Human immunodeficiency virus and oral microbiota: mutual influence on the establishment of a viral gingival reservoir in individuals under antiretroviral therapy.

The role of the oral microbiota in the overall health and in systemic diseases has gained more importance in the recent years, mainly due to the systemic effects that are mediated by the chronic inflammation caused by oral diseases, such as periodontitis, through the microbial communities of the mouth. The chronic infection by the human immunodeficiency virus (HIV) interacts at the tissue level (e.g. gut, genital tract, brain) to create reservoirs; the modulation of the gut microbiota by HIV infection is a good example of these interactions. The purpose of the present review is to assess the state of knowledge on the oral microbiota (microbiome, mycobiome and virome) of HIV-infected patients in comparison to that of HIV-negative individuals and to discuss the reciprocal influence of HIV infection and oral microbiota in patients with periodontitis on the potential establishment of a viral gingival reservoir. The influence of different clinical and biological parameters are reviewed including age, immune and viral status, potent antiretroviral therapies, smoking, infection of the airway and viral coinfections, all factors that can modulate the oral microbiota during HIV infection. The analysis of the literature proposed in this review indicates that the comparisons of the available studies are difficult due to their great heterogeneity. However, some important findings emerge: (i) the oral microbiota is less influenced than that of the gut during HIV infection, although some recurrent changes in the microbiome are identified in many studies; (ii) severe immunosuppression is correlated with altered microbiota and potent antiretroviral therapies correct partially these modifications; (iii) periodontitis constitutes a major factor of dysbiosis, which is exacerbated in HIV-infected patients; its pathogenesis can be described as a reciprocal reinforcement of the two conditions, where the local dysbiosis present in the periodontal pocket leads to inflammation, bacterial translocation and destruction of the supporting tissues, which in turn enhances an inflammatory environment that perpetuates the periodontitis cycle. With the objective of curing viral reservoirs of HIV-infected patients in the future years, it appears important to develop further researches aimed at defining whether the inflamed gingiva can serve of viral reservoir in HIV-infected patients with periodontitis.

Epstein-Barr Virus Promotes Inflammatory Cytokine Production in Human Gingival Fibroblasts.

BACKGROUND: Periodontitis is one of the most common chronic oral inflammatory diseases. Over the past decade, herpes viruses, particularly Epstein-Barr virus (EBV), have been considered promising pathogenic candidates for periodontitis. However, the specific mechanism by which EBV contributes to the development of periodontitis is still unknown. This study aimed to explore the mechanism of EBV underlying the inflammatory response in human gingival fibroblasts (HGFs). MATERIALS AND METHODS: HGFs were stimulated with different concentrations of EBV (104, 105, 106, 107, and 108 DNA copies/mL) for 0, 8, 24, or 48 hours. The mRNA levels of interleukin (IL)-1ß, tumour necrosis factor-α (TNF-α), IL-8, monocyte chemoattractant protein-1 (MCP-1), and Toll-like receptor 9 (TLR9) were measured using quantitative real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assays (ELISAs) were performed for determining the mRNA and protein levels of IL-1ß, TNF-α, IL-8, and MCP-1. Real-time PCR and ELISA were performed to determine the protein levels of IL-1ß, TNF-α, IL-8, and MCP-1. Activation of the TLR9/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway was evaluated using western blotting. RESULTS: The expressions of IL-1ß, TNF-α, IL-8, and MCP-1 were significantly upregulated in HGFs under EBV stimulation in a concentration- and time-dependent manner. EBV promoted TLR9 and MyD88 expression and induced NF-κB transcription. On the contrary, the upregulation of these factors and the activation of NF-κB pathway were drastically inhibited by TLR9 antagonists. CONCLUSIONS: Our findings demonstrate that EBV promotes the production of inflammatory cytokines IL-1ß and TNF-α and chemokines IL-8 and MCP-1 in HGFs through the TLR9/MyD88/NF-κB pathway.

