Herpesviralâbacterial interactions in periodontal diseases

Periodontitis is one of the most complex infectious diseases of the human body. Individual periodontal lesions may harbor millions of genomic copies of herpesviruses (179) as well as papillomaviruses, human immunodeficiency virus (HIV), human T-lymphotropic virus type 1, torquetenovirus, and hepatitis B and C viruses (190). Herpesvirus-infected periodontal sites tend to exhibit more breakdown than herpesvirus-free sites, and a herpesviral active infection is associated with an elevated risk of progressive periodontal disease (189). Furthermore, the oral cavity supports more than 700 bacterial species, and the periodontal pocket area harbors more than 400 bacterial species (148). Periodontopathic bacteria, such as Porphyromonas gingivalis and Tannerella forsythia, possess virulence factors involved in colonizing periodontal sites, neutralizing local host defenses and destroying periodontal tissues (78, 192). The host immune response attempts to control both pathogenic viruses and bacteria in periodontal sites. However, it is unclear if various immune mediators, such as certain cytokines and chemokines, exert primarily a protective or a destructive role in periodontal disease. Also, some immune mechanisms that are active against viruses may diminish antibacterial immune responses, and vice versa. It may be that periodontitis is the result of extensive and partly opposing immune responses against viral–bacterial combined infections (179). Major advances in the diagnosis, prevention and treatment of periodontitis probably depend upon a better understanding of the pathogenic infections and the associated host responses. This review article presents evidence that viruses and bacteria in aggregate produce a greater pathogenic effect than the sum of the individual agents, and discusses how the concept of a herpesviral–bacterial combined infection may change our understanding of the pathogenesis, and possibly the management, of destructive periodontal disease. Emphasis is placed on synergistic interactions between periodontal Epstein–Barr virus and cytomegalovirus, and major suspected periodontopathic bacteria. Respiratory disease represents the best-known example of serious viral–bacterial co-infections (113, 129). Worldwide, respiratory infections give rise to more disease and death than any other human infection and are one of the leading causes of morbidity and mortality (121). Seasonal influenza in the USA is estimated to result in more than 200,000 hospitalizations and 36,000 deaths annually (218). The great ‘Spanish flu’ influenza pandemic of 1918–1919 caused 20–50 million deaths worldwide and an estimated 675,000 deaths in the USA, affecting mostly young adults and not the usual children and old individuals. At the peak, half of the world’s population was clinically infected. During the influenza pandemic years of 1918–1919 (type A influenza, subtype H1N1) and 1957–1958 (type A influenza, subtype H2N2), the incidence of secondary bacterial pneumonia, the most common cause of excess mortality during influenza outbreaks, varied between 2 and 18% in different populations (123). The influenza A (swine flu) pandemic of 2009 involved a novel H1N1 strain. The main bacteria in influenza-associated pneumonia are Streptococcus pneumoniae, alpha-hemolytic streptococci, Haemophilus influenzae, Staphylococcus aureus and Moraxella (Branhamella) catarrhalis (123). A combined infection with influenza viruses and S. aureus causes particularly severe and fatal pneumonia in both children and adults (29). Respiratory viruses other than the influenza viruses can also interact pathogenically with bacterial pathogens, for example human parainfluenza virus with S. pneumoniae (50) and adenovirus with Bordetella pertussis (185). A study of community-acquired childhood pneumonia revealed that mixed viral–bacterial infections, which comprised about 75% of the pneumonias studied, resulted in more severe inflammation and clinical illness than single viral or bacterial infections (87). Viruses can induce alterations in the respiratory tract, which allow resident bacteria to multiply to levels capable of inducing inflammation (6). Respiratory tract viruses can inhibit the ciliary activity of the respiratory epithelium and thereby increase the risk for bacterial superinfection (6). Respiratory cells infected with influenza A virus, respiratory syncytial viruses or adenoviruses show enhanced bacterial adherence in both in vitro and in