Conceptual Emergence of Human Herpesvirus 8 (Kaposi’s Sarcoma-associated Herpesvirus) as an Oral Herpesvirus

Abstract

Recognition of the various clinico-epidemiologic forms of Kaposi’s sarcoma, a disease putatively caused by an infectious agent, did not provide ready clues as to how that agent might be transmitted, although fecal and sexual routes were implicated. Application of serologic and genome-detection assays, and cell-culture studies following the identification of human herpesvirus 8 as the causative agent now implicate that virus as one that is orally shed. While oral transmission of the virus might account for the viral endemicity in Africa and Mediterranean countries, why it is particularly prevalent among male homosexuals in the West remains more difficult to explain. Such explanation may be sought from behavioral studies into the role saliva plays in sexual interactions.

Introduction

This review traces the progression in the development of the concept of human herpesvirus (HHV)-8, or Kaposi’s sarcoma-associated herpesvirus, as an oral herpesvirus. An “oral herpesvirus” is defined here as one that persists in the mouth and is shed in the oral cavity, but may not necessarily lead to the development of an oral disease. Thus, the following may be classified as bona fide oral herpesviruses: cytomegalovirus and Epstein-Barr virus, which cause disease; and HHV-6 and HHV-7, which do not. I would not consider human simplex viruses 1 and 2 and varicellazoster virus as true oral viruses, since they do not establish persistence in the mouth, and are shed there only following ganglionic activation.

Kaposi’s Sarcoma: HHV-8’s Hallmark Disease

Clinico-epidemiological Varieties

Four epidemiological forms of KS, based on clinical and epidemiological differences, are recognized: classic KS, endemic or African KS, iatrogenic KS, and epidemic or AIDS-associated KS. Classic KS occurs most frequently in elderly patients of Mediterranean, Jewish, or Arabic descent and is more common in men than in women. It is a relatively mild disease with little internal organ involvement. Nodules, firm and purple-blue or reddish-brown, occur on the legs or arms and increase slowly over years or decades (Safai, 1984).

African KS had persisted in the sub-Saharan regions for many decades preceding the HIV epidemic. It is usually more aggressive than the classic form. Both adults and children are affected, lymph node involvement being a common feature (Safai, 1984; Ziegler and Katongole-Mbidde, 1996).

The third form, iatrogenic KS, is directly related to drug-induced immunosuppression, and reduction or cessation of immunosuppressive drug therapy often leads to remission of KS disease. People from certain ethnic backgrounds, including those of Mediterranean or Middle-Eastern origin, are at increased risk of acquiring post-transplantation KS, and continue to be at increased risk even if they are born in or migrate to a region of lower incidence (Franceschi and Geddes, 1995; Al-Khader and Shaheen, 2004).

AIDS-associated KS became prominent in the wake of the AIDS epidemic in KS non-endemic countries. It was first described by Friedman-Kien (1981) as a new and aggressive form of the disease, affecting a large number of previously healthy young homosexual men in New York City. HIV-infected men who had sex with men (MSM) showed a 50% greater lifetime risk of developing KS at the start of the AIDS epidemic than did other HIV-infected persons (Katz et al., 1994). In the West, AIDS-KS is most common among MSM, less so in HIV-positive heterosexuals, and virtually non-existent in persons who contract HIV parenterally (Beral et al., 1990). In Africa, however, AIDS-KS is widespread (Boshoff and Weiss, 2001), its prevalence varying according to geographic region but affecting men more often than women. Regardless of its geographical occurrence, the disease course is often short, with patients surviving only weeks to months following the onset of symptoms.

HHV-8 as the Causative Agent of KS

Chang et al.(1994) reported the discovery, by representational difference analysis, of DNA sequences belonging to a unique herpesvirus (HHV-8) in KS lesional tissues. DNA unique to this virus was sought by PCR from KS lesional tissues taken from patients in North America (Ambroziak et al., 1995), Asia (Su et al., 1995), Europe (Boshoff et al., 1995a; Dupin et al., 1995), and Africa (Schalling et al., 1995), thereby linking the presence of KSHV DNA with all epidemiological forms of KS.

HHV-8 DNA has been amplified in the peripheral blood mononuclear cells (PBMCs) of HIV-positive individuals, and its presence predicts the subsequent development of KS. Moore et al.(1996) studied paired PBMC samples taken before and after KS onset from AIDS-KS patients, high-risk HIV-positive MSM patients, and single PBMC samples from low-risk HIV-positive hemophiliacs, and detected HHV-8 DNA in 52% of AIDS-KS patients prior to the onset of KS, and in only 9–13% of members in the control groups. Another study showed that 55% of anti-HIV- and anti-HHV-8-positive patients without KS developed the disease within 30 months of follow-up, vs. 9% of HHV-8-seronegative/HIV-seropositive patients (Whitby et al., 1995). Furthermore, the HHV-8 detection rate in patients with KS was improved as the patient became more immunosuppressed. Such dynamic relationships between HHV-8 detectability and the development of KS supported the hypothesis that HHV-8 was causally linked to KS.

Is KS a True Sarcoma?

