Oral Epstein-Barr Virus Research Articles

Evidence linking the association of periodontal viruses and pentraxins in periodontitis patients with coronary artery disease.

Although there is ample evidence in the literature supporting a significant positive association between key periodontal pathogens and established inflammatory markers of periodontitis and coronary artery disease (CAD), their exact role remain unclear. Especially, the role of viruses in the etiology and specific biomarkers have not been validated. Thus, the current study aims to evaluate the role of periodontal viruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), and herpes simplex virus (HSV), as well as the inflammatory marker pentraxin-3 (PTX3), and to analyze their association with CAD. The study included 240 patients divided into four groups of 60 patients each: nonperiodontitis + noncardiac (NP+NC) group, periodontitis + noncardiac patients (P+NC) group, nonperiodontitis + cardiac patients (NP+C) group, and periodontitis + cardiac (P+C) group. The cardiac surgery group (C-S) was a subgroup of NP+C and P+C. It consisted of 60 patients from the abovementioned two cardiac groups in whom coronary artery bypass graft (CABG) was indicated. Demographic variables, cardiac parameters, and periodontal parameters were recorded. The viruses (EBV, CMV, and HSV) and the inflammatory marker PTX3 were evaluated in the subgingival plaque samples of all the four groups and atheromatous plaque samples of the C-S using reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (qPCR), respectively, and were compared between the groups. The results were obtained and statistically analyzed. The demographic variables did not differ significantly between the groups, except for age. Systolic blood pressure, diastolic blood pressure, low-density lipoprotein, and random blood sugar were significantly higher in NP+C and P+C, whereas high-density lipoprotein was significantly lower (p≤0.05) in the same. Plaque index (PI), probing pocket depth (PPD), and clinical attachment level (CAL) were significantly higher (p ≤ 0.05) in P+NC and P+C. PTX were significantly elevated in P+C among the four groups. On evaluating the subgingival plaque samples, EBV and CMV were significantly higher in the two periodontitis groups P+NC and P+C (p=0.000). HSV was significantly higher in the two cardiac groups (NP+C and P+C) (p ≤ 0.05). Cardiac EBV and CMV were significantly elevated in the P+C group with a p value of 0.004 and 0.033, respectively. Cardiac HSV was found in the NP+C group with statistical insignificance (p=0.410) between the groups. On correlation, oral PTX were significantly associated with bleeding index (BI), PPD, and CAL (p=0.000). Similarly, cardiac PTX showed significant association with PI, BI, PPD, and CAL (p=0.000). Oral and cardiac PTX also showed significant correlation with each other. Multiple logistic regression analysis revealed a significant association between CAL and oral EBV (p ≤ 0.05). Similarly, cardiac EBV showed a significant association with CAL and oral EBV (p ≤ 0.05). Multiple logistic regression analysis also revealed that both cardiac and oral PTX showed a significant association only with oral EBV, CMV, and HSV. The results of the current study suggest that the clinical severity of periodontitis (CAL), etiology of periodontitis (EBV and CMV), and inflammatory marker of periodontitis (PTX3) were found to be significantly elevated in CAD. These findings suggests that periodontal diseases may be a risk factor that could influence the progression of CAD.

Prevalence of oral Epstein-Barr virus and Porphyromonas gingivalis and their association with periodontal inflamed surface area: A cross-sectional study.

We previously reported that oral herpesviruses, such as Epstein-Barr virus (EBV), are associated with periodontitis. However, the relationship between oral EBV or dual oral EBV and Porphyromonas gingivalis infections and periodontal inflammation severity remains unclear. We conducted this study to determine the relationship between oral EBV and P gingivalis prevalence and the periodontal inflamed surface area (PISA) in middle-aged and older adults. We analyzed 205 patients (median age, 70 years) who visited Hiroshima University Hospital. Tongue swab samples were used to investigate the presence of EBV and P gingivalis DNA using real-time PCR. Probing pocket depth and bleeding on probing were measured at 6 sites per tooth. PISA scores were calculated based on the results of probing pocket depth and bleeding on probing. Propensity scores were calculated via logistic regression analysis of 8 clinical factors: age, sex, smoking status, remaining teeth, denture use, hypertension, diabetes, and hyperlipidemia. EBV DNA was present in 41 of the 205 participants (20.0%). Thirty-seven EBV-positive or -negative participants in 74 matched pairs after propensity-score matching were examined via univariate analysis. EBV-positive participants exhibited higher plaque control record scores and PISAs than did EBV-negative participants. EBV DNA was significantly associated with plaque control record scores and PISA (both P = .04). Of the 205 participants, 111 were positive for P gingivalis (54.1%). Nineteen participants (9.3%) were infected with both oral EBV and P gingivalis. Logistic regression analysis revealed that dual infection with EBV and P gingivalis was significantly associated with diabetes (odds ratio = 3.37, 95% confidence interval: 1.13-10.1; P = .03). Oral EBV prevalence is associated with oral hygiene and the spread of inflamed periodontal tissue. Diabetes may be a risk factor for dual infection with oral EBV and P gingivalis.

