Sabara Marta I, Larence June E
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.
J Virol Methods. 2003 Jan;107(1):9-14. doi: 10.1016/s0166-0934(02)00207-0.
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.
描述了一种使用日本鹌鹑纤维肉瘤细胞系（QT-35）检测、分离和滴定禽肺病毒的蚀斑测定法。在用属于A、B或C亚组的代表性禽偏肺病毒感染的细胞单层上施加琼脂糖或羧甲基纤维素（CMC）覆盖物后会产生蚀斑。病毒蚀斑可通过光学显微镜或染色后轻松观察到。影响蚀斑外观的参数包括：细胞接种浓度、病毒株、覆盖物组成以及感染后的孵育时间。蚀斑形成的最佳条件是，当使用1.5% CMC覆盖物时，以每平方厘米40,000个细胞接种QT-35细胞；当使用1.0%或0.8%琼脂糖覆盖物时，以每平方厘米100,000个细胞接种。在这两种情况下，细胞单层在接种后24小时用病毒感染，6天内会形成清晰可见的蚀斑。由于这些细胞的特性稳定，感染后的孵育时间可延长至最长13天，以便产生更大的蚀斑。
