Infection and Persistence of Rhesus Monkey Rhadinovirus in Immortalized B-Cell Lines

Infection and Persistence of Rhesus Monkey Rhadinovirus in Immortalized B-Cell Lines

A TaqMan based real-time PCR assay was developed for rapid detection and quantitation of herpes B virus (Cercopithecine herpesvirus 1) in clinical samples. In a statement issued by Kazutaka Osawa of Nagasaki University, the infected monkeys (between 3 and 13 years old) were captured near residential areas between July and November. A previous report from our laboratory described a comprehensive PCR-based method for the detection and species-level identification of all known human herpesviruses (1). To clarify this issue, we performed polymerase chain reaction (PCR) analysis to amplify BV sequences from trigeminal ganglia of 30 Japanese macaque monkeys that were seropositive for BV. Using the SEAP and GFP recombinant RRVs, assays were developed to monitor RRV infection, neutralization, and replication. HHV-8 is strongly implicated as a necessary cofactor in human Kaposi’s sarcoma (reference 1 and references therein). Sexual contact is a significant, but not predominant, mode of B virus transmission between monkeys.

The A, B, and C capsids are stable enough to be readily isolated from the nuclei of HSV-1-infected cells or, alternatively, from the extracellular media after cell lysis. Thus, productive replication of RRV is unlinked to that of rhEBV; factors that influence activation to productive replication act separately on RRV and rhEBV, even within the same cell. Of the 84 ORFs in RRV26-95, 83 contain sequences similar to the recently determined sequences of the independent RRV isolate 17577. These results establish a naturalistic cell culture system for the study of infection and persistence by RRV in rhesus monkey B cells. Yerkes Regional Primate Center, where a young worker who received an ocular exposure with contaminated body fluids from a CeHV-1–positive macaque became ill and subsequently died (6). The vast majority of the open reading frames present in HHV-8 have at least one corresponding homolog in RRV, and there is a close sequence similarity in corresponding genes. Unlike HHV-8, RRV can be grown lytically and to high titers in permissive monolayer cells (6).

The purpose of the present study was to collect ovarian and reproductive data (hormones and menstrual cyclicity) from female vervet monkeys of known age (6 d to older than 25 y) by using archived ovarian tissues and blood samples, vaginal swabbing, and offspring data. This situation parallels HHV-8 in humans, where the B lymphocyte has also been found to be the principal cell type that harbors the virus during persistent infection (11, 14, 17). Here we describe a rhesus monkey B-cell culture system for the study of RRV persistence and latency. The gamma-2 herpesviruses not only persist in lymphoid cells but often immortalize or otherwise alter the growth properties of these lymphoid target cells (4, 9, 21-23). To examine if RRV infection alters the growth of rhesus monkey B cells, we selected three rhesus monkey B-cell lines, 211-98, 260-98, and 309-98, for RRV infection. These three rhesus monkey B-cell lines have been described previously (18); they were established by immortalization with the Epstein-Barr virus (EBV)-related virus of rhesus monkeys (rhEBV; also called rhesus lymphocryptovirus [LCV]) (15, 18). Survival Outside Host: Storage of CHV-1 in tissue culture medium (pH 7.2, 4°C) was shown to result in a slight loss in viability after 8 weeks (12).

(I have a vivid memory of swinging a camera case at a monkey in Barbados that for some reason seemed to be less than impressed with my existence). Some patients develop lymphadenopathy proximal to the site of inoculation. It is not possible to directly investigate the causal relation between IM and MS in humans, since over 90% of the population is already infected with EBV and for obvious ethical reasons, virus negative humans cannot be infected with the virus. The gamma-2 herpesviruses, also called rhadinoviruses, are a distinct subfamily of the lymphotropic herpesviruses. Rhesus monkey and baboon LCVs have been reported to be incapable of immortalizing human B cells (10). No differences in growth properties were detected in the RRV-infected versus the uninfected B-cell lines maintained in the presence of 10% FBS. At the 2% serum concentration, the RRV-infected 260-98 cell line displayed a slightly reduced growth rate compared to that of its uninfected counterpart (Fig.