Abstract 2264: Co-infection with Epstein-Barr virus impacts oral HPV persistence

Abstract Rates of oropharyngeal cancer (OPC) associated with human papillomavirus (HPV) infection are increasing in men. The obligate precursor to OPC is a persistent infection with an oncogenic HPV type that is unable to be cleared via natural immunity alone. However, the factors that influence HPV persistence at the oral cavity are unknown. It is plausible that long-term infection with other pathogens at the same anatomic site indicates an overall inability to clear an oral infection. Epstein-Barr virus (EBV) is a saliva-transmitted gamma herpesvirus that infects 90% of the population by adulthood and replicates in the epithelial cells of the oropharynx. This study aimed to investigate whether oral EBV shedding, a marker of active viral replication, could serve as a biomarker for persistent, oncogenic oral HPV infection in men. Men with an HPV16 or 18 oral infection present at the baseline visit of the HPV Infection in Men (HIM) Study were included (n=63). Briefly, men from Tampa, FL, U.S.; Cuernavaca, Mexico; and Sao Paolo, Brazil were followed every 6 months for a median of 4.1 years. Men completed a risk factor questionnaire and provided an oral gargle sample at each visit. For this study, the baseline, 6-month, and 12-month oral specimens were HPV genotyped using the SPF10 PCR-DEIA-LiPA25 system. Presence of EBV was evaluated in the baseline specimen using PCR to amplify and quantify the EBV DNA on the Rotor-Gene Q MDx instrument. Men with the same oral HPV type present at the 6-month and 12-month visit were categorized as 6-month and 12-month oral HPV persistent, respectively. The association between EBV and persistent HPV infection was investigated using a logistic regression model adjusted for age, country, and smoking. Among 63 men with oral HPV 16/18 infection at baseline, 44 HPV infections did not persist, 19 persisted to at least 6-months and 11 persisted to at least 12 months. Among the total sample, 32 also had EBV detected in the oral gargle at baseline. Among men who did not have a persistent HPV infection, 21 (47.7%) had EBV detected at baseline while 11 (57.9%) men with an HPV infection that persisted at least 6 months had detectable EBV at baseline; and 9 (81.8%) of the men who persisted to 12 months had EBV detected. Mean EBV viral load was highest among men who persisted to at least 12 months (5.0 log copies/ml) and was significantly higher than men who did not persist (2.5 log copies/ml; p=0.047). Detection of oral EBV at baseline was significantly associated with elevated risk of ≥12-month oral HPV 16/18 persistence (aOR: 9.90; 95% CI: 1.38-70.96), but not ≥6-month HPV 16/18 persistence (aOR: 1.62; 95% CI: 0.52-5.06).These data support the hypothesis that EBV reactivation may be a biomarker of poor immune control leading to increased risk of oral HPV persistence. The use of a biomarker that identifies men whose HPV infection may persist would be a useful tool to allow for screening and early detection of OPC. Citation Format: Brittney L. Dickey, Anna R. Giuliano, Bradley Sirak, Martha Abrahamsen, Eduardo Lazcano-Ponce, Luisa L. Villa, Anna E. Coghill. Co-infection with Epstein-Barr virus impacts oral HPV persistence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2264.

Control of Epstein-Barr Virus (EBV) in the Oral Cavity Is Associated With Persistence of Oral Human Papillomavirus (HPV)16/18 Among Men From the HPV Infection in Men Study.