vivo model systems (69). The influenza virus may predispose to secondary bacterial pneumonia by denudating the respiratory epithelium and exposing basement membrane elements (e.g. fibrinogen) to which bacteria can attach (47, 123). An influenza virus infection can also cause prolonged desensitization of lung sentinel cells to toll-like receptor ligands, resulting in a reduced presence of neutrophils and an increased secondary bacterial load (39). In addition, alteration of neutrophil functions by the influenza virus may decrease the clearance of pulmonary bacteria (97, 104). Acute otitis media has been linked to synergistic interactions between viruses and bacteria (7). Otitis media is often associated with viruses, such as rhinovirus, respiratory syncytial virus, adenovirus, coronavirus, influenza virus (232), cytomegalovirus (238) and other herpesviruses (20), as well as with bacteria, including H. influenzae, S. pneumoniae, M. catarrhalis, and Prevotella and Peptostreptococcus species (19). Acute otitis media with a tympanostomy tube showed S. pneumoniae, S. aureus, H. influenzae, Pseudomonas aeruginosa and yeast (166). Viruses were detected in 65% of acute otitis media samples that were positive for H. influenzae, in 77% of samples that were positive for S. pneumoniae and in 73% of samples that were positive for M. catarrhalis (170). Tympanic membrane changes with acute otitis media are more severe in patients co-infected with respiratory viruses and bacteria than in patients who have a single infection with either of the two types of infectious agents (237). Viruses interact with bacteria in acute otitis media to boost the local inflammatory process and also have a profound adverse effect on resolution of the disease (75). Other human diseases seem also to have a combined viral–bacterial etiopathogeny. Recurrent bacterial sinusitis may develop as a complication to viral colds (5). Infectious mononucleosis caused by infection with Epstein–Barr virus can give rise to tonsillar upgrowth of Prevotella intermedia and Fusobacterium nucleatum, and subsequently pharyngotonsillitis (18). The Lemierre’s syndrome, which is characterized by severe pharyngitis and sepsis, can occur as a sequel to Epstein–Barr virus-induced mononucleosis and is frequently associated with an overgrowth of Fusobacterium necrophorum (53). Gastroenteritis is often associated with mixed infections of viruses (rotavirus, adenovirus, norovirus, astrovirus) and bacteria (pathogenic Escherichia coli, Salmonella, Shigella, Campylobacter jejuni), and the combined viral–bacterial infection can lead to enhanced pathosis (117). Appendicitis has been associated with measles virus, adenovirus and herpesviruses, as well as with Bacteroides fragilis, E. coli, Yersinia, Salmonella and Shigella (91). In Helicobacter pylori-associated gastritis, the activation of a latent Epstein–Barr virus infection by monochloramine from infiltrating neutrophils can aggravate the gastric disease (128). In organ transplant recipients, cytomegalovirus and human herpesviruses type 6 or type 7 can induce serious opportunistic infections with bacteria, fungi, protozoa and other viruses, probably as a result of immunomodulation by the herpesviruses (12, 137, 150). A renal transplant patient developed a subhepatic abscess as a result of infection with cytomegalovirus and P. gingivalis (103). Vascular diseases occur at a higher frequency as a result of a combined infection with herpes simplex virus and Chlamydia pneumoniae (165) or periodontopathic bacteria (224) than from a single infection with either of these agents. Rheumatic heart disease and autoimmune myocarditis may involve dual infection with coxsackievirus and group A streptococci, which have the potential to dysregulate immune mechanisms and trigger an autoimmune reaction (167). HIV infection causes a variety of severe bacterial, fungal and viral infections (46, 100, 174, 242). Moreover, P. gingivalis may up-regulate expression of the HIV-1 R5-specific co-receptor CCR5 in oral keratinocytes and thereby increase the risk for oral infection and dissemination of R5-tropic HIV-1 strains (58). Also, various gram-positive and gram-negative bacteria of the periodontitis biofilm have the capacity to re-activate HIV in latently infected T cells, macrophages and dendritic cells (80), and the vaginosis bacteria Prevotella bivia and Peptostreptococcus asaccharolyticus, but no other vaginal bacteria studied, are potential activators of HIV expression in monocytoid cells and T-lymphocytic