Cell culture experiments

All experiments were carried out on three immortalized mammalian kidney cell lines: Vero (African green monkey), RoNi/7.1 (Rousettus aegyptiacus) (Kühl et al., 2011; Biesold et al., 2011) and PaKiT01 (Pteropus alecto) (Crameri et al., 2009). The species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (Kühl et al., 2011; Biesold et al., 2011; Crameri et al., 2009). Vero cells were obtained from ATCC.

Monolayers of each cell line were grown to 90% confluency (~9×10⁵ cells) in 6-well plates. Cells were maintained in a humidified 37 °C, 5% CO₂ incubator and cultured in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA), and 1% penicillin-streptomycin (Life Technologies). Cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing.

Previous work has demonstrated that all cell lines used are capable of mounting a type I IFN response upon viral challenge, with the exception of Vero cells, which possess an IFN-β deficiency (Desmyter et al., 1968; Rhim et al., 1969; Emeny and Morgan, 1979). RoNi/7.1 cells have been shown to mount idiosyncratic induced IFN defenses upon viral infection (Pavlovich et al., 2018; Kuzmin et al., 2017; Arnold et al., 2018; Kühl et al., 2011; Biesold et al., 2011), while PaKiT01 cells are known to constitutively express the antiviral cytokine, IFN-α (Zhou et al., 2016). This work is the first documentation of IFN signaling induced upon challenge with the particular recombinant VSVs outlined below. We verified known antiviral immune phenotypes via qPCR. Results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in RoNi/7.1 versus PaKiT01 cells.

Replication-capable recombinant vesicular stomatitis Indiana viruses, expressing filovirus glycoproteins in place of wild type G (rVSV-G, rVSV-EBOV, and rVSV-MARV) have been previously described (Wong et al., 2010; Miller et al., 2012). Viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell’s entry receptor. These interactions ranged from the total absence of evolutionary history in the case of rVSV-G infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rVSV-MARV infections on PaKiT01 cell lines.

To measure infectivities of rVSVs on each of the cell lines outlined above, so as to calculate the correct viral dose for each MOI, NH₄Cl (20 mM) was added to infected cell cultures at 1–2 hr post-infection to block viral spread, and individual eGFP-positive cells were manually counted at 12–14 hr post-infection.

Previously published work indicates that immortalized kidney cell lines of Rousettus aegyptiacus (RoNi/7.1) and Pteropus alecto (PaKiT01) exhibit different innate antiviral immune phenotypes through, respectively, induced (Biesold et al., 2011; Pavlovich et al., 2018; Kühl et al., 2011; Arnold et al., 2018) and constitutive (Zhou et al., 2016) expression of type I interferon genes. We verified these published phenotypes on our own cell lines infected with rVSV-G, rVSV-EBOV, and rVSV-MARV via qPCR of IFN-α and IFN-β genes across a longitudinal time series of infection.

Specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at MOIs of 0.0001 and 0.001—with the exception of rVSV-MARV on PaKiT01 cell lines, for which infection was only performed at MOI = 0.0001 due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). All experiments were run in duplicate on 6-well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two MOI = 0.0001 wells and two MOI = 0.001 wells, excepting PaKiT01 plates, which had two control and four MOI = 0.0001 wells at a given time. We justify this PaKiT01 exemption through the expectation that IFN-α expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower MOI should also be present at the higher MOI.

For these gene expression time series, four 6-well plates for each cell line–virus combination were incubated with virus for one hour at 37 °C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. Plates were then harvested sequentially at timepoints of roughly 5, 10, 15, and 20 hr post-infection (exact timing varied as multiple trials were running simultaneously). Upon harvest of each plate, agar overlay was removed, and virus was lysed and RNA extracted from cells using the Zymo Quick RNA Mini Prep kit, according to the manufacturer’s instructions and including the step for cellular DNA digestion. Post-extraction, RNA quality was verified via nanodrop, and RNA was converted to cDNA using the Invitrogen Superscript III cDNA synthesis kit, according to the manufacturer’s instructions. cDNA was then stored at 4 °C and as a frozen stock at −20 °C to await qPCR.

We undertook qPCR of cDNA to assess expression of the type I interferon genes, IFN-α and IFN-β, and the housekeeping gene, β-Actin, using primers previously reported in the literature (Supplementary file 6). For qPCR, 2 µl of each cDNA sample was incubated with 7 µl of deionized water, 1 µl of 5 UM forward/reverse primer mix and 10 µl of iTaq Universal SYBR Green, then cycled on a QuantStudio3 Real-Time PCR machine under the following conditions: initial denaturation at 94 °C for 2 min followed by 40 cycles of: denaturation at 95 °C (5 s), annealing at 58 °C (15 s), and extension at 72 °C (10 s).