Human papillomavirus (HPV)-related oropharyngeal cancer (OPC) incidence is increasing among men. Biomarkers that can identify oral HPV16/18 infections likely to persist, the obligate precursor for HPV-OPC, are needed. We assessed the association between oral Epstein-Barr virus (EBV) and oral HPV16/18 persistence among 63 men in the HPV Infection in Men Study who tested positive for HPV16/18 at the baseline visit. Control of oral coinfections, including EBV, could serve as a biomarker of immune competence and the ability to control oral HPV. Detection of oral EBV was significantly associated with oral HPV16/18 ≥12-month persistence. Detection of oral EBV deserves evaluation as a biomarker for oral HPV persistence and HPV-related OPC.

Prognostic Value of Oral Epstein-Barr Virus DNA Load in Locoregionally Advanced Nasopharyngeal Carcinoma.

Background: Plasma Epstein–Barr virus (EBV) DNA load has been widely used for nasopharyngeal carcinoma (NPC) prognostic risk stratification. However, oral EBV DNA load, a non-invasive biomarker that reflects the EBV lytic replication activity, has not been evaluated for its prognostic value in NPC yet. Methods: A total number of 1,194 locoregionally advanced NPC (LA-NPC) patients from south China were included from a prospective observational cohort (GARTC) with a median follow-up of 107.3 months. Pretreatment or mid-treatment mouthwashes were collected for EBV DNA detection by quantitative polymerase chain reaction (qPCR). The difference of pre- and mid-treatment oral EBV DNA load was tested by the Wilcoxon signed-rank test. The associations of oral EBV DNA load with overall survival (OS), progression-free survival (PFS), distant metastasis–free survival (DMFS), and locoregional relapse-free survival (LRFS) were assessed using the log-rank test and multivariate Cox regression. Results: The high level of the oral EBV DNA load (>2,100 copies/mL) was independently associated with worse OS (HR = 1.45, 95% CI: 1.20–1.74, p < 0.001), PFS (HR = 1.38, 95% CI: 1.16–1.65, p < 0.001), DMFS (HR = 1.66, 95% CI: 1.25–2.21, p = 0.001), and LRFS (HR = 1.43, 95% CI: 1.05–1.96, p = 0.023). Similar and robust associations between oral EBV DNA load and prognosis were observed for patients in both the pretreatment and mid-treatment stages. The detection rate (71.7 vs. 48.6%, p < 0.001) and the median load of oral EBV DNA (13,368 vs. 382 copies/mL, p < 0.001) for patients in the pretreatment stage were significantly higher than those in the mid-treatment stage. The combination of the oral EBV DNA load and TNM staging provided a more precise risk stratification for the LA-NPC patients. Conclusion: Oral EBV DNA load was an alternative non-invasive predictor of prognosis and may facilitate risk stratification for the LA-NPC patients.

Association of oral Epstein-Barr virus with periodontal health in Japanese adults.

Previous studies have demonstrated that oral Epstein-Barr virus (EBV) is associated with periodontitis. However, the relationship between periodontitis and oral EBV has not been fully elucidated by reducing the effects of confounding factors. The aim of the present study was to clarify the association between oral Epstein-Barr virus (EBV) and oral health status among middle-aged and older Japanese individuals. A total of 124 patients (46 males and 78 females; mean age, 69.2 years; age range, 35-90 years) who visited Hiroshima University Hospital between October 2018 and December 2019 were recruited into the present study. EBV DNA positivity was determined in 124 oral rinse samples using quantitative PCR. Periodontal disease-related bacteria were also detected by PCR analysis. EBV DNA was determined as positive in 16 of the 124 enrolled patients (12.9%). No significant difference was identified between EBV DNA and clinical factors (sex, age, remaining teeth, denture use, smoking or medical history). Of the 38 patients with periodontal pockets ≥6 mm, 10 were EBV DNA positive (26.3%). There was a significant association between EBV DNA positivity and probing depth (P=0.01). Additionally, a significant association was identified between bleeding on probing (BOP) and EBV DNA positivity (P=0.03). To investigate the relationship between EBV and periodontal health status, propensity score-matching was determined between participants without ≥4 mm periodontal pockets and BOP (participants with good periodontal health) and those with ≥4 mm periodontal pockets, BOP or both (participants with poor periodontal health). A total of 35 matched pairs were identified among the patients. Patients with poor periodontal health exhibited a higher EBV DNA positivity rate (25.7%) than those with good periodontal health (0.0%). Additionally, there was a significant association between EBV DNA positivity and periodontal health status (P=0.001). T. denticola-positive participants exhibited a higher EBV DNA positivity rate than negative participants (17.6 vs. 9.6%). However, there was no significant difference. The results indicated that oral EBV may be markedly associated with periodontitis in middle-aged and older Japanese individuals.

