Chapter 1 Herpesviruses in Periodontal Disease

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1 Chapter 1 Herpesviruses in Periodontal Disease Miriam Ting 1 and Jørgen Slots 2 * 1 Specialty Clinic in Periodontology, USA 2 University of Southern California, USA * Corresponding Author: Jørgen Slots, University of Southern California, CA, USA, jslots@usc.edu First Published March 30, 2017 Copyright: 2017 Miriam Ting and Jørgen Slots. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source. Abstract Periodontitis is one of the most complex infectious diseases of the human body. Individual periodontal lesions can harbor millions of genomic copies of herpesviruses and equal numbers of bacteria. Herpesvirus-infected periodontal sites exhibit more breakdown than herpesvirusfree sites, and a herpesvirus active infection is associated with elevated risk of periodontal disease progression. Various immune mechanisms that are active against herpesviruses may diminish anti-bacterial immune responses, and vice versa. We propose that severe periodontitis is the result of extensive and partly opposing immune responses against herpesvirus-bacterial combined infections. The herpesvirus-bacterial model of periodontitis provides a rationale for considering an anti-viral pharmacotherapeutic approach to periodontal disease prevention and treatment. Introduction The periodontium includes gingiva, alveolar bone, root cementum and a collagenous ligament. Periodontal tissue in health is firm, pink, and forms a tight cuff around teeth. In gingivitis, the gingiva is swollen, edematous, red, and bleeds easily on contact, and may form pseudopockets around teeth, but it is not associated with loss of alveolar bone or periodontal ligament. Periodontitis is an extension of gingivitis with loss of supportive bone and 2 3

2 ligament, leading to pocket formation and potentially loss of teeth. Probing measurements of periodontal pocket depth and radiographic assessment of alveolar bone loss are used to determine the severity of the periodontal disease. Basic periodontal therapy focuses on plaque and calculus removal, and include oral hygiene instruction, scaling and root planing, and perhaps periodontal surgery. Gingivitis affects approximately 82% of US adults, which is a similar prevalence as that found in other parts of the world [1]. Slight to moderate types of periodontitis is also widespread globally. However, severe periodontitis affects only 8-15% of the global population [1]. Additional risk factors besides plaque and calculus are probably involved in the most severe forms of periodontal disease. Periodontal disease is associated with specific bacteria, and cellular and humoral immune responses, but the precise role of the periodontopathic species and the associated immune response is not fully understood. Periodontal infections in some patients might lead to massive loss of alveolar bone, whereas periodontal infections in other patients are limited to gingiva with hardly any bone loss [1]. Although detection of key periodontal pathogens may identify patients at elevated risk for periodontitis, it may not always predict outcomes. Thus, the search continues for additional etiologic factors that may exacerbate periodontal disease. Since the mid-1990s, studies have shown a high prevalence of cytomegalovirus, Epstein-Barr virus and herpes simplex virus type 1 in severe periodontitis [1]. Available evidence supporting a periodontopathogenic role of herpesviruses comes from association studies and immunology-based research [2]. Though, the specific molecular mechanisms by which herpesviruses are involved in periodontitis are still incompletely understood. Herpesvirus-Bacteria Interaction in Periodontitis Severe periodontal lesions harbor a combination of herpesviruses and specific bacterial pathogens. Cytomegalovirus, Epstein-Barr virus type 1 or herpes simplex virus type 1 were found to be statistically associated with Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens, Dialister pneumosintes, Treponema denticola, Campylobacter rectus, and Aggregatibacter actinomycetemcomitans [3]. Those bacteria are gram-negative anaerobic species. Severe periodontitis in adults show particularly strong relationships between cytomegalovirus, Epstein-Barr virus, P. gingivalis and T. forsythia. Periodontitis lesions in adolescents yield mainly cytomegalovirus and A. actinomycetemcomitans or P. gingivalis [3]. In localized aggressive periodontitis, a disease of adolescence, the presence of cytomegalovirus or Epstein-Barr virus increased the odds for detecting A. actinomycetemcomitans by 31.8-fold and by 9.3-fold, respectively. The individual odds ratios for cytomegalovirus and 4 5

