Plaque assay for human coronavirus NL63 using human colon carcinoma cells.
Plaque assay for human coronavirus NL63 using human colon carcinoma cells.
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DOI：
10.1186/1743-422x-5-138
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发表时间：
2008-11-12
期刊：
影响因子：
4.8
通讯作者：
Mueller, Marcel A.
中科院分区：
文献类型：
--
作者：
Herzog, Petra;Drosten, Christian;Mueller, Marcel A.
冠状病毒在动物和人类中引起广泛的疾病。人类冠状病毒（hCoV）NL 63与高达10%的普通感冒有关。病毒空斑测定能够表征病毒感染性并允许纯化病毒储备液。它们对于药物筛选至关重要。用于hCoV-NL 63的细胞培养物显示出低水平的病毒复制和弱的和扩散的致细胞病变作用。目前还不可能建立针对这种重要的人类病原体的切实可行的空斑测定法。测试了12种不同的细胞培养物对hCoV-NL 63感染的易感性。人结肠癌细胞（CaCo-2）复制病毒的效率比常用的非洲绿色猴肾细胞（LLC-MK 2）高100倍以上。CaCo-2细胞在感染后4天表现出细胞病变效应。Avicel，琼脂糖和羧甲基纤维素覆盖证明适合空斑测定。Avicel获得了最佳结果，从感染第4天开始产生大而清晰的斑块。证实了琼脂糖覆盖的空斑试验用于纯化病毒，从而使病毒感染性增加1 log 10 PFU/mL。CaCo-2细胞比LLC-MK2细胞更好地支持hCoV-NL 63，并且能够进行致细胞病变空斑测定。Avicel覆盖有利于空斑定量，琼脂糖覆盖优选用于空斑纯化。感染性增加的HCoV-NL 63病毒储备将有益于抗病毒筛选、疾病的动物建模和其他实验任务。
Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV) NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen. 12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2) replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2). CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4th day of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL. CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.