Epstein-Barr virus-infected plasma cells in periodontitis lesions

Growing evidence supports that the Epstein-Barr virus (EBV) is a putative periodontal pathogen, but little is known regarding EBV behavior in periodontitis. Here, EBV infection was monitored in saliva and periodontal pocket (PP), at baseline and 3 months after periodontal non-surgical therapy (p-NST) in 20 patients diagnosed with periodontitis. After the treatment, the patients with the improved periodontal condition (good responders) showed a significant decrease in salivary EBV load. In contrast, in poor responders, EBV load was slightly increased. Moreover, after the therapy, most patients showed clear signs of EBV infection in a deep PP (≥5 mm) selected as a study site. To investigate how EBV can persist in a PP, we further investigate cellular sites of viral replication in PP. We identified large amounts of infiltrated EBV-infected cells, mostly overlapping with CD138+ plasma cells (PC). EBV-infected PCs formed high-density clusters within the infiltrate and along the periodontal epithelium which were commonly associated with CD3+ T-cells and CD20+ B-cells to evoke diffuse ectopic lymphoid-like structures. Taking together, this study provides new insights to support a model where the periodontal condition may play a major role in oral EBV shedding. Since PC harbors the late productive phases of EBV replication, the periodontal condition may favor B-cell differentiation with possible amplification of periodontal EBV infection and viral spreading. PCs have long been recognized as pathogenic markers in inflammatory lesions. Our finding sheds new light on the role of EBV infection and PC in periodontitis.

Associations between environmental factors and serological Epstein‐Barr virus antibodies in patients with nasopharyngeal carcinoma in South China

Epstein‐Barr virus (EBV) reactivation, reflected by aberrantly increased levels of various serological antibodies, has been suggested to be an early indicator of nasopharyngeal carcinoma (NPC) onset and progression. We have previously suggested that certain lifestyle and dietary factors were associated with elevated serological levels of the antibody against various EBV antigens namely VCA, Zta, EBNA1, and oral EBV DNA loads among healthy population. It remains unclear whether these potential environmental factors would also influence EBV serological antibodies in NPC patients. We conducted an epidemiological study to evaluate the associations between such environmental factors and EBV antibody levels among 1701 NPC patients in South China. Pretreatment serums were collected and examined for VCA‐IgA and EA‐IgA by immunoenzymatic assays and antienzyme rate (AER) of EBV DNase‐specific neutralizing antibody. We found that consumption of Canton‐style herbal tea was significantly correlated with increased serological antibody levels of VCA‐IgA and EA‐IgA, with adjusted ORs of 1.35 (95% CI: 1.03‐1.76) and 1.32 (95% CI: 1.01‐1.73), respectively, in the weekly intake frequency stratum, while not related to AER of EBV DNase‐specific neutralizing antibody. Smoking was found to be not only an apparent risk factor for higher antibody levels of AER in stage III‐IV patients (OR = 1.60, 95% CI: 1.11‐2.30), but also associated closely with NPC stage at diagnosis (OR = 2.17, 95% CI: 1.47‐3.22), with dose‐response effects. In conclusion, we found consumption of Canton‐style herbal tea and cigarette smoking were in positive associations with elevated EBV antibodies in NPC patients, which may be of public health significance for the primary prevention of EBV‐associated diseases especially NPC.

Association Between Environmental Factors and Oral Epstein-Barr Virus DNA Loads: A Multicenter Cross-sectional Study in China.

Oral Epstein-Barr virus (EBV) status reflects host EBV activity and potentially links to EBV-associated diseases, however, factors influencing oral EBV loads or reactivation, such as environmental exposures or host factors, are not fully understood. A 2-stage, multicenter, cross-sectional study of 6558 subjects from 21 administrative cities of southern China and 3 populations from representative geographical areas in China (referred to as the south, north, and northeastern populations) was performed. The relationships between demographical factors and environmental exposures to EBV loads were analyzed by logistic regression models. Current smoking, with a dose-response effect, was found to be strongly associated with higher oral EBV loads in the pooled data, with an odds ratio of 1.58 (95% confidence interval, 1.39-1.79), as well as in each of the separate populations. The odds ratio increased to 3.06 when current smokers in southern China were compared to never smokers in northern China. Additionally, higher oral EBV loads tended to be detected in older participants, male participants, and participants in southern China. This study provided evidence linking the effect of host-environmental factors, particularly smoking, to oral EBV activity. It could strengthen our understanding of the possible causal roles of EBV-related diseases, which may help to prevent or mitigate EBV-associated diseases.

