Department of Food Science and Technology, College of Food, Agricultural and Environmental Sciences, The Ohio State University, Columbus, OH 43210, USA.
J Virol Methods. 2012 Oct;185(1):61-8. doi: 10.1016/j.jviromet.2012.05.030. Epub 2012 Jun 6.
The genus Metapneumovirus within the subfamily Pneumovirinae and family Paramyxoviridae includes only two viruses, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), which cause respiratory disease in humans and birds, respectively. These two viruses grow poorly in cell culture and other quantitation methods, such as indirect immuno-staining and immuno-fluorescent assays, are expensive, time consuming, and do not allow for plaque purification of the virus. In order to enhance research efforts for studying these two viruses, a direct plaque assay for both hMPV and aMPV has been developed. By optimizing the chemical components of the agarose overlay, it was found that both hMPV with a trypsin-independent F cleavage site and aMPV formed clear and countable plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) after 5 days of incubation. The plaque forming assay has similar sensitivity and reliability as the currently used immunological methods for viral quantitation. The plaque assay is also a more simple, rapid, and economical method compared to immunological assays, and in addition allows for plaque purification of the viruses. The direct plaque assay will be a valuable method for the quantitation and evaluation of the biological properties of some metapneumoviruses.
副黏病毒科肺病毒亚科中的呼肠孤病毒属只包括两种病毒，即人类偏肺病毒（hMPV）和禽偏肺病毒（aMPV），它们分别导致人类和鸟类的呼吸道疾病。这两种病毒在细胞培养中生长不良，其他定量方法，如间接免疫染色和免疫荧光检测，既昂贵又耗时，且不允许对病毒进行噬菌斑纯化。为了加强对这两种病毒的研究，已经开发了一种用于 hMPV 和 aMPV 的直接噬菌斑测定法。通过优化琼脂糖覆盖物的化学成分，发现具有非依赖胰蛋白酶 F 裂解位点的 hMPV 和 aMPV 在孵育 5 天后，在一些哺乳动物细胞系（如 Vero-E6 和 LLC-MK2 细胞）中形成清晰且可计数的噬菌斑。噬菌斑形成测定法与目前用于病毒定量的免疫学方法具有相似的灵敏度和可靠性。与免疫学检测相比，噬菌斑检测法也是一种更简单、快速和经济的方法，此外还允许对病毒进行噬菌斑纯化。直接噬菌斑测定法将成为定量和评估某些呼肠孤病毒生物学特性的一种有价值的方法。