1B, top), but this effect was marginal for 211-98 and 309-98 (Fig. Nonetheless, the sensitivity is comparable to that of other herpes B virus-specific PCR assays, i.e., 100 or 700 genome copies, respectively (3, 4). All cell lines were maintained for an additional 8 weeks post RRV infection and then analyzed again for their growth properties. In this report, we describe the generation of overlapping cosmid clones for reconstitution of the RRV26-95 genome and their use in producing recombinant RRV by cotransfection. 1A, B, and C, bottom). No differences in growth properties were detected in RRV-infected versus uninfected B-cell lines independent of the serum concentration. Briefly, a 3-μl aliquot of purified RRV capsids was applied to a Quantifoil R 2/1 grid (Quantifoil Micro Tools Gmbh, Jena, Germany), quickly blotted with filter paper, and plunged into liquid nitrogen-cooled ethane so that the capsid particles were embedded in a thin layer of vitreous ice across the holes of the supporting film.

(Top) Immortalized rhesus B-lymphocyte cell lines were infected with RRV and cultured in the presence of 10% or 2% FBS starting with a cell density of 0.1 × 106 cells per ml of medium. Live-cell counts were determined for each culture up to day 15 postinfection. On day 7 (arrows), cultures were arbitrarily diluted 1:6 for cultures supplemented with 10% FBS and 1:3 for cultures supplemented with 2% FBS. Most of their caloric intake is from daily provisions provided by temple workers and food given to them by visitors. Although RRV has been associated with lymphomas in the setting of simian immunodeficiency virus-induced immunodeficiency at one primate center (24), we were not able to detect any alterations in the growth potential of the B cells in our present study, even at reduced serum concentrations. It is, of course, possible that any growth-altering properties of RRV were overshadowed by the potent rhEBV cell growth transformation. Interobserver CV and correlations for primordial follicles were CV = 21.2%, r = 0.99, and 95% CI = 0.971 to 0.998; for primary follicles were CV = 25.8%, r = 0.82, and 95% CI = 0.453 to 0.946; and for secondary follicles were CV = 24.2%, r = 0.97, and 95% CI = 0.908 to 0.993 (P < 0.01 for all correlations). At this time point, clarified supernatants from RRV-infected B-cell lines were analyzed for RRV production and supernatants from parental cell cultures were used in parallel as negative controls. As additional controls, RRV titers in supernatant from lytically infected rhesus fibroblast cells and uninfected parental cells were determined in parallel. Fifty percent tissue culture infective dose (TCID50) endpoint titers were determined in 48-well plates in duplicate by serial 10-fold dilution on a rhesus fibroblast line until complete cell lysis by 2 weeks (Table 1). All RRV-infected B-cell lines produced infectious RRV at titers of 103 to 104 per ml, indicating that the rhesus B cells were persistently infected with RRV. These titers were lower by a factor of 1,000 to 10,000 than titers obtained from infected fibroblast cultures (typically, 107 TCID50/ml). Additional precautions should be considered with work involving animal activities. No overt cytopathic effects were observed in the RRV-infected B-cell lines. The prevalence of shedding of B virus is increased among primates that are stressed, breeding, immunosuppressed, or ill. Superimposed on the response against the B-LCL we observed proliferation against the pulsed peptides in several monkeys, provided that these were presented in the ex vivo assays by autologous B-LCL. These results provide support for the conserved use of Eph by both KSHV and RRV for entry into B cells and endothelial cells. Filters were washed at 50°C with 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 