Pediatric oral Epstein-Barr virus associated self-remitting CD30+ lymphoproliferative disorder: A distinct entity

Epstein-Barr virus (EBV) has a well-known association with lymphoproliferative disorders of B and T cell origin. EBV-related B cell lymphoproliferative disorders include Hodgkin and Burkitt lymphomas, lymphomatoid granulomatosis, EBV positive diffuse large cell B cell lymphoma of the elderly, as well as B cell lymphomas associated with solid organ transplantation and methotrexate use. EBV-related T cell disorders are primarily represented by NK/T- cell lymphoma. In a subset of patients, EBV has been implicated in CD30 positive B cell lymphoproliferative disorders of the oral mucosa falling under the rubric of the mucocutaneous ulcer of the oral cavity. We previously reported on an index series of endogenous CD30 positive T cell lymphoproliferative disorder of the oral cavity resembling borderline type C lymphomatoid papulosis. The clinical manifestation of type C oral lymphomatoid papulosis is that of a recurrent self-remitting ulcer of the oral mucosa, which histologically resembles anaplastic large cell lymphoma. Such cases can be misdiagnosed as aggressive lymphoma leading to unnecessary treatment with aggressive chemotherapeutic regimens. Whereas none of the patients in our index series exhibited EBV positivity, here we discuss a very unique example of a 14-year-old girl diagnosed with EBV positive CD30 positive lymphoproliferative disorder strongly resembling the cases of intra-oral type C lymphomatoid papulosis. The patient was initially diagnosed by a senior hematopathology consultant as having EBV positive aggressive NK/T-cell lymphoma. The significance of raising physician awareness regarding pediatric oral EBV associated CD30 positive lymphoproliferative disease of the oral cavity lies in preventing inadvertent exposure to toxic chemotherapeutic agents intended for treatment of aggressive look-alikes, namely anaplastic large cell lymphoma. Additionally, we include a literature review of similar reports of pediatric intra-oral EBV positive CD30 positive T cell lymphoproliferative disease.

Decreased oral Epstein-Barr virus DNA loads in patients with nasopharyngeal carcinoma in Southern China: A case-control and a family-based study.

The link of nasopharyngeal carcinoma (NPC) with Epstein‐Barr virus (EBV) has been established for decades. Although an abnormal high level of EBV sero‐antibody spectrum and cell‐free circulating EBV DNA loads were exhibited in NPC patients, oral EBV DNA loads, which are primarily responsible for the EBV transmission, has not been previously studied in NPC patients. We conducted an epidemiological study to measure the oral EBV loads, viral components, and the relationship with the serum antibody titers in a large case‐control population (968 cases and 1656 controls) and a family‐based population (91 cases and 165 unaffected family members). EBV DNA loads were detected by quantitative PCR approach targeting the BamHI‐W region. Although a large individualized variation existed, we still observed a decreased oral EBV DNA loads in the population of NPC patients compared to that of healthy controls (ORs were 1.00, 0.69, 0.62, 0.33 classified by the quartiles of viral loads, P trend < .001) and family members. In contrast, the elevated levels of oral EBV loads were present in asymptomatic males and elders, suggesting a different important source for EBV transmission. Notably, oral EBV loads were inversely associated with serum antibody titers of VCA‐IgA, EA‐IgA (All P trend < .001) in the cases but not in the controls. Our study provides the first epidemiological data of oral EBV loads and viral components in NPC patients and controls in the highest risk area of Southern China, indicating that NPC status is unlikely to be an important determinant of EBV transmission.

Epstein-Barr Virus DNA in Parental Oral Secretions: A Potential Source of Infection for Their Young Children.

