González-Hernández Mariam Bernadette, Perry Jeffrey William, Wobus Christiane E
Department of Microbiology and Immunology, Graduate Program of Immunology, University of Michigan, Ann Arbor, MI, USA.
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA.
Bio Protoc. 2013 Apr 5;3(7).
Neutral red (NR) is a dye that must be actively imported into the cell, and, therefore, the dye has been used for decades to selectively stain living cells. In addition, NR can also be incorporated into virus particles, although the mechanism behind this is poorly understood. Once encapsulated into the virion, NR, a light sensitive dye, can be photoactivated to inactivate the virus. The proposed mechanism explaining this observation is that activation of NR allows the dye to cross-link viral genome to viral capsid and thus preventing viral uncoating and infection. To study the early events of murine norovirus (MNV)-host interaction, light-sensitive NR-containing MNV is used to distinguish between input virus (, NR-containing virus) and replicated virus (, NR-free virus). This protocol describes the incorporation of NR into MNV capsids and the use of these virions for detection of viral replication in a mouse and in tissue culture by standard plaque assay. The same technique is also used for study of poliovirus replication (1-3). Thus, there is the potential that this technique can be used for additional non-enveloped viruses. However, this has to be tested on a case-by-case basis as unpublished data on feline calicivirus suggests not all viruses may be able to stably incorporate NR into their capsid (J. Parker, personal communication).
中性红（NR）是一种必须被主动转运进入细胞的染料，因此，几十年来该染料一直用于选择性地对活细胞进行染色。此外，NR也可以掺入病毒颗粒中，尽管其背后的机制尚不清楚。一旦被包裹进病毒粒子，NR这种光敏染料就可以被光激活以灭活病毒。解释这一现象的推测机制是，NR的激活使该染料能够将病毒基因组与病毒衣壳交联，从而阻止病毒脱壳和感染。为了研究小鼠诺如病毒（MNV）与宿主相互作用的早期事件，含光敏NR的MNV被用于区分输入病毒（即含NR的病毒）和复制病毒（即不含NR的病毒）。本方案描述了将NR掺入MNV衣壳的方法，以及通过标准噬斑测定法利用这些病毒粒子检测小鼠和组织培养中病毒复制的方法。同样的技术也用于脊髓灰质炎病毒复制的研究（1 - 3）。因此，该技术有可能用于其他无包膜病毒。然而，由于关于猫杯状病毒的未发表数据表明并非所有病毒都能将NR稳定地掺入其衣壳中（J. 帕克，个人交流），所以必须逐例进行测试。