Epstein-Barr Virus Promotes the Production of Inflammatory Cytokines in Gingival Fibroblasts and RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells.

Periodontitis is an inflammatory condition that causes the destruction of the supporting tissues of teeth and is a major public health problem affecting more than half of the adult population worldwide. Recently, members of the herpes virus family, such as the Epstein-Barr virus (EBV), have been suggested to be involved in the etiology of periodontitis because bacterial activity alone does not adequately explain the clinical characteristics of periodontitis. However, the role of EBV in the etiology of periodontitis is unknown. This study aimed to examine the effect of inactivated EBV on the expression of inflammatory cytokines in human gingival fibroblasts (HGFs) and the induction of osteoclast differentiation. We found that extremely high levels of interleukin (IL)-6 and IL-8 were induced by inactivated EBV in a copy-dependent manner in HGFs. The levels of IL-6 and IL-8 in HGFs were higher when the cells were treated with EBV than when treated with lipopolysaccharide and lipoteichoic acid. EBV induced IκBα degradation, NF-κB transcription, and RAW264.7 cell differentiation into osteoclast-like cells. These findings suggest that even without infecting the cells, EBV contributes to inflammatory cytokine production and osteoclast differentiation by contact with oral cells or macrophage lineage, resulting in periodontitis onset and progression.

Cigarette Smoke Stimulates SARS-CoV-2 Internalization by Activating AhR and Increasing ACE2 Expression in Human Gingival Epithelial Cells.

A large body of evidence shows the harmful effects of cigarette smoke to oral and systemic health. More recently, a link between smoking and susceptibility to coronavirus disease 2019 (COVID-19) was proposed. COVID-19 is due to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which uses the receptor ACE2 and the protease TMPRSS2 for entry into host cells, thereby infecting cells of the respiratory tract and the oral cavity. Here, we examined the effects of cigarette smoke on the expression of SARS-CoV-2 receptors and infection in human gingival epithelial cells (GECs). We found that cigarette smoke condensates (CSC) upregulated ACE2 and TMPRSS2 expression in GECs, and that CSC activated aryl hydrocarbon receptor (AhR) signaling in the oral cells. ACE2 was known to mediate SARS-CoV-2 internalization, and we demonstrate that CSC treatment potentiated the internalization of SARS-CoV-2 pseudovirus in GECs in an AhR-dependent manner. AhR depletion using small interference RNA decreased SARS-CoV-2 pseudovirus internalization in CSC-treated GECs compared with control GECs. Our study reveals that cigarette smoke upregulates SARS-CoV-2 receptor expression and infection in oral cells. Understanding the mechanisms involved in SARS-CoV-2 infection in cells of the oral cavity may suggest therapeutic interventions for preventing viral infection and transmission.

Cetylpyridinium chloride blocks herpes simplex virus replication in gingival fibroblasts.

Infections with herpes simplex viruses are lifelong and highly prevalent worldwide. Individuals with clinical symptoms elicited by HSVs may suffer from occasional or recurrent herpetic lesions in the orofacial and genital areas. Despite the existence of nucleoside analogues that interfere with HSV replication, such as acyclovir, these drugs are somewhat ineffective in treating skin lesions as topical formulations only reduce in one or few days the duration of the herpetic ulcers. Cetylpyridinium chloride (CPC) is a quaternary ammonium compound present in numerous hygiene products, such as mouthwashes, deodorants, aphtae-treating formulations and oral tablets as an anti-septic to limit bacterial growth. Some reports indicate that CPC can also modulate host signaling pathways, namely NF-κB signaling. Because HSV infection is modulated by NF-κB, we sought to assess whether CPC has antiviral effects against HSVs. Using wild-type HSV-1 and HSV-2, as well as viruses that are acyclovir-resistant or encode GFP reporter genes, we assessed the antiviral capacity of CPC in epithelial cells and human gingival fibroblasts expanded from the oral cavity and its mechanism of action. We found that a short, 10-min exposure to CPC added after HSV entry into the cells, significantly limited viral replication in both cell types by impairing viral gene expression. Interestingly, our results suggest that CPC blocks HSV replication by interfering with the translocation of NF-κB into the nucleus of HSV-infected cells. Taken together, these findings suggest that formulations containing CPC may help limit HSV replication in infected tissues and consequently reduce viral shedding.