A potential source of primary Epstein-Barr virus (EBV) infection for young children is parental oral secretions. If parents who identify with racial/ethnic categories other than white have a higher prevalence of oral EBV DNA, this difference could explain why their children acquire primary EBV infection at an earlier age than white children. To test this hypothesis, we recruited parents who brought their children <8 years old to routine clinic visits, and tested the parents' oral washes for EBV DNA by real-time polymerase chain reaction. Positive samples were assayed for encapsidated EBV DNA, which is potentially infectious, versus naked EBV DNA, which is not infectious. Overall, 221/800 parents (28%) had EBV DNA in their oral washes. Oral EBV DNA was more prevalent in parents who identified as non-white as compared with white parents (P = .0004), and was more prevalent in male vs female parents (P = .04). The mean quantity of EBV DNA in positive samples was 5000 copies/mL. Encapsidated viral DNA comprised 40.3% of the total EBV DNA found in parental oral secretions. Our data support the hypothesis that parents could be a source of virus for their young children, because 28% of parents had a mean of 5000 EBV copies/mL of oral wash and 40.3% of the EBV DNA was encapsidated. The higher prevalence of EBV DNA in non-white parents could explain why their children acquire EBV at an earlier age than children of white parents.

Natural Variations in BRLF1 Promoter Contribute to the Elevated Reactivation Level of Epstein-Barr Virus in Endemic Areas of Nasopharyngeal Carcinoma

Background: Epstein-Barr virus (EBV) infection is a crucial risk factor for nasopharyngeal carcinoma (NPC), but the mechanism for its elevated activation level in NPC endemic areas remains unclear. This study aims to identify the EBV natural variations contributed to the different reactivation potential between NPC endemic and non-endemic areas. Methods: 1030 subjects were recruited in China, including 303 healthy individuals from two NPC non-endemic areas, 483 healthy people from three endemic areas and 244 NPC patients. Among which, saliva DNA samples from 244 participants were sequenced for the EBV immediate early (IE) genes of BRLF1 and BZLF1, their promoters were included; the rest 786 subjects were used for the validation of significant variations among three different populations. Haplotype and population structure analysis were conducted. Dual-luciferase assay was used to detect the promoter activity. Results: A total of 246 distinct variations were detected, 29 showed significant difference in the frequencies between healthy people from NPC endemic area and non-endemic area. Population structure analysis clustered EBV strains into 9 subgroups mostly in accordance with the geographical origin of samples. Interestingly, two EBV genotypes, Rp-V1 and Rp-V2, were identified according to the linkage relationship of the variations in BRLF1 promoter (Rp). Rp-V1 has higher frequency in NPC endemic areas than in non-endemic areas (52.38% vs 18.15%, P=2.07×10-14), and was associated with higher oral EBV DNA levels (adjusted OR=1.64, 95% CI=1.21-2.24, P=0.002), suggesting a more powerful activation ability of Rp-V1 than that of the wild type Rp-WT of the EBV strain; On the contrary, Rp-V2 has higher frequency in NPC non-endemic areas than in endemic areas (18.48% vs 0.38%, P=1.17×10-7), might represent a reduced activation potential of EBV. Further dual-luciferase assay showed Rp-V1 has higher promoter activity while compared with Rp-V2 (P<0.0001). Notably, Rp-V1 impaired the transcription repression effect of YY1 while Rp-V2 strengthened the transcription repression effect of EBF1 on Rp. In addition, significant differences of Rta 393-407 CTL epitope which may influence the recognition of Rta by CD8 T cells were detected between healthy people from NPC endemic area and non-endemic area. Conclusions: This study identified natural variations in cis-acting elements (YY1 and EBF1) of EBV Rp altering Rp transcription activities, which may contribute to the elevated EBV activation level in NPC endemic areas than non-endemic areas. Funding Statement: This work was supported by the National Key Research and Development Program of China [grant number: 2016YFC1302704], the Sino-Sweden joint research program [grant number: 81861138006], the Science and Technology Planning Project of Guangzhou City, China [grant number: 201804020094] and the National Natural Science Foundation of China [grant number: 81502056, 81325018 and 81220108022]. The funder had no role in the study design, participant recruitment, data collection, data analysis, data interpretation, or writing of the report. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was reviewed and approved by the Human Ethics Approval Committee at Sun Yat-sen University Cancer Center.

Valganciclovir for the Suppression of Epstein-Barr Virus Replication.

