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Oral EBV and KSHV Infection in HIV

J. Webster-Cyriaque^*, K. Duus, C. Cooper, and M. Duncan

Dental Research Center CB#7455, Room 113, University of North Carolina, Chapel Hill, NC 27599, USA

Correspondence: ^* corresponding author, Jennifer_webster-cyriaque{at}med.unc.edu

	Abstract

TOP Abstract Introduction Materials and Methods Results Oral Epithelial and Lymphoid... Discussion References

 
The gamma herpesviruses, Kaposi’s-sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are tightly associated with the development of AIDS-associated oral disease and malignancy during immune suppression. The objective of this investigation was to characterize oral infection and pathogenesis in healthy and immune-suppressed individuals. To characterize oral EBV and KSHV infection, we examined throat washings and oral epithelial cells from HIV-positive and HIV-negative individuals. Quantitative/real-time polymerase-chain-reaction (PCR) assays, transmission electronmicroscopy, immunostaining, and sequence analysis were used to identify viral infection. Virus was isolated from throat-wash samples and was used to infect epithelial and lymphoid cell lines. We detected EBV and KSHV in the oral cavity in healthy and immune-suppressed individuals. Viral strain analysis of KSHV K1 in multiple clones from the oral cavities of healthy persons and immunosuppressed patients detected several strains previously detected in KS lesions, with minor strain variation within individuals. Immunoelectron microscopy for multiple viral antigens detected consistent expression of viral proteins and oral epithelial specimens. In oral epithelial cells infected with wild-type KSHV in vitro, the K8.1 glycoprotein associated with lytic KSHV infection was detected in both primary and telomerase immortalized oral epithelial cultures by 24 hours post-infection. Virions were detected, subsequent to infection, by scanning electron microscopy. Oral epithelial cells were also infected in vitro with wild-type EBV originating from throat washes. Analysis of these data suggests that, like EBV, KSHV infection is present in the oropharynx of healthy individuals, is transmissible in vitro, and may be transmitted by saliva.

KEY WORDS: Oral infection • EBV • KSHV • virus

	Introduction

TOP Abstract Introduction Materials and Methods Results Oral Epithelial and Lymphoid... Discussion References

 
Herpesviruses are generally transmitted by saliva and are highly prevalent in the general population. There are two known human gamma herpesviruses, human herpesvirus 8 and human herpesvirus 4, or Kaposi’s-sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), respectively. These organisms are tumorigenic herpesviruses. EBV, a widely studied virus, is closely related to KSHV and is carried as a persistent infection by more than 90% of adults (Kieff and Rickinson, 2001). Likewise, KSHV is a lymphotrophic virus that infects multiple cell types. Most persistently infected people produce EBV in their saliva, and transmission occurs through close contact. The exact mode of transmission of KSHV is unclear, although it appears to be spread by both sexual and non-sexual mechanisms, depending on the background prevalence in the populations under study. Serologic evidence shows that the prevalence of KSHV infection in general populations appears to be low, but varies considerably by geographic location. In populations with endemic KS, the seroprevalence is generally higher than in other populations. The high frequency of KS in HIV-infected homosexual men suggests that the agent, like EBV, can be sexually transmitted (Kedes et al., 1996). Interestingly, KSHV DNA levels in saliva were considerably higher than those measured in semen. Transmissible KSHV has recently been detected in the saliva of men with a history of KS (Pauk et al., 2000). Similar to places where KS is endemic, in horizontal models of EBV infection, infection generally occurs early in life, and is often spread among family members (Cook et al., 2002).

Opportunistic lesions and malignancies of the oral lymphoid and epithelial tissue associated with EBV and KSHV cause significant morbidity and mortality. Each of these maladies is associated with low CD4 counts and high HIV viral loads. From a public health perspective, EBV- and KSHV-associated diseases are particularly significant, given the high burden of HIV infection globally. EBV-associated oral malignancies in AIDS include non-Hodgkins lymphoma and Burkitt’s lymphoma. Hairy leukoplakia (HLP) is the only pathologic manifestation of EBV permissive infection. HLP, a lesion most often found on the side of the tongue of immunocompromised individuals, is characterized histologically by intracellular edema, epithelial acanthosis, lack of inflammmatory infiltrate, and hyperkeratosis. HLP displays focal viral infection in superficial layers of epithelium, with overt EBV replication and perhaps continual re-infection of adjacent epithelial cells (Greenspan et al., 1985). The frequent association of the HLP lesion with HIV infection and its rare occurrence in normal individuals usually instigate the search for immunosuppression, particularly secondary to HIV. Kaposi’s sarcoma is universally established as the most common AIDS-associated malignancy, and as being among the most important of these neoplasms (Chang et al., 1994). Its etiologic agent, KSHV, alters specific cellular pathways to induce KS and other malignancies (pleural effusion lymphomas). Although KS is an AIDS-defining lesion, it occurs in other populations, albeit at low frequency. The expression of virus-encoded transforming genes, and the ability to modulate host gene expression such that the immune evasion occurs, result in the success of these pathogens in disease development.