cells (72). Animal models have been used to study viral–bacterial interactions. Respiratory pathosis was enhanced in mice when the influenza virus or other viruses co-infected with bacteria (198). In a mouse model of pathogenetic synergy, an influenza virus infection antecedent to an S. pneumoniae challenge caused pneumonia and led to 100% mortality, whereas an infection with S. pneumoniae prior to an influenza virus infection provided protection from influenza and improved survival rates (123). Immune mediators, including cytokines and chemokines, through toll-like receptors/mitogen-activated protein kinase pathways, seem to play important roles in the pathogenesis of the influenza virus–S. pneumoniae co-infection (184). Calves dually infected with bovine respiratory syncytial virus and Haemophilus somnus, both bovine respiratory pathogens, contracted lung disease and harbored H. somnus in the lungs, whereas the lungs of monoinfected calves showed neither pathosis nor H. somnus infection (57). Acute otitis media was induced in 63% of mice that had been stably colonized by S. pneumoniae and then infected with the influenza virus, whereas all mock-infected mice remained free of disease (122). Mice infected with herpes simplex virus, adenovirus or vaccinia virus revealed enhanced susceptibility to E. coli pyelonephritis (59). Mice inoculated intraperitoneally with murine cytomegalovirus together with P. aeruginosa, S. aureus or Candida albicans exhibited mortality rates of 80–100%, whereas immunization against murine cytomegalovirus abrogated the synergistic effect on mortality for all combinations of the infectious agents studied (70). In immunocompromised rats, a symptomatic infection with rat cytomegalovirus induced severe neutropenia and subsequently a high rate of enteric gram-negative rod bacteremia (201). A markedly higher mortality rate was found in mice co-infected with murine cytomegalovirus and P. gingivalis than in mice mono-infected with either cytomegalovirus or P. gingivalis (203). A significantly lower level of systemic interferon-gamma was detected in the dually infected mice than in the mono-infected mice, suggesting that P. gingivalis was able to reduce the antiviral interferon-gamma response and thus increase the pathogenicity of the co-infecting cytomegalovirus (203). For completeness, a latent herpesvirus infection may, in some experimental settings, up-regulate the activation state of the innate immunity, thereby providing immune benefits for the host. Mice latently infected with murine Epstein–Barr virus or cytomegalovirus can show resistance to infection with the bacterial pathogens Listeria monocytogenes and Yersinia pestis (10). However, the herpesvirus-induced protection against bacterial infection seems to be transient, lasting only a few months (236). Collectively, numerous studies of natural diseases and experimental infections conclude that viral–bacterial co-infections produce more disease than single infections with either of the two types of infectious agents. Early studies on viral–bacterial synergism emphasized a decreased clearance or an enhanced pathogenicity of the bacteria involved in the infectious complex, but more recent investigations also point to the possibility of an increased virulence of the virus. As millions of herpesviruses and bacteria can inhabit individual periodontitis sites (179), it is reasonable to assume that herpesviral–bacterial interactions also play a role in the development of human periodontitis. Studies from various countries have found genomic copies of Epstein–Barr virus and cytomegalovirus to be present in most periodontitis lesions and to be less prevalent in gingivitis and healthy periodontal sites (Table 1). Study-to-study variation in the detection of periodontal herpesviruses may be a result of (i) the clinical status of the study subjects, (ii) the viral diagnostic methods utilized, or (iii) true geographical/ethnic differences in herpesviral occurrence. Identification of periodontal herpesviruses is influenced by the sample site and method utilized. Because pathogen loads typically peak during periods of active disease, herpesvirus recovery from periodontal sites depends greatly on the accuracy of the clinical diagnosis. The difficulty of defining periodontitis for the purpose of research was recently pointed out (156). The use of patient age as a main criterion for distinguishing between ‘aggressive’ and ‘chronic’ periodontitis may not be a reliable indicator of active or stable