Flow Virometry: a Powerful Tool To Functionally Characterize Viruses

Abstract

For several decades, flow cytometry has been a common approach to analyze cells and sort them to near-purity. It enables one to probe inner cellular molecules, surface receptors, or infected cells. However, the analysis of smaller entities such as viruses and exocytic vesicles has been more difficult but is becoming mainstream. This has in part been due to the development of new instrumentation with resolutions below that of conventional cytometers. It is also attributed to the several means employed to fluorescently label viruses, hence enabling them to stand out from similarly sized particles representing background noise. Thus far, more than a dozen different viruses ranging in size from 40 nm to giant viruses have been probed by this approach, which was recently dubbed "flow virometry." These studies have collectively highlighted the breadth of the applications of this method, which, for example, has elucidated the maturation of dengue virus, served as quality control for vaccinia vaccines, and enabled the sorting of herpes simplex virus discrete viral particles. The present review focuses on the means employed to characterize and sort viruses by this powerful technology and on the emerging uses of flow virometry. It similarly addresses some of its current challenges and limitations.

Keywords: FACS; HSV; exosomes; flow cytometry; flow virometry; herpes; herpes simplex virus; hsv; nanoparticles; review; sorting; viral particles.

FIG 1

Technological developments of flow cytometers. A schematic representation of a FACS apparatus, including the fluidic, optic, and electronic components, is drawn. Various viruses (differently colored) are also portrayed. Current improvements to the cytometers (in blue boxes) include filtering of the sheath buffer with a 0.1-μm-pore-size filter, optional filtering of the viral sample with a 0.45-μm-pore-size filter (for viruses smaller than that), better hydrodynamic focusing, more-powerful lasers, and forward light scatter (FSC) detectors with greater sensitivity and reduced wide-angle capabilities. Note that standard FSC detectors typically monitor light in the 0.5° to 15° range, where most of the background signal is found (red line) and where less light is emitted by nanoparticles (green lines). The reduced wide-angle FSC detector instead blocks any light below 15° and records the signal from 15° to 70°, greatly improving signal-to-noise ratios. SSC, side light scatter.

FIG 2

Efficient means to label viral particles for flow cytometry. The typical constitution of a virus is depicted (an enveloped virus is shown here). The viral genome is enclosed in a proteinaceous capsid, which can be surrounded by a protein layer called a matrix or tegument depending on the given virus. For enveloped virions, a cell-derived envelope that contains viral glycoproteins is additionally present. The figure indicates the current methods used to perform flow virometry, which includes genetic tagging of the diverse virion constituents, including the capsid, matrix/tegument, or viral glycoproteins (fFP; GFP or alternative fluorescent tags). It is also possible to detect the viruses with antibodies directed against surface molecules (glycoproteins for the enveloped viruses or capsid proteins for nonenveloped viruses). Some laboratories couple the antibodies to nanoparticles for greater detectability by light scattering. A third approach is to employ fluorescent lipophilic markers in the case of enveloped viruses (DiD, DiO, DiI) with the possible use of PKH67. The use of DiR, a member of the same family of dyes but with excitation/emission profiles in the infrared spectrum, should also be an option. Finally, one can label the RNA/DNA viral genome with several different dyes (Syto13, Syto62, SyBr green-I, YOYO-1, TOTO-1, and PicoGreen). Note that this list is not exhaustive, as many other dyes may also perform well.