In this paper, we focus on the detection and pathogenesis of EBV and KSHV in the oral cavity both in vivo and in vitro.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Oral Epithelial and Lymphoid... Discussion References

 
Cells and Media
BJAB and BCBL1 cell lines were cultured in RPMI-1640 supplemented with 10% FBS, and containing penicillin, streptomycin, sodium pyruvate, and L-glutamine (GIBCO-BRL, Carlsbad, CA, USA). Primary oral epithelial (P-EPI) cells were generated as previously described (Duus et al., 2004). The cell pellet was washed once in KGM, re-suspended in 2 mL fresh KGM, plated in a 60-mm culture dish, and incubated at 36°C in 5% CO2. One volume of fresh, warm medium was added every 48 hrs, and the medium was changed completely after 5–6 days of culture. The cells began to adhere to the dish after 4–5 days in culture, and became 50% confluent after an additional 10–15 days. P-EPI monolayers were split 1:3 when 40–60% confluent. All cells stained positive for epidermal growth factor receptor, indicating their epithelial origin. P-EPI cells from passages 1 and 2 were immortalized by transduction with a retrovirus vector containing the human telomerase gene (h-tert).

Virus Isolation
Throat-wash (TW) samples in 10 mL sterile PBS were obtained with informed consent from normal, HIV-negative, and KS-negative individuals. Wild-type KSHV was isolated from these samples as previously described (Vieira et al., 1997).

Conditions of Infection
Cell monolayers were trypsinized, washed, re-suspended in fresh culture medium, mixed with the re-suspended WT virus, BCBL virus, or BJAB culture medium (mock/endog), and plated in 8-well chamber slides (BT Falcon, BT Biosciences, San Jose, CA, USA) or 35-mm well culture plates at a concentration of 2.5 x 10⁵ cells/mL. At various hours post-infection (hpi), the monolayers were further processed for IFA, or DNA isolation, as described below. Replication of KSHV was blocked by the addition of 2.5 mg/mL ganciclovir (Roche Laboratories, Inc., Novi, MI, USA) or 10 mg/mL acyclovir (Sigma-Aldrich Corp., St. Louis, MO, USA) to the culture medium at the time of infection. Infection of cells was blocked by pre-incubation of the re-suspended virus pellet with 60 µg/mL neutralizing anti-gB antibody or the UK-218 control antibody (a gift from B. Chandran, University of Kansas, Kansas City, KS, USA), as described previously (Akula et al., 2001).

Immunofluorescence Assays (IFA)
Detection of KSHV antigens in infected cell monolayers and in oropharyngeal cells isolated from TW samples was performed as previously described (Webster-Cyriaque et al., 2000).

Immunoblotting
Cell pellets were processed as previously described. Following SDS-PAGE, protein samples were transferred to nitrocellulose, blocked, and probed with a virus-specific monoclonal as previously described.

Imaging by SEM
Primary oral epithelial cell monolayers were infected with BCBL1-derived or WT virus as described above and seeded into 8-well chambered borosilicate coverslips (Nalge Nunc International). At 48 hpi, the monolayers were washed with PBS, fixed in PBS containing 1% glutaraldehyde, and shipped overnight on ice to the University of Iowa Central Microscopy Research Facility. High-resolution SEM was performed by methods specifically optimized for visualization of herpesviral particles on the surfaces of infected cells (Duus et al., 2004).

DNA Isolation and PCR Amplification
DNA was isolated from throat-wash cells and from infected cell monolayers by use of the DNEasy kit protocol recommended by the manufacturer (Qiagen, Inc., Valencia, CA, USA). Cellular DNA was used for PCR amplification or nested PCR for the detection of EBV LMP1, KSHV K1, K9, K15, and ORF 26 DNA. Briefly, a 300-ng quantity of cellular DNA was used as a template for the first-round PCR, with an outer primer pair in a 50-µL reaction volume, and a 5-µL quantity of the first-round reaction was use as template DNA for the second round, with an inner primer pair. DNA from uninfected BJAB and latently infected BCBL1 cells was used as negative and positive controls, respectively. A 300-ng quantity of cellular DNA was also used for real-time PCR detection of KSHV ORF 73 with primer and probe sequences (Qiagen Operon Technologies, Inc., Alameda, CA, USA), as previously described by Fakhari and Dittmer (2002). Real-time PCR was performed in an ABI Prism 7000 Sequence Detection System, with Taqman Universal PCR Master Mix, No AmpErase^®UNG (Applied Biosystems, Foster City, CA, USA), as previously described.