It is not established whether KS is a true cancer or a reactive hyperplasia. Although the spindle-shaped cells are thought to be neoplastic, they lack aneuploidy (Delli Bovi et al., 1986). Furthermore, very few spindle-like cells are present in the early patch/plaque stage. Complete remission of disease can occur, even in severely immunocompromised patients, if immunosuppression is relieved, a feature not characteristic of a true sarcoma. Clonality is often used to define a neoplasm, since a true malignancy originates from a single cell. X-linked inactivation assays have been attempted to establish clonality, but findings have varied. Thus, Rabkin et al.(1997) showed that KS is a monoclonal cancer and that the dissemination of KS in a patient arises from a monoclonal population of circulating neoplastic cells. However, Gill et al.(1998) found multiple lesions in a single patient to be of different clonal origin, and discovered some evidence of polyclonal inactivation patterns in other lesions. Judde et al.(2000), in studying the number of terminal repeats contained in the fused terminal repeat region of HHV-8 genomes carried in KS lesions, demonstrated HHV-8 clonality in nodular lesions of KS, indicating that HHV-8 was present prior to tumor growth. If one views these various findings together, it would appear that KS in its early stages is a non-clonal proliferation of endothelial cells which then evolves into a true clonal disease.

KS Cell Types Infected by HHV-8 and the Histogenesis of KS

Endothelial cells are the primary infected cells in KS lesions (Boshoff et al., 1995b). HHV-8 DNA and RNA transcripts have been detected, by in situ hybridization, in the spindle-shaped cells of KS lesions. HHV-8 DNA was detected, by in situ PCR, in all nodular KS tumors but not in the surrounding epidermis and dermis or in other control tissues (Boshoff et al., 1995b). RNA transcripts have been detected in all plaque/patch and nodular stages of KS in spindle cells of the tumor, but not in surrounding healthy tissue or control tissue from other proliferative disorders (Staskus et al., 1997), although macrophages can also be found to carry HHV-8-specific RNA and antigens (Blasig et al., 1997). Most cells in late KS tumors appear to be latently infected, as is evidenced by the expression of latency-associated transcripts and antigens in endothelial and spindle cells in the KS lesions (Sturzl et al., 1997; Kellam et al., 1999).

In early lesions, epithelial cells may be found to be infected by HHV-8. Thus, in an early, developing KS lesion, the HHV-8 latent nuclear antigen could be observed to be expressed in ductal epithelial cells within the lesion (Webster-Cyriaque, 2002). The endothelial cells in KS also bear antigenic markers that are selectively expressed by lymphatic endothelium (Kahn et al., 2002; Xu et al., 2004). Such expression appears to be due to HHV-8 infection of vascular endothelial cells, which drives them to differentiate to lymphatic endothelial cells (Carroll et al., 2004; Wang et al., 2004). Nonetheless, the initial cell types infected by HHV-8 that set the stage for an endothelium-dominant lesion like KS to develop are unknown. Competing models have been put forward, one based on the initial infectablity of B-lymphoctyes (Corey et al., 2002), and the other of oral epithelial cells (Duus et al., 2004). The latter is based on firmer evidence, as will be discussed in more detail later in this review.

Epidemiological Studies of KS and HHV-8 Infection

Early Studies

Beral et al.(1990) published pivotal findings on the epidemiology of KS among patients with AIDS. KS in the United States was observed to be at least 20,000 times more common in persons infected with HIV than in the general population. Furthermore, AIDS patients were 300 times more likely to be affected by KS than were other groups of immunosuppressed individuals. Moreover, North American homosexual men who reported sexual contact with men from the US AIDS epicenters (San Francisco, Los Angeles, and New York) were more likely to acquire KS (Beral et al., 1990; Archibald et al., 1992). The evidence pointed to an infectious agent, other than HIV, that was responsible for the development of KS. Enquiries into sexual practices of MSM in London and several cities in the United States identified insertive oral-anal sexual contact to be the main risk factor for KS; this activity tended to be associated with other activities, such as receptive and insertive anal intercourse, and receptive and insertive fisting (Beral et al., 1992; Darrow et al., 1992; Peterman et al., 1992). It was hypothesized that the agent of KS is a microbe transmitted primarily by fecal contact. Findings from other MSM cohorts (Lifson et al., 1990), however, do not support this hypothesis.

Seroprevalence and Behavioral Studies

The report of Chang et al.(1994) on the discovery of HHV-8 was soon followed by that on the sequence and organization of the full HHV-8 genome (Russo et al., 1996), and also on body-cavity-associated B-cell lymphoma (BCBL) (also referred to as primary effusion lymphoma) as being associated with the virus (Cesarman et al., 1995). Knowledge of the viral genomic organization permitted the prediction of the nature of viral proteins that could be expressed, paving the way for the production of viral antigens for antibody assays (Simpson et al., 1996). The observation that BCBL cells propagated in vitro could continue to carry replicating HHV-8 was followed up by the use of these cells as ready sources of antigens for immunofluorescence antibody assays (Gao et al., 1996; Kedes et al., 1996; Chatlynne et al., 1998). A flurry of reports on antibody prevalences quickly followed.

In Europe and North America, the anti-HHV-8 seroprevalence rate in HIV-seropositive homosexual men was observed to be the highest of any risk group. A study of men in the San Francisco area, from samples dating back to 1984, reported a seroprevalence of 48% among homosexual or bisexual men who were HIV-seropositive but were unaffected by KS (Martin et al., 1998). A similar study conducted in Amsterdam between 1984 and 1996 reported a baseline HHV-8 incidence of 30% among homosexual men who were also HIV-seropositive but had not developed KS (Dukers et al., 2000). Similar anti-HHV-8 seroprevalence values for HIV-1-seropositive homosexual men (~ 30%) have been described elsewhere in North America and Western Europe (Gao et al., 1996; Kedes et al., 1996; Simpson et al., 1996). HIV-1-uninfected homosexual men consistently show a lower seroprevalence of HHV-8 infection than do HIV-1-infected homosexual men (Kedes et al., 1996; Martin et al., 1998; Melbye et al., 1998). These levels of seropositivity were higher than what was being observed among blood donors in Europe and North America. The findings, in aggregate, indicated a strong propensity for HHV-8 to be transmitted via activities of MSM.

