• O1: A total of 54 participants were recruited including 27 healthy (H) volunteers & 27 patients with active periodontitis (PA). To test for the presence of EBV, the EBV R-gene kit was used to detect EBV DNA in the gingival tissues of participants. Following the EBV detection, all the 54 samples were categorized as EBV- or EBV+. EBV was prevalent in 75.9% of the total. Meanwhile, EBV was detected in PA at a higher frequency than in H (81.5% vs 70.4%). The high prevalence of EBV in both H & PA tissues may suggest that EBV spreading is common in gingival tissues even in the absence of inflammatory lesions. To establish the relationship between EBV infection & dysbiosis, several pathogenic bacterial species were detected in EBV- & EBV+ gingival tissues using RT-qPCR. The results were inconclusive to delineate the association between EBV & pathogenic bacteria & further experiments will be required.
• O2: Microarray transcriptional profiling was used to provide a comprehensive description of differential gene expression signatures & biological processes in H & PA-affected tissues that were modulated by EBV infection. 3 scenarios were observed: comparison of the states of H & PA disease, EBV effect in H & PA-affected tissues. In summary, in periodontally-diseased tissues, the host bioprocesses linked to excessive immune/inflammatory responses & tissue degradation were documented. While in the presence of EBV in periodontally-diseased tissues these processes were affected dramatically as EBV augmented the deleterious state of the disease by worsening the inflammatory condition, promoting the B (BC) & plasma (PC) cell infiltration into the lesion & advancing the epithelial tissue degradation. In contrast, in H tissues EBV provoked typical host immune responses against the viral challenge. Overall, this study provides the first evidence to support EBV as a significant pathogenic contributor to periodontitis. These results were presented at the EMBO Workshop as a poster presentation. Also in preparation for submission to the JCI.
• O3: In vitro model was established for BC sequential differentiation into PC as a multi-step 11-day culturing process. At the same time, this BC-to-PC transition was accompanied by the infection of BCs with EBV. Generated various cell populations were further analyzed for the gene expression regarding BC-to-PC development markers, EBV infection & cytokine profile using RT-qPCR. For determination of BC phenotype, the expression of PAX5 & CD21 were examined showing a progressive decrease from D1 to D11 in non-infected and infected cultures. For PC profile, the expression of BLIMP1, CD38 & CD138 were studied. The CD38 was increased from D1 to D11 in non-infected & infected cultures. The fate of BLIMP1 & CD138 were ambiguous in our experiments. As the EBV infection indicator, the expression of 2 latent & 3 lytic genes were monitored. In non-infected cultures there was no expression of all EBV genes, while in infected cultures from iD4 to iD11 the expression of 5 EBV genes dropped down progressively. A broad literature review is required to interpret these observations. For cytokine profile, IL1β, IL6, IL8 & TNFα were monitored. The IL1β increased from D1 to D11 in non-infected & infected cultures, IL6 & TNFα were the highest in D1, while IL8 was similarly high & stayed stable in all the non-infected & infected cultures. For the understanding of cytokine profile, a broad literature search is also needed following which a possible research article is feasible.
• O4 was not achieved because of the complexity & lack of time related to COVID19 restrictions. Instead, an attempt was made to investigate EBV infection of junctional epithelial cells (JECs) isolated from periodontal tissues. JECs & EBV infection of JECs were detected by immunofluorescent staining of cytokeratin 19 & EBNA1, respectively. In addition, EBV gene expression profiles were determined by RT-qPCR for EBNA1, LMP2 & BZLF1. The micrographs from non-infected cells showed no fluorescence signal from EBNA1, while infected JECs were positive for EBNA1. Regarding the EBV gene expression in JECs, there was no expression of EBV genes in non-infected JECs, while all EBV transcripts were detected in EBV-infected JECs in the following decreasing order: LMP1>BZLF1>EBNA1. These promising results that JECs may be a possible target of EBV infection could be an important section of an imminent publication.
