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Methods. 2016 Apr 1;98:82-90. doi: 10.1016/j.ymeth.2015.11.002. Epub 2015 Nov 2.

Visual detection of Flavivirus RNA in living cells.

Author information

1
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
2
IFOM - Istituto FIRC di Oncologia Molecolare, via Adamello 16, 20139 Milan, Italy.
3
Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy. Electronic address: marcello@icgeb.org.

Abstract

Flaviviruses include a wide range of important human pathogens delivered by insects or ticks. These viruses have a positive-stranded RNA genome that is replicated in the cytoplasm of the infected cell. The viral RNA genome is the template for transcription by the virally encoded RNA polymerase and for translation of the viral proteins. Furthermore, the double-stranded RNA intermediates of viral replication are believed to trigger the innate immune response through interaction with cytoplasmic cellular sensors. Therefore, understanding the subcellular distribution and dynamics of Flavivirus RNAs is of paramount importance to understand the interaction of the virus with its cellular host, which could be of insect, tick or mammalian, including human, origin. Recent advances on the visualization of Flavivirus RNA in living cells together with the development of methods to measure the dynamic properties of viral RNA are reviewed and discussed in this essay. In particular the application of bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are analysed in the context of tick-borne encephalitis virus replication. Conclusions driven by this approached are discussed in the wider context Flavivirus infection.

KEYWORDS:

FLIP; FRAP; Flavivirus; Innate immunity; Live imaging; RNA; Replication

PMID:
26542763
DOI:
10.1016/j.ymeth.2015.11.002
[Indexed for MEDLINE]

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