Prevalence of some herpesviruses in gingival crevicular fluid

Journal of Clinical Virology 34 (2005) 147–152

Prevalence of some herpesviruses in gingival crevicular fluid P. Klemenc a,∗ , U. Skaleriˇc b , B. Artnik c , P. Nograˇsek b , J. Marin a b

a Institute of Microbiology and Immunology, Medical Faculty, Zaloˇska 4, 1000 Ljubljana, Slovenia Department of Oral Medicine and Periodontology, Medical Faculty, Hrvatski trg 6, 1000 Ljubljana, Slovenia c Department of Public Health, Medical Faculty, Zaloˇska 4, 1000 Ljubljana, Slovenia

Received 18 October 2004; received in revised form 10 January 2005; accepted 1 March 2005

Abstract Background: The herpesviruses, ancient pathogens which have co-evoluted with human, are etiologically associated with a number of diseases, from asymptomatic to oncogenic and mortal diseases. It seems that some of them have also an important role in the pathogenesis of human periodontal disease. Objective: This study aimed to determine the prevalence of Epstein–Barr virus (EBV), human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8) and human cytomegalovirus (HCMV) in gingival crevicular fluid (GCF) and, eventually, to find the correlation between specific virus types and clinical parameters which are important in periodontitis, like plaque index (PI), gingival index (GI) and probing depth (PD). Study design: A polymerase chain reaction (PCR) and digestion of PCR products with restriction endonuclease were employed to identify the presence of EBV, HHV-6, HHV-8 and HCMV. Results: Out of 66 samples of GCF taken from the patients with periodontal disease, EBV was found in 29 (43.9%), HHV-6 in 16 (24.2%) and HCMV in 2 (3%) samples, while in the samples of healthy persons, these viruses were not found. HHV-8 was detected neither in the patients with periodontitis nor in healthy control group. More positive results were found in clinical samples taken from people with higher PI and GI and in the samples taken from the patients with medium PD (PD = 3–6 mm). In all HHV-6 positive samples, we found only variant A; as for EBV positive samples, type A and type B were identified and also co-infection with the two types. It seems that there is a correlation between PI, PD and EBV types, but no correlation was found between EBV types and GI or HHV-6 types and PI, PD, GI. Conclusions: The present findings confirm some association between herpesviruses and human periodontitis. © 2005 Elsevier B.V. All rights reserved. Keywords: Epstein–Barr virus (EBV); EBV type A; EBV type B; Human herpesvirus 6 (HHV-6); HHV-6 variant A; HHV-6 variant B; Human herpesvirus 8 (HHV-8); Human cytomegalovirus (HCMV); Periodontal disease; Gingival crevicular fluid (GCF); Plaque index (PI); Gingival index (GI); Probing depth (PD)

1. Introduction Periodontitis is an infectious disease that involves the activity of specific bacteria, yeasts and probably herpesviruses. Theoretically, viral infections may facilitate the destruction of periodontal tissues by immunosuppression, immunemediated tissue destruction or by lytic activity against periodontal cells. Human cytomegalovirus (HCMV) and Epstein–Barr virus type A (EBV type A) assume particularly close relationship with human periodontitis. Epstein–Barr virus type B (EBV type B), human herpesvirus 6 (HHV-6) and herpes simplex virus (HSV) seem to exhibit less important ∗

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or no association with most types of destructive periodontal diseases (Contreras et al., 1997, 1999, 2000; Contreras and Slots, 1996; Hanookai et al., 2000; Parra and Slots, 1996). But the latest studies report that periodontium might constitute the site of infection or reservoir for human herpesvirus 6, 7 and 8 (HHV-6, -7, -8) (Mardirossian et al., 2000). With improved molecular diagnostics, today we cannot only identify different viruses, but also different virus strains or types (Bornkamm et al., 1980). So, in the early 1980s two different types of EBV, designated either as type A and B (or type 1 and 2), have been described. These strains can be distinguished from each other on the basis of sequence polymorphism in their EBNA 2, 3, 4, and 6 genes. Serological and PCR investigations indicated that both types have a worldwide distribution, although type A appears to

