Nano-Flow Cytometry-Guided Discrimination and Separation of Human Cytomegalovirus Virions and Extracellular Vesicles

Abstract

Accurate quantification and physical separation of viral particles and extracellular vesicles (EVs) produced by virus-infected cells presents a significant challenge due to their overlapping physical and biochemical properties. Most analytical methods provide information on a particle mixture as a whole, without distinguishing viral particles from EVs. By utilising nano-flow cytometry (nFC), a specialised form of flow cytometry adapted for the investigation of nanoparticles, we developed a simple, nucleic acid staining-based method for discrimination and simultaneous quantification of the human cytomegalovirus (HCMV) virions, dense bodies and EVs, within extracellular particle mixtures produced by HCMV-infected cells. We show that nucleic acid staining allows for discrimination of the individual particle types based on their distinct fluorescence/side scatter profiles, assessed at single-particle level by nFC. Following this, we optimised a method for physical separation of EVs from viral particles, based on high-speed centrifugation through density cushions, using nFC as a tool to evaluate the purity of the isolated EVs. The methods introduced here have the capacity to circumvent common difficulties associated with the co-investigation of EVs and viruses.

Keywords: cytomegalovirus; extracellular vesicles; nanoscale flow cytometry; nano‐flow cytometry; nucleic acid staining; particle purification; virus.

FIGURE 1

Graphical representation of the main extracellular particle types produced by uninfected and HCMV‐infected HFF cells. Created in BioRender. Bokun, V. (2025) https://BioRender.com/c88l755.

FIGURE 2

Label‐free particle concentration and size measurements in uninfected and HCMV‐infected HFF conditioned media. Conditioned medium was collected from uninfected and HCMV‐infected HFF cells, clarified by centrifugation, fixed with 4% PFA and diluted in DPBS before being analysed by nFC. Particles were excited at 488 nm and side scatter signal measured through a 488 ± 5 nm filter. Particle concentration was determined based on a concentration standard comprising silica nanospheres of a defined concentration. Particle size was estimated based on side scatter intensities, using silica nanosphere sizing standards. Calibration materials and control samples are shown in Figure S1. (A and B) Representative burst trace plots of the uninfected (A) and HCMV‐infected (B) HFF conditioned medium particles (sample dilutions are indicated in parentheses). Pulses of high side scatter were more frequently seen in HCMV‐infected HFF conditioned media. (C) A scatter plot of the particle concentrations in uninfected and HCMV‐infected HFF conditioned media (mean ± SEMs; two‐tailed ratio t‐test, p = 0.001). (D and E) Representative size distributions of particles in the uninfected HFF (D) and HCMV‐infected (E) HFF conditioned medium. EV peaks were observed in both samples at about 50 nm, while HCMV virions were observed as a peak at about 140 nm, only from infected cells. Data are from eight experiments. SS = side scatter; ms = milliseconds; nm = nanometers.

FIGURE 3

Measurement of HCMV replication kinetics by nFC and qPCR. HFF cells were infected at MOIs of 0.01, 0.1 and 1.0, or left uninfected. Conditioned medium was collected at indicated timepoints, fixed with 4% PFA, incubated with 2 µM SYTO 13 in DPBS, and analysed by nFC by excitation at 488 nm and measurement of emitted light through 488 ± 5 nm (side scatter) and 525 ± 20 nm (SYTO 13) bandpass filters. HCMV virions were identified as a distinct population, gated, and quantified using a concentration standard. The same samples (unfixed) were analysed by qPCR to obtain HCMV genome concentrations (Figure S4). (A and B) Dot plots of SYTO 13‐stained uninfected (A) and HCMV‐infected (B) HFF conditioned medium particles analysed by nFC at 2, 4 and 6 days. HCMV virions were detected as a distinct population (gate shown in red), and their quantity increased over the time course. The HCMV virion population was not observed from uninfected cells. (C) Virion and genome copy number concentrations measured by nFC and qPCR, respectively, were consistent across the timepoints and MOIs tested. (D) Virion percentages across the timepoints and MOIs, as measured by nFC, by gating on the virion population. d = days.

FIGURE 4

Discrimination of HCMV virions, dense bodies and EVs by nucleic acid staining. Extracellular particles secreted into conditioned media were purified by size exclusion chromatography, fixed with 4% PFA, and stained with SYTO 13, SYBR Safe or SYBR Green I. Samples were analysed by nFC by excitation at 488 nm and measurement of side scatter through the 488 ± 5 nm filter and emitted fluorescence through the 525 ± 20 nm filter. The populations of interest (HCMV virions, EVs and DBs) identified in the dot plots based on their distinct side scatter/fluorescence signatures were gated and quantified using a concentration standard. Control samples are shown in Figure S5. The same samples were visualised by transmission electron microscopy (TEM), to identify the different particle types observed by nFC. (A) Dot plots of uninfected HFF extracellular particles stained with the three nucleic acid dyes. Particles exhibited similar staining patterns with all three dyes, with a proportion of labelled EVs visualised above the unstained background levels. (B) Dot plots of HCMV‐infected HFF extracellular particles stained with the three nucleic acid dyes revealed three major populations: the HCMV virions, the DBs, and the EVs. The HCMV and DB populations were only seen from HCMV‐infected cells. (C) A table summarising mean concentrations of gated HCMV virions, DBs and EVs (N = 4), and the virion population MFIs for each of the three dyes. Coefficients of variation (C.V.) are shown for each of the measured parameters (mean, N = 4). (D and E) TEM imaging of extracellular particles produced by uninfected (D) and HCMV‐infected (E) HFF cells. EVs were the only particle type observed from uninfected cells, as expected, while large numbers of HCMV virions and DBs were also noted from HCMV‐infected cells.

