A Cytopathic Effect-Based Tissue Culture Method for HCoV-OC43 Titration Using TMPRSS2-Expressing VeroE6 Cells.
A Cytopathic Effect-Based Tissue Culture Method for HCoV-OC43 Titration Using TMPRSS2-Expressing VeroE6 Cells.
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DOI：
10.1128/msphere.00159-21
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发表时间：
2021-05-12
期刊：
影响因子：
4.8
通讯作者：
Nakaya T
中科院分区：
文献类型：
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作者：
Hirose R;Watanabe N;Bandou R;Yoshida T;Daidoji T;Naito Y;Itoh Y;Nakaya T
HCoV-OC 43在感染细胞系中很少显示细胞病变效应（CPE），因此通过CPE观察的空斑和TCID 50试验不适用于滴定;而是使用间接免疫过氧化物酶试验（IPA）。然而，IPA相对复杂，耗时，昂贵，不适合同时滴定许多样品。人冠状病毒（HCoV）-OC 43在感染各种细胞系后很少显示细胞病变效应（CPE），而间接免疫过氧化物酶测定（IPA）是一种相对复杂的方法，长期以来一直被用作替代测定。由于HCoV-OC 43使用细胞表面跨膜蛋白酶丝氨酸2（TMPRSS 2）进入细胞，因此表达TMPRSS 2的VeroE 6细胞在HCoV-OC 43感染后可能显示出明显的CPE。本研究的目的是基于使用VeroE 6/TMPRSS 2细胞的CPE评价，构建HCoV-OC 43的50%组织培养感染剂量（TCID 50）试验。VeroE 6/TMPRSS 2细胞在低滴度HCoV-OC 43感染后3至4天显示出清晰的CPE。病毒动力学的评价表明，在感染的早期阶段，VeroE 6/TMPRSS 2细胞的培养上清中的病毒滴度高于其他细胞。相比之下，基于CPE和基于IPA（即，参考滴度）方法中，使用VeroE 6/TMPRSS 2细胞在感染后4至5天通过CPE评价测量的滴度显示出与使用其它细胞测量的参考滴度相比小得多的差异。因此，使用VeroE 6/TMPRSS 2细胞进行CPE评价的TCID 50测定提供了正确的滴度值，将极大地有助于HCoV-OC 43的未来研究。重要性HCoV-OC 43在感染细胞系中很少显示细胞病变效应（CPE），因此通过CPE观察的空斑和TCID 50试验不适用于滴定;而是使用间接免疫过氧化物酶试验（IPA）。然而，IPA相对复杂，耗时，昂贵，不适合同时滴定许多样品。我们开发了一种TCID 50测定法，使用表达TMPRSS 2的VeroE 6/TMPRSS 2细胞进行CPE评价，其准确度与传统的基于IPA的病毒滴定法相同，并且不需要使用抗体或底物进行任何染色程序。这种滴定方法将大大有助于未来的研究HCoV-OC 43允许简单，低成本，准确的滴定这种病毒。
HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43. IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.