Possible linkages between sexual behavior and HHV-8 seropositivity have been investigated. Martin et al.(1998) collected data regarding sexual practices from men participating in the San Francisco Men’s Health Study, and confirmed that the prevalence of HHV-8 infection was higher in homosexual men, both with and without pre-existing HIV infection, than in the general population. This increased seroprevalence correlated with the degree of homosexual activity reported by the participants in the preceding five years. The association between the number of sexual partners and HHV-8 infection was supported by findings from a study of young homosexual men recruited from the San Francisco Young Men’s Health Study, which showed that the risk for HHV-8 infection was positively correlated with the number of sexual partners (Blackbourn et al., 1999). Various sexual practices have been found to be associated with the increased risk for HHV-8 infection among MSM: anal-genital sexual contact (Melbye et al., 1998), insertive oral-anal sexual contact (Grulich et al., 1999), insertive and receptive orogenital contact (Dukers et al., 2000), and deep kissing (Pauk et al., 2000). The Amsterdam Cohort study (Dukers et al., 2000) revealed that oral sex among homosexual men was a more important predictor of HHV-8 infection than was the number of sexual partners. Nonetheless, these studies exemplify the difficulty in pinpointing any one sexual practice as being associated with HHV-8 transmission (Martin and Osmond, 2000). It has been considered that, since oral-genital contact and deep kissing are common practices among MSM, saliva would be an inefficient vehicle of transmission; otherwise, HHV-8 infection would be universal in MSM as well as among heterosexuals (Martin and Osmond, 2000; Dukers and Rezza, 2003).

Persons attending Western sexually transmitted disease clinics, including heterosexual men and women, show slightly increased seroprevalence when compared with the general population (Melbye et al., 1998; Whitby et al., 1999). Such studies are consistent with sexual HHV-8 transmission between heterosexuals, but also indicate that heterosexual sexual activity carries significantly less risk of transmission than male homosexual activity.

While some correlation between number of sexual partners and HHV-8 infection has been found in African studies (Sitas et al., 1999), the high prevalence of HHV-8 infection in children is indicative of a significant non-sexual route of transmission. Gessain et al.(1999) found that in Cameroon, HHV-8 infection was common among children. The overall seroprevalence in children and adolescents was 28%. From the age of 4 years, the seroprevalence was observed to increase to 39% among 12- to 14-year-olds, and then to 48% in children over 15 years of age, approaching the level reported in adults.

A Zambian study also discovered that the pattern of HHV-8 infection was different from that of a sexually transmitted infection. During 1985, the rate of HIV infection was highest in persons aged 20–29 years and was not observed in any person over 50 years of age. In contrast, HHV-8 infection was already high (47%) in adolescents between the ages of 14 and 19 years, increasing with age (Olsen et al., 1998). This pattern of HHV-8 infection is consistent with childhood non-sexual transmission co-existing with sexual transmission throughout adulthood.

Gessain et al.(1999) found that vertical transmission between mother and child was rare in Cameroon, while Calabro et al.(2000) reported that all infants born to HHV-8-seropositive mothers who tested positive for HHV-8 antibodies at 3 months became seronegative by 24 months. Therefore, it is likely that children acquire infection through casual contact in the home and community, rather than via vertical transmission (Plancoulaine et al., 2000; Bourboulia et al., 2001).

Plancoulaine et al.(2000) studied a population of villagers of African origin in French Guiana, and found a high correlation in anti-HHV-8 seroprevalence between mother and child, and between siblings, suggesting the predominance of intra-household horizontal transmission. No significant correlation in anti-HHV-8 serostatus between spouses was identified, however. In South Africa, transmission between mothers and their children occurs at a rate of 30% or more, and the probability of HHV-8 transmission was increased in mothers with a high HHV-8 antibody titer (Sitas et al., 1999). In that population, very few children of anti-HHV-8-seronegative mothers under the age of 10 years are anti-HHV-8-seropositive, positing the mother as the source of most transmissions.

In Mediterranean and Middle Eastern countries, evidence of HHV-8 infection in children also supports non-sexual transmission, although the route of transmission is less clear than that for Equatorial and Southern Africa. In Egyptian children, the anti-HHV-8 seroprevalence is as high as 45%, the seroprevalence increasing steadily up to 10 years of age, stabilizing thereafter (Andreoni et al., 1999). Primary HHV-8 infection, which may be associated with a febrile illness and skin rash in Egyptian children, has been positively associated with close contact with at least two other children in the community (Andreoni et al., 2002).

In Italy, ~ 5% of children tested were carrying HHV-8 antibodies, with no significant variation noted between children living in the North and those living in the South of Italy (Whitby et al., 2000). Clustering of HHV-8 seroprevalence among spouses, children, and siblings was demonstrated in a Sardinian study sample, without an apparent mother-child predominance, indicating horizontal HHV-8 in the family along broader, non-sexual routes (Angeloni et al., 1998).

The picture emerging from sero-epidemiological studies in African and Mediterranean HHV-8-endemic regions is that there is significant non-sexual horizontal HHV-8 transmission in subpopulations at no, or little, risk of sexually transmitted infection, and transmissions occur commonly in the household context. Wojcicki (2003) has suggested that, in the African context, ethnographic practices that often involve salivary exchanges likely play a significant role in HHV-8 transmission.

Body Fluids and Discharges That May Transmit HHV-8

HHV-8 genomic DNA was sought by PCR techniques from gastrointestinal tissues of MSM with KS, and could be found in a substantial proportion (46%) of rectal tissues (Thomas et al., 1996). Nonetheless, HHV-8 DNA could not be amplified from fecal samples of patients with AIDS-associated KS (Whitby et al., 1995).