Immunofluorescence
Frozen tissue sections were cut to 5-µm thickness and placed on poly-L-lysine-coated slides. Tissue sections and cell lines were fixed in a chilled 1:1 mixture of methanol and acetone, blocked with 20% normal goat serum, stained with primary antibody, then stained with FITC-conjugated goat anti-mouse IgG. Slides were mounted with coverslips using Vectashield (Vector Laboratories), then subjected to confocal microscopy.

	Results

TOP Abstract Introduction Materials and Methods Results Oral Epithelial and Lymphoid... Discussion References

 
Oral KSHV Infection
To determine the role of KSHV in oral infection, a cohort of HIV-positive and -negative men and women with and without KS was tested for the presence of KSHV DNA in oropharyngeal samples, by polymerase chain-reaction for multiple regions of the KSHV genome, including K1, K15, and ORF 26. All members of the cohort were from North America. Persons in the cohort were healthy adults, AIDS patients, HIV-negative immunosuppressed persons, or HIV-positive persons with oral KS. Examination of matched blood samples from these same individuals detected viral DNA more readily in the oropharyngeal fluids. Furthermore, multiple strains were detected that were distinct from the laboratory isolates that represent these strains. Multiple independently amplified PCR products from the KSHV K1 region of viral DNA isolated from the oral cavities of persons in our cohort were cloned and sequenced from each throat-wash sample, thus ensuring that sequence variations did not reflect mutations that arose during PCR amplification. Multiple clones from the same patient yielded similar results, and were the same strain with minimal variance (data not shown). This minor variance occurred in hypervariable regions. These sequences were compared with previously published sequences of Kaposi’s sarcoma lesions or KSHV-infected cell lines (Zong et al., 1999). Several variants were detected in our population. Sequence analysis and subsequent phylogenetic analysis of the K1 region of the genome suggested that the virus that infects the general population is not different from the virus detected in actual oral KS lesions and in previously reported KS lesions. In the segment of the population that we examined by sequence and subsequent phylogenetic analysis, KSHV types A and C were predominant, consistent with the geographic predominance previously identified in Kaposi’s sarcoma lesions from patients in North America (Fig. 1). As in US AIDS KS samples, our population demonstrated primarily A1, A4, and C3 variants. This result strongly suggests that KSHV is truly opportunistic and, in the face of severe immune suppression, may trigger KS lesion development.

View larger version (86K): [in this window] [in a new window]   Fig. 1 - Detection of KSHV K1 from throat-washes by conventional PCR designed to distinguish strain KSHV K1A from KSHV K1C. Lane M, Marker; lane 1, dH20 control; lane 2, BCBL cell DNA. Lanes 3–5, DNA isolated from donor TW samples: lane 3, healthy; lane 4, HIV-positive; lane 5, HIV-negative, immune-suppressed.

View larger version (123K): [in this window] [in a new window]   Fig. 2 - Detection of the lytic KSHV glycoprotein gB in oral epithelial cells from buccal scrapings. Cells were fixed and stained with a gB-specific polyclonal antibody against gB. Rhodamine-conjugated secondary antibodies were used to detect the primary bound to the KSHV protein. Shown is a representative gB immunofluorescence assay of buccal scrapings from a healthy individual. The white arrow indicates a positive cytoplasmic staining (red). Nuclei were stained with DAPI (blue).

