Skip Navigation

High Prevalence of Epstein-Barr Virus Type 2 among Homosexual Men Is Caused by Sexual Transmission

  1. Debbie van Baarle1,2,
  2. Egbert Hovenkamp2,
  3. Nicole H. T. M. Dukers5,
  4. Neil Renwick3,
  5. Marie José Kersten4,
  6. Jaap Goudsmit3,
  7. Roel A. Coutinho3,5,
  8. Frank Miedema2,3 and
  9. Marinus H. J. van Oers1
  1. 1Department of Hematology, CLB, and Laboratory for Experimental and Clinical Immunology, Amsterdam, The Netherlands
  2. 2Department of Clinical ViroImmunology, CLB, and Laboratory for Experimental and Clinical Immunology, Amsterdam, The Netherlands
  3. 3Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
  4. 4Department of Immunology and Medical Oncology, Netherlands Cancer Institute, Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, The Netherlands
  5. 5Department of Public Health, Municipal Health Service, Amsterdam, The Netherlands
  1. Reprints or correspondence: Dr. D. van Baarle, Dept. of Clinical Viro-Immunology CLB, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands (d_van_baarle{at}clb.nl).
 
Next Section

Abstract

To investigate whether Epstein-Barr virus (EBV) type 2 infection is highly prevalent among homosexual men, the prevalence of EBV type 2 was studied among homosexual and heterosexual white men who were at high and low risk for sexually transmitted diseases; these data were correlated with sexual behavior. The prevalence of EBV type 2 among homosexual men was significantly higher than it was among heterosexual men (39% vs. 6%). Among high-risk heterosexual men, prevalence was significantly higher than it was among low-risk heterosexual men (15% vs. 0). In univariate analyses, EBV type 2 infection in homosexual men was significantly associated with human immunodeficiency virus (HIV) seropositivity, increased numbers of intercourse partners, non-Dutch nationality, and human herpesvirus 8 seropositivity. In multivariate analyses, an independent association with EBV type 2 was observed only for HIV seropositivity and number of sex partners. These data support the conclusion that EBV type 2 infection is more prevalent among white homosexual men and is caused by sexual transmission.

Epstein-Barr virus (EBV), a γ-herpesvirus, causes widespread, persistent infection of the human population [1]. Two EBV types exist, 1 and 2, which differ in the genes encoding transformation-associated EBV nuclear antigen (EBNA)-2 [2, 3] and EBNA-3A, -3B, and -3C [4] and, therefore, display distinct biologic differences. Both virus types can be transmitted orally and infect B lymphocytes in vivo. In addition, EBV can be transmitted via a hematogenous route, through sexual contact.

In healthy persons, it has been shown that only a single EBV strain is present in peripheral blood B lymphocytes [5]. Type 1 strains are more prevalent in white and Asian populations, whereas both types are common in Africa and New Guinea. However, within the white population, human immunodeficiency virus (HIV) type 1-infected persons are exceptional with regard to their EBV status; they frequently are infected with EBV type 2, and a high percentage of AIDS patients is super-infected with types 1 and 2 [6]. This has been ascribed to immunodeficiency.

Interestingly, among HIV-infected patients with hemophilia, the prevalence of EBV type 2 (super)infection was found to be much lower than that among HIV-infected homosexual men [7], which suggests that HIV-induced immunosuppression is not the principal explanation for EBV superinfection. Recently, we used a type-specific polymerase chain reaction (PCR) directly on peripheral blood mononuclear cells (PBMC) to study EBV type prevalence in different groups of HIV-1-infected persons [8], including those progressing to AIDS who developed non-Hodgkin's lymphoma, opportunistic infections, or Kaposi's sarcoma (AIDS-KS), as well as long-term asymptomatic persons. We found high prevalence of EBV type 2 in all groups (50%–62%). However, infection with EBV type 2 was not related to the degree of immunodeficiency and frequently was already found early in HIV-1 infection. Therefore, these results might be a reflection of high EBV type 2 prevalence among homosexual men. In addition, our finding that patients with AIDS-KS, in particular, had a high prevalence of EBV type 2 infection suggested that type 2 infections might be associated with specific sexual behavior, because the Kaposi's sarcoma-inducing human herpesvirus 8 (HHV-8) has been shown to be sexually transmitted, probably by orogenital contact [9, 10]. To further test this hypothesis, we extended the EBV type analysis to groups of HIV-1-seronegative homosexual and heterosexual men and correlated the results with more-detailed data on sexual behavior.

