Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro
Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro
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DOI：
10.1016/s0166-0934(98)00071-8
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发表时间：
1998-09-01
影响因子：
3.1
通讯作者：
Osterhaus, ADME
中科院分区：
文献类型：
--
作者：
Rimmelzwaan, GF;Baars, M;Osterhaus, ADME
需要快速、灵敏的方法来监测体外流感病毒的复制，以解决一些研究问题。比较了基于不同原理的四种方法。血凝 (HA) 测定、培养上清液中病毒感染滴度的测量、使用地高辛 (DIG) 标记的 RNA 探针通过免疫荧光和 RNA 杂交技术计数受感染的细胞。为此，用最近的甲型流感病毒（A/Netherlands/18/94 H3N2）以不同的感染复数（moi）感染MDCK细胞，并用这四种测定法监测病毒复制的动力学。在高moi下，感染后12小时通过HA测定检测到释放到感染细胞的培养物上清液中的病毒，而在较低moi（小于或等于0.01）下，在感染后24小时之前未检测到第一个HA活性。培养物上清液中感染性病毒的测量被证明更为灵敏，因为感染后4-12小时根据所使用的moi检测到新产生的病毒。这一发现与使用核蛋白特异性抗体制剂进行免疫荧光测定获得的结果一致：感染后 4 小时即可检测到单个感染细胞。此时，对NP基因片段进行mRNA/cRNA特异性杂交时也获得阳性信号，但对病毒NP RNA或血凝素特异性RNA则没有获得阳性信号，这些信号仅在感染后较晚的时间点检测到。因此，除了直接测量感染性病毒和免疫荧光之外，RNA杂交被证明是监测流感病毒体外复制的灵敏测定方法。 (C) 1998 Elsevier Science B.V. 保留所有权利。
Rapid and sensitive methods for the monitoring of influenza virus replication in vitro are needed to address several research questions. Four methods based on different principles were compared. the hemagglutination (HA) assay, the measurement of virus infectivity titers in culture supernatants, the enumeration of infected cells by immunofluorescence and RNA hybridization techniques using digoxigenin (DIG) labeled RNA probes. To this end, MDCK cells were infected at different multiplicities of infection (moi) with a recent influenza A virus (A/Netherlands/18/94 H3N2) and the kinetics of virus replication were monitored with these four assays. At high moi, virus released into the culture supernatant of infected cells was detected by the HA assay 12 h post infection, whereas at lower moi (less than or equal to 0.01) the first HA activity was not detected before 24 h post infection. The measurement of infectious viruses in the culture supernatant proved to be more sensitive, since 4-12 h post infection newly produced virus was detected depending on the moi used. This finding was in agreement with results obtained by the immunofluorescence assay using an antibody preparation specific for the nucleoprotein: single infected cells could be detected as early as 4 h post infection. At this time point, positive signals were also obtained when mRNA/cRNA specific hybridization was carried out for the NP gene segment, but not for viral NP RNA or RNA specific for the hemagglutinin, which were only detected at later time points after infection. Thus, besides direct measurement of infectious virus and immunofluorescence, RNA hybridization proved to be a sensitive assay for monitoring influenza virus replication in vitro. (C) 1998 Elsevier Science B.V. All rights reserved.