HHV-8 DNA can be detected in prostate tissue biopsies of HIV-infected men (Diamond et al., 1998). The HHV-8 genome, however, is rarely detected in the semen of patients with KS, and almost never in healthy semen donors (reviewed in Boshoff and Weiss, 2001). Seminal transmission of HHV-8 therefore seems to contribute little to HHV-8 endemicity.

Urinary shedding of HHV-8 has been reported (Cattani et al., 1999; Cannon et al., 2003; Beyari et al., 2004). The frequency of shedding is consistently low (~ 5%) across the different geographical regions studied so far and independent of the HIV serostatus of the subjects sampled. While objects contaminated with HHV-8-carrying urine might plausibly act as vehicles of transmission if licked or ingested, it seems unlikely that HHV-8 endemicity would be enhanced by such routes.

While HHV-8 may be transmitted by blood or blood products (Blackbourn et al., 1997; Cannon et al., 2001), transfusion-related transmissions are rare. Moreover, the prevalence of HHV-8 infection is not remarkably higher among injecting drug users than in the general population (Rezza et al., 1998). Blood is therefore unlikely to play an important contributory role to endemicity, in both KS endemic and non-endemic regions.

The findings of Dukers et al.(2000), pointing to oral sexual activity as the most significant practice associated with HHV-8 infection, implicated saliva as a vehicle of transmission among MSM. Indications that HHV-8 might be tropic for the oral mucosa came from a report by Di Alberti et al.(1997), revealing the frequent amplification by PCR of HHV-8 DNA from tissue biopsies taken at various sites in HIV-infected patients. Later studies revealed that salivary HHV-8 DNA may be detected in American HIV-infected/KS-positive, HIV-infected/KS-negative, and HIV-uninfected/KS-positive persons (Koelle et al., 1997; Blackbourn et al., 1998). Moreover, HHV-8 in the cell-free fraction of saliva was determined to be infectious (Vieira et al., 1997). Subsequently, Pauk et al.(2000) reported the localization, in mucosal samples obtained from American homosexual men with no apparent KS disease, of HHV-8 DNA and RNA to the epithelial cells of the buccal mucosa and, to a lesser extent, to those of the genital tract. It is also significant that HHV-8 DNA was detected at higher titers in saliva than in samples from other body sites, and that some men shed virus into saliva at consistently high titers, despite showing no signs of clinical KS disease. The dominance of oral shedding among MSM has been confirmed by later studies (Gandhi et al., 2004; Marcelin et al., 2004).

Investigations have shown that in Africa, too, HHV-8 has a predilection to be shed in saliva. A study of Egyptian children with acute fever not due to a specific viral exanthem showed that, in anti-HHV-8-seropositive children, HHV-8 DNA sequences could be amplified from saliva in 30%, compared with 8% in whom sequences could be amplified from plasma only, while in seronegative children, HHV-8 DNA could be detected in saliva of 12% compared with 4% in plasma (Andreoni et al., 2002); thus, the children preferentially carried HHV-8 in saliva than in blood. A study conducted in Malawi of asymptomatic family members of patients with KS found HHV-8 DNA in mouthrinses of 27% of the study individuals, but not in any of their blood samples (Cook et al., 2002). Further work in this study group showed that orally shed HHV-8 may originate from the buccal mucosa, palatal mucosa, and parotid glands (Beyari et al., 2003). Subsequent studies conducted in other African communities have confirmed the relatively high rate of oral HHV-8 shedding (Brayfield et al., 2004; Mbulaiteye et al., 2004; Taylor et al., 2004).

Cell Culture Studies

Early in vitro studies have cast doubts on whether epithelial cells would be able to support HHV-8 replication fully. Foreman et al.(1997) co-cultivated cells from primary KS lesions with cells of an epithelial cell line, and found that 1% of the cells could support productive HHV-8 infection; furthermore, virus could be recovered after serial passage. However, Blackbourn et al.(2000) examined 25 lines for infectibility by HHV-8, and while finding that 7 supported HHV-8 entry and gene expression, could identify only one (of endothelial origin) from which infectious virus could be recovered. Renne et al.(1998) examined 39 cell lines, and found that 11 could support at least low-level spontaneous lytic replication, but were unable to identify one that could support efficient serial passage. Another study showed that primary human keratinocytes could be infected by HHV-8 and support some degree of productive replication, but their support of replication was transient (Cerimele et al., 2001). Subsequently, Bechtel et al.(2003) found that most adherent cell lines they studied, regardless of species of origin or tissue lineage, were permissive for viral entry, but could not support full lytic replication.

More recently, primary and immortalized oral epithelial cells have been found to be susceptible to infection by laboratory-adapted and wild-type HHV-8 (Duus et al., 2004). Transmission electronmicroscopy revealed abundant production of HHV-8 virions in buccal cells derived from healthy donors. The reason why previous cell culture studies utilizing epithelial cell lines failed is attributed to the inoculation of virus after the cells have formed a monolayer. Duus et al.(2004) inoculated virus while the cells were still in the process of adhering, i.e., during the phase in the cellular life cycle when the cellular receptors for the virus (integrin α₃β₁ and heparan sulphate) (Akula et al., 2001, 2002) would be expressed to high levels. The findings of Duus et al.(2004) are significant, providing strong evidence that oral epithelial cells indeed constitute a reservoir of HHV-8 infection, consistent with the earlier observations of Di Alberti et al.(1997) and Pauk et al.(2000).

