Plaque assay for avian metapneumovirus using a Japanese quail fibrosarcoma cell line (QT-35)

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40,000 cells per cm(2) when using a 1.5% CMC overlay or 100,000 cells per cm(2) when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.