Epstein-Barr virus (EBV) causes infectious mononucleosis and can lead to lymphoproliferative diseases. We evaluated the effects of valganciclovir on oral EBV shedding in a randomized, double-blind, placebo-controlled study. Twenty-six men received oral valganciclovir or daily placebo for 8 weeks, followed by a 2-week "washout period" and then 8 weeks of the alternative treatment. Valganciclovir reduced the proportion of days with EBV detected from 61.3% to 17.8% (relative risk, 0.28; 95% confidence interval [CI], .21-.41; P < .001), and quantity of virus detected by 0.77 logs (95% CI, .62-.91 logs; P < .001). Further investigations into the impact of valganciclovir on EBV-associated diseases are needed.

Prospective studies of infectious mononucleosis in university students.

We performed an intensive prospective study designed to obtain as much data as possible on the incubation and early illness periods of primary Epstein–Barr virus (EBV) infection. Undergraduate students who lacked EBV antibody and oral EBV DNA (EBV-naive) were seen every 2 weeks during their freshman year. Clinical and behavioral data, oral washes and venous blood were obtained. EBV antibodies were quantified by enzyme immunoassay and viral loads by PCR. During a median 8 months of observation, 14/85 subjects experienced primary EBV infections (24 cases/100 person-years). The only significant risk factor for acquisition of EBV infection was deep kissing (P=0.02). Eleven subjects had infectious mononucleosis with a median duration of 21 days. Two subjects were hospitalized. Infections were initially identified in 12 subjects by finding EBV DNA in oral cells before onset of symptoms and in 2 subjects by symptom reporting. EBV DNA and viral capsid antigen (VCA) IgM and gp350 IgG antibodies were present in the blood before onset of illness. To provide a more robust evaluation of primary EBV infection in undergraduate university students, we combined data on risk factors and antibody responses from this and an earlier study that used the exact same clinical and laboratory methods. The observation that the only significant risk factor for acquisition of EBV infection was deep kissing was confirmed. Most importantly, higher amounts of gp350 antibody correlated significantly with a lower severity of infectious mononucleosis (P<0.0001), which strengthens the rationale for a gp350-based prophylactic EBV vaccine.

Expression of cyclin-dependent kinase inhibitor 2A 16, tumour protein 53 and epidermal growth factor receptor in salivary gland carcinomas is not associated with oncogenic virus infection.

It is known that human papillomavirus (HPV) infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) and tumour protein p53 (TP53). Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas (n=51) and benign tumours (n=26) were analysed by polymerase chain reaction (PCR) analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus (CMV) was performed by immunohistochemistry. Epstein–Barr virus (EBV) was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

Epstein-Barr virus in oral shedding of children with multiple sclerosis

To investigate Epstein-Barr virus (EBV) oral shedding frequency and EBV genetic diversity in pediatric patients with multiple sclerosis (MS). This was a prospective case-control study. We used PCR-based assays to detect viral DNA in the monthly mouth swabs of 22 pediatric patients with MS and 77 age- and sex-matched healthy controls. EBV-positive samples were further analyzed for sequence variation in the EBV BCRF1 (ebvIL-10) gene using direct DNA sequencing methods, and in the EBV LMP1 gene by mass spectrometry. Nineteen of the 22 (86.4%) children with MS were seropositive for remote EBV infection compared to 35 out of 77 (45.5%) healthy controls (p = 0.008). Baseline analysis of mouth swabs revealed a higher proportion of EBV-positive samples from EBV-seropositive patients with MS compared to EBV-seropositive healthy controls (52.6% vs 20%, p = 0.007). Longitudinal analysis of monthly swabs revealed average EBV detection rates of 50.6% in patients with MS and 20.4% in controls (p = 0.01). The oral shedding frequencies of Herpesviruses herpes simplex virus-1, cytomegalovirus, human herpesvirus (HHV)-6, and HHV-7 did not differ between groups. Changes in the predominant EBV genetic variants were detected more frequently in patients with MS; however, no specific EBV genetic variant was preferentially associated with MS. Children with MS demonstrate abnormally increased rates of EBV viral reactivation and a broader range of genetic variants, suggesting a selective impairment in their immunologic control of EBV.