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be more prevalent in Europe, Japan and America, and EBV type B in Africa and New Guinea. A co-infection with the two types is not uncommon (Abdel-Hamid et al., 1992; Apolloni and Sculley, 1994; Barrle et al., 1999; Borisch et al., 1993; Buck et al., 1999; Epstein et al., 2001; Klemenc, 2000; Sixbey et al., 1989). A very similar situation regarding EBV exists for HHV6 which appears in two variants, variant A and B, between which there is a 1–5% nucleotide sequence variation in conserved core genes. It seems that HHV-6B is more common associated with exanthema subitum (ES) and other pediatric febrile illnesses. On the other side, HHV-6A is frequently detected in the patients with chronic fatigue syndrome (Braun et al., 1997). To delineate the possible relationship between different herpesviruses (EBV, CMV, HHV-6 and HHV-8) and clinical parameters, which are important in defining periodontitis, like plaque index (PI), gingival index (GI), and probing depth (PD), we wanted to determine the prevalence of these viruses in the gingival crevicular fluid (GCF) of the patients with progressive periodontitis and in GCF of the people without sings of periodontal disease. After that we wanted to find out in EBV and HHV-6 positive samples if virus types and variants correlate with PI, GI and PD. 2. Material and methods 2.1. Clinical samples Of the patients with periodontitis (aged from 31–74 years, mean 50.95), 66 GCF samples were taken from randomly

chosen teeth. In all of them, three clinical parameters–PI, GI and PD were defined. Thirty specimens of persons with no clinical signs of gingivitis or periodontitis (aged from 16–24 years, mean 19.43) were taken as negative control material. The specimens were taken by sterile paper points inserted into the pockets and retained for 30 s. After sampling, the paper points were put in 50 ␮l of phosphate buffered saline (PBS) and homogenized by vortex mixing. The paper points were than removed and eluted samples were stored at −70 ◦ C prior to analysis. 2.2. DNA isolation and PCR The samples were thawed and genomic DNA was extracted by QIAamp DNA Mini Kit (QIAGEN, GmbH, Germany). The isolation procedure was checked by PCR, using the primers designed for beta-globin gene (PC03, PC04). EBV DNA was identified by PCR with the primers designed for conserved region of EBV genome–gp 350/220 region. A nested PCR method was used to define the type of EBV. We used EBNA-2.1.A and EBNA-2.1.B as outer primers; inner primers for EBV type A were EBNA-2.2.A and EBNA-2.2.B, and for EBV type B EBNA-2.2.C and EBNA2.2.D (Borisch et al., 1993). All the primers used are listed in Table 1. In brief, each PCR reaction test tube contained 10× PCR buffer, 0.2 mM mixture of dNTP, 30 pmol each of primers, 1.25 U of Taq polymerase, various concentrations of MgCI2 , 10 ␮l of DNA, and water to the final volume of 50 ␮l. PCR amplification was performed in a thermal cycler Mastercycler® gradient (Eppendorf, Germany). The first am-

Table 1 Sequences of primers used to amplify EBV, HHV-6, HHV-8 and CMV DNA with product size Nucleotide sequence (5 –3 )

Product size (bp)

ACA CAA CTg TgT TCA CTA gC CAA CTT CAT CCA CgT TCA CC

123

EBV gp220 A gp220 B EBNA-2A.1 EBNA-2.1.B EBNA-2.2.A EBNA-2.2.B EBNA-2.2.C EBNA-2.2.D

ggC Tgg TgT CAC CTg TgT TA CCT Tag gAg gAA CAA gTC CC agg gAT gCC Tgg ACA CAA gTg CTg gTg CTg CTg gTg g TCT TgA TAg ggA TCC gCT Agg ATA ACC gTg gTT CTg gAC TAT CTg gAT C AgA CTT AgT TgA TgC CCT Ag AgA CTT AgT TgA TgC CCT Ag

