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Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a member of the gamma-2 herpesvirus family of lymphotropic viruses and is currently accepted to be the etiological agent associated with the development of Kaposi's sarcoma (KS) and B-cell lymphoproliferative disorders (LPD) such as primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV DNA has been found in almost all forms of KS (5), localized specifically in the endothelial and spindle cells (3) and in monocytes that comprise the lesion (2), as well as in DNA isolated from patients with PEL and MCD (4, 9). Moreover, KSHV DNA has also been found in CD19⁺ B cells and monocytes from peripheral blood of individuals with or without KS and in KS patients with or without human immunodeficiency virus (HIV) infection (1, 2, 12). Detection of KSHV DNA in peripheral blood is thought to be predictive of the development of KS in HIV-infected individuals.

Related gamma-2 herpesviruses have been previously identified in other animal species, most recently in rhesus monkeys (referred to as rhesus rhadinovirus [RRV]) (6). DNA sequence analysis of the unique long region of the RRV genome reveals it is essentially colinear with KSHV and that it encodes several of the cellular homologues hypothesized to mediate host immune response or alter cell growth and cell cycle regulation (8). Although much can now be illuminated from the DNA sequence of RRV, little is currently known about the biology of RRV infection. Results from experimental RRV infections of naive and simian immunodeficiency virus (SIV)-infected monkeys indicate that RRV induces B-cell hyperplasia and persistent lymphadenopathy that resembles MCD in immunocompromised but not immunocompetent monkeys, suggesting the virus is associated with LPD (13). In this brief report, we provide evidence to demonstrate that in rhesus monkeys naturally infected with RRV, the virus can be found in peripheral blood mononuclear cells (PBMCs) and that further fractionation reveals that B lymphocytes are a major site of virus persistence and latency.

To identify rhesus monkeys naturally infected with RRV, we utilized an enzyme-linked immunosorbent assay generated with whole RRV-infected cell lysate (13) and randomly sampled monkeys for prior exposure to RRV as revealed by the presence of reactive antibodies. Our studies corroborated those of Desrosiers et al. (6) in finding that greater than 90% of rhesus monkeys over 2 years of age and group housed were strongly positive for antibodies to RRV and that infants 6 to 9 months of age and individually housed with their mothers or hand reared were the least likely to possess antibodies reactive to RRV-infected cells (data not shown).

To determine if RRV is present in the blood as cell-associated virus in naturally infected monkeys, we generated a set of nested PCR primers and a hybridization probe to amplify and detect DNA sequences specific to the RRV DNA polymerase gene (Table 1). Nested PCR analysis, rather than single-round PCR, was developed, as naive monkeys experimentally infected with RRV strain 17577 become PCR negative 2 to 4 weeks postinoculation (13).

Peripheral blood samples from seropositive and seronegative rhesus monkeys were collected by standard venipuncture, and the mononuclear cells were isolated by density gradient centrifugation over Ficoll. DNA from purified PBMCs was isolated with a Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.) and subjected to 30 cycles of PCR amplification with the first-round primers under the following conditions: 94°C for 1 min (1 cycle) followed by 94°C for 1 min, 50°C for 2 min, and 72°C for 1.5 min (30 cycles). Each first-round PCR used 0.1 µg of genomic DNA, 100 pmol of each primer, 2.5 U of Vent polymerase, 200 µM each deoxynucleotide triphosphate, and 2 mM MgSO₄ in a 50 µl reaction. For nested PCR, 5 µl of the first-round reaction mixtures was added to the inner primer set and amplified for an additional 30 cycles under identical conditions. Nested PCR products were resolved by agarose gel electrophoresis, transferred to nitrocellulose, hybridized to the oligonucleotide probe overnight, and washed at 55°C in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate prior to being exposed to X-ray film. Analysis of the Southern blot hybridizations shown in Fig. 1 indicates that all five seropositive monkeys are nested PCR positive and the two seronegative monkeys are negative. Moreover, the nested primer set and probe are specific for RRV, as DNAs from rhesus cytomegalovirus (11) and rhesus lymphocryptovirus (7) were also negative.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   RRV DNA detection by nested PCR amplification of PBMCs obtained from healthy rhesus monkeys. Total DNA from purified PBMCs was amplified by nested PCR, and the products were analyzed by Southern hybridization. Lanes 1 to 3, PCR reagent controls containing water, 50 pg of rhesus cytomegalovirus DNA, or 100 ng of rhesus lymphocryptovirus-infected cell DNA, respectively; lanes 4 to 10, reaction mixtures containing PBMC DNA from healthy seropositive monkeys (lanes 4, 7, 8, 9, and 10) or seronegative monkeys (lanes 5 and 6).

To evaluate which lymphocyte subset of PBMCs harbored viral genomes, we collected blood samples from five monkeys, one seronegative and four seropositive. All five monkeys were 1.5 years or older, and the seropositive monkeys had been seropositive for at least 6 months. PBMCs were negatively selected by immunomagnetic bead depletion to generate two populations of cells, B-cell or T-cell enriched, and subsequently stained with fluorescein isothiocyanate (FITC)-labeled antibodies. Table 2 shows that two of five T-cell-enriched populations were depleted of CD20⁺ B cells, while the three remaining fractions exhibited nearly twofold depletion. The immunomagnetic bead procedure appeared to be more effective for enriching for B cells, as significant depletion of T cells was observed in four of five B-cell-enriched populations. Failure to achieve complete depletion in some of these fractions is most likely due to the high percentage of cells prior to negative selection or the accidental washing of cells off the magnetic beads.

The results in Table 2 also indicate that enrichment of B cells coincided with the isolation of infectious virus in three of four fractions from seropositive monkeys, whereas no virus was recovered in PBMCs or the T-cell-enriched fractions. Similar results have been reported for the related gamma-2 herpesvirus MHV-68 (10). Confirmation was further evident when enriched-cell populations were analyzed by nested PCR as described above. Analysis of the PCR products by Southern blot analysis revealed that virus was present in the PBMCs as well as in the B-cell-enriched fraction but less evident or absent in the T-cell-enriched fractions. The slight signal observed in three of four T-cell-enriched fractions is most likely due to the small percentage of CD20⁺ B cells remaining after negative selection (Fig. 2).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Detection of RRV DNA in enriched lymphocyte fractions. Total DNA from PBMCs, T-cell-enriched (T cell) or B-cell-enriched (B cell) fractions, obtained from the monkeys indicated in Table 2, were amplified by nested PCR, and the products were analyzed by Southern hybridization.

Our data demonstrate that rhesus B lymphocytes are a major site for RRV persistence. Moreover, this is consistent with the B-cell involvement observed in SIV-infected rhesus monkeys experimentally inoculated with RRV strain 17577 (13). Interestingly, although B-cell hyperplasia was evident in the peripheral blood compartment of SIV-RRV-infected monkeys, we did not detect a substantial increase in cell-associated virus. This would suggest that a subpopulation of B lymphocytes are infected or that other cell types in the peripheral blood also harbor virus. Further experiments are under way to identify the subpopulation of B lymphocytes persistently infected and whether peripheral blood monocytes are also infected. Nonetheless, the data suggest that RRV and KSHV share similar biological properties and indicate that RRV infection of rhesus monkeys may serve as an accessible animal model for elucidating the biology of KSHV infection and associated disease.