	Oral Epithelial and Lymphoid Cells Can Be Infected with KSHV and with EBV in vitro

TOP Abstract Introduction Materials and Methods Results Oral Epithelial and Lymphoid... Discussion References

 
Since virus was detected from oral specimens, we next sought to determine whether these wild-type viruses from immunocompetent persons were capable of oral epithelial cell infection in vivo. Primary cells derived from oral epithelia and telomerase-immortalized oral epithelial cell lines were developed. Infection of primary oral epithelial cells (P-EPI) and telomerase-immortalized epithelial cells (T-EPI) with WT KSHV isolated from healthy individuals resulted in productive viral expression. These cell lines were infected with either virus derived from throat wash or BCBL1 virus. Nuclear staining for LANA was detected 48 hrs post-infection. These in vitro-infected cells displayed cytoplasmic expression of the gB lytic antigen 38 hrs post-infection with wild-type virus and with prototype BCBL1 virus, and a minority of cells expressed LANA. The percentage of cells latently infected was approximately –.05–2%, while the percentage of lytically infected cells was approximately 15–20%. Latently infected BCBL1 or TPA-induced BCBL1 cells served as controls for expression of lytic and latent antigens K8.1 and LANA, respectively. Further, these immortalized human keratinocytes were infected with wild-type isolates (TW-R), and viral DNA was detected in these cells after 72 hrs by ORF 26 real-time PCR, but not in mock-infected cells (Duus et al., 2004). RNA was isolated and cDNA synthesized from P-EPI cells infected with throat-wash virus. Reverse-transcriptase real-time PCR was performed with ORF 73 primers that spanned an intron to confirm expression at the level of mRNA.

We next went on to determine whether throat washes containing wild-type isolates were capable of infecting cell lines in vitro with EBV as well. Infection of primary oral epithelial cells (P-EPI) and telomerase-immortalized epithelial cells (T-EPI) with WT EBV isolated from the throat washes of healthy individuals resulted in productive viral replication. PCR and sequence analysis of the LMP1 region of the viral genome showed that virus present in the throat wash was the same strain as that isolated from infected oral epithelial cells (data not shown).

To determine whether KSHV virions were detected on the membranes of primary oral epithelial cells, we performed scanning electron microscopy experiments (SEM) on P-EPI-3 passage 3 monolayers endogenously infected, as well as on P-EPI-3 monolayers infected with either BCBL1-derived or TW-derived WT virus. Infection of naïve epithelial cells with endogenous WT KSHV virions was detected, suggesting that oral epithelial cells may constitute an infectious reservoir for KSHV, as has previously been documented for Epstein-Barr virus and murine homologues (Stewart et al., 1998). When cells were infected in vitro with WT throat-wash virus or with BCBL1-derived virus, an abundance of virions was detected in association with the epithelial cell membrane. Considerably more virions were present on the WT and BCBL1 virus-infected monolayers than on the endogenously infected monolayers (Duus et al., 2004). The virion surfaces were pleomorphic, a common finding among herpesviruses, particularly those considered to be more cell-associated. These results documented the presence of KSHV virions on the surfaces of endogenously infected primary oral epithelial cells.

Lymphoid infection has been performed as well. Primary tonsil cells and tonsil explants were infected with both wild-type isolates and prototype virus, and while the incidence of infected cells was low, lytically infected cells were detected, as determined by cytoplasmic staining for the KHSV lytic antigen K8.1. Both epithelial and lymphoid infections were blocked by the addition of neutralizing antibody to gB, and viral replication was inhibited by the addition of antiviral drugs such as ganciclovir and acyclovir.

	Discussion

TOP Abstract Introduction Materials and Methods Results Oral Epithelial and Lymphoid... Discussion References

 
In these studies, we have determined that KSHV, like EBV, infects oral epithelial cells and is present in the oropharynx of both healthy and immune-suppressed persons. Mucosal epithelial cells are frequently the site of lytic gamma herpesviral infection, as determined by infection of murine lung epithelial cells and gastric epithelial cells with murine herpesvirus-68 (MHV-68) (Stewart et al., 1998). The AIDS-associated oral hairy leukoplakia lesion, the only pathologic manifestation of permissive Epstein Barr virus infection, exhibits abundant viral replication within oral epithelial cells. The oral cavity is also a reservoir for EBV infection in healthy individuals (Sitki-Green et al., 2002). We and others have shown that WT KSHV is present in the oral cavity of healthy individuals. Similar to other herpesviruses, in particular Epstein-Barr virus, frequent detection of KSHV from oral secretions in African children suggests that the horizontal route is the most common way that the virus is acquired during childhood (Gao et al., 1996; Sarmati, 2004). Phylogenetic comparisons of Malawian families detected identical and nonidentical viral sequences in mouthrinses of first-degree family members of patients with KS (Cook et al., 2002). Based on our detection of lytic antigens in vivo, this virus is capable of permissive infection of oral epithelial cells (Duus et al., 2004). Oral KSHV detection rates in some studies were lower than those of others and those detected by our group. Differing detection rates between different studies may reflect differences in collection methods, in the primer pairs used, and in the numbers of times the assays were repeated. For example, we have found that there is a considerable difference in the detection of virus in throat-washings as compared with that in saliva. Work from the Levy group corroborates this, in that virus was more readily detected from nasal washings than from nasal cells (Blackbourne et al., 1998). The most powerful argument for oral KSHV infection in vivo is supported by the data from our study where KSHV antigens were detected in these cells, confirming that oral epithelial cells are susceptible to KSHV infection. Similar to other gamma herpesviruses, expression of viral gene products in oral epithelial cells suggests that the oral mucosa may represent the predominant reservoir of infectious virus for KSHV transmission. Other oropharyngeal cells may contribute as well. Recently, KSHV was detected in tonsil cells morphologically characterized as lymphoid. In a subset of cases, tonsillar epithelial cells were also positive (Chagas et al., 2006). The detection of KSHV in oropharyngeal epithelial cells suggests that these cells may serve as a reservoir of virus and may contribute to KSHV in the saliva. Analysis of these data, collectively, suggests that sexual transmission may not be the primary mode of transmission, and that infection in North America is apparently prevalent among the general population and is not restricted to men who have sex with men. Thus, one route of KSHV transmission likely occurs via saliva in both heterosexual and homosexual populations.

