RSV plaque assay

Lung and nose samples were determined via plaque assay on HEp-2 cells. Briefly, samples were diluted and added in duplicate to 24-well or to 96-well plates as described.^56,57 For studies conducted using the traditional 24-well plaque assay, HEp-2 cells were pre-seeded and incubated at 37 °C on the day before the assay. On the assay day, virus was added to confluent cell monolayersfor 1 h at 37 °C, the liquid was aspirated and 0.75% methylcellulose was added. Following 5 days at 37 °C, cells were fixed and stained with crystal violet/glutaraldehyde solution. Viral plaques were counted, and titers were expressed as pfu/g of tissue. We also used the higher throughput 96-well assay described in Wen et al.⁵⁷. In this assay format, HEp-2 cells and virus were added to the wells of a 96-well plate at the same time and were incubated for 3 days. Plaques were visualized by immunostaining using a cocktail of anti-F and anti-N antibodies. Primary antibodies were incubated with the fixed cells for 1 h before anti-mouse IgG Alex488 conjugated secondary antibodies (Invitrogen) were added. Viral plaques were then imaged and counted using an EnSight imager reader 2.02 (PerkinElmer). Virus titers were expressed as pfu/g of tissue. Both assay methods were tested on the same set of samples from a cotton rat challenge study and yielded comparable titers.