Fig. 2.

Analysis of SHFV replication kinetics, cytokine production and tissue factor expression in Japanese macaques. (A) Virus infectivity titers in plasma. Plasma samples collected at the indicated times after infection were titrated for infectivity by plaque assay in MA104 cells. Each data point is the average of duplicate titrations. (B) Viral RNA copy numbers in PBMCs. Total cell RNA was extracted from PBMCs collected at the indicated times after infection and plus-sense, genome and subgenomic viral RNA was amplified by real-time RT-PCR and quantified using a standard curve generated with a known concentration of viral RNA. (C, D) Relative quantification (RQ) of intracellular IFNβ mRNA in PBMCs by real-time quantitative RT-PCR. PBMC RNA was used to measure IFNβ mRNA levels. IFNβ mRNA levels are expressed as the fold change in the levels of IFNβ mRNA in PBMCs from an infected animal versus the level in the same animal at day zero. The amount of IFNβ mRNA in each sample was normalized to the level of 18S rRNA in the same sample. Values shown are the averages of assays done in triplicate. Error bars represent standard error (E–I) IFNα and pro-inflammatory cytokine levels in the plasma were quantified by multiplexed ELISA. Values shown are the averages of assays done in triplicate. Error bars represent standard deviation. (J) Tissue factor expression in PBMCs collected on different days after infection was analyzed by Western blotting using anti-tissue factor antibody. Actin was used as a loading control. Representative gels are shown.