Previous SectionNext Section

Materials and Methods

Study population

We analyzed blood samples from 85 HIV-1-seropositive homosexual men and 113 HIV-1-seronegative homosexual men who all were participants of the Amsterdam Cohort studies on AIDS and HIV-1 infection. These persons at risk for HIV-1 infection visited the Municipal Health Services every 3 months, when a medical history and physical examination were carried out, blood samples were collected for HIV-1 serologic and immunologic studies, and PBMC were cryopreserved. At entry and every 6 months, participants completed a standardized behavioral questionnaire.

Furthermore, we studied blood samples from 108 healthy blood bank donors, 54 men and 54 women (“low-risk heterosexuals”), as well as 60 randomly selected heterosexual men who visited the Amsterdam outpatient clinic for sexually transmitted diseases (“high-risk heterosexuals”). The median age was 39 years (range, 21–66 years) and did not differ between groups.

Lymphocyte isolation and B cell lines

PBMC were isolated from heparinized blood by ficoll-hypaque density centrifugation. B cells were purified by positive selection by use of CD19 micro-beads and MiniMacs (Milteny Biotec, Bergisch Gladbach, Germany) according to the protocol described by the manufacturer.

The B95.8 and Ag876 cell lines were used as sources of EBV type 1 and EBV type 2, respectively. The EBV-negative B cell line BJAB was used as a negative control.

DNA extraction and PCRfor EBV typing

B cells were lysed by addition of L6 lysis buffer. Genomic DNA was extracted by precipitation with isopropanol, and 1–2 μg was amplified in a nested PCR in 50-μL reactions containing 5 μL of 10 × PCR buffer, 1.5 mM MgCl2, 10 mM dNTPs, and 1 U of DNA Taq polymerase, as described elsewhere [8], with the use of non-type-discriminating EBNA-2 primers in the first reaction. The region within the EBNA-2 gene that discriminates EBV type 1 from EBV type 2 was amplified with type-specific nested primers. After PCR, the identity of the amplified EBV fragment was confirmed by Southern blot analysis with γ32P(dATP)-labeled type-specific probes.

The assay was shown to be type specific and could detect as few as 5 EBV copies/104 EBV-negative human diploid genomes or genomes containing the other EBV type.

HHV-8 serology

Serum samples were tested for antibodies to recombinant HHV-8 lytic-phase capsid (open-reading frame [ORF] 65) antigen and latency-associated nuclear (ORF73) antigen by EIA, as described elsewhere [11].

Statistical analysis

For comparison of EBV type 2 prevalence between homosexual and high- and low-risk heterosexual men, χ2 tests were used. Among homosexual men, we assessed risk factors for EBV type 2 infection. Several demographic and sexually related variables were examined. Because the number of sex partners is the number during the 5 years that preceded study entry through the date of EBV typing and, thus, depends on the time the participant was in follow-up, we represented this variable as the number of sex partners corrected for time since follow-up. Logistic regression was used to obtain univariate and multivariate odds ratios and 95% confidence intervals, to quantify variation in estimates. Multivariate analyses were done with a stepwise forward procedure (SPSS, version 7.5, for Windows; SPSS, Chicago).

