Figure 2.

Common Powassan virus focus forming assay issues

Representative images of focus forming assays performed with lineage 1 Powassan virus LB in duplicate depicting some common issues.

(A) These images show the results of incubation times that are too long (48 h, left) or too short (18 h, right). For incubations that are too long, foci will run together and will not be individually discernable. For incubations that are too short, foci will appear too small for detection, or will not appear at all.

(B) These images show the results of plating cell densities that are too high (4 × 10⁴ cells per well, left) or too low (2.5 × 10³ cells per well, right). For plating an excess of cells, background staining or ‘stacking’ of cells vertically (i.e., not on the cell monolayer) is possible. An excess of cells also increases the likelihood of washes damaging the cell monolayer (well A2). For plating too few cells, gaps in the cell monolayer may be visible, and viral titer will likely appear lower than expected.

(C) Representative image showing a focus forming assay with a cell dilution of 2.5 × 10⁴ cells and 24 h incubation, suitable for viral titer determination, as well as compound screening or neutralizing antibody quantification, resulting in distinct and individually discernible foci.