Figure 1.
Quantitation of yellow fever virus by focus forming assay (FFA).
This representative photograph shows the results of a FFA performed using uninfected Vero cells (top row) or Vero cells infected with YFV-17D (bottom left) or YFV-Dakar (bottom right). At 3 days post-infection, the monolayers were fixed and stained with anti-YFV Mab, 3A8.B6, and infectious foci were identified by addition of detection reagents as described in the methods. No focus forming units (FFU) could be detected in the YFV-Dakar-infected wells at 3, 7, or 10 days after infection with >100 infectious units of virus as determined by TC-LDA. The data is based on 4 experiments, each with similar results.
Figure 2.
Outline of YFV tissue culture LDA (TC-LDA) methodology.
For each washing step, the plates are centrifuged at 250× g for 3 minutes after which the solution is rapidly decanted into a waste container and the cell pellets are dislodged and resuspended by gentle vortexing before adding the next solution/reagent. Dilution of the biotinylated MAb 3A8.B6 and Strep-Avidin-R-phycoerythrin (SA-PE) were optimized for each lot. For instance, in the studies shown here, the biotinylated MAb 3A8.B6 was used at 8 µg/ml (4 µg/ml, final) and SA-PE was used at a final dilution of 1∶500.
Figure 3.
In (A), representative flow cytometry dot plots show the results of staining uninfected C6/36 cells or YFV-17D-infected C6/36 cells (MOI = 5, 3 days post-infection) with biotinylated YFV-specific antibodies. Background staining with the secondary reagent, streptavidin-PE (SA-PE) in the absence of a primary YFV-specific antibody was low (<0.004%). Up to 85% of YFV-infected cells could be detected with biotinylated YFV-hyperimmune polyclonal mouse IgG (PAb + SA-PE) but about 98% of YFV-infected C6/36 cells could be detected with biotinylated MAb 3A8.B6 (MAb + SA-PE). In (B), the titer of two strains of YFV (YFV-17D and YFV-Dakar) were determined by TC-LDA (using MAb 3A8.B6) at 1, 3, 7, or 10 days after infection to determine the optimal incubation time required for sensitive quantitation of replicating virus. The data in (B) is based on the average of two independent experiments.
Table 1.
Quantitation of infectious yellow fever virus by plaque assay and TC-LDA.
Figure 4.
Independent analysis of YFV-infected cells by flow cytometry and RT-PCR.
Dilutions of serum from a YFV-Dakar infected rhesus macaque (day 5 after infection with 1,000 infectious units of YFV-Dakar) were incubated with C6/36 cells (10 wells per dilution) for 10 days prior to removing 10% of the cells from each well for qRT-PCR analysis and then the remaining cells were fixed/stained for YFV antigen as described. The percentage of YFV-positive events shown in each dotplot represents the percentage of cells residing in the gated region that was set based on positive and negative controls included in the assay. Wells were considered positive by flow cytometry if the percentage of positive events were at least 4-fold over the background number of events observed in uninfected wells as indicated by (+) or (−). The titer of YFV-Dakar used in this experiment was calculated to be 4.1×109 infectious units/mL and the sample dilutions performed in these representative dotplots (108, 109, 1010) is shown on the right-hand side of the figure. Wells that scored positive by YFV RT-PCR (PCR+) or negative by YFV RT-PCR (PCR-) are shown in the figure. There was 100% concordance between these two independent approaches to YFV detection.
Figure 5.
Correlation between serum YFV titers determined by TC-LDA and quantitative RT-PCR.
The titer of YFV-Dakar in the serum of 12 infected rhesus macaques (4–6 days post-infection) were determined by TC-LDA, an approach that measures infectious virus, and by quantitative RT-PCR (qRT-PCR), an approach that measures the number of virus genome equivalents. The correlation (R2 = 0.89, p<0.0001) between these two independent and quantitative approaches indicates a nearly 1∶1 relationship between infectious virus and viral RNA genome equivalents when measuring in vivo infection by YFV-Dakar.