Previous SectionNext Section

Results

EB V types in homosexual and heterosexual men

EBV type was analyzed among 85 HIV-1-infected homosexual men, 113 HIV-1-seronegative homosexual men, and 114 HIV-1-seronegative heterosexual men by means of a type-specific nested PCR assay that could detect as few as 5 EBV copies/104 human diploid genomes. Within these groups, EBV sequences could be detected in 97% (n = 81), 80% (n = 90), and 82% (n = 93) of the subjects, respectively. As shown in figure 1A, the proportion of total EBV type 2 infection (type 2 and dual infection with type 1) was significantly lower among HIV-negative homosexual men (39%) than it was among HIV-positive homosexual men (67%; P < .001). In addition, EBV type 2 prevalence in the total group of HIV-negative heterosexual men was 6%, significantly lower than that among HIV-negative homosexual men (P <. 001).

Figure 1
View larger version:
Figure 1

Epstein-Barr virus (EBV) type prevalence among EBV-positive human immunodeficiency virus (HIV)-1-infected homosexual men and EBV-positive, HIV-seronegative homosexual and heterosexual men (A) and among EBV-positive, HIV-negative heterosexual men at high and low risk for sexually transmitted diseases (B). EBV typing was done by use of type-specific polymerase chain reaction analysis on purified B cells. *Significantly different (χ2, P <. 001).

As shown in figure 1B, none of the heterosexual men who were at low risk for sexually transmitted diseases (n = 53) were infected with EBV type 2. The same result was obtained for 54 HIV-negative heterosexual women who were at low risk (data not shown). In contrast, within the group of heterosexual men who were at high risk for sexually transmitted diseases (n = 40), 10% harbored EBV type 2 only and an additional 5% had a dual infection. The EBV type 2 prevalence among high-risk heterosexual men (15%) was significantly higher than that among low-risk heterosexual men (0%; P <. 001).

EBV-2 infection in homosexual men in relation to sexual behavior

Data on sexual behavior and HHV-8 serologic studies were available for the 171 homosexual men (table 1). Subjects who harbored an EBV type 2 strain (n = 88) were compared with those without EBV type 2 infection (n = 83). Univariate analysis showed that EBV type 2 prevalence increased with the number of sex partners. Also, HIV-seropositive men, HHV-8-seropositive men, and men with non-Dutch nationality had a higher prevalence of EBV type 2 infection. No association was found with specific sexual techniques, although there was a borderline significant trend toward higher EBV type 2 prevalence with higher numbers of partners engaging in oroanal sex (active, receptive, or both).

Table 1
View larger version:
Table 1

Prevalence of Epstein-Barr virus (EBV) type 2 infection among 171 homosexual men, by sexual behavior and other characteristics.

In multivariate analysis, EBV type 2 infection was statistically significantly associated with HIV infection and the number of sex partners. Homosexual men with >500 sex partners had a nearly 10 times greater risk of being infected with EBV type 2 than did men with <100 sex partners. HIV-infected homosexual men had an almost 4 times greater risk of having an EBV type 2 infection (table 1).

Previous SectionNext Section

Discussion

Until now, analysis of EBV strains in B lymphoblastoid cell lines has shown that 1%–3% of healthy white EBV carriers in Europe harbor a type 2 strain [6]. To our knowledge, this is the first study in which EBV type prevalence was investigated among white homosexual men and was compared with that among heterosexual individuals by use of direct EBV type analysis of peripheral blood B lymphocytes. We observed a high prevalence of EBV type 2 infection among homosexual men, compared with that among heterosexual men, and obtained evidence that EBV type 2 is sexually transmitted, at least among homosexual men.

Our study provides evidence that, in western Europe, EBV type 2 is transmitted by sexual contact. First, among homosexual men, we found a significant association between EBV type 2 infection and a higher number of partners. This high EBV type 2 prevalence could not be attributed to a specific sexual technique, although there was a trend in the number of partners with whom oroanal sex was practiced. Second, HIV-positive homosexual men have higher EBV type 2 prevalence than do HIV-negative homosexual men. This was not due to the degree of immunodeficiency, as shown in our previous study [8]. A more likely explanation may be that HIV-positive persons tend to have sex with HIV-seroconcordant persons, leading to more EBV type 2 exposure for those who are HIV-positive. This also has been suggested as an explanation for HHV-8 prevalence [10].

