Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

What is in a Viral Stock: Perspectives and Current Limitations of Flow Virometry

Version 1 : Received: 13 November 2020 / Approved: 16 November 2020 / Online: 16 November 2020 (10:38:44 CET)

How to cite: Soh, T.K.; Bosse, J.B. What is in a Viral Stock: Perspectives and Current Limitations of Flow Virometry. Preprints 2020, 2020110409 (doi: 10.20944/preprints202011.0409.v1). Soh, T.K.; Bosse, J.B. What is in a Viral Stock: Perspectives and Current Limitations of Flow Virometry. Preprints 2020, 2020110409 (doi: 10.20944/preprints202011.0409.v1).

Abstract

Herpesviruses produce a plethora of pleomorphic and heterogeneous particle populations. The composition and biological role of these is not understood. Detailed analysis has been challenging due to the lack of multidimensional identification and purification methodologies. Fluorescence-activated cell sorting (FACS), originally developed to sort objects with at least ten thousand-fold larger volumes, has recently been applied to cellular exosomes as well as viral particles and has been dubbed nanoscale flow cytometry or “flow virometry”. In comparison to other nanoparticles, herpesvirus concentrations can be measured with high precision using simple culturing methods. Here, we used this unique capability to evaluate a standard FACS sorter. We demonstrate that detection and separation capabilities were insufficient to distinguish infectious fluorescent viral populations from populations lacking fluorescence and infectivity. Moreover, fluorescent populations did not contain single virus particles but mostly aggregates. On top, analysis of viral samples by flow cytometry was confounded by swarm detection, as multiple objects are measured simultaneously and interpreted as a single object. Despite these technical difficulties, comparison of crude supernatant to gradient purified HCMV revealed that infectious virus is a minor proportion of the particles released from infected cells. Our data stress the need for a set of standardized controls and protocols when applying FACS to biological nanoparticles and highlights technical challenges that need to be solved before flow virometry can achieve its full potential.

Subject Areas

HSV-1; HCMV; flow virometry; extracellular vesicles

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