disease. The term ‘normal’ periodontal site is sometimes used to suggest absence of attachment loss rather than inflammation-free gingiva. As herpesviruses may enter even slightly inflamed periodontal sites, sensitive polymerase chain reaction (PCR) techniques may show viruses in ‘unhealthy’ control sites. Conversely, periodontal therapy can markedly reduce herpesvirus subgingival counts and thus cause a significant underestimation of the viral load associated with untreated disease (177). Reliable data on the prevalence and quantity of periodontal herpesviruses may require study of virgin lesions with a well-defined disease-activity status. Studies that examine a small number of periodontal sites in each subject will inevitably underestimate the prevalence of herpesviruses in the study population and the total number of herpesvirus genome-copies in the entire periodontium. Studies of herpesviruses in subgingival sites will also not account for the substantial herpesvirus counts residing within inflamed gingival tissue (98, 168). The era of relying upon in vitro cell culture for routine laboratory diagnosis of viral infections has truly passed. Viral isolation indicates an active and possibly disease-producing infection, but isolation of viruses is difficult, costly and time-consuming. High-fidelity PCR-based techniques have become the standard for identification and quantification of periodontal herpesviruses (99, 146), and the transcription of late herpesvirus genes for structural proteins is understood to signify productive viral replication and is commonly used to indicate an active herpesvirus infection (60, 124). PCR identification of oral herpes simplex virus may yield two- to fourfold more positive samples than viral culture (42, 227). Nested PCR may unveil more periodontal sites that are positive for cytomegalovirus than viral culture or real-time PCR (16) or end-point detection PCR (23). Nested PCR technology is particularly efficient in detecting low viral loads (168). However, PCR primers that amplify templates of microbial communities at different efficiencies may yield biased results (86). Misamplification and false-positive herpesvirus results may emerge when there are shared regions of nucleotide sequences between herpesvirus species and unknown infectious agents. Nevertheless, as periodontal Epstein–Barr virus and cytomegalovirus have been identified using a variety of PCR primers in platforms of end-point detection PCR, nested PCR, reverse transcription PCR and real-time PCR, the risk of a systematic misidentification of these viruses is small. However, different PCR primers and protocols may detect herpesviruses with a varying degree of proficiency (28), and technical expertise and quality assurance methods vary among laboratories (102, 144, 183). Preparation of the target nucleic acid constitutes a particularly vulnerable stage of PCR protocols. The genotype distribution and seroprevalence of Epstein–Barr virus (82, 175) and cytomegalovirus (147, 168) differ among populations. Similarly to medical infections (157), some herpesvirus subtypes may exhibit increased periodontopathogenicity. Glycoprotein B (gB) homologues within the herpesvirus family participate in virus entry and cell-to-cell spread, and the encoding gene is commonly used in genotyping (145). The cytomegalovirus gB-II genotype seems to predominate in chronic periodontitis, whereas the cytomegalovirus gB-I genotype is more closely related to gingivitis and a healthy periodontium (235). In addition, herpesviruses occur at a higher prevalence in low-income countries than in affluent countries (3), and the herpesvirus seroprevalence in high-income countries exhibits significant racial, educational and socioeconomic disparities, starting at an early age and persisting into middle age (43, 44, 243). Epstein–Barr virus and cytomegalovirus genomes have been identified in periodontitis lesions with prevalences ranging from a few per cent to more than 80% (Table 1). Cytomegalovirus seropositivity was identified in 95% of patients with periodontitis but in only 74% of patients with gingivitis (P = 0.057) (88). Epstein–Barr virus (105) and cytomegalovirus (240) are also common inhabitants of peri-apical lesions of endodontic origin. Cytomegalovirus was detected in 32% of peri-apical abscesses exhibiting spontaneous pain (23). To assess, in more detail, the linkage between herpesviruses and periodontitis, patients have been grouped according to disease severity. Table 1 reveals the presence