1% sodium dodecyl sulfate (SDS). Protein concentrations were measured by using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). For the B-cell lines, 20-μg samples of whole-cell lysates were analyzed by Western blotting and compared to 10 μg of fibroblast-derived lysates. Viral proteins were detected with a polyclonal rabbit serum raised against purified Triton X-100-denatured RRV particles (Fig. 2A). While RRV-infected fibroblast cells displayed a complex pattern of RRV polypeptide expression, only one or two of the bands were detected in the RRV-infected B cells. Schematic representation of the cosmids derived from RRV26-95. Two bands with the same mobility were detected in column-purified RRV virion preparations with the rabbit anti-RRV serum but not with normal rabbit control serum (Fig. 2B, right and left sides respectively). ). Eight peptides matched the gene product of open reading frame 52 (Orf52), and one peptide matched Orf47 of RRV. Orf52 of RRV is a homologue of BLRF2 of EBV, which constitutes one component of the highly immunogenic viral capsid antigen complex (8, 10). All sera from RRV-infected rhesus monkeys that were tested showed good reactivity to p13 by Western blotting (data not shown). Expression of RRV proteins in persistently infected B-cell lines. (A) RRV-infected rhesus B-cell lines and their parental cell lines (211-98, 260-98, and 309-98) were lysed in RIPA buffer, and 20 μg of total cellular protein was loaded per lane. As controls, uninfected rhesus fibroblast cells (Rf 388-93) and fibroblast cells that had been infected for 81 h were lysed in parallel and 10 μg of total cellular protein was loaded per lane. Pearson correlations between the square root of mean follicle numbers and age, among follicle types, between the percentage of female vervets with offspring and age, and between AMH and age were determined by using JMP software (version 9, SAS Institute, Cary, NC). (B) Detection of RRV proteins in virus particle preparations. Column-purified RRV particles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RRV proteins were detected by Western blotting with an RRV-specific rabbit serum (α-RRV) or normal rabbit serum (NRS) as a control. We showed previously that in situ hybridization with pooled rhEBV cosmid probes can reliably identify cells that are lytically or latently infected with rhEBV (12, 18). An analogous approach was used here to detect cells infected with RRV. E., Hilliard, J. The RRV cosmid library was established by subcloning genomic RRV DNA into modified cosmid vector pSuperCos1 (Stratagene, La Jolla, CA). The person who has been exposed should be aware that his or her personal physician is unlikely to have any knowledge of or experience in treating or preventing B virus infection, and the occupational health care provider should make written information about B virus available to the physician. B-LCL from animals in group A were pulsed with MOG34–56, B-LCL from animals in group with CMVmcp981–1003 and B-LCL from animals in group C with cMOG34–56 (Table 1). The immobilized proteins were then incubated with equal amounts of lysates from 293T cells transfected with expression constructs for the respective gH (V5 epitope-tagged)/gL complexes, followed by washing and Western Blot analysis. Proliferation of human and rhesus monkey peripheral blood lymphocytes after infection with EBV, rhesus monkey LCV, or baboon LCV. The percentage of cells that stained positively for the RRV genome ranged from 5 to 40%, depending on the B-cell line (Fig. 3 and Table 2). Most positive cells contained fine, punctate nuclear signals (Fig. 3A), a pattern consistent with the presence of viral episomes in latently infected cells (16, 18, 19). Infection and Persistence of Rhesus Monkey Rhadinovirus in Immortalized B-Cell Lines