FIGURE 5

HCMV virion sizing by nFC, TEM and cryo‐EM. Extracellular particles purified by size exclusion chromatography were analysed by nFC and negative stain TEM for virion size. Virion sizing by nFC was performed on untreated virions and those treated with 1% Triton X‐100, which were labelled with SYBR Green I, and gated as before. Additional sizing standards and different side scatter decay settings were also compared (Figure S6). Viral particles prepared by gradient ultracentrifugation were also analysed for virion size by cryo‐EM. Representative micrographs of the HCMV virions included in the sizing analyses by negative stain TEM and cryo‐EM are shown in Figure S7. (A) HCMV virion sizing by nFC. Size distributions were generated for all extracellular particles as well as gated virions only (untreated and treated with Triton X‐100). The mean virion diameter was found to be 131.9 nm, decreasing to 115.1 nm upon Triton X‐100 treatment. Data are representative of three independent experiments. (B) Statistical summary of the HCMV virion sizing results obtained by the three techniques, including mean diameter, standard deviation, median diameter and the number of data points included in the analysis. d = diameter.

FIGURE 6

Separation of EVs from viral particles in the HCMV‐infected HFF conditioned medium. Conditioned medium was clarified by centrifugation and 0.45‐µm filtration, and then centrifuged at 25,000 × g over underlaid 16% iodixanol cushions. The resulting supernatants and pellets as well as the starting conditioned medium were analysed for particle concentration and size by nFC following SYBR Green I labelling, and by plaque assays for infectivity. (A) Illustration of the methodological approach for the separation of EVs from viral particles and medium contaminants. (B and C) Dot plots and size distributions obtained by nFC following SYBR Green I labelling of uninfected (B) and HCMV‐infected (C) HFF conditioned medium particles, before and after the separation step. Dilutions of samples for the nFC analysis and their total volumes are noted in the top right corner of each dot plot. No major changes were observed between the pre‐ and post‐centrifugation samples in the uninfected HFF samples, with very few particles found in the pellets. In contrast, nFC revealed a total depletion of HCMV virions and DBs from the supernatants, and a concomitant enrichment in the pellets, following the high‐speed centrifugation step. All plots in the figure are representative of four independent experiments. Numerical results are shown in Table 1. Transmission electron microscopy of the 25,000 × g pellets is shown in Figure S8. Graphical elements of the figure were created in BioRender. Bokun, V. (2025) https://BioRender.com/d12r423.

FIGURE 7

Size exclusion chromatography purification of EVs from post‐25,000 × g centrifugation supernatants. EVs remaining in the supernatants following high‐speed centrifugation were concentrated by 100‐kDa ultrafiltration, and fractionated on a custom Sepharose CL‐2B column. (A) EV/protein elution profiles as measured by nanoparticle tracking analysis (NTA) and Bradford assay show a robust separation of EVs from the contaminating protein. (B) Silver staining of fractions 6–35 from the HCMV‐infected cells, including the starting conditioned medium (CCM), and the non‐conditioned medium (NCM). Bovine serum albumin (BSA), the most abundant component of the fetal bovine serum (FBS) in the complete growth medium, appeared in fraction 19 and peaked in fraction 31 (indicated with red rectangles). (C) ELISA for CD63 and β‐actin shows positivity for these two EV‐associated proteins in the particle‐containing fractions. Matching NTA and Bradford assay data obtained from non‐conditioned complete medium is shown in Figure S9A while the matching silver staining data for uninfected cells is shown in Figure S9B. Data in panel A are presented as means ± SEMs (N = 3), with all measurements performed in triplicate. Data included in panels B and C are from single experiments. ELISA measurements were performed in duplicate. F = fraction; M = molecular weight markers.

FIGURE 8

Characterisation of uninfected and HCMV‐infected HFF EVs. EVs were purified from 25,000 × g supernatants by SEC and concentrated by ultrafiltration. EVs were examined by TEM and nFC. (A and B) TEM imaging of HFF EVs revealing characteristic circular/oval EV morphology. No HCMV virions were observed in EV preparations from infected cells. Additional widefield micrographs are included in Figure S10. (C and D) Representative nFC dot plots showing SYBR Green I labelling of uninfected and HCMV‐infected HFF EVs. The mean percentage of events in the virion gate is indicated above. (E and F) Representative size distributions of purified HFF EVs generated by nFC. The data in panels (A) and (B) are representative of two independent experiments, and the data in panels (C–F) are representative of five biological replicates.