3 P. gingivalis in localized aggressive periodontitis were 4.6 and 7.8, respectively, but the cytomegalovirus-p. gingivalis co-infection occurred with an odds ratio as high as Those findings suggest that herpesviruses and specific bacteria collaborate synergistically, not just additively, in the development of aggressive periodontitis [3]. The herpesvirus-bacterial interaction is bidirectional. Herpesviruses can lower periodontal immunity and induce an overgrowth of pathogenic bacteria, and bacterial inflammatory factors can activate periodontal herpesvirus. The bacterial overgrowth may be triggered by herpesvirus-mediated release of pro-inflammatory cytokines, which may suppress anti-inflammatory cytokines and antibody production against bacterial pathogens [3]. Epstein-Barr virus reactivation can trigger overgrowth of P. gingivalis, and P. gingivalis has the potential to reactivate Epstein-Barr virus. Porpyromonas gingivalis can reactivate Epstein-Barr virus by epigenetic mechanisms, and P. gingivalis lipopolysaccharide may activate intracellular signaling pathways, such as nuclear factor-kappab, which may facilitate herpesviral entry into periodontal fibroblasts. This vicious cycle of increasing pathogenicity of both herpesviruses and pathogenic bacteria can result in periodontal breakdown if the herpesvirus-bacterial interaction is not swiftly controlled by the host defense or periodontal treatment [4]. Herpesvirus counts usually peak during advancing periodontal disease and may be low or undetectable in a disease state of remission. Therefore, to associate herpesviruses with periodontal breakdown, it is important to study periodontitis lesions that are actively progressing. Study of periodontal herpesviruses should preferably also include individuals with no history of professional periodontal treatment. Periodontal herpesviruses may not be detected post-treatment in 50-90% of periodontal lesions that were virus-positive pre-treatment [5]. Herpesvirus Periodontopathogenicity Herpesviruses can exert direct cytopathic effects on fibroblasts, keratinocytes, endothelial cells, osteocytes and inflammatory cells. In periodontitis sites, cytomegalovirus and Epstein Barr virus can infect and alter monocyte, macrophage and lymphocyte functions, and can cause defects in polymorphonuclear leukocyte adherence, chemotaxis, phagocytosis and bactericidal activities. Active Epstein-Barr virus infection can produce anti-neutrophilic antibodies resulting in neutropenia [5]. Herpesviruses can also up-regulate mononucleocyte gene expression of pro-inflammatory cytokines such as interleukin-1beta and tumor necrosis factor-alpha, which are associated with periodontal tissue breakdown. Pro-inflammatory cytokines and chemokines may suppress the antibacterial host defense, stimulate bone-resorption, increase matrix 6 7

4 metalloproteinase (collagenase) release and decrease metalloproteinase inhibitors [4]. Periodontal Herpesviruses in Immunocompetent Individuals Periodontal Health and Gingivitis Herpesviruses occur in approximately 8% of healthy periodontal sites and in 20-33% of gingivitis sites, and are more frequent in such sites of periodontitis patients than of generally healthy periodontal subjects. Herpesviruses in periodontal health and gingivitis occur typically in a non-transcriptional state and in low copy-counts of 1,000 20,000 [5,6]. However, Epstein-Barr virus is commonly present in pregnancy gingivitis. Periodontal Epstein Barr virus may mediate overgrowth of P. gingivalis and P. intermedia in pregnant women, and the exacerbated inflammation may generate further influx and proliferation of Epstein-Barr virus [2]. Herpesviruses also contribute to necrotizing ulcerative gingivitis. In Nigeria, cytomegalovirus was detected in 59%, Epstein-Barr virus-1 in 27%, and herpes simplex virus type 1 in 23% of necrotizing ulcerative gingivitis lesions of non-hiv-infected 3-14 year-old malnourished