A small minority of cells showed intense staining confined to one or several large nuclear foci, a pattern consistent with productive replication (Fig. Overlay medium was then aspirated, and a staining solution (0.8% crystal violet in 50% ethanol) was applied for 10 min. No hybridization was observed in the parental cell lines that were not infected with RRV (Fig. 3C). and Fig. Detection of RRV genomes in persistently infected B-cell lines by FISH. RRV-infected B-cell lines and corresponding parental cell lines were fixed and hybridized in situ with a SpectrumRed-labeled RRV pooled-cosmid probe that covered 90% of the unique coding region.

Cell nuclei were counterstained with DAPI (blue). Results are shown for the RRV-infected 211-98 cell line (A and B) and the parental RRV-negative cell line (C). Staining patterns observed were consistent with latent persistence of RRV (A) and, at a lower frequency, productive replication (B). No RRV-specific staining was observed in the RRV-negative 211-98 parental cell line (C). Ovarian histologic sections illustrating decreasing primordial follicle populations with age. The predominant pattern that was observed was the joint presence of both RRV and rhEBV signals in a punctate nuclear pattern (Fig. 4A), consistent with latent coinfection.

Punctate hybridization to RRV in latently infected cells was predominantly distinct from that of rhEBV, with little or no colocalization. Productive replicative infection by RRV or rhEBV, as identified by FISH, was observed at a much lower frequency and never in the same cell (Fig. 4B and C, respectively). In R. Again, all cells exhibiting a productive RRV staining pattern showed a latent staining pattern for rhEBV. However, a positive culture or PCR result indicating the presence of viral DNA either at a site not directly associated with the exposure (e.g., the conjunctiva, in the case of a bite), or in a wound or at a site of exposure concurrent with symptoms compatible with B virus disease should be considered indicative of infection. Fluorescent cells were analyzed using an LSRII equipped with FACSDiva software (BD).

Murine EphA2-Fc was used in order to utilize identically prepared proteins from the same supplier since human EphA2-Fc was not available. This result indicates that simian LCVs are capable of infecting human B cells and suggests that a species restriction for efficient B-cell immortalization is likely to occur at a step beyond virus infection and penetration. RRV-infected B-cell lines and corresponding parental cell lines were fixed and hybridized in situ simultaneously with a SpectrumRed-labeled RRV probe and a SpectrumGreen-labeled rhEBV probe. Cell nuclei were counterstained with DAPI (blue). Results are shown for the RRV-infected 211-98 cell line. Panels: A, latent coinfection of RRV and rhEBV; B, productive replication of RRV in the presence of latent rhEBV; C, productive replication of rhEBV in the presence of latent RRV infection. Note that in panel B, three rhEBV-positive cells are present that are apparently not infected by RRV.

The diluted serum was allowed to flow through the column. The vast predominance of cells with a small number of these dots for RRV is not consistent with productive, replicative infection but rather is entirely consistent with one form or another of latent or repressed infection. Furthermore, the intense diffuse nuclear staining pattern observed in a small percentage of the cells was associated with intranuclear inclusions, further evidence for productive, replicative infection in these intensely staining cells. At least six spherical shells of densities can be distinguished before the densities gradually become featureless with further progression toward the center (Fig. It has been used convincingly in previous studies to identify single EBV genomes within cells, for example, in defining the single site of chromosomal integration of EBV DNA in the Namalwa cell line (16). Human B-cell lines that are latently infected with episomal EBV DNA give FISH signals consisting of punctate dots that correspond to individual EBV genomes, whose number can vary from cell line to cell line and even from cell to cell within an individual cell line (16, 19). Kutok et al.

(12) have previously shown that lytic replication of rhEBV in epithelial cells is associated with a pattern of intense diffuse nuclear staining that is identical to the pattern observed in the small subset of rhEBV- and RRV-infected B cells that we describe here. Thus, a punctate staining pattern has been shown in previous publications to correspond to individual viral genomes in latent infection, the copy number of latent viral genomes can be variable, and the intense diffuse nuclear staining pattern is associated with productive replicative infection. Our analysis does not, however, specify the nature or level of repressed expression and latent infection with RRV in these cell lines. Depicted in are the percentages of female vervets with offspring according to the following age categories: 4 to 10 y (n = 148), 11 to 15 y (n = 53), 16 to 20 y (n = 35), and older than 20 y (n = 11). RRV-GFP was generated and its titer was determined in rhesus monkey fibroblasts as described by Bilello et al. (3). At day 4 postinfection, triplicate cultures of LCL211-98 cells, either uninfected or infected with increasing numbers of PFU of RRV-GFP per cell, were analyzed by fluorescence-activated cell sorting (FACS) to determine the percentage of cells that were GFP positive at each MOI.

As displayed in Fig. 5A, the percentage of GFP-positive cells was MOI dependent, ranging from approximately 0.2% to 47% GFP positive at day 4 postinfection, with MOIs of 0.009 and 0.6 PFU/cell, respectively. B., & Wright, A. Each day postinfection, cultures were examined for GFP positivity by FACS analysis until day 17. We are currently unable to accurately quantify the risk associated with all exposures. It has been well established that EBV infection of immunocompetent humans induces a brisk response of CD8+ T-cells, resulting in expansion to about 40 to 60% of the CD8+ T-cell pool in blood [26], [27]. We found that inhibition of vesicle acidification strongly interfered with entry of RRV and KSHV into both R8 endothelial cells and rhesus fibroblasts (Fig.

