Abstract
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 (i.e., NR-containing virus) and replicated virus (i.e., 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 However, this has to be tested on a case-by-case basis as unpublished data on feline calicivirus suggests not all viruses may be able to stably incorporate NR into their capsid (J. Parker, personal communication).
Materials and Reagents
Equipment
Procedure
Figure 1. Representative image of a plaque assay plate after 48 h of MNV-1 infection. Plaques are observed after staining with a neutral red solution overlay for 1-3 h. The image shows duplicate wells of three 10-fold viral dilutions: 10-1, 10-2 and 10-3 dilutions. Arrows indicate the formation of plaques and the viral titer of the sample is indicated below. Answer: 11+9= 20 x 102 = 2 x 103 PFU/ml.
Recipes
Acknowledgments
This neutral red assay protocol for murine norovirus was adapted thanks to the original work for poliovirus that was previously described by Brandenburg et al. (2007) and Kuss et al. (2008). Additionally, this protocol was adapted from the work performed by Perry et al. (2010); Perry et al. (2012) and Gonzalez-Hernandez et al. (2012). Our work was funded by start-up funds from the University of Michigan and NIH grant AI080611 to C.E.W. M.B.G.-H. was funded by the University of Michigan Experimental Immunology Training Grant (NIH T32 A1007413-16), by the Molecular Mechanisms of Microbial Pathogenesis Training Grant (NIH T32 A1007528), and by the Herman and Dorothy Miller Fund for Innovative Immunology Research. J.W.P. was funded by the University of Michigan Human Genetics training grant (grant NIH T32 GM 07544) and the Molecular Mechanisms of Microbial Pathogenesis training grant (NIH T32 AI 007528).
References
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