4). Panel (b) illustrates a cluster of EBV-infected PCs showing colocalization between EBER and CD138 (magnification ×100). Panel (c) is indicative of colocalization between EBV-infected cells (EBER), PCs (CD138), B cells (CD20) and T cells (CD3) (magnification ×10). Reprinted from [62] with permission from Elsevier.