EBV LMP1 in Gingival Epithelium Potentially Contributes to Human Chronic Periodontitis via Inducible IL8 Production.

BACKGROUND/AIM: Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBV-encoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. MATERIALS AND METHODS: Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. RESULTS: Extremely high IL8 production was induced by LMP1 in time- and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. CONCLUSION: Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation.

Screening for Epstein-Barr virus (EBV) infection status in university freshmen: acceptability of a gingival swab method.

Prophylactic vaccines against Epstein-Barr virus (EBV) are under development. EBV-naïve college freshmen are ideal candidates for an efficacy trial, because their incidence of infectious mononucleosis (mono) during freshman year is as high as 20%. To assess perceptions about mono and a mono vaccine, and to learn if EBV immune status could be determined using a gingival swab rather than phlebotomy, we performed a cross-sectional study of 235 healthy students at the beginning of their freshman year. Subjects completed questionnaires and donated oral washes, gingival swabs and venous blood. Overall, 90% of students found the swab easy to use and 80% preferred the swab over venepuncture. Of the 193 students with sufficient samples, 108 (56%) had EBV antibodies in blood vs. 87 (45.1%) in the gingival swab. The sensitivity and specificity of the swab compared with blood for detecting EBV antibodies was 75.9% and 94.1%, respectively, with an accuracy of 89.3%. EBV DNA was detected in the oral wash and swab of 39.2% and 30.4% of blood-antibody-positive individuals, respectively. In conclusion, 44% of our freshmen were EBV-naïve and thus vaccine candidates, the gingival swab was an acceptable alternative to phlebotomy for detecting EBV antibody but needs improved sensitivity, and the perceived value of EBV vaccine was high (72% believed they would benefit).

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma arising in patient with a history of EBV-positive mucocutaneous ulcer and EBV-positive nodal polymorphous B-lymphoproliferative disorder.

Elderly patients with Epstein-Barr virus (EBV) infection are at increased risk for developing B-cell lymphoproliferative disorder (B-LPD) due to immunosenescence. Here, we describe a case of a 75-year-old man who developed an EBV-positive (EBV+) mucocutaneous ulcer (EBVMCU) in the gingiva with spontaneous regression. Eighteen months after regression, he had a cervical lymph node enlargement that was diagnosed as EBV+ nodal polymorphous B-LPD, Ann Arbor stage IA. Clinicians decided to observe his clinical course without any treatment. Fourteen months later, the patient developed EBV-positive diffuse large B-cell lymphoma (DLBCL), Ann Arbor stage IIA, and received six courses of age-adjusted dose chemotherapy and achieved a complete remission. No evidence of a clonal relationship was found among these three lesions by standard polymerase chain reaction (PCR) analysis for immunoglobulin heavy chain. However, they all had expression of PD-L1 in the EBV+ large B-cells and Hodgkin Reed-Sternberg-like cells. This is the first case report of a PD-L1-positive (PD-L1+) EBVMCU and the development of multiple EBV-driven B-LPDs in the setting of immunosenescence within a 32-month period.

Hepatitis C Infection and Periodontal Disease: Is there a Common Immunological Link?

Hepatitis C virus (HCV) infections could have an important impact on the oral health status of patients, favoring conditions such as periodontal disease and oral cancer. The review of the existing scientific literature written in English was performed, searching for oral and periodontal manifestations of HCV infection and its impact on the oral fluids. HCV infection can determine direct extrahepatic manifestations at the oral and periodontal level including oral lichen planus, Sjögren-like sialadenitis, and oral cancer. The changes caused by the infection in the subjects' immune system, diet, and lifestyle can facilitate the development of oral conditions such as periodontal disease. Important changes also occur in the composition of the infected patients' saliva and gingival fluid. HCV-infected patients need to be carefully monitored in terms of oral health since the infection with the virus can result in oral complications. The cellular and molecular particularities of the gingival fluid of HCV-infected patients can answer some questions regarding its impact upon periodontium impairment and whether this refers to a possible bidirectional relationship, with hepatic biomarker adjustments being induced by the periodontal patients' inflammatory status.