We report simple δ-Ct values for each run, with raw Ct of the target gene of interest (IFN-α or IFN-β) subtracted from raw Ct of the β-Actin housekeeping gene in Figure 1—figure supplement 6. Calculation of fold change upon viral infection in comparison to mock using the δ-δ-Ct method (Livak and Schmittgen, 2001) was inappropriate in this case, as we wished to demonstrate constitutive expression of IFN-α in PaKiT01 cells, whereby data from mock cells was identical to that produced from infected cells.

After being grown to ~90% confluency, cells were incubated with pelleted rVSVs expressing eGFP (rVSV-G, rVSV-EBOV, rVSV-MARV). Cell lines were challenged with both a low (0.0001) and high (0.001) multiplicity of infection (MOI) for each virus. In a cell monolayer infected at a given MOI (m), the proportion of cells (P), infected by k viral particles can be described by the Poisson distribution: R0, such that the number of initially infected cells in an experiment equals: R0>1. We assumed that a ~90% confluent culture at each trial’s origin was comprised of ~9x10⁵ cells and conducted all experiments at MOIs of 0.0001 and 0.001, meaning that we began each trial by introducing virus to, respectively, ~81 or 810 cells, representing the state variable ‘E’ in our theoretical model. Low MOIs were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture.

Six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and 2–3 wells each at MOI 0.001 and 0.0001 were incubated simultaneously on the same plate. In total, we ran between 18 and 39 trials at each cell-virus-MOI combination, excepting r-VSV-MARV infections on PaKiT01 cells at MOI = 0.001, for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. Cells were incubated with virus for one hour at 37 °C. Following incubation, virus was aspirated off, and cell monolayers were washed in PBS, then covered with a molten viscous overlay (50% 2X MEM/L-glutamine; 5% FBS; 3% HEPES; 42% agarose), cooled for 20 min, and re-incubated in their original humidified 37 °C, 5% CO₂ environment.

After application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of GFP expression were witnessed (~6–9.5 hr post-infection, depending on the cell line and virus under investigation). From that time forward, a square subset of the center of each well (comprised of either 64- or 36-subframes and corresponding to roughly 60% and 40% of the entire well space) was imaged periodically, using a CellInsight CX5 High Content Screening (HCS) Platform with a 4X air objective (ThermoFisher, Inc, Waltham, MA). Microscope settings were held standard across all trials, with exposure time fixed at 0.0006 s for each image. One color channel was imaged, such that images produced show GFP-expressing cells in white and non-GFP-expressing cells in black (Figure 1—figure supplement 1).

Wells were photographed in rotation, as frequently as possible, from the onset of GFP expression until the time that the majority of cells in the well were surmised to be dead, GFP expression could no longer be detected, or early termination was desired to permit Hoechst staining.

In the case of PaKiT01 cells infected with rVSV-EBOV, where an apparently persistent infection established, the assay was terminated after 200+ hours (8+ days) of continuous observation. Upon termination of all trials, cells were fixed in formaldehyde (4% for 15 min), incubated with Hoechst stain (0.0005% for 15 min) (ThermoFisher, Inc, Waltham, MA), then imaged at 4X on the CellInsight CX5 High Content Screening (HCS) Platform. The machine was allowed to find optimal focus for each Hoechst stain image. One color channel was permitted such that images produced showed live nuclei in white and dead cells in black.

Hoechst stain colors cellular DNA, and viral infection is thought to interfere with the clarity of the stain (Dembowski and DeLuca, 2015). As such, infection termination, cell fixation, and Hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. Due to uncertainty over the exact epidemic state of Hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from GFP-expressing images and used Hoechst stain images as a post hoc visual check on our fit only (Figure 5; Figure 5—figure supplements 1–2).

Images recovered from the time series above were processed into binary (‘infectious’ vs. ‘non-infectious’ or, for Hoechst-stained images, ‘live’ vs. ‘dead’) form using the EBImage package (Pau et al., 2010) in R version 3.6 for MacIntosh, after methods further detailed in Supplementary file 7. Binary images were then further processed into time series of infectious or, for Hoechst-stained images, live cells using a series of cell counting scripts. Because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. As such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line–virus-MOI combinations (Figure 1—figure supplements 2–3), as well as for declining live cell counts from control well data derived from the Hoestch time series (Supplementary file 1; Supplementary file 7; Figure 1—figure supplement 7). All original and processed images, image processing and counting code, and resulting time series data are freely available for download at the following FigShare repository: DOI: 10.6084/m9.figshare.8312807.