Third, EBV type 2 prevalence among high-risk heterosexual men was significantly higher than that among low-risk heterosexual men, suggesting that EBV type 2 infection also is related to sexual behavior in heterosexual contacts.

Interestingly, EBV type 2-infected homosexual men had a higher prevalence of HHV-8 infection than did EBV-1-infected homosexual men, in the univariate analysis. Because HHV-8 prevalence also increases with an increase in the number of sex partners, this suggests that EBV type 2 may resemble HHV-8 in its mode of sexual transmission [9, 10].

The fact that EBV type 2 prevalence among HIV-negative homosexual men is higher than that among HIV-negative heterosexual men probably was not caused by differences in EBV load (recent data in our laboratory show no difference in EBV load between persons infected with type 1 or type 2) and supports the recently proposed idea that EBV type 2 infection is endemic among white homosexual men [7]. This could be explained by the fact that EBV type 2 has been introduced into this community by sexual contacts with persons from areas where EBV type 2 is endemic and is further transmitted by multiple sexual contacts.

Sexual transmission of EBV is quite likely, because EBV is shed from many mucosal sites, including both female and male genital tracts [12]. One group reported that, among homosexual men, a higher percentage shed virus from the genital tract (50%) than from the oropharynx (25%) [13]. In addition, EBV has been detected in the anal region of sexually active homosexual men [14]. The fact that EBV type 2 is difficult to contract, despite the high prevalence among homosexual men, argues against kissing as the main route of transmission, and sexual transmission is likely in this study group. In parts of Africa, where EBV type 2 prevalence is high among the adult population, as well as among children with Burkitt's lymphoma, EBV type 2 probably is acquired by oral transmission during childhood.

The fact that homosexual men with >500 partners had a nearly 10-fold-greater risk of EBV type 2 infection, whereas this infection was only moderately increased in homosexual men with 100–400 sex partners, indicates that EBV type 2 is not easily transmitted and, therefore, requires high exposure. Although the mechanism of this relatively poor efficiency of transmission of EBV type 2 remains to be established, it may explain why EBV type 2 infection is highly prevalent among persons with multiple sex partners and virtually is not found among the general white population.

Previous SectionNext Section

Acknowledgments

This study was part of the Amsterdam Cohort Studies on AIDS and human immunodeficiency virus (HIV) type 1 infection, a collaboration of the Municipal Health Service, the Academic Medical Center, and the CLB. We thank H. Fennema for collecting blood samples from HIV-seronegative heterosexual white men and I. Spijkerman for initial analyses.

Previous SectionNext Section

Footnotes

  • Financial support: Dutch Cancer Society (grant 96–1168); Dutch AIDS Fund (grant 1007).

  • Received December 6, 1999.
  • Revision received February 24, 2000.
Previous Section
 

