Dengue virus’ evaluation with flow virometry

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Flow virometry used to address the maturation stage of individual virions

Text by Sonia Zicari


Dengue virus (DENV) is a small positive single-stranded RNA enveloped Flavivirus that is formed as a non-infectious particle, which undergoes maturation by cleavage of a protein (prM) on its surface. Different prMs are cleaved at different time-points/locations, resulting in viral heterogeneity both at the population and at the individual particle levels. Bulk analyses cannot capture this heterogeneity. Using our new technology, flow virometry, we analyzed single virions and evaluated the heterogeneity of the surface proteins that determine viral pathogenicity. We showed that uncleaved protein (prM) was present on about 50% of infectious DENV particles produced by BHK-21 cells. In contrast if DENV is produced by LoVo cells, which lack the furin-protease mediating the cleavage of prM, this precursor was present on ~85% of virions. Flow virometry opens the way to analyze individual proteins on individual virions.


We think that in any population the biological objects are heterogeneous and their individual features determine biological properties. At the cell level it was appreciated by centuries of morphological observations and was formalized with flow cytometry using fluorescent antibodies defining Cluster of Differentiation (CD). However, because viruses are too small for the majority of these techniques they are analyzed predominantly in bulk and their individualities are lost. To overcome this, our laboratory developed a nano-technique, flow virometry, that permits analysis of single virions stained with fluorescent antibodies (Arakelyan et al, JCI, 2013). Initially this technique was applied to HIV and is now used to study maturation of individual DENVs. Virions were stained with anti-prM antibodies, captured by magnetic nanopartocles (MNPs) coupled with anti-E protein antibodies, the unbound antibodies were washed off on magnetic columns, and eluted viruses-MNPs complexes were analyzed using flow cytometers thresholding on fluorescence. Our main idea was to show that we can detect differences in individual DENVs even if they had been produced by the same cells and, more generally, that flow virometry can be used to address the heterogeneity of envelope proteins of various viruses.


Unlike HIV, DENVs are highly pH-dependent and easily aggregate. First, we applied to DENV the protocol we developed for HIV, but we encountered problems of steric hindrance and aspecificity. To overcome these problems we incubated the virus first with detection antibodies then with MNPs and to avoid aspecificity we used mouse IgG. Eventually we created a protocol that suits DENV analysis.


Figure legend

Maturation state of DENV virions. DiI labeled DENV produced either in BHK-21 or in LoVo cells were stained for prM protein with Alexa Fluor647-labeled 2H2 or with its isotype control IgG2a, and then captured with Alexa Fluor 488 3H5-1-MNPs. (A) DENV produced in BHK-21 cells, stained with specific 2H2 antibody. (B) Isotype control to A. (C) DENV produced in LoVo cells, stained with specific 2H2 antibody. (D) Isotype control to C. Here is shown a representative experiment out of four. Note a higher incidence of prM-positive virions produced in furin-deficient LoVo cells.

Introducing the authors

Zicari_author picture complete

Pictured from left to right: Sonia Zicari, Anush Arakelyan, Leonid Margolis, Wendy Fitzgerald, Leonid Chernomordik, Elena Zaitseva and Jean-Charles Grivel. Dr. Grivel is now the Director of the Deep Phenotyping Core of Sidra Medical and Research Center, in Doha, Qatar.

About the research

Evaluation of the maturation of individual Dengue virions with flow virometry

Sonia Zicari, Anush Arakelyan, Wendy Fitzgerald, Elena Zaitseva, Leonid V. Chernomordik, Leonid Margolis, Jean-Charles Grivel

Virology, Volume 488, 15 January 2016, Pages 20–27