HHV-8 as a bona fide Oral Virus

Initial findings into the epidemiology of KS among MSM wrongly implicated the causative agent of KS to be fecally transmitted. It has now become clearer—following epidemiological, serological, and virological studies into other MSM populations infected by KS, MSM infected by HHV-8, and non-MSM populations in Africa and the Mediterranean—that saliva is the principal vehicle of HHV-8 transmission. The virus is shed into saliva from multiple sites of the mouth and at higher titers than into any other body fluid. Cell culture studies are pointing to the oral epithelium as being highly susceptible to HHV-8 infection and carriage. HHV-8 legitimately joins the ranks of EBV, CMV, and HHV-6 and-7 as a true oral herpesvirus.

An unusual question is how saliva facilitates the transmission of HHV-8 among MSM, since salivary exchanges are common among heterosexuals as well. It is possible that such exchanges are conducted more frequently and intensely between MSM than among heterosexuals. More refined behavioral studies could be carried out into the practices preceding, accompanying, and following sexual contact that might contribute to the high HHV-8 endemicity among MSM. In non-MSM populations living in HHV-8-hyperendemic areas, the insight into HHV-8 as an oral herpesvirus, and therefore one whose spread cannot be curtailed by behavioral change or the prophylactic administration of antiviral drugs, should lead to fresh impetus for the development of vaccines against HHV-8. It will be by vaccination that the near-universal spread of HHV-8 can be controlled and the adverse consequences of its infection prevented from developing.

Footnotes

Presented at the Fifth World Workshop on Oral Health and Disease in AIDS, Phuket, Thailand, July 6–9, 2004, sponsored by Prince of Songkla University, Thailand, the International Association for Dental Research, the World Health Organization, the NIDCR/National Institutes of Health, USA, and the University of California-San Francisco Oral AIDS Center.

References

Akula SM, Pramod NP, Wang FZ, Chandran B (2001). Human herpesvirus 8 envelope-associated glycoprotein B interacts with heparan sulfate-like moieties. Virology 284:235–249.

Akula SM, Pramod NP, Wang FZ, Chandran B (2002). Integrin alpha3beta1 (CD 49c/29) is a cellular receptor for Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells. Cell 108:407–419.

Al-Khader AA, Shaheen FA (2004). Posttransplant complications encountered in renal transplantation in the Middle East. Transplant Proc 36:180–183.

Ambroziak JA, Blackbourn DJ, Herndier BG, Glogau RG, Gullett JH, McDonald AR, et al. (1995). Herpes-like sequences in HIV-infected and uninfected Kaposi’s sarcoma patients. Science 268:582–583.

Andreoni M, El-Sawaf G, Rezza G, Ensoli B, Nicastri E, Ventura L, et al. (1999). High seroprevalence of antibodies to human herpesvirus-8 in Egyptian children: evidence of nonsexual transmission. J Natl Cancer Inst 91:465–469.

Andreoni M, Sarmati L, Nicastri E, El Sawaf G, El Zalabani M, Uccella I, et al. (2002). Primary human herpesvirus 8 infection in immunocompetent children. J Am Med Assoc 287:1295–1300.

Angeloni A, Heston L, Uccini S, Sirianni MC, Cottoni F, Masala MV, et al. (1998). High prevalence of antibodies to human herpesvirus 8 in relatives of patients with classic Kaposi’s sarcoma from Sardinia. J Infect Dis 177:1715–1718.

Archibald CP, Schechter MT, Le TN, Craib KJ, Montaner JS, O’Shaughnessy MV, et al. (1992). Evidence for a sexually transmitted cofactor for AIDS-related Kaposi’s sarcoma in a cohort of homosexual men. Epidemiology 3:203–209.

Bechtel JT, Liang Y, Hvidding J, Ganem D (2003). Host range of Kaposi’s sarcoma-associated herpesvirus in cultured cells. J Virol 77:6474–6481.

10.

Beral V, Peterman TA, Berkelman RL, Jaffe HW (1990). Kaposi’s sarcoma among persons with AIDS: a sexually transmitted infection? Lancet 335:123–128.

11.

Beral V, Bull D, Darby S, Weller I, Carne C, Beecham M, et al. (1992). Risk of Kaposi’s sarcoma and sexual practices associated with faecal contact in homosexual or bisexual men with AIDS. Lancet 339:632–635.

12.

Beyari MM, Hodgson TA, Cook RD, Kondowe W, Molyneux EM, Scully CM, et al. (2003). Multiple human herpesvirus-8 infection. J Infect Dis 188:678–689.

13.

Beyari MM, Hodgson TA, Kondowe W, Molyneux EM, Scully CM, Porter SR, et al. (2004). Genotypic profile of human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus) in urine. J Clin Microbiol 42:3313–3316.

14.

Blackbourn DJ, Ambroziak J, Lennette E, Adams M, Ramachandran B, Levy JA (1997). Infectious human herpesvirus 8 in a healthy North American blood donor. Lancet 349:609–611.

15.

Blackbourn DJ, Lennette ET, Ambroziak J, Mourich DV, Levy JA (1998). Human herpesvirus 8 detection in nasal secretions and saliva. J Infect Dis 177:213–216.

16.

Blackbourn DJ, Osmond D, Levy JA, Lennette ET (1999). Increased human herpesvirus 8 seroprevalence in young homosexual men who have multiple sex contacts with different partners. J Infect Dis 179:237–239.

17.

Blackbourn DJ, Lennette E, Klencke B, Moses A, Chandran B, Weinstein M, et al. (2000). The restricted cellular host range of human herpesvirus 8. AIDS 14:1123–1133.

18.

Blasig C, Zietz C, Haar B, Neipel F, Esser S, Brockmeyer NH, et al. (1997). Monocytes in Kaposi’s sarcoma lesions are productively infected by human herpesvirus 8. J Virol 71:7963–7968.

19.