Effects of long‐term use of antiretroviral therapy on the prevalence of oral Epstein–Barr virus

The objectives of this study were to determine (i) the prevalence of oral Epstein-Barr virus (EBV) in HIV-infected subjects compared to non-HIV controls and (ii) the effects of long-term use of antiretroviral therapy (ART) on the prevalence of oral EBV. A cross-sectional study was performed in HIV-infected subjects with and without ART, and non-HIV individuals. DNA in saliva samples was extracted and used as a template to detect EBV BamH1W and EBNA1 by quantitative polymerase chain reaction. Student t-test and ANOVA test were performed to determine the prevalence rates among groups. Forty-nine HIV-infected subjects: 37 on ART (age range 23-54 year, mean 37 year), 12 not on ART (age range 20-40 year, mean 31 year), and 20 non-HIV controls (age range 19-53 year, mean 31 year) were enrolled. The numbers of EBV BamH1W in saliva were found to be significantly higher in HIV-infected subjects than non-HIV controls (80% vs. 20%, mean = 12118 vs. 134 copies/10(5) cells, P < 0.001). HIV-infected subjects who were on ART had significantly lower numbers of EBV BamH1W than those who were not (mean = 4102 vs. 138613 copies/10(5) cells, P = 0.011). The numbers were significantly lower in those who received long-term ART compared with short-term (mean = 1401 vs. 11124 copies/10(5) cells, P = 0.034). No significant difference was observed between the groups when using EBNA1 primers. Prevalence of oral EBV was significantly higher in HIV-infected subjects than non-HIV-controls. The numbers of the virus were significantly decreased by ART. Long-term use of ART did not increase oral EBV.

Oral EBV and KSHV Infection in HIV

The gamma herpesviruses, Kaposi's-sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are tightly associated with the development of AIDS-associated oral disease and malignancy during immune suppression. The objective of this investigation was to characterize oral infection and pathogenesis in healthy and immune-suppressed individuals. To characterize oral EBV and KSHV infection, we examined throat washings and oral epithelial cells from HIV-positive and HIV-negative individuals. Quantitative/real-time polymerase-chain-reaction (PCR) assays, transmission electronmicroscopy, immunostaining, and sequence analysis were used to identify viral infection. Virus was isolated from throat-wash samples and was used to infect epithelial and lymphoid cell lines. We detected EBV and KSHV in the oral cavity in healthy and immune-suppressed individuals. Viral strain analysis of KSHV K1 in multiple clones from the oral cavities of healthy persons and immunosuppressed patients detected several strains previously detected in KS lesions, with minor strain variation within individuals. Immunoelectron microscopy for multiple viral antigens detected consistent expression of viral proteins and oral epithelial specimens. In oral epithelial cells infected with wild-type KSHV in vitro, the K8.1 glycoprotein associated with lytic KSHV infection was detected in both primary and telomerase immortalized oral epithelial cultures by 24 hours post-infection. Virions were detected, subsequent to infection, by scanning electron microscopy. Oral epithelial cells were also infected in vitro with wild-type EBV originating from throat washes. Analysis of these data suggests that, like EBV, KSHV infection is present in the oropharynx of healthy individuals, is transmissible in vitro, and may be transmitted by saliva.

Lack of evidence for local immune activity in oral hairy leukoplakia and oral wart lesions

Oral warts, caused by human papillomavirus (HPV), and oral hairy leukoplakia (OHL) caused by Epstein-Barr virus (EBV), are common oral manifestations in HIV-infected persons. Although both conditions occur most often with reduced blood CD4+ T-cell numbers, oral warts and OHL rarely occur simultaneously, suggesting that dysfunctions in other secondary local immune parameters are also involved. The present study evaluated tissue-associated proinflammatory and T-helper cytokine and chemokine mRNA expression and the presence of T cells in each lesion. Biopsies were taken from lesion-positive and adjacent lesion-negative sites of HIV+ persons with oral warts or OHL and lesion-negative sites from HIV+ persons who were oral HPV or EBV DNA-positive (matched controls). Cytokine/chemokine mRNA expression was quantified by real-time polymerase chain reaction. CD3, CD4, and CD8 cells were identified by immunohistochemistry. No differences were detected in tissue-associated cytokine/chemokine mRNA expression in warts or OHL when compared to lesion-negative sites. Immunohistochemical analysis of T cells showed CD8+ cells exclusively, but few cells were present in either lesion. No differences were detected between lesion-positive and -negative control sites of each pathologic condition. Little evidence was found for local immune reactivity to either oral warts and OHL, suggesting that CD4+ T cells are a primary host defense against both oral warts and OHL, but with nonimmune factors potentially responsible for the divergent prevalence of each.