References

  1. 1.↵
    1. Fields BN,
    2. Knipe DM
    1. Miller G
    . Epstein-Barr virus: biology, pathogenesis and medical aspects. In: Fields BN, Knipe DM, editors. Fields' virology. 2d ed. New York: Raven Press; 1990. p. 1921-58.
  2. 2.↵
    1. Adldinger HK,
    2. Delius H,
    3. Freffe UK,
    4. Clarke J,
    5. Bornkamm GW
    . A putative transforming gene of the Jijoye virus differs from that of Epstein-Barr virus prototypes. Virology 1985;141:221-34.
    CrossRefMedlineWeb of Science
  3. 3.↵
    1. Aitken C,
    2. Sengupta SK,
    3. Aedes C,
    4. Moss DJ
    . Heterogeneity within the Epstein-Barr virus nuclear antigen 2 gene in different strains of Epstein-Barr virus. J Gen Virol 1994;75:95-100.
    Abstract/FREE Full Text
  4. 4.↵
    1. Sample J,
    2. Young LS,
    3. Martin B,
    4. Chatman T,
    5. Kieff E,
    6. Rickinson AB
    . Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes. J Virol 1990;64:4084-92.
    Abstract/FREE Full Text
  5. 5.↵
    1. Yao QY,
    2. Rowe DT,
    3. Martin B,
    4. Young LS,
    5. Rickinson AB
    . The Epstein-Barr virus carrier state: dominance of a single growth-transforming isolate in the blood and in the oropharynx of healthy virus carriers. J Gen Virol 1991;72:1579-90.
    Abstract/FREE Full Text
  6. 6.↵
    1. Sculley TB,
    2. Apolloni A,
    3. Hurren L,
    4. Moss DJ,
    5. Cooper DA
    . Coinfection with A- and B-type Epstein-Barr virus in human immunodeficiency virus-positive subjects. J Infect Dis 1990;162:643-8.
    Abstract/FREE Full Text
  7. 7.↵
    1. Yao QY,
    2. Croom-Carter D,
    3. Tierney RJ,
    4. et al
    . Epidemiology of infection with Epstein-Barr virus types 1 and 2: lessons from the study of a T cell-immunocompromised hemophilic cohort. J Virol 1998;72:4352-63.
    Abstract/FREE Full Text
  8. 8.↵
    1. van Baarle D,
    2. Hovenkamp E,
    3. Kersten MJ,
    4. Klein MR,
    5. Miedema F,
    6. Van Oers MHJ
    . Direct Epstein-Barr virus (EBV) typing on peripheral blood mononuclear cells: no association between EBV type 2 infection or superinfection and the development of acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma. Blood 1999;93:3949-55.
    Abstract/FREE Full Text
  9. 9.↵
    1. Martin JN,
    2. Ganem DE,
    3. Osmond DH,
    4. Page-Shafer KA,
    5. Macrae D,
    6. Kedes DH
    . Sexual transmission and the natural history of human herpesvirus 8 infection. N Engl J Med 1998;338:948-54.
    CrossRefMedlineWeb of Science
  10. 10.↵
    1. Dukers NHTM,
    2. Renwick N,
    3. Prins M,
    4. et al
    . Risk factors for human herpesvirus 8 seropositivity and HHV-8 seroconversion in a cohort of homosexual men. Am J Epidemiol 2000;151:213-24.
    Abstract/FREE Full Text
  11. 11.↵
    1. Renwick N,
    2. Halaby T,
    3. Weverling GJ,
    4. et al
    . Seroconversion for human herpesvirus 8 during HIV infection is highly predictive of Kaposi's sarcoma. AIDS 1998;12:2481-8.
    CrossRefMedlineWeb of Science
  12. 12.↵
    1. Naeher H,
    2. Gissmann L,
    3. Freese UK,
    4. Petzoldt D,
    5. Helfrich S
    . Subclinical Epstein-Barr virus infection of both the male and female genital tract—indication for sexual transmission. J Invest Dermatol 1992;98:791-3.
    CrossRefMedlineWeb of Science
  13. 13.↵
    1. Israele V,
    2. Shirley P,
    3. Sixbey JW
    . Excretion of the Epstein-Barr virus from the genital tract of men. J Infect Dis 1991;163:1341-3.
    Abstract/FREE Full Text
  14. 14.↵
    1. Naeher H,
    2. Lenhard B,
    3. Wilms J,
    4. Nickel P
    . Detection of Epstein-Barr virus in anal scrapings from HIV-positive homosexual men. Arch Dermatol Res 1995;287:608-11.
    CrossRefMedlineWeb of Science
| Table of Contents

This Article

  1. J Infect Dis. (2000) 181 (6): 2045-2049. doi: 10.1086/315521
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most