Wild-Type Kaposi's Sarcoma-Associated Herpesvirus Isolated from the Oropharynx of Immune-Competent Individuals Has Tropism for Cultured Oral Epithelial Cells

Abstract

Based on the observation that wild-type Kaposi's sarcoma-associated herpesvirus (KSHV) DNA can be detected in the oral cavity of healthy, immunocompetent individuals, we hypothesized that epithelial cells could be infected in vitro by wild-type (WT) KSHV isolated from immunocompetent individuals. Primary oral epithelial (P-EPI) cells and telomerase-immortalized oral epithelial cells were generated from human gingival tissue and were then infected in vitro with WT KSHV isolated from throat wash samples. Markers of lytic and latent KSHV infection were detected in cultures by 24 h postinfection by immunofluorescence confocal microscopic assays. The infectivity of the WT and BCBL virus was blocked by neutralizing antibodies against KSHV gB. The presence of KSHV DNA in these cells was confirmed by real-time PCR amplification of different regions of the viral genome. The significant in vitro viral replication that had occurred was inhibited by ganciclovir and by neutralizing antibodies against gB. When infected cultures were examined by scanning electron microscopy, thousands of KSHV particles were clearly visible across the surfaces of P-EPI cells. The detection of enveloped particles indicated that the infectious cycle had proceeded through assembly and egress. We thus demonstrated that oral WT KSHV isolated from immunocompetent individuals was able to infect and replicate in vitro in a relevant primary cell type. Furthermore, our results provide compelling evidence for KSHV transmission within infected oral epithelial cells derived from healthy, immunocompetent populations.Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8 ) is the etiologic agent of Kaposi's sarcoma (KS) (12) and peripheral effusion lymphoma (PEL) (10). The human herpesviruses are generally ubiquitous among human populations, are generally shed in the oral cavity, and are primarily transmitted via the salivary route. However, current serological tests for KSHV have determined that the seroprevalence within the general U.S. population is low, suggesting that, unlike the other human herpesviruses (with the exception of herpes simplex virus type 2 [HSV-2]), KSHV is not ubiquitous among healthy human populations.The primary mode of KSHV transmission remains unresolved, as extensive evidence exists for both sexual (8,25,30,31) and oral (5, 29, 34) transmission routes in immunosuppressed individuals. However, recent epidemiology studies suggested that oral transmission of KSHV does occur among healthy populations (14,15). Complicating the transmission issue, the identities of the cell types harboring KSHV in vivo in the oral cavity and producing the virus detected in saliva are not known. Endothelial cells are the primary infected cells in KS lesions (7). Recently, KSHV antigens were detected in oral epithelial cells of an early KS lesion (44). In addition, the ability of the virus to infect primary human keratinocytes has also been demonstrated (3, 9). Based on the above evidence, we hypothesized that the wild-type (WT) isol...

X Box Binding Protein XBP-1s Transactivates the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) ORF50 Promoter, Linking Plasma Cell Differentiation to KSHV Reactivation from Latency

“…XBP-1s fulfills such criteria by potentially allowing KSHV lytic reactivation from latently infected B cells as they terminally differentiate into plasma cells at mucosal surfaces. The lymphoid tissue of the nasopharynx that comprises the Waldeyers ring is infected by KSHV (7,11), and the virus is able to lytically infect oral epithelial cells (21) and is detectable in saliva samples of infected individuals. Together, this suggests that KSHV is present as a latent infection in an as-yet-undefined B-cell compartment in the oral mucosa, is triggered into lytic replication as the B cells differentiate, and is shed into saliva.…”

KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine

“…We do know that periodic "spontaneous" reactivation from latency occurs regularly, both in cell culture (6) and in vivo (72,81,82). In the human host, the principal site of lytic virus replication is the oropharynx, most likely in B cells of tonsillar or other pharyngeal lymphoid tissue, though growth in pharyngeal epithelium is another possibility (83). Careful clinical studies show that shedding of KSHV virions, reflecting periodic bouts of lytic reactivation, is intermittent and generally asymptomatic (81,82).…”

Development of hemophagocytic lymphohistiocytosis in triplets infected with HHV-8

“…9,10 Briefly, DNA was isolated from each subject sample (DNeasy; Qiagen, Valencia, CA), then the HHV-8-specific 336-base pair (bp) open reading frame 26 (ORF-26) region (nucleotide position 890 to 1226) was amplified by nested PCR using an outside primer pair (5Ј-ATCTATCCAAGTGCACACTGC-3Ј and 5Ј-CTGGGAACCAAGGCTGATAGG-3Ј) and an inside primer pair (5Ј-GATGATCCCTCTGACAACCT-3Ј and 5Ј-GGATCCGTGTTGTCTACG-3Ј). The product of the nested PCR was resolved on a 1.5% agarose gel.…”

“…The BC-3 and Mewo cell lines were used for positive and negative controls, respectively. [9][10][11] …”