children; the herpesviruses were present in 5% of healthy periodontal sites of the same children. Herpesvirus infection in early childhood, coupled with an impaired cellular defense due to malnutrition and an abundance of virulent periodontal bacteria, seemed to be risk factors for necrotizing ulcerative gingivitis in African children. Acute herpetic gingivostomatitis in children and older individuals is caused by herpes simplex virus type 1 and occasionally by herpes simplex virus type 2 [5]. Chronic Periodontitis Periodontal pockets of chronic periodontitis lesions contain subgingival herpes simplex virus in a prevalence of %, Epstein-Barr virus in a prevalence of 3-89% and cytomegalovirus in a prevalence of %. In gingival biopsies of chronic periodontitis lesions, 57% yielded herpes simplex virus-1, 79% Epstein-Barr virus type 1, and 86% cytomegalovirus; herpesviruses were found at a low frequency in normal periodontal sites of the same individuals. Chronic periodontitis sites having a low probability of disease progression typically harbor a latent rather than an active cytomegalovirus infection [7]. Patients co-infected by Epstein-Barr virus and cytomegalovirus exhibited deeper periodontal pockets and increased bone loss [5]. Herpesvirus antibodies are also associated with chronic periodontitis. High occurrence of gingival crevicular fluid antibodies against Epstein-Barr virus and cytomegalovirus have been detected in chronic periodontitis lesions. Most herpesvirus antibodies in gingival crevicular fluid seem to originate from local synthesis rather than from passive transudation of blood [4]. Herpesvirus- 8 9

5 es have also been associated with bone destruction around dental implants ( peri-implantitis ). Aggressive Periodontitis Localized aggressive periodontitis is characterized by onset around puberty of attachment loss limited to the permanent first molars and incisors. The disease is related to and probably caused by herpesviruses in co-infection with specific bacteria like A. actinomycetemcomitans or P. gingivalis. In Afro-Caribbean adolescents with classical localized aggressive periodontitis, cytomegalovirus and P. gingivalis synergistically influenced the incidence and the degree of periodontal disease. In localized aggressive periodontitis, as stated above, the odds ratio of cytomegalovirus infection alone was 6.6, and the odds ratio of P. gingivalis alone was 8.7. However, in localized aggressive periodontitis individuals with a co-infection of cytomegalovirus and P. gingivalis, the odds ratio of this co-infection increased exponentially to 51.4 [2]. The hormonal changes associated with puberty may play a role in the activation of herpesviruses in localized aggressive periodontitis. In US adolescence, transcription of the major cytomegalovirus capsid protein indicative of viral re-activation was detected in deep periodontal pockets in all five of the cytomegalovirus-positive patients with early periodontitis years of age, but only in one of the three cytomegalovirus-positive patients older than 14 years of age [8]. Presence of this major capsid protein was detected in deep periodontal pockets, but not in any cytomegalovirus-infected shallow gingival crevices. Cytomegalovirus transcription of the major capsid protein was identified in periodontitis sites with an absence of radiographic crestal alveolar lamina dura indicative of ongoing periodontal breakdown, but not in disease-stable periodontitis sites showing radiographic crestal alveolar lamina dura [8]. Hypothetically, a primary cytomegalovirus infection at the time of root formation of permanent incisors and first molars can give rise to a defective periodontium with cemental hypoplasia, which may accelerate future disease progression [8]. The hormonal changes at the onset of puberty may re-activate a latent cytomegalovirus periodontal infection causing local immune suppression and bacterial overgrowth and tissue invasion by exogenous bacteria like certain genotypes of A. actinomycetemcomitans. Electron microscopic identification of herpesvirus-like virions in aggressive periodontitis lesions provides support for an active viral infection. Localized aggressive periodontitis sites with an active cytomegalovirus infection are more heavily colonized by A. actinomycetemcomitans compared to periodontal sites with latent cytomegalovirus infection [8]. In that scenario, localized aggressive periodontitis is caused by herpesvirus-induced suppression of the periodontal host defense and gingival tissue invasion by bacterial pathogens