Boshoff C, Weiss RA (2001). Epidemiology and pathogenesis of Kaposi’s sarcoma-associated herpesvirus. Phil Trans R Soc Lond Biol Sci 356:517–534.

20.

Boshoff C, Whitby D, Hatziioannou T, Fisher C, van der Walt J, Hatzakis A, et al. (1995a). Kaposi’s-sarcoma-associated herpesvirus in HIV-negative Kaposi’s sarcoma. Lancet 345:1043–1044.

21.

Boshoff C, Schulz TF, Kennedy MM, Graham AK, Fisher C, Thomas A, et al. (1995b). Kaposi’s sarcoma-associated herpesvirus infects endothelial and spindle cells. Nat Med 1:1274–1278.

22.

Bourboulia D, Whitby D, Boshoff C, Newton R, Beral V, Carrara H, et al. (2001). Serologic evidence for mother-to-child transmission of Kaposi sarcoma-associated herpesvirus infection. J Am Med Assoc 280:31–32.

23.

Brayfield BP, Kankasa C, West JT, Muyanga J, Bhat G, Klaskala W, et al. (2004). Distribution of Kaposi sarcoma-associated herpesvirus/ human herpesvirus 8 in maternal saliva and breast milk in Zambia: implications for transmission. J Infect Dis 189:2260–2270.

24.

Calabro ML, Gasperini P, Barbierato M, Ometto L, Zanchetta M, De Rossi A, et al. (2000). A search for human herpesvirus 8 (HHV-8) in HIV-1 infected mothers and their infants does not suggest vertical transmission of HHV-8. Int J Cancer 85:296–297.

25.

Cannon MJ, Dollard SC, Smith DK, Klein RS, Schuman P, Rich JD, et al. (2001). Blood-borne and sexual transmission of human herpesvirus 8 in women with or at risk for human immunodeficiency virus infection. N Engl J Med 344:637–643.

26.

Cannon MJ, Dollard SC, Black JB, Edlin BR, Hannah C, Hogan SE, et al. (2003). Risk factors for Kaposi’s sarcoma in men seropositive for both human herpesvirus 8 and human immunodeficiency virus. AIDS 17:215–222.

27.

Carroll PA, Brazeau E, Lagunoff M (2004). Kaposi’s sarcoma-associated herpesvirus infection of blood endothelial cells induces lymphatic differentiation. Virology 328:7–18.

28.

Cattani P, Capuano M, Cerimele F, La Parola IL, Santangelo R, Masini C, et al. (1999). Human herpesvirus 8 seroprevalence and evaluation of nonsexual transmission routes by detection of DNA in clinical specimens from human immunodeficiency virus-seronegative patients from central and southern Italy, with and without Kaposi’s sarcoma. J Clin Microbiol 37:1150–1153.

29.

Cerimele F, Curreli F, Ely S, Friedman-Kien AE, Cesarman E, Flore O (2001). Kaposi’s sarcoma-associated herpesvirus can productively infect primary human keratinocytes and alter their growth properties. J Virol 75:2435–2443.

30.

Cesarman E, Moore PS, Rao PH, Inghirami G, Knowles DM, Chang Y (1995). In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi’s sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood 86:2708–2714.

31.

Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, et al. (1994). Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266:1865–1869.

32.

Chatlynne LG, Lapps W, Handy M, Huang YQ, Masood R, Hamilton AS, et al. (1998). Detection and titration of human herpesvirus-8-specific antibodies in sera from blood donors, acquired immunodeficiency syndrome patients, and Kaposi’s sarcoma patients using a whole virus enzyme-linked immunosorbent assay. Blood 92:53–58.

33.

Cook RD, Hodgson TA, Waugh AC, Molyneux EM, Borgstein E, Sherry A, et al. (2002). Mixed patterns of transmission of human herpesvirus-8 (Kaposi’s sarcoma-associated herpesvirus) in Malawian families. J Gen Virol 83:1613–1619.

34.

Corey L, Brodie S, Huang ML, Koelle DM, Wald A (2002). HHV-8 infection: a model for reactivation and transmission. Rev Med Virol 12:47–63.

35.

Darrow WW, Peterman TA, Jaffe HW, Rogers MF, Curran JW, Beral V (1992). Kaposi’s sarcoma and exposure to faeces [letter]. Lancet 339:685.

36.

Delli Bovi P, Donti E, Knowles DM, Friedman-Kien A, Luciw PA, Dina D, et al. (1986). Presence of chromosomal abnormalities and lack of AIDS retrovirus DNA sequences in AIDS-associated Kaposi’s sarcoma. Cancer Res 46:6333–6338.

37.

Di Alberti L, Ngui SL, Porter SR, Speight PM, Scully CM, Zakrewska JM, et al. (1997). Presence of human herpesvirus 8 variants in the oral tissues of human immunodeficiency virus-infected persons. J Infect Dis 175:703–707.

38.

Diamond C, Brodie SJ, Krieger JN, Huang ML, Koelle DM, Diem K, et al. (1998). Human herpesvirus 8 in the prostate glands of men with Kaposi’s sarcoma. J Virol 72:6223–6227.

39.

Dukers NH, Rezza G (2003). Human herpesvirus 8 epidemiology: what we do and do not know. AIDS 17:1717–1730.

40.

Dukers NH, Renwick N, Prins M, Geskus RB, Schulz TF, Weverling GJ, et al. (2000). Risk factors for human herpesvirus 8 seropositivity and seroconversion in a cohort of homosexual men. Am J Epidemiol 151:213–224.

41.

Dupin N, Gorin I, Deleuze J, Agut H, Huraux JM, Escande JP (1995). Herpes-like DNA sequences, AIDS-related tumors, and Castleman’s disease [letter]. N Engl J Med 333:798; author reply 798–799.

