Titration and neutralizing antibody quantification by focus forming assay for Powassan virus

Abstract

The development of high-throughput assays measuring Powassan virus (POWV) lineage I and II represents an important step in virological and immunological studies. By adapting focus-forming assays previously optimized for West Nile virus and Zika virus, this protocol is able to determine viral load, evaluate antivirals, and measure neutralizing antibodies. Although limited by its requirement of a detection antibody, this protocol includes a rapid and high-throughput assay for measuring viral titer. By utilizing a baby hamster kidney cell line and a 96-well plate format, this protocol allows for more sensitivity in the detection of POWV lineage I. For complete details on the use and execution of this protocol, please refer to Stone et al. (2022).

Keywords: Antibody; Cell-based Assays; Microbiology.

Graphical abstract

Figure 1

Baby hamster kidney cells should be used in Powassan virus titration (A) A representative focus forming assay showing the results of plating identical ten-fold serial dilutions of Powassan virus Spooner lineage (Spo, blue) or Powassan virus LB lineage (LB, orange) on Vero WHO cells (left) or baby hamster kidney 21 clone 13 cells from the ATCC (right). Boxed in blue and orange are the serial dilutions that could be used to determine the viral titer. (B) Quantification of POWV infectious viral titer images presented in (A). Data are reported as the mean foci for the two replicates.

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.