6 Herpesviruses occurred more frequently in periodontitis sites of adults from Greece with actively progressing periodontitis. Cytomegalovirus was detected in 59% of disease-active sites compared to 13% of stable sites, Epstein-Barr virus was detected in 44% of active sites compared to 13% of stable sites, and herpes simplex virus type 1 was detected in 35% of active sites compared to 9% of stable sites. Co-infection with any of the three herpesviruses was detected in 44% of active sites compared to 3% of stable sites. Those findings provide further evidence that cytomegalovirus, Epstein-Barr virus, and especially Epstein-Barr virus-cytomegalovirus co-infection are significantly associated with disease-active periodontitis [2]. Generalized aggressive periodontitis affecting most teeth in a dentition is also related to herpesvirus infections. Cytomegalovirus was detected in 49%, Epstein-Barr virus in 45%, and herpes simplex virus type 1 in 63% of aggressive periodontitis lesions. In young military recruits from Turkey with generalized aggressive periodontitis, advanced periodontitis lesions harbored cytomegalovirus, Epstein-Barr virus and herpes simplex virus type 1 in 73 78% of pooled samples. This is compared to a low or undetectable herpesvirus occurrence in recruits with a healthy periodontium [5]. Periodontal Abscess A periodontal abscess is an accumulation of pus limited to the marginal periodontium, and can arise in association with both minor and severe periodontal breakdowns. The development of a periodontal abscess includes tissue invasion and proliferation of Fusobacterium species, P. intermedia, Prevotella nigrescens, P. gingivalis and A. actinomycetemcomitans. Epstein-Barr virus was detected in 72%, cytomegalovirus in 67%, and co-infection with the two viruses in 56% of the periodontal abscesses studied. Post-treatment, the abscess sites or the surrounding healthy periodontium revealed no herpesviruses. Tooth hypermobility was present in 90% of the abscessed teeth infected by two or more herpesviruses. An active herpesvirus infection impairing periodontal host defenses may facilitate bacterial pathogen invasion into gingival tissue with subsequent development of a periodontal abscess [5]. Surgical Complications Herpesviruses may interfere with wound healing. Periodontal surgery, tooth extraction and other types of surgical intervention to the periodontium may trigger herpesvirus reactivation, resulting in impaired clinical healing or acute inflammation. In guided tissue regeneration procedures, barrier membrane-treated sites infected with cytomegalovirus or Epstein-Barr virus showed mm less clinical attachment gain than herpesvirusnegative sites. Herpes simplex virus can also compromise periodontal regenerative and plastic treatment, and produce dry socket pathosis following tooth extraction [5]

7 Periodontal Herpesviruses in Systemically Compromised Individuals Medically compromised patients tend to exhibit advanced types of periodontitis, which may be related to repeated and prolonged herpesvirus re-activation. HIV-induced immunosuppression can trigger herpesvirus re-activation, and active herpesviruses can in turn activate latent HIV. Cytomegalovirus was recovered from 81% of HIV-associated periodontitis lesions, compared to 50% of non-hiv-associated periodontitis lesions, and from acute periodontitis, periodontal abscess formation, mandibular osteomyelitis, and refractory chronic sinusitis of HIV-infected individuals. Herpesvirus-like virions were identified by electron microscopy in 56% of gingival tissue of HIV-associated necrotizing ulcerative periodontitis. Epstein-Barr virus type 1 was identified in 72% of subgingival sites in HIV-positive patients compared to 48% in subgingival sites of HIV-negative patients. Epstein-Barr virus type 2 was detected in 57% of biopsies from HIV-associated periodontitis lesions, but was not found in non-hiv-associated periodontitis biopsies. In individuals with no clinical signs of Kaposi sarcoma, human herpesvirus-8 was detected in 24% of HIV-infected periodontitis