240

HHV-6 661 662 663 664

CAAgCCCTAACTgTgTATgT TCTgCAATgTAATCAgTTTC CTgggCggCCCTAATAACTT ATCgCTTTCACTCTCATAAg

325 or 553

HHV-8 KS1 KS2

AgCCgAAAgTCCACCAT TCCgTgTTgTCTACgTCCAG

233

HCMV MIE4 MIE5

CCAAgCggCCTCTgATAACCAAgCC CAgCACCATCCTCCTCTTCCTCTgg

Name PC03 PC04

600 497 140

195 or 423

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plification round included an initial denaturation step at 95 ◦ C for 4 min, followed by 35 cycles of denaturation at 95 ◦ C for 1 min, primer annealing at 66 ◦ C for 2 min, and an extension step at 72 ◦ C for 1 min, followed by final extension step at 72 ◦ C for 5 min. Nested PCR was used for the detection and subtyping of HHV-6 according to previous report (Wang et al., 1996). The basis for this PCR was an insert of 228 bp fragment in the putative immediate-early (IE) region of HHV-6 variant B, which was lacking in HHV-6 variant A. The PCR protocol was the same as for EBV, except the concentration of outer primers 661 and 662 which was 50 pmol. Inner primers 663 and 664 were used at the concentration of 30 pmol. The PCR program for the two PCR runs consisted of 30 cycles for both rounds, consisting of 1 min at 94 ◦ C, 1 min at 50 ◦ C (first round) or at 60 ◦ C (second round), and 1 min at 72 ◦ C. After the last cycle, the extension step was extended to 10 min at 72 ◦ C. PCR analysis for HHV-8 was done with primers KS1 and KS2, which amplified 233 bp DNA fragment (Minhui et al., 2001). To test the specificity of PCR, the amplified PCR product was digested for 2 h at 37 ◦ C with 4U Pstl restriction endonuclease (Promega, Madison, USA). The endonuclease digestion products resulted in 95 bp and 138 bp fragments. Primers MIE4 and MIE5 at the concentration of 40 pmol were used for the detection of HCMV immediate early region (Ratnamohan et al., 1992); the protocol and program were the same as described for EBV PCR. In all PCR runs, negative and positive controls were included: DNA free water as negative control, positive control for EBV was DNA isolated from cell lines B95-8 for type A and Ag876 for EBV type B, for HHV-6 DNA isolated from control virus obtained from ATCC, for HCMV DNA purified from the urine of the CMV positive patient after liver transplantation diagnosed in our laboratory, and for HHV-8, DNA from a human Kaposi’s sarcoma biopsy lesion containing HHV-8, also diagnosed in our laboratory. Amplicons were identified by electrophoresis in 2% agarose gel (Sigma, GmbH, Germany) containing 0.5 ␮g/ml of ethidium bromide.

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2.3. Statistical analysis χ2 -test was used to compare differences in EBV, HHV-6, HHV-8 and HCMV occurrence between different values of PI, GI and PD. P-values equal to or below 0.05 were considered statistically significant.

3. Results An adequate DNA template for PCR and absence of PCR inhibitors we were confirmed in all clinical materials with amplification of 123 bp fragment of beta-globin gene. All 66 clinical samples taken from the patients with periodontitis were analyzed according to PI, GI, PD and for the presence of DNA of EBV, HHV-6, HHV-8 and HCMV. DNA of EBV was confirmed in 29 (43.9%) out of 66 samples, DNA of HHV-6 in 16 (24.2%) clinical materials, and HCMV DNA in 2 (3%). HHV-8 DNA was not detected. The clinical materials of control group were negative for EBV, HHV-6, HHV-8 and HCMV DNA. On the basis of PI and GI, the samples were divided into two groups. In the group of samples which were taken from the patients with PI values 2 and 3, a higher percentage of positive results for EBV, HHV-6 and HCMV were observed as compared to the samples from the second group that included the patients with PI values of 0 or 1. This difference between the two groups was statistically significant (P < 0.05) (Table 2). A higher percentage of positive results–58.3% for EBV, 33.3% for HHV-6 and 5.6% for HCMV was also found in the samples of the patients with GI = 2 when compared to the samples obtained from the areas with mild inflammation or none at all (GI = 0 or 1) (Table 2). Regarding to PD, the samples were divided into 6 groups as shown in Table 2. EBV DNA was found in all groups–starting with one positive case (12.5%) in the PD ≤ 2 mm group and with increasing number of positive cases in the PD = 3 and 4 mm groups where the prevalence of EBV was the highest (up to 73.3%). A decreased number

Table 2 Presence of EBV, HHV-6 and CMV DNA in 66 samples and correlation to PI, GI and PD Number of samples

Number of positive samples (%)

P-value

EBV

CMV

HHV-6

EBV

CMV

HHV-6

PI

0 or 1 2 or 3

43 23

9 (21%) 20 (87%)

0% 2 (8.7%)

3 (7%) 13 (56.5%)

0.05

0.05

0.0005

GI

0 or 1 2

30 36

8 (26.7%) 21 (58.3%)

0% 2 (5.6%)

4 (13.3%) 12 (33.3%)

0.01

>0.05

>0.05

PD

≤2 mm 3 mm 4 mm 5 mm 6 mm 7 mm

8 13 15 9 7 14

1 (12.5%) 6 (46.1%) 11 (73.3%) 5 (55.6%) 2 (28.6%) 4 (28.6%)

0% 0% 1 (6.7%) 1 (11.1%) 0% 0%

0% 0% 5 (33.3%) 7 (77.8%) 4 (57.1%) 0%

P0–2/4 = 0.05*

*

Range of values which are statistically significant.

