Single-cell herpes simplex virus type 1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics

Abstract

Single-cell analyses of viral infections reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of herpes simplex virus type 1 (HSV-1) infection of neurons at the single-cell level. We used micrometer-scale Matrigel beads, termed microgels, to culture individual murine superior cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are encapsulated in individual media-in-oil droplets with a dual-fluorescent reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. These techniques for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations.

Fig. 1.. Experimental schematic for the embedding, growth, and infection of individual neurons within microfluidic drops.

(A) SCG neurons are suspended in liquid Matrigel and emulsified in oil. The drops are incubated at 37°C for 35 min for gelation. The microgels and neurons are washed and placed in media for 7 days for neuronal maturation. (B) After 7 days, the neurons are co-flowed with viral inoculum and emulsified in fluorinated oil. (C) A dual-fluorescent HSV-1 recombinant is used to visualize infection. Initiation of viral gene expression is reported by YFP detection. Late gene expression is reported by RFP detection. (D) Individual cells can be tracked over time to observe the progression of FP detection.

Fig. 2.. Impacts of microgels on infection.

(A) Model of Vero cell culture in microgels, on microgels, or in suspension for co-flow inoculation. (B to E) Representative phase contrast, YFP, and merged images of infected cells. Scale bars, 25 μm. (B) In microgels with an internally positioned cell. (C) In microgels with a peripherally positioned cell. (D) On microgel. (E) In suspension. (F) Bar graphs showing the percentage of YFP-positive cells. Vero cells cultured as indicated and co-flow inoculated with 10 PFU per drop of dual-reporter HSV-1. (G) Bar graphs showing the percentage of YFP-positive cells from Vero cells cultured on microgels inoculated with 1, 10, and 100 PFU per drop. This trend was statistically significant (one-way ANOVA, P = 1.3 × 10⁻⁵). All infections in (F) and (G) were performed in triplicate with an average number of 200 cells per condition per replicate. Error bars show SDs.

Fig. 3.. HSV-1 virions cannot enter Matrigel.

(A) Representative images of time-lapse confocal microscopy of mRFP-labeled HSV-1 virions diffusing next to a disc of Matrigel (right side of image). Images were acquired every 10 min for 90 min in bright field and mRFP (false-colored green). (B) Nanoparticles (green) were added to the solution surrounding the Matrigel disc. Scale bar, 15 μm; t = 60 min.

Fig. 4.. Microgels support neuronal maturation and infection.

(A) Representative image of a mature SCG neuron in a Matrigel microgel after 7 days in culture. Cells were stained with calcein AM, false-colored purple. Scale bar, 25 μm. (B) Mature SCG neuron grown in a microgel immunostained for phosphorylated neurofilament H (red) and nuclei (blue). Scale bar, 25 μm. (C) Bar graph showing the percentage of YFP-positive neurons following infection at 1, 10, and 100 PFU per drop. Infections were performed in triplicate with an average of 158 cells per replicate per condition. Statistical significance evaluated by one-way ANOVA (P = 5.9 × 10⁻⁴). Error bars show SD. (D) Representative phase contrast, YFP, and merged images of an infected SCG neuron. Scale bar, 25 μm.

Fig. 5.. The effect of inoculating dose on neuronal infection progression and kinetics.

(A) Schematic of experimental design for temporal tracking of YFP and RFP. (B) Representative images from time-lapse microscopy of an infected neuron expressing YFP and RFP in a DropSOAC chamber (31). Scale bar, 50 μm. (C) Normalized intensities of YFP (open green squares) and RFP (filled red circles) for the representative cell in (B). Dashed lines represent the threshold value above which cells are considered positive for FP detection. (D) Timing of YFP and RFP detection plotted with the mean and SD. Each data point represents quantitation from single neurons. Statistical significance was evaluated by one-way ANOVA (P = 1.1 × 10⁻⁷). Error bars show SD. Infections were performed three to four times and pooled together for analysis with a total of 174 cells analyzed. (E) Correlation of YFP versus RFP detection time for RFP-positive neurons (1 PFU per drop, blue X; 10 PFU per drop, purple square; 100 PFU per drop, black circle). A linear regression fit to evaluate the significance of correlation is plotted as a dashed line with the fit.

Fig. 6.. HSV-1 replication kinetics in Vero cells.

(A) Timing of YFP and RFP expression in individual Vero cells plotted with the mean and SD. Timing of YFP detection decreased with increased inoculating dose (one-way ANOVA, P = 1.9 × 10⁻³⁵). Error bars show SD. Infections were performed three times and pooled together for analysis with a total of 669 cells analyzed. (B) Correlation of YFP versus RFP detection time for RFP-positive Vero cells (1 PFU per drop, blue stars; 10 PFU per drop, purple squares; 100 PFU per drop, black circle) (linear regression: t_RFP+ = 6.4 + 0.9 × t_YFP+, P = 1.1 × 10⁻⁷, R² = 0.22). (C) Representative images of Vero cells infected in drop on microgels. White circles outline the microgel. Scale bar, 50 μm.