Herpesvirus-encoded microRNAs detected in human gingiva alter host cell transcriptome and regulate viral infection.

MicroRNAs (miRNAs) are small, non-coding RNAs of ~18-25 nucleotides that have gained extensive attention as critical regulators in complex gene networks including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and function. Many viruses encode miRNAs that directly downregulate the expression of factors of the innate immune system, which includes proteins involved in promoting apoptosis and recruitment. In this study, we examined the expression profiles of three previously identified viral miRNAs (v-miRs) from the human herpesvirus (HHV) family, HSV-1 (miR-H1), KSHV (miR-K12-3-3p), and HCMV (miR-US4) in healthy and diseased periodontal tissues and observed increased levels of v-miRs in diseased tissues. To understand the significance of this increase, we overexpressed v-miRs in human oral keratinocytes (HOK), a common target for various HHV, and analyzed the impact of miR-H1 and miR-K12-3-3p on the host transcriptome. More than 1300 genes were altered in HOK overexpressing miR-H1 and miR-K12-3-3p. Global pathway analysis of deregulated genes identified several key cellular pathways that may favor viral persistence. Using bioinformatic analysis, we predicted hundreds of potential v-miR binding sites on genes downregulated by miR-H1 and miR-K12-3-3p and validated three novel target v-miR sites suggesting widespread direct and indirect modulation of numerous host genes/pathways by a single v-miR. Finally, in vitro HSV-1 infection assays showed that miR-H1 can regulate viral entry and infection in human oral keratinocytes (HOK). Overall, our results demonstrate clinical and functional relevance of pathogenic viral molecules viz., v-miRs that regulate both host and viral functions and may contribute to the pathogenesis of inflammatory oral diseases.

Detection of a new species of torque teno mini virus from the gingival epithelium of patients with periodontitis.

We describe a novel species of torque teno mini virus called TTMV-204, which was isolated from the gingival epithelium of patients with periodontitis and characterized using viral metagenomics. The sequence of the full genome is 2824 nt in length. Phylogenetic analysis and genetic analyses show classic Betatorquevirus species organization with less than 40% amino acid similarity in ORF1. The prevalence of TTMV-204 in the periodontitis patient population was 18.75% (15/80), which was higher than in periodontally healthy individuals (10.00%, 10/80). However, the difference of the TTMV-204 prevalence between two groups was not statistically significant (p = 0.115). Further investigation is required to determine whether this new virus is associated with inflammation.

Herpes Simplex 1 and Periopathogen Role in Peri-implantitis.

INTRODUCTION: The objectives of this study were to compare the qualitative and quantitative profiles of herpes simplex virus type I (HSV-1) in implant surfaces between participants with peri-implantitis (PI) and Healthy peri-implant tissues and to quantitatively assess the relation between HSV-1 and periopathogens inside the microbiological profile associated with PI. MATERIALS AND METHODS: A total of 40 patients with PI and 40 with healthy peri-implant tissues (HI) were recruited. Plaque samples from peri-implant sulcus and internal implant connections were analyzed using quantitative real-time polymerase chain reaction to detect and quantify HSV-1 and periodonto-pathogens. Frequencies of detection and levels of microorganisms were compared between PI and HI; the frequencies and levels of periodontopathogens were compared between HSV-1+ and HSV-1- PI to assess qualitative relations between HSV-1 and bacteria. Correlation between HSV-1 and periodontopatho-gens levels was assessed in PI and HI. RESULTS: A total of 77 dental implants affected by PI, and 113 HIs were included. The HSV-1 prevalence was slightly higher in PI compared with controls (33.3 vs 23.8%; p > 0.05); HSV-1 was detected in external samples more frequently compared with internal samples. The HSV-1-positive patients revealed higher median loads of Prevotella intermedia (Pi) and Campylobacter rectus (Cr) compared with HSV-1-negative patients. In the PI group, a significant positive correlation was evidenced between HSV-1 and Tannerella forsythia, Parvimonas micra (Pm), Fusobacterium nucleatum, and Cr levels, while in the HI, positive correlation between HSV-1 and Aggregatibacter actinomycetemcomitans, Pi, and Pm was established. CONCLUSION: The HSV-1 prevalence cannot be used to identify PI. The HSV-1 was found in similar levels of PI and HI patients after an average of 6 years of loaded implants. The HSV-1 prevalence cannot be used to identify implants with or without the presence of PI. CLINICAL SIGNIFICANCE: Although HSV-1 is detected in PI site, HSV-1 may represent an unspecific indicator for the host response to the bacterial challenge observed in PI.

Detection of EBV, CMV and HSV-1 in subgingival samples of HIV positive and negative patients with chronic periodontitis / Detección de VEB, CMV y VHS-1 en muestras subgingivales de pacientes VIH positivos y negativos con periodontitis crónica

Abstract: objective: to detect the presence of infection by EBV (Epstein-Barr Virus), CMV (Cytomegalovirus) and HSV-1 (Herpes Simplex Virus type 1) in subgingival samples from HIV- positive patients under HAART (High Activity Antiretroviral Therapy), HIV- positive patients without HAART, HIV-negative patients with chronic periodontitis and healthy controls. Methodology: Crevicular fluid samples of 11 HIV+ patients on therapy were evaluated, 6 without antiretroviral therapy, 7 HIV- negative subjects with chronic periodontitis and 7 periodontally-healthy controls. PI (Plaque index), GI (Gingival Index), PD (probing depth) and CAL (Clinical Attachment Loss) were registered at six sites per each tooth in all teeth and subgingival plaque samples of a tooth were collected per quadrant. Nested PCR was used to detect EBV and endpoint PCR to detect infection by CMV and HSV-1. Results: Clinical parameters showed statistically significant differences between HIV-positive patients and subjects with chronic periodontitis compared with the control group (p<0.05). DNA of EBV was detected mainly in HIV-positive patients under HAART, 91 percent (10/11). DNA of CMV was detected mainly in patients without HAART, 67 percent (4/6), while HSV-1 was observed in 27 percent (3/11) of patients under HAART. In the control group no virus was detected. Coinfection was observed in 50 percent of HIV patients without HAART, 36 percent of HIV patients with HAART and 14 percent of HIV-negative with chronic periodontitis. Conclusion: Viral infection was prevalent in HIV patients under HAART and EBV was the primary viral infection detected in HIV-positive patients with chronic periodontitis.

Resumen: detectar la presencia de infección por VEB (Virus Epstein-Barr), CMV (Citomegalovirus) y VHS-1 (Virus Herpes simple tipo 1) en muestras subgingivales de pacientes VIH-positivos bajo HAART (Terapia Anti Retroviral de Alta Actividad), VIH-positivos sin HAART, pacientes VIH-negativos con periodontitis crónica y controles sanos. Metodología: Se evaluaron muestras de fluido crevicular de 11 pacientes VIH+ bajo terapia, 6 sin terapia antiretroviral, 7 sujetos VIH–negativo con periodontitis crónica y 7 controles periodontalmente sanos. Se registró el IP (Índice de placa), IG (Índice Gingival), PS (Profundidad del Sondaje) y NIC (Nivel de Inserción Clínica) en seis sitios por diente en todos los dientes y se recolectaron muestras de placa subgingival de un diente por cuadrante. Se empleó PCR anidada para detectar VEB y PCR punto final para identificar la infección con CMV y VHS-1. Resultados: Los parámetros clínicos mostraron diferencias estadísticamente significativas entre pacientes VIH-positivos y sujetos con periodontitis crónica comparados con el grupo control (p<0.05). El ADN de EBV fue detectado principalmente en pacientes VIH-positivos bajo HAART con 91 por ciento (10/11). El ADN de CMV se detectó principalmente en pacientes sin HAART, 67 por ciento (4/6), mientras que VHS-1 se observó en 27 por ciento (3/11) de los pacientes bajo HAART. En el grupo control no se detectó ningún virus. La coinfección fue observada en 50 por ciento de los pacientes VIH sin HAART, 36 por ciento de los VIH con HAART y 14 por ciento de los VIH negativos con periodontitis crónica. Conclusión: La infección viral fue predominante en los pacientes VIH bajo HAART y VEB fue la principal infección viral detectada en los pacientes VIH positivos y con periodontitis crónica.

A novel species of torque teno mini virus (TTMV) in gingival tissue from chronic periodontitis patients.

A new species of torque teno mini virus, named TTMV-222, was detected in gingival tissue from periodontitis patients using a viral metagenomics method. The 2803-nucleotide genome of TTMV-222 is closely related to TTMV1-CBD279, with 62.6% overall nucleotide similarity. Genetic analyses of the new virus genome revealed a classic genomic organization but a weak identity with known sequences. The prevalence of TTMV-222 in the periodontitis group (n = 150) was significantly higher than that in the healthy group (n = 150) (p = 0.032), suggesting that the new virus may be associated with inflammation in chronic periodontitis patients. However, this finding requires further investigation.

Detection of human papillomavirus in dental biofilm and the uterine cervix of a pregnant adolescent / Detecção do papilomavírus humano em biofilme dental e no colo uterino de uma adolescente grávida

CONTEXT: Adolescence and pregnancy are considered to be risk factors for human papillomavirus (HPV) infection. The relationship between this infection in the uterine cervix and oral HPV infection is controversial. CASE REPORT: This report describes a case of a pregnant 16-year-old adolescent who presented HPV infection in the uterine cervix and the mouth. Smears were collected from the cervix and the tongue/palate. Dental biofilm samples were also collected. The microarray technique was used to detect HPV. The HPV 56 subtype was observed in the cervical smear and HPV 6 in dental biofilm. CONCLUSION: In this pregnant adolescent, HPV infection was present in both the cervix and the mouth, but the HPV subtypes infecting these two areas were different.

CONTEXTO: A adolescência e a gestação são consideradas fatores de risco para a infecção pelo papilomavírus humano (HPV). A relação entre essa infecção no colo do útero e na boca num mesmo paciente é controversa. RELATO DE CASO: Descrever o caso de uma adolescente grávida de 16 anos que apresentou a infecção pelo HPV no colo do útero e na boca. Esfregaços foram realizados no colo do útero e em língua/palato. Amostras de biofilme dental também foram coletadas. Para detectar o HPV, foi utilizada a técnica do microarranjo. O HPV 56 foi o subtipo encontrado no esfregaço cervical e o tipo HPV 6 no biofilme dental. CONCLUSÕES: Observamos, nessa adolescente grávida, a presença do HPV na boca e no colo do útero, mas os subtipos virais que infectavam essas duas regiões eram distintos.

Detection of human papillomavirus in dental biofilm and the uterine cervix of a pregnant adolescent.

CONTEXT: Adolescence and pregnancy are considered to be risk factors for human papillomavirus (HPV) infection. The relationship between this infection in the uterine cervix and oral HPV infection is controversial. CASE REPORT: This report describes a case of a pregnant 16-year-old adolescent who presented HPV infection in the uterine cervix and the mouth. Smears were collected from the cervix and the tongue/palate. Dental biofilm samples were also collected. The microarray technique was used to detect HPV. The HPV 56 subtype was observed in the cervical smear and HPV 6 in dental biofilm. CONCLUSION: In this pregnant adolescent, HPV infection was present in both the cervix and the mouth, but the HPV subtypes infecting these two areas were different.