P4/6 = 0.05* P4/7 = 0.05*

P2/5 = 0.01* P2/6 = 0.05* P3/4 = 0.05* P3/5 = 0.005* P3/6 = 0.01* P4/5 = 0.05*

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Table 3 Correlation between EBV types and different values of PI, GI and PD Type A

Type B

Type A and B

PI

0 or 1 2 or 3

4 (44%) 5 (25%)

2 (22%) 12 (60%)

3 (33%) 3 (15%)

GI

0 or 1 2

3 (37.5%) 6 (28.6%)

2 (25%) 7 (33.3%)

3 (37.5%) 8 (38.1%)

PD

≤2 mm 3 mm 4 mm 5 mm 6 mm 7 mm

1 (100%) 0% 0% 2 (40%) 1 (50%) 4(100%)

0% 4 (67%) 7 (64%) 3 (60%) 1 (50%) 0%

0% 2 (33%) 4 (36%) 0% 0% 0%

of positive results was observed in the PD = 5, 6 and 7 mm groups. So, we found 55.6% (5 out of 9) of samples taken from the patients with PD = 5 mm to be EBV positive. In the last two groups of samples (PD = 6 and 7 mm), the percentage of EBV positive samples was lower. In these two groups, we found 28.6% of EBV positive samples. In cases with low PD (PD ≤ 3 mm), HHV-6 and HCMV DNA were not found. HCMV DNA was found only in one case with PD = 4 mm and in one case with PD = 5 mm, but this difference is not significant (P > 0.05) in comparison to other PD groups. HHV-6 DNA was found more often in cases with PD = 4, 5 and 6 mm (Table 2). So, we found HHV-6 DNA in 77.8% of patients with PD = 5 mm and in 57.1% of patients with PD = 6 mm. In all clinical samples identified as EBV or HHV-6 positive, the type of EBV and HHV-6 variants were defined as described earlier (Table 1) and looked for the correlation with PD, PI and GI. The correlation of EBV types with PI, GI and PD is illustrated in Table 3. The results suggest that there is a relationship between the type of EBV and both PI and PD. There was no correlation found between EBV types and GI. In the two groups of patients regarding GI values, very similar percentages of EBV type A, EBV type B and even co-infection with the two types were found (Table 3). Overall, type B prevailed in the group of patients with higher PI values (PI = 2/3). It was confirmed in 12 (60%) out of 20 samples obtained from the patients with PI = 2/3. Furthermore, co-infection with type A was proved in 3 (15%) clinical materials taken from the patients with PI = 2/3. Type A solely was found in 5 (25%) samples. At lower values of PI, we did not reveal any observable discrepancy in EBV type distribution. If we compare the group of samples with PI = 0/1 and the group with PI = 2/3, we can see that the percentage of type B is higher in PI = 2 or 3 group, while type A alone and co-infection with type B were more frequently found in PI = 0 or 1 group (Table 3). Regarding EBV types and PD, we realized that the two EBV types were detected only in the samples with a medium grade of PD. These samples were obtained from the patients with PD values of 3 and 4 mm. In the group with PD = 3 mm, we found 2 (33.3%) out of 6 samples which harbored the two EBV types and 4 (36.4%) out of 11 samples of the patients

with PD = 4. PD 5 mm was the border at which type A and B were separately present in equal number of samples. From that point on, the percentage of type A showed an increasing tendency (Table 3). But, despite the fact that it seems that there is some correlation between PI, PD and EBV types, no correlation has been found between PI, PD, GI and HHV-6 variants, as only variant A was determined in HHV-6 positive samples.