of Epstein–Barr virus in 65% of aggressive periodontitis lesions and in 49% of chronic periodontitis lesions and of cytomegalovirus in 44% of aggressive periodontitis lesions and in 44% of chronic periodontitis lesions. Although the prevalence of Epstein–Barr virus and cytomegalovirus was similar in aggressive and chronic periodontitis, the herpesvirus infection may differ qualitatively in the two diseases. An active (‘productive’) cytomegalovirus infection tends to be associated with periodontal sites showing no radiographic evidence of alveolar crestal lamina dura (211, 219), a finding consistent with disease-active periodontitis (158). An active cytomegalovirus infection has also been linked to increased gingival inflammation in immunosuppressed organ-transplant recipients (141). An acute herpes simplex virus-type 1 infection in a 26-year-old patient caused, within a few hours, extensive gingival recession (155). By contrast, a latent (‘nonproductive’) herpesvirus infection is generally associated with chronic periodontitis sites exhibiting little propensity for disease progression (16). Quantitatively, significantly more herpesvirus genomic copies inhabit progressive and untreated periodontitis lesions than stable or treated periodontitis sites (89, 90, 179). As many as 8.3 × 108 Epstein–Barr virus DNA copies and 4.6 × 105 cytomegalovirus DNA copies have been detected in samples of individual periodontal pockets (179), and even higher viral loads may reside within the gingival tissue of periodontitis lesions (98). Sunde et al. (208) identified up to one million genome-copies of Epstein–Barr virus in periodontal sites of a 63 year-old patient with refractory periodontitis. Herpesviruses other than Epstein–Barr virus and cytomegalovirus (110, 118, 138, 152, 168), as well as papillomavirus and other viruses (189), can also inhabit periodontitis lesions. Indeed, the total count of viruses approaches that of bacteria in some advanced periodontitis lesions. The abundance of herpesvirus genomic copies in individual periodontitis lesions translates into a heavy viral load for the entire periodontium of patients with severe and widespread periodontal disease. The high number of herpesviral copies in progressive periodontitis has implications for our understanding of the etiology of the disease. Table 2 summarizes statistical relationships between periodontal herpesviruses and bacteria. A study in China found that 17% of Epstein–Barr virus-positive periodontitis lesions, and 54% of cytomegalovirus-positive periodontitis lesions, contained as many as six to eight species of major periodontopathic bacteria (40). Periodontal Epstein–Barr virus and cytomegalovirus seem to be most closely associated with P. gingivalis and T. forsythia (179), two bacteria with high periodontopathic potential (78). The link between cytomegalovirus and P. gingivalis appears to be particularly strong (193). Cytomegalovirus and P. gingivalis were linked to localized aggressive (juvenile) periodontitis in Jamaica with odds ratios of 4.6 and 7.8, respectively, but the cytomegalovirus–P. gingivalis dual infection was linked to the disease with an odds ratio as high as 51.4 (125). The significantly higher odds ratio of the cytomegalovirus-P. gingivalis co-infection than of the sum of the individual pathogens is suggestive of a pathogenetic synergy between the infectious agents. A study, in the USA, of 140 adults with gingivitis or periodontitis, linked Epstein–Barr virus-1 and cytomegalovirus to elevated occurrence of the pathogens P. gingivalis, T. forsythia, P. intermedia, Prevotella nigrescens and Treponema denticola (Table 3). In a study from Japan, P. gingivalis comprised 0.25% of total salivary bacterial counts in Epstein–Barr virus-positive periodontitis patients but only 0.02% (a in Epstein–Barr patients Periodontal Epstein–Barr virus and cytomegalovirus have also been related to the subgingival presence of the major An between herpesviruses and bacteria has also been in pathosis simplex virus may participate in periodontal disease in a of 138, simplex in with P. gingivalis, T. forsythia, P. intermedia or has been linked to periodontitis in with T. T. forsythia or to periodontitis and a of studies of populations are to the of a between various herpesviral–bacterial and periodontal disease severity. is also to the to which herpesviral–bacterial co-infections in periodontitis significant interactions or an of each of the infectious agents. 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