DOI: 10.5061/dryad.g4f4qrfx6
title: Single-cell Herpes simplex virus type-1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics

description: 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 HSV-1 infection of
 neurons at the single-cell level. We used micron-scale Matrigel beads,
 termed microgels, to culture individual murine Superior Cervical ganglia
 (SCG) neurons or epithelial cells. Microgel-cultured cells are
 subsequently 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. The innovative
 techniques presented here for cell culture and infection in drops provide
 a foundation for future virology and neurobiology investigations.
description: The microfluidic device containing hydrogel microgels with
 infected mature neurons were placed in a microscope stage top incubation
 chamber at 37°C. Images in Phase Contrast/YFP/RFP were taken every 15 min
 for 16 h.  <em>Percentage of YFP Positive
 Cells</em>. To quantify the percentage of YFP positive cells,
 end-point images were analyzed using Fiji ImageJ. Cells were found in
 brightfield. The background intensity of the YFP channel was subtracted
 from the image, and the percentage of YFP positive cells was quantified.
 Experiments were repeated in triplicate. Cell counts for Vero cells
 embedded in-microgels ranged from 79 -280 cells/replicate, for Vero cells
 grown on-microgels from 165 - 390 cells/replicate, for Vero cells
 in-suspension from 228 - 456 cells/replicate, and for SCG neurons from 84
 - 209 cells/replicate. Multiple comparison tests and one-way ANOVAs were
 performed in Matlab. <em>The Timing of YFP and
 RFP Detection</em>. To quantify the timing of YFP and RFP detection,
 time-lapse images were analyzed using Fiji ImageJ. Cells were difficult to
 identify in brightfield, so cells were identified by finding YFP positive
 cells at 16 hpi. Once located, a 30 μm diameter circular region of
 interest (ROI) was drawn around each individual cell. The maximum pixel
 intensity in YFP and RFP for the ROI was then measured for each
 time-frame. The frame 1 maximum pixel intensity of each ROI was subtracted
 from the subsequent frames for that ROI. The noise threshold was found to
 be 50 arbitrary units (a.u.) for YFP and 75 a.u. for RFP. The timing of
 YFP and RFP detection were defined as the first time-frame the YFP and RFP
 pixel intensities in each ROI were greater than the noise threshold for
 two consecutive frames. For SCG time-lapse studies, experiments were
 repeated 3-4 times with a total of 174 cells analyzed. For Vero time-lapse
 studies, experiments were repeated 3 times with a total of 669 cells
 analyzed. One-way ANOVAs and linear regressions were performed in
 Matlab.
description: # Single-cell Herpes Simplex Virus type-1 infection of neurons using
 drop-based microfluidics reveals heterogeneous replication kinetics
 ## Description of the data
 and file structure 1.) The dataset titled
 "Infection_percentage_raw_data_2-20-24.xlsx" describes the
 infection condition, trial number, and number of YFP positive and negative
 cells counted. 2.) The dataset titled
 "Raw_Timelapse_Data_2_27_24_V2.xlsx" is the raw fluorescence
 data of the infected cells, organized by by the date of the experiment,
 the cell type analyzed, the inoculation concentration, the fluorescence
 channel (YFP or RFP) analyzed, the time point at which the image was
 taken, and the cell number analyzed. Data has not been background
 subtracted. Time is in hours post infection (hpi). NA values are
 timepoints where images were not acquired.