4. Discussion The present findings confirm and extend the previous data from other authors associating herpesviruses with destructive periodontal disease (Contreras et al., 2000; Parra and Slots, 1996). This study demonstrated a relatively higher prevalence of EBV (43.9%) and HHV-6 (24.2%) in GCF of the patients with periodontitis than in clinically healthy people who did not harbor a detectable amount of viral DNAs. HCMV DNA was found in two subjects with periodontitis, while HHV-8 DNA was not found. The correlation between EBV and HHV-6 positivity and clinical parameters, which are important in defining periodontitis, proved a possible role of some herpesviruses in pathogenesis of periodontal disease. Namely, the presence of EBV and HHV-6 DNA was established in much higher percentage in the samples with greater PI and GI values. The study yielded somehow different results concerning PD, where EBV and HHV-6 were confirmed in higher percentage when investigating samples with a medium PD values range (PD = 3–6 mm) than in the samples with higher or lower PD values (Table 2). It is possible to conclude that this is the site at which viruses beside bacteria are an additional factor which triggers initiation and facilitates exacerbation of the disease. At PD of lower values, viruses were found only in few samples. When PD was higher (PD = 6, 7 mm), we suppose that there might have been an ideal condition for bacterial growth which was probably essential for the progression of periodontitis. All herpesviruses after primary infection persist in different parts of human body for lifetime, but certain circumstances, like decreased immune response, stress and/or different diseases, can trigger the reactivation of latent virus. Therefore, gingival lesions occur either directly, on account of lytic viral activity or indirectly, through immune system (Contreras and Slots, 1996; Parra and Slots, 1996). The lytic activity of virus might result in the lesions of epithelium which cause digression of the epithelium from the tooth cement and the formation of periodontal pockets which enhance epithelial attachment loss. Viral proteins expressed on damaged epithelial cells can act as binding sites for bacteria. These conditions in periodontal pockets (PD = 4, 5 mm) induce binding, and subsequently, the colonization of anaerobic and other pathogenic bacteria. Thus, the late phase of disease (PD > 5 mm) offers very

P. Klemenc et al. / Journal of Clinical Virology 34 (2005) 147–152

good conditions for a vigorous replication and growth of bacteria. Secondly, if viruses are involved in the pathogenesis of periodontal disease through the host’s immune system, elevated numbers of bacteria in dental plaque (assessed by plaque index) induce an extensive immune response. A part of immune response is also the inflammation process which results in infiltration of lymphocytes, macrophages and monocytes. Therefore, much more immunocompetent cells, especially EBV-infected lymphocytes are found in the sites of colonization (Contreras et al., 1999; Hanookai et al., 2000). These lymphocytes are incapable to eliminate bacteria and their products (such as endotoxins); therefore, the overgrowth of bacteria leads to exacerbation of periodontal disease. In the next step of our study, we wished to determine a possible role of a specific virus type in the progression of periodontal disease. Our findings showed that, in GCF, we can find either EBV type A or B and even co-infection; the prevalence of single type depends especially on two critical clinical parameters in periodontitis, PI and PD. Quite different situation was at HHV-6 positive samples were we found only HHV-6 variant A. The persistence of EBV in a healthy immunocompetent person has been thought to be characterized by the dominance of a single EBV type. Sixbey et al. were the first to report the finding of both EBV types by PCR in the throat wash of healthy viral carriers (Sixbey et al., 1989). They suggested that both types of EBV compete effectively at mucosa surface (Sixbey et al., 1989), but the two types were also demonstrated in PMNC of healthy viral carriers (Falk et al., 1997; Klemenc, 2000; Sculley et al., 1990; Srivastava et al., 2000). Today, we know that the deletions in EBNA 2 gene of type B contribute to a decreased transformation ability of lymphocytes B. But it seems that the decreased transformation ability can be compensated by an increased lytic activity of type B that allows this virus type to survive in vivo (Buck et al., 1999). The lytic activity of virus is one of the primary factors in the pathogenesis of periodontal diseases, which tends to explain the dominance of type B over type A in the progressive periodontal disease. It leads to the destruction of gingival tissue and permits the establishment of conditions for overgrowth of bacteria. Besides, lytic lesions provoke slight oral immunosuppression, which additionally triggers the reactivation of latent EBV (Contreras et al., 1999; Hanookai et al., 2000; Parra and Slots, 1996) and thereby creates conditions for actively replicating type B of EBV. Turning to PD values, we can conclude that, at a certain point (PD = 4–5 mm), the dominance of type B declines and type A takes prevalence. This situation is probably the result of lower transformation efficiency, growth rate and saturation density of EBV type B (Buck et al., 1999). In the future, controlled clinical and virological studies, which should include quantitative PCR and other methods,

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