42.

Duus KM, Lentchitsky V, Wagenaar T, Grose C, Webster-Cyriaque J (2004). Wild-type Kaposi’s sarcoma-associated herpesvirus isolated from the oropharynx of immune-competent individuals has tropism for cultured oral epithelial cells. J Virol 78:4074–4084.

43.

Foreman KE, Friborg J Jr, Kong WP, Woffendin C, Polverini PJ, Nickoloff BJ, et al. (1997). Propagation of a human herpesvirus from AIDS-associated Kaposi’s sarcoma. N Engl J Med 336:163–171.

44.

Franceschi S, Geddes M (1995). Epidemiology of classic Kaposi’s sarcoma, with special reference to Mediterranean population. Tumori 81:308–314.

45.

Friedman-Kien AE (1981). Disseminated Kaposi’s sarcoma syndrome in young homosexual men. J Am Acad Dermatol 5:468–471.

46.

Gandhi M, Koelle DM, Ameli N, Bacchetti P, Greenspan JS, Navazesh M, et al. (2004). Prevalence of human herpesvirus-8 salivary shedding in HIV increases with CD4 count. J Dent Res 83:639–643.

47.

Gao SJ, Kingsley L, Hoover DR, Spira TJ, Rinaldo CR, Saah A, et al. (1996). Seroconversion to antibodies against Kaposi’s sarcoma-associated herpesvirus-related latent nuclear antigens before the development of Kaposi’s sarcoma. N Engl J Med 335:233–241.

48.

Gessain A, Mauclere P, van Beveren M, Plancoulaine S, Ayouba A, Essame-Oyono JL, et al. (1999). Human herpesvirus 8 primary infection occurs during childhood in Cameroon, Central Africa. Int J Cancer 81:189–192.

49.

Gill PS, Tsai YC, Rao AP, Spruck CH III, Zheng T, Harrington WA Jr, et al. (1998). Evidence for multiclonality in multicentric Kaposi’s sarcoma. Proc Natl Acad Sci USA 95:8257–8261.

50.

Grulich AE, Olsen SJ, Luo K, Hendry O, Cunningham P, Cooper DA, et al. (1999). Kaposi’s sarcoma-associated herpesvirus: a sexually transmissible infection? J Acquir Immune Defic Syndr Hum Retrovirol 20:387–393.

51.

Judde JG, Lacoste V, Briere J, Kassa-Kelembho E, Clyti E, Couppie P, et al. (2000). Monoclonality or oligoclonality of human herpesvirus 8 terminal repeat sequences in Kaposi’s sarcoma and other diseases. J Natl Cancer Inst 92:729–736.

52.

Kahn HJ, Bailey D, Marks A (2002). Monoclonal antibody D2-40, a new marker of lymphatic endothelium, reacts with Kaposi’s sarcoma and a subset of angiosarcomas. Mod Pathol 15:434–440.

53.

Katz MH, Hessol NA, Buchbinder SP, Hirozawa A, O’Malley P, Holmberg SD (1994). Temporal trends of opportunistic infections and malignancies in homosexual men with AIDS. J Infect Dis 170:198–202.

54.

Kedes DH, Operskalski E, Busch M, Kohn R, Flood J, Ganem D (1996). The seroepidemiology of human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus): distribution of infection in KS risk groups and evidence for sexual transmission. Nat Med 2:918–924.

55.

Kellam P, Bourboulia D, Dupin N, Shotton C, Fisher C, Talbot S, et al. (1999). Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8. J Virol 73:5149–5155.

56.

Koelle DM, Huang ML, Chandran B, Vieira J, Piepkorn M, Corey L (1997). Frequent detection of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) DNA in saliva of human immunodeficiency virus-infected men: clinical and immunologic correlates. J Infect Dis 176:94–102.

57.

Lifson AR, Darrow WW, Hessol NA, O’Malley PM, Barnhart JL, Jaffe HW, et al. (1990). Kaposi’s sarcoma in a cohort of homosexual and bisexual men. Epidemiology and analysis for cofactors. Am J Epidemiol 131:221–231.

58.

Marcelin AG, Gorin I, Morand P, Ait-Arkoub Z, Deleuze J, Morini JP, et al. (2004). Quantification of Kaposi’s sarcoma-associated herpesvirus in blood, oral mucosa, and saliva in patients with Kaposi’s sarcoma. AIDS Res Hum Retroviruses 20:704–708.

59.

Martin JN, Osmond DH (2000). Invited commentary: determining specific sexual practices associated with human herpesvirus 8 transmission. Am J Epidemiol 151:225–229; discussion 230.

60.

Martin JN, Ganem DE, Osmond DH, Page-Shafer KA, Macrae D, Kedes DH (1998). Sexual transmission and the natural history of human herpesvirus 8 infection. N Engl J Med 338:948–954.

61.

Mbulaiteye SM, Pfeiffer RM, Engels EA, Marshall V, Bakaki PM, Owor AM, et al. (2004). Detection of Kaposi sarcoma-associated herpesvirus DNA in saliva and buffy-coat samples from children with sickle cell disease in Uganda. J Infect Dis 190:1382–1386.

62.

Melbye M, Cook PM, Hjalgrim H, Begtrup K, Simpson GR, Biggar RJ, et al. (1998). Risk factors for Kaposi’s-sarcoma-associated herpesvirus (KSHV/HHV-8) seropositivity in a cohort of homosexual men, 1981–1996. Int J Cancer 77:543–548.

63.

Moore PS, Kingsley LA, Holmberg SD, Spira T, Gupta P, Hoover DR, et al. (1996). Kaposi’s sarcoma-associated herpesvirus infection prior to onset of Kaposi’s sarcoma. AIDS 10:175–180.