Figure 5(top) shows results from representative cell lines, and data are also summarized in Table 2. 5) was similar to the number of RRV-positive cells obtained by FISH in separate experiments (41% in Table 2). Dose and time dependence. (A) The percentage of GFP-positive LCL211-98 cells after infection with RRV-GFP is MOI dependent. LCL211-98 cells were infected with increasing MOIs of RRV-GFP. The number of PFU of RRV-GFP was determined on rhesus monkey fibroblasts. Whereas transfection of whole genomic viral DNA directly into RFs routinely produced infectious virus, transfection of the cosmid combinations into RFs repeatedly failed to yield infectious virus.

The results show the percentage of viable GFP-positive cells along with the standard deviation at the indicated number of PFU per cell. Where no error bar is shown, the error falls within the size of the symbol. ). Triplicate cultures of LCL211-98 cells were mock infected or infected with RRV-GFP at 0.037 PFU/cell. The number of PFU of RRV-GFP was determined on rhesus monkey fibroblasts. Each day postinfection, 1 ml of the suspension culture was removed and replaced with 1 ml of medium. The aliquoted cells were pelleted by centrifugation and examined by FACS analysis to determine the percentage of GFP-positive cells.

Bilello et al. (3) have recently described a neutralization assay in which sera from RRV-positive monkeys, but not RRV-negative monkeys, were able to block infection of rhesus fibroblasts by RRV-GFP. In addition, there was no evidence of oophoritits. Following this 3-h incubation, LCL211-98 cells were inoculated with the virus-serum mixture. At day 4 postinfection, the percentage of GFP-positive cells was determined by FACS analysis. Sera from RRV-negative monkeys 288-94 and 232-03 did not neutralize RRV-GFP infection of LCL211-98 cells (Fig. 6).

However, sera from rhesus monkeys 541-03, 140-83, and 488-03, which were naturally infected with RRV, neutralized RRV-GFP infection by 99%, 77%, and 63%, respectively, at a 1:20 dilution (Fig. 6). Sera from naturally infected monkeys 288-03 and 526-91 did not show neutralizing activity in this B-cell assay. 974]. Evident neurological deficits were not observed. 9A, left panels). By Western blotting, the cell lines derived from rhesus monkey lymphocytes cocultivated with LCL15 were shown to express a protein similar in size to baboon LCV EBNA-2, consistent with recovery of baboon LCV from the coinfected cell line LCL15.

RRV-GFP (MOI = 0.3 PFU/cell) was incubated with either medium alone (No Antibody) or sera from RRV-negative rhesus monkeys (Mm 288-94 and 232-03) or rhesus monkeys naturally infected with RRV (Mm 526-91, 488-03, 541-03, 140-83, and 288-03) at a dilution of 1:20 or 1:100. The number of PFU of RRV-GFP was determined on rhesus monkey fibroblasts. After incubating of the virus-serum mixture for 3 h at 37°C with gentle rocking, LCL211-98 cells were inoculated with either medium alone (No Virus) or the virus-serum mixture. At day 4 postinfection, cultures were examined for GFP expression by FACS analysis. The results shown are the percentage of GFP-positive cells along with the standard deviation for each serum dilution tested. The fragments with altered mobility in the SphI and/or BbvCIA/B digests of the RRV-GFP and RRV-SEAP genomes resulted from the insertion of the CMV-SEAP or CMV-GFP reporter gene cassettes into the RRV genome. Persistence of RRV and Kaposi sarcoma-associated herpesvirus in human B-cell lines has recently been described (5, 7).

One line of investigation that will be made possible by the availability of this system is the extent to which different RRV glycoproteins and different cellular receptors may be used for infection of B cells versus fully permissive fibroblasts. Although the triplexes have similar overall structures, there are some minor differences among them because of their different (or quasi-equivalent) local environments.

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