(B) Cells were stained with anti-human FITC-CD83, and the percentage of positive cells was determined by flow cytometry

(B) Cells were stained with anti-human FITC-CD83, and the percentage of positive cells was determined by flow cytometry. determining the outcome of some viral infections (7, 8). We hypothesized that JUNV targets hpDC in order to modulate the host’s response. hpDC were isolated from the buffy coats of 14 donors by immunomagnetic negative selection (catalog number 130-092-207; Miltenyi, Germany). According to flow cytometry analysis, the percentage of CD123+ (specific marker) (9) cells ranged from 95 to 99%, with a cell viability of 97% (FACScan flow cytometer and FCS Express V3 software). Viral stocks of the attenuated Candid#1 (C#1) and virulent P3441 (P) strains of JUNV (10, 11) Lep were made and quantified as described previously (12). Briefly, monolayers of Picrotoxin BHK21 cells were infected with each strain and clarified supernatants (5.000 = x. values of 0.05 after Student’s paired test or one-way analysis of variance were considered to be statistically significant. The level of infective virus in the supernatants increased in a time- and MOI-dependent manner, as Picrotoxin confirmed by infectivity assays (Fig. 1A) in concordance with the viral RNA levels detected by quantitative PCR (qPCR) (Fig. 1B). C#1 and P nucleoprotein (NP) was detected in 70 to 95% of the cells, respectively (Fig. 1B) with a monoclonal antibody (clone AB06-B610) (14), followed by Alexa 488-conjugated anti-mouse IgG and a phycoerythrin (PE)-conjugated rabbit anti-human CD123 (BD Biosciences, NJ). Analysis was conducted by confocal microscopy (11). Picrotoxin Negative and positive controls were uninfected hpDC and infected Vero cells, respectively. The results demonstrated that hpDC are susceptible to both JUNV strains, and in agreement with previous studies, the P strain elicited higher values than did C#1 (15, 16). Open in a separate window FIG 1 hpDC are susceptible to JUNV infection and replication. (A) PFU counts in supernatants at the time points indicated were quantified with infectivity titration assays on Vero cells (= 5). ND, not detected. (B) Viral mRNA levels were detected by reverse transcription (RT)-qPCR in hpDC infected with JUNV strain C#1 or P at the time points indicated (= 3). AU, arbitrary units. (C) Mock-, C#1-, or P-infected hpDC were costained with the specific marker CD123 (red) and anti-JUNV protein antibody (green) at 2 dpi. Original magnification, 600. The bar graph shows the percentages of JUNV+ cells in the Picrotoxin CD123+ population as determined by manual scoring of at least 200 cells (= 4). JUNV failed to induce cell apoptosis or necrosis at any time point or MOI (Fig. 2A), as established by nuclear morphology after acridine orange and ethidium bromide staining (11). The Picrotoxin percentages of apoptotic cells were similar in mock- and C#1-infected hpDC, while the P strain inhibited spontaneous apoptosis over time and in an MOI-dependent manner, even preventing apoptosis induced by fetal bovine serum (FBS) deprivation (data not shown). Flow cytometry analysis of JUNV+ cells indicated that P, but not C#1, reduced caspase-3 activation and phosphatidylserine exposure (detected by annexin V binding) (Fig. 2B and ?andC).C). In this regard, a recent study demonstrated retinoic acid-inducible gene 1 (RIG-I)-mediated IFN-I-independent apoptosis in a cell- and viral strain-dependent manner (17). Moreover, the NP of JUNV interfered with the activity of caspase-3, inhibiting apoptosis (18). Finally, the Z protein of pathogenic, but not nonpathogenic, arenaviruses interacts with the RIG-I-like receptor, resulting in inhibition of IFN-I production (19) and associated apoptosis (20). It remains necessary to clarify if any of these mechanisms are involved with both of the JUNV strains studied here. Open in a separate window FIG 2 JUNV failed to cause cytopathic effects, and strain P suppressed spontaneous hpDC apoptosis. (A) Cells were double stained with acridine orange and ethidium bromide, and the percentage of apoptotic cells in noninfected (mock-infected) hpDC or those infected with C#1 or P was analyzed by fluorescence microscopy. Serum-deprived hpDC (no FBS) were used as a positive control (= 5)..