KSHV has been shown to infect multiple cell types, including epithelial cells. Virus from patients with KS has been shown to infect 293 cells (Cerimele et al., 2001), and it has previously been shown that virus induced with phorbol esters to replicate within the prototype BCBL1 cell line can infect primary epithelial cells from foreskin keratinocytes. Since virus had been detected in the throat-washes of healthy persons, we then went on to infect primary and established epithelial and lymphoid cell lines with these wild-type KSHV isolates. Scanning electron microscopy experiments were performed that detected infection of naïve epithelial cells with endogenous WT KSHV virions, suggesting that oral epithelial cells may constitute an infectious reservoir for KSHV, as has previously been documented for Epstein-Barr virus and murine homologues (Stewart et al., 1998; Kieff and Rickinson, 2001). We initially postulated that this herpesvirus would be more difficult to detect than the other herpesviruses, because of the reported low titers of KSHV in healthy people. Surprisingly, KSHV particles were visualized as easily as the other herpesviruses, by SEM. Importantly, the markedly increased numbers of virions present on both BCBL1- and WT virus-infected oral epithelial cell membranes confirmed the fact that viral replication and virion assembly were occurring in these cells. Oral epithelial cells were readily infectable, with replication differences apparently dependent on whether the source of the virus was WT from immune-competent individuals or laboratory-adapted BCBL1-derived virus (Duus et al., 2004). Oral epithelial cells more readily sustained infection by WT isolates than by BCBL1-derived virus, revealing potential differences in pathogenicity (Duus et al., 2004).

We have demonstrated, for the first time, that normal human oral epithelial cells can be productively infected by KSHV in vivo and are capable of infection transfer in vitro. While the early B-cell is the principally infected cell type in the peripheral blood of KSHV-seropositive patients with KS, analysis of our data suggests that expression of the integrin receptor might render oral epithelium a cellular portal for KSHV infection via oral mucosal exposure (Akula et al., 2002).

Likewise, Epstein-Barr virus (EBV) is transmitted through saliva, but the cellular source is controversial. Putative reservoirs include oral epithelium and salivary glands. In situ hybridization detected EBV replication in a small percentage of tongue mucosal samples and not at all in salivary glands (Frangou et al., 2005). It has also been suggested that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo (Pegtel et al., 2004). Recently, tonsillar epithelial cells have been postulated as a likely site for EBV infection in vivo. Primary epithelial cell cultures, generated from tonsil explants, were latently infected upon co-culture with EBV-releasing cell lines. EBV gene products have been detected in cultures from EBV-positive tonsil donors. Analysis of our data supports EBV oropharyngeal epithelial cell infection and shows that EBV present in throat-washes is capable of infecting oral epithelial cell lines in vitro.

In conclusion, both EBV and KSHV viruses are capable of oral epithelial cell infection, and the viruses from the oral cavity are capable of transmission. Upon infection, these opportunists modulate cellular pathways to facilitate their pathogenesis, resulting in significant disease in the immune-suppressed individual.

	Acknowledgments

 
This investigation was supported in part by USPHS Research Grants K23DE00460-01 and RO3DE1444-01 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

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This Article Abstract Full Text (PDF) Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Webster-Cyriaque, J. Articles by Duncan, M. Search for Related Content PubMed PubMed Citation Articles by Webster-Cyriaque, J. Articles by Duncan, M.