64.

Olsen SJ, Chang Y, Moore PS, Biggar RJ, Melbye M (1998). Increasing Kaposi’s sarcoma-associated herpesvirus seroprevalence with age in a highly Kaposi’s sarcoma endemic region, Zambia in 1985. AIDS 12:1921–1925.

65.

Pauk J, Huang ML, Brodie SJ, Wald A, Koelle DM, Schacker T, et al. (2000). Mucosal shedding of human herpesvirus 8 in men. N Engl J Med 343:1369–1377.

66.

Peterman TA, Friedman-Kien AE, Jaffe HW, Beral V (1992). Kaposi’s sarcoma and exposure to faeces. Lancet 339:685–686.

67.

Plancoulaine S, Abel L, van Beveren M, Tregouet DA, Joubert M, Tortevoye P, et al. (2000). Human herpesvirus 8 transmission from mother to child and between siblings in an endemic population. Lancet 356:1062–1065.

68.

Rabkin CS, Janz S, Lash A, Coleman AE, Musaba E, Liotta L, et al. (1997). Monoclonal origin of multicentric Kaposi’s sarcoma lesions. N Engl J Med 336:988–993.

69.

Renne R, Blackbourn D, Whitby D, Levy J, Ganem D (1998). Limited transmission of Kaposi’s sarcoma-associated herpesvirus in cultured cells. J Virol 72:5182–5188.

70.

Rezza G, Lennette ET, Giuliani M, Pezzotti P, Caprilli F, Monini P, et al. (1998). Prevalence and determinants of anti-lytic and anti-latent antibodies to human herpesvirus-8 among Italian individuals at risk of sexually and parenterally transmitted infections. Int J Cancer 77:361–365.

71.

Russo JJ, Bohenzky RA, Chien MC, Chen J, Yan M, Maddalena D, et al. (1996). Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc Natl Acad Sci USA 93:14862–14867.

72.

Safai B (1984). Kaposi’s sarcoma: a review of the classical and epidemic forms. Ann NY Acad Sci 437:373–382.

73.

Schalling M, Ekman M, Kaaya EE, Linde A, Biberfeld P (1995). A role for a new herpes virus (KSHV) in different forms of Kaposi’s sarcoma. Nat Med 1:707–708.

74.

Simpson GR, Schulz TF, Whitby D, Cook PM, Boshoff C, Rainbow L, et al. (1996). Prevalence of Kaposi’s sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen. Lancet 348:1133–1138.

75.

Sitas F, Carrara H, Beral V, Newton R, Reeves G, Bull D, et al. (1999). Antibodies against human herpesvirus 8 in black South African patients with cancer. N Engl J Med 340:1863–1871.

76.

Staskus KA, Zhong W, Gebhard K, Herndier B, Wang H, Renne R, et al. (1997). Kaposi’s sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells. J Virol 71:715–719.

77.

Sturzl M, Blasig C, Schreier A, Neipel F, Hohenadl C, Cornali E, et al. (1997). Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi’s sarcoma. Int J Cancer 72:68–71.

78.

Su IJ, Hsu YS, Chang YC, Wang IW (1995). Herpesvirus-like DNA sequence in Kaposi’s sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet 345:722–723.

79.

Taylor MM, Chohan B, Lavreys L, Hassan W, Huang ML, Corey L, et al. (2004). Shedding of human herpesvirus 8 in oral and genital secretions from HIV-1-seropositive and -seronegative Kenyan women. J Infect Dis 190:484–488.

80.

Thomas JA, Brookes LA, McGowan I, Weller I, Crawford DH (1996). HHV8 DNA in normal gastrointestinal mucosa from HIV seropositive people. Lancet 347:1337–1338.

81.

Vieira J, Huang ML, Koelle DM, Corey L (1997). Transmissible Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) in saliva of men with a history of Kaposi’s sarcoma. J Virol 71:7083–7088.

82.

Wang HW, Trotter MW, Lagos D, Bourboulia D, Henderson S, Makinen T, et al. (2004). Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat Genet 36:687–693.

83.

Webster-Cyriaque J (2002). Development of Kaposi’s sarcoma in a surgical wound. N Engl J Med 346:1207–1210.

84.

Whitby D, Howard MR, Tenant-Flowers M, Brink NS, Copas A, Boshoff C, et al. (1995). Detection of Kaposi sarcoma associated herpesvirus in peripheral blood of HIV-infected individuals and progression to Kaposi’s sarcoma. Lancet 346:799–802.

85.

Whitby D, Smith NA, Matthews S, O’Shea S, Sabin CA, Kulasegaram R, et al. (1999). Human herpesvirus 8: seroepidemiology among women and detection in the genital tract of seropositive women. J Infect Dis 179:234–236.

86.

Whitby D, Luppi M, Sabin C, Barozzi P, Di Biase AR, Balli F, et al. (2000). Detection of antibodies to human herpesvirus 8 in Italian children: evidence for horizontal transmission. Br J Cancer 82:702–704.

87.

Wojcicki JM (2003). Traditional behavioural practices, the exchange of saliva and HHV-8 transmission in sub-Saharan African populations. Br J Cancer 89:2016–2017.

88.

Xu H, Edwards JR, Espinosa O, Banerji S, Jackson DG, Athanasou NA (2004). Expression of a lymphatic endothelial cell marker in benign and malignant vascular tumors. Hum Pathol 35:857–861.

89.

Ziegler JL, Katongole-Mbidde E (1996). Kaposi’s sarcoma in childhood: an analysis of 100 cases from Uganda and relationship to HIV infection. Int J Cancer 65:200–203.