Junín Virus Infection of Human Hematopoietic Progenitors Impairs In Vitro Proplatelet Formation and Platelet Release via a Bystander Effect Involving Type I IFN Signaling
Figure 4
JUNV replication in CD34+ cells.
CD34+ cells were inoculated with UV-irradiated JUNV or JUNV and stimulated with TPO for ten days. Viral replication was assayed by RT-PCR, immunofluorescence and flow cytometry. (A) RT-PCR studies. (B) To detect JUNV antigens by immunofluorescence cells were washed, cytocentrifuged on silanized glasses, fixed, permeabilized and incubated first with a pool of specific mAbs against JUNV and a rabbit-anti-human vWF polyclonal Ab to identify megakaryocytes, and then with FITC-conjugated anti-rabbit (green) and Cy3-conjugated anti-mouse Igs (red). The slides were counterstained with DAPI and photographed at 1000× magnification. (C) Cells were stained as in B and then analyzed by flow cytometry. (D) As a positive control, JUNV-susceptible Vero-76 cells were inoculated with JUNV and seven days later were stained with the pool of specific mAbs against JUNV followed by Cy3-anti-mouse Igs. Negative controls in B and C were performed by incubating cells only with secondary Abs. Panels show a representative experiment of three similar replicates.