Imaging flow cytometry reveals the mechanism of equine arteritis virus entry and internalization

Figures

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  Fig. 1. Imaging flow cytometry in tracking viral entry. Imaging flow cytometry allows visualisation of the early stages of viral infection by exploiting the optical capabilities of the instrument (left) in conjunction with simultaneous quantitative measurements (right). Images of cells fixed at different time points after infection (0 h, 1 h, 3 h) correspond to different stages of viral entry via endocytosis, including attachment, internalization and uncoating. In addition, parameters indicative of the extent of viral internalization during these early infection events were extracted through analysis using the IDEAS software.

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  Fig. 2. EAV entry into Vero cells visualised by confocal microscopy. Vero cells were synchronously infected with EAV or left uninfected. The cells were then fixed and subjected to immunostaining at different time points—0, 1 and 3 h post infection. An anti-N antibody was used to detect the viral nucleocapsid protein, allowing specific visualisation of the virus on or in the cells (green). To further visualise cell morphology and structure, actin filaments were stained with phalloidin (red) and nuclei with DAPI (blue). The pictures were taken with a confocal microscope (Zeiss Cell Observer SD, 63 × oil objective). Each scale bar represents 20 µm.

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  Fig. 3. Impact of selected inhibitors on EAV replication in Vero cells. Vero cells were treated with the inhibitors and infected by EAV. The percentage of EAV positive cells were determined using conventional flow cytometry 36 h post infection. (A) Representative dot plot showing gating strategies for conventional flow cytometry. Vero cells were first gated to exclude debris and then the percentage of Alexa Fluor 647 positive (EAV infected) cells was determined. (B) Example overlay histograms from conventional flow cytometry showing fluorescence intensity in the Alexa Fluor 647 channel, reflecting virus concentration in Vero cells across different samples. (C) The graph presented the percentage of EAV-infected cells (36 h post infection) after treatment with selected inhibitors. The groups with inhibitors were each compared with infected control (EAV + no inhibitor). Statistical significance was detected between infected cells (EAV + no inhibitor) and mock (no infected cells) or EAV + NH4Cl (Alpha = 0,05, p < 0,0001) (****). In the case of the remaining inhibitors, no statistical significance was achieved (ns). (D) Cytotoxicity of inhibitors determined using the Cell Counting Kit-8…

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  Fig. 4. Schematic of the gating strategy with representative cell images for ImFC analysis.(A) Single Vero cells were gated from the total population using the Area and Aspect Ratio features on the BF images. (B) Focused cells were then selected using the Gradient RMS feature (BF channel). (C) Finally, Alexa Fluor 647-stained Vero cells (EAV-positive cells) were gated based on their Intensity and Max Pixel features.

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  Fig. 5. ImFC as a tool for differentiating effective from ineffective virus entry inhibitors. (A) A graphical representation illustrating the absence and presence of viral uncoating inhibition, with corresponding images from immunofluorescence microscopy (ImFC) taken 3 h post-infection. The cells were pre-treated with either NH4Cl or camostat, alongside a non-infected control, then fixed, permeabilized, blocked, and immunostained. ImFC analysis was conducted, and the captured images were presented as bright field (BF), AF647 fluorescence, and merged composites. In the control and camostat-treated cells, endocytosis is performed normally, and the fluorescent signal from the anti-N EAV antibody (red) is absent due to successful viral uncoating (left image). In contrast, NH4Cl -treated cells display a visible signal, indicating inhibition of endocytosis as a result of impaired acidification (right image). (B) The histogram highlights differences between positive and negative inhibition. The inhibition effect of NH4Cl was statistically significant (t-test, n = 4, p = 0.0265, two-tailed), while camostat showed no significant effect on uncoating (t-test, n = 4, p = 0.3586, two-tailed), when…

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  Fig. 6. Investigation of equine arteritis virus (EAV) entry into Vero cells using imaging flow cytometry (ImFC). (A) The entry of equine arteritis virus (EAV) into Vero cells via endocytosis is illustrated by representative fluorescence images of infected cells at 0 h, 1 h, and 3 h post-infection. (B) Measurements of mean fluorescence intensity (MFI) showed a decreasing trend in fluorescence signals at later infection times (1 h and 3 h) compared to the initial time point (0 h). Mock-infected cells exhibited similar levels of fluorescence at 3 h. No statistical significance was observed. (C-D) Virus uptake, evaluated at 0 h and 1 h, revealed statistically significant differences in the percentage of cells with internalized EAV virions (t- test, n = 4, p < 0,0001, two- tailed). (E-F) The spot count parameter, calculated for the 0 h and 1 h stages, indicated a statistically significant difference in the number of virions per cell based on median (t-test, n = 3, p = 0,0249, two-tailed). At 0 h, more virions were detected (on the cell surface), while at 1 h, a lower number of viral particles were found (inside the cells). Bars and dots represent standard deviations and replicates,…