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.
Sabara
Marta I
MI
Canadian Food Inspection Agency, The Canadian Science Center for Human and Animal Health, National Center for Foreign Animal Disease, 1015 Arlington Street, Winnipeg, Man, Canada R3E 3M4. msabara@inspection.gc.ca
Larence
June E
JE
D000818:Animals; D003370:Coturnix; D005354:Fibrosarcoma / Q000821:virology; D029121:Metapneumovirus / Q000254:growth & development* / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D010948:Viral Plaque Assay / Q000379:methods*