lesions, but was not present in periodontitis sites of non-hiv-infected individuals. Cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and human herpesvirus-8 genomes are also frequently recovered from saliva of HIV-infected individuals. In summary, herpesviruses play important roles in oral pathoses associated with HIV-infection Malnourished, psychosocially stressed and other immunocompromised individuals can develop necrotizing ulcerative gingivitis or periodontitis. In Europe and USA, necrotizing ulcerative gingivitis/periodontitis occurs typically in HIV-infected individuals. In developing countries, however, necrotizing gingivitis is more common and can expand beyond the periodontium and manifest as the lifethreatening condition termed noma or cancrum oris [1]. Papillon-Lefèvre syndrome is an autosomal recessive palmoplantar keratodermal disorder associated with severe periodontitis in both the primary and the permanent dentition. In the permanent dentition, Papillon-Lefèvre periodontitis can resemble localized aggressive periodontitis. Cytomegalovirus, Epstein-Barr virus, A. actinomycetemcomitans, P. gingivalis and other periodontopathic bacteria were detected in deep periodontal lesions of an 11-year-old girl with Papillon-Lefèvre syndrome. Fanconi anemia is an autosomal-recessive disease caused by a defect in DNA repair with increased chromosomal instability, resulting in congenital abnormalities, bone marrow failure, and predisposition to squamous cell carcinomas in the head, neck, and anogenital regions. Patients with Fanconi anemia are also more prone to head-and-neck infections and periodontitis. A dual infection of cytomegalovirus and herpes simplex virus was detected in the per

8 iodontitis sites of an 11-year-old boy with Fanconi anemia [2]. Trisomy 21 subjects (Down syndrome) show high prevalence of periodontitis, even in childhood. Herpesviruses were identified in 84% of periodontitis lesions and in 5% of normal periodontal sites of Trisomy 21 young adults. Periodontitis and Non-Dental Diseases Periodontitis and Non-Oral Diseases Periodontitis has been linked statistically with 43 non-oral diseases including cardiovascular disease, stroke, pregnancy complications and rheumatoid arthritis. Cytomegalovirus and herpes simplex virus have the potential to promote the inflammatory and procoagulant conditions associated with atherosclerosis. Cytomegalovirus can change the functionalities of macrophages, smooth muscle cells, and endothelial cells, and can in vascular cells induce the expression of adhesion proteins, cytokine receptors, and major histocompatibility complex molecules. Similarly, herpes simplex virus together with periodontopathic bacteria have been linked to vascular disease [2]. Thus, cytomegalovirus and herpes simplex virus may contribute to the pathogenesis of both periodontal and vascular diseases. Periodontitis has been associated with premature birth, late miscarriage, and pre-eclampsia. Individuals of low socioeconomic position exhibit increased risk of both periodontitis and premature birth, and such women are also more prone to cytomegalovirus and herpes simplex virus infections [2]. Periodontitis has also been associated with rheumatoid arthritis, renal diseases, and premature death from neoplasms, vascular and digestive diseases. Rheumatoid arthritis in turn has been linked to Epstein- Barr virus and cytomegalovirus. Renal diseases have been associated with cytomegalovirus and other viruses. Premature death from neoplasms, vascular and digestive diseases have been linked to a herpesvirus etiology [2]. Periodontal herpesviruses entering the systemic circulation may be risk factors for those medical diseases. Porphyromonas gingivalis is also implicated in non-oral diseases. However, because periodontal herpesvirus infections can stimulate an overgrowth of periodontal P. gingivalis, the P. gingivalis-medical disease linkage may be a consequence of a periodontal herpesvirus infection [3]. High serum levels of C-reactive protein and various cytokines are present in periodontal as well as in medical diseases [1]. These inflammatory mediators may in part occur in periodontitis and medical diseases as a result of a shared herpesvirus infection. Periodontitis and Oral Diseases Epstein-Barr virus and papillomavirus type 18 coinfection has been linked to tongue carcinoma. Periodon

9 titis sites may constitute the reservoir for oncogenic herpesviruses and papillomaviruses. Periodontitis lesions in intimate contact with the tongue can also be a source of Epstein-Barr virus and cytomegalovirus for the initiation of hairy leukoplakia of the tongue and of non-neoplastic peripheral giant cell granuloma around teeth [7]. Clinical Implications Treatment that aims at decrease periodontal herpesvirus load can help in the management of severe periodontitis. Scaling and root planing can reduce high copy counts of subgingival herpesvirus which may stay suppressed for several months. Subgingival application of povidone-iodine or dilute sodium hypochlorite (bleach) can further suppress periodontal infections and are effective against both viruses and bacteria. Sodium hypochlorite at low concentrations can also interfere with nuclear factor-kappab cellular signaling and pro-inflammatory gene activation. In severe periodontitis, systemic antiviral therapy may be required to affect herpesviruses colonizing within gingival tissue. Valacyclovir (500 mg twice daily for 10 days) can suppressed high copy counts of periodontal Epstein-Barr virus to undetectable levels for at least a year, with a concomitant reduction in gingival bleeding and periodontal pocket depth [1]. The established relationships between periodontitis and cardiovascular disease, diabetes, preterm birth, aspiration pneumonia and several other medical diseases can have, as discussed above, a herpesvirus component. The intermittent shedding of herpesvirus virions from periodontitis lesions into the general circulation may cause disease in extra-oral sites, especially in immunocompromised individuals. Disease-active periodontitis lesions harboring high loads of active herpesviruses may be a particularly important source of infectious virions. Periodontopathic bacteria whose numbers increase during periods of periodontal herpesvirus reactivation may also contribute to systemic diseases [4]. Thus, decreasing periodontal herpesvirus loads by periodontal treatment may reduce the risk of non-oral disease. Surveillance testing for periodontal herpesviruses and targeted anti-herpesvirus therapy may be warranted for periodontitis patients at high risk for herpesvirus-related systemic diseases. Conclusion and Future Implications The etiopathogenesis of periodontitis includes virulence factors of herpesviruses and bacteria, host immune responses against herpesviruses and bacterial pathogens, and alteration of host-cell metabolism. Herpesviruses may induce periodontitis by activating specific tissue-destroying pathways of the immune system and by predisposing to bacterial carriage and increased bacterial load. The increased understanding of the importance of herpesviruses in periodontitis has already resulted in more effective disease management, including the use of anti-herpesvirus agents such as acyclovir and valacyclovir. Future vaccination against herpesviruses may provide the long-awaited 18 19

10 breakthrough in periodontal disease prevention. Elimination of herpesviruses from periodontal sites can improve the periodontal health status but also decrease the risk of transmitting the viruses via the general circulation to extra-oral sites or by saliva to other individuals. It seems important to treat and prevent herpesvirus infections of the periodontium for both periodontal and medical reasons. References (Readers are recommended to refer the Periodontology 2000 reviews for more detailed information on the original research findings cited in this article) 1. Slots J. Periodontal herpesviruses: prevalence, pathogenicity, systemic risk. Periodontol ; 69: Slots J. Human viruses in periodontitis. Periodontol ; 53: Slots J. Herpesviral-bacterial interactions in periodontal diseases. Periodontol ; 52: Slots J, Contreras A. Herpesviruses: a unifying causative factor in periodontitis? Oral MicrobiolImmunol. 2000; 15: Slots J. Herpesviruses in periodontal diseases. Periodontol ; 38: Slots J, Slots H. Bacterial and viral pathogens in saliva: disease relationship and infectious risk. Periodontol ; 55: Slots J. Oral viral infections of adults. Periodontol ; 49: Ting M, Contreras A, Slots J. Herpesvirus in localized juvenile periodontitis. J Periodontal Res. 2000; 35: