Filovirus glycoproteins and distribution of fruit bats believed to serve as natural reservoir.

<p>(A) Phylogenetic tree based on the amino acid (aa) sequences of the glycoproteins (GP) of different filoviruses was generated using MEGA 6 (version 6.06). Viruses from which GPs were examined in the present study are written in bold. Viruses were named as follows: Filovirus species (abbreviation)/Country where the sample specimen originates from (abbreviation)/year of sampling/isolate-specific name (if available). In addition to GPs from viruses that have caused infection in humans and non-human primates, GP sequences of one Reston virus from a pig (diamond), as well as four Marburg virus-related and two Ravn virus-related GP sequences from bats (circles) were included. Construction of the tree was performed by the neighbor joining method with 1,000 bootstrap replications, using the MEGA 6 software (version 6.06). Small numbers at the nodes and the scale bar indicate bootstrap values and the number of aa substitutions per site, respectively. GenBank accession numbers for all GPs are given in brackets after the virus name. (B) Schematic drawing of the wildtype (wt) Ebola virus (EBOV) GP and EBOV-GP mutants lacking the mucin-like domain and the furin cleavage site. (Abbreviations: SP = signal peptide; RBD = receptor binding domain; FP = internal fusion peptide; HR1/2 = heptad repeat 1/2; TD = transmembrane domain). (C) Maps of the distribution of the four different fruit bat species from which the cell lines used in this study originated: <i>Rousettus aegyptiacus</i> (RoNi/7), <i>Hypsignathus monstrosus</i> (HypNi/1.1), <i>Eidolon helvum</i> (EidNi/41), <i>and Epomops buettikoferi</i> (EpoNi/22.1). The data on the bat distribution have been obtained from <a href="http://www.iucnredlist.org/" target="_blank">www.iucnredlist.org</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149651#pone.0149651.ref056" target="_blank">56</a>].</p

Differential transduction of fruit bat cells lines by different filovirus glycoproteins.

<p>Vesicular stomatitis virus (VSV)-based pseudotypes (VSVpp) harboring the indicated filovirus glycoproteins or the glycoprotein of VSV (VSV-G; positive control), or no glycoprotein (pCAGGS; negative control) were used to inoculate human (HEK-293T), non-human primate (Vero) and fruit bat cell lines (RoNi/7, HypNi/1.1, EidNi/41, EpoNi/22.1). Mock-treated cells that received only fresh culture medium served as additional negative controls (mock). The VSVpp decorated with the respective GPs or VSV-G were either infectivity-normalized (A) or applied undiluted (B) to the target cells. At 18 h post inoculation, the activity of virus-encoded firefly luciferase (given in counts per second; cps) as an indicator for transduction efficiency was quantified. The results of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in three independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations (SD).</p

Information on the fruit bat cell lines used in this study.

<p>Information on the fruit bat cell lines used in this study.</p

Glycoprotein-mediated entry into human and fruit bat cells relies on the same host cell factors.

<p>Equal volumes of vesicular stomatitis virus (VSV)-based pseudotypes (VSVpp) harboring the indicated glycoproteins were used to inoculate human (HEK-293T) and fruit bat (EpoNi/22.1, EidNi/41) cell lines pre-incubated with the indicated inhibitors for 3 h. Cells treated with solvent (water or dimethyl sulfoxide) alone served as controls (vehicle control, VC). At 18 h post inoculation, the activity of virus-encoded firefly luciferase as an indicator for transduction efficiency was quantified and normalized against the values of the respective VC (x-fold changes). The results of a representative experiment carried out with quadruplicate samples are shown and were confirmed in an independent experiment, conducted with a separate pseudotype batch. The following inhibitor concentrations were used: Mannan (final concentration: 25 μg/ml), ammonium chloride (NH₄⁺; 50 mM), bafilomycin A1 (50 nM), tetrandrine (2 μM), E-64d (50 μM), MDL28170 (50 μM), camostat mesylate (100 μM), U18666A (20 μM). Error bars indicate SD. An unpaired student’s t-test was used to test statistical significance (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).</p

Furin cleavage and mucin-like domain are dispensable for EBOV-GP-driven transduction of fruit bat cells.

<p>Equal volumes of vesicular stomatitis virus (VSV)-based pseudotypes (VSVpp) harboring the indicated glycoproteins were inoculated onto human (HEK-293T), primate (Vero) and fruit bat cell lines (RoNi/7, HypNi/1.1, EidNi/41, EpoNi/22.1). EBOV1976-GP(∆Cleav) cannot be cleaved by furin while EBOV1976-GP(∆MLD) does not contain the mucin-like domain. VSVpp that did not harbor a viral glycoprotein at all served as negative controls (pCAGGS). At 18 h post inoculation, the activity of virus-encoded firefly luciferase in cell lysates was quantified as an indicator for transduction efficiency. Transduction mediated by the tested GPs is shown relative to transduction mediated by wt EBOV1976-GP, which was set as 1. The average of three independent experiments with separate pseudotype preparations is shown. Error bars indicate standard error of the mean. A paired student’s t-test was performed to test statistical significance (* = p < 0.05).</p

Surface glycoproteins of batFLUAV enable pseudotype entry into different bat but not human, simian and canine cell lines.

<p>Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring no or the indicated viral glycoproteins were either left untreated (white bars) or treated with trypsin (black bars), before they were inoculated onto mammalian cell lines of human (HEK-293T, Huh7), simian (Vero) and canine (MDCK) origin (A) or bat cell lines (RoNi/7, EidNi/41, HypNi/1.1, EpoNi/22.1, CpKd) representing five different bat species (B). At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase (given as counts per second, cps, on a logarithmic scale). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in four independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance (* = p < 0.05).</p

HAL of batFLUAV are robustly expressed and incorporated into rhabdoviral particles.

<p>(A) BHK-21 cells were transfected with the indicated HAL proteins harboring a C-terminal FLAG antigenic tag and protein expression was analyzed by immunofluorescence microscopy of permeabilized cells (magnification, 10x). Cells transfected with an empty expression vector served as negative control. Nuclei were visualized by DAPI staining. Similar results were obtained in a separate experiment. (B) For analysis of HAL incorporation into rhabdoviral particles, vesicular stomatitis virus (VSV)-based pseudotypes were pelleted though a 20% sucrose cushion and analyzed by SDS-PAGE and Western blotting with antibodies against the FLAG tag (α-FLAG), VSV glycoprotein (α-VSV-G) and matrix protein (α-VSV-M). The numbers on the left side of the blots indicate the molecular weight in kilo Daltons (kDa). The results were confirmed in an independent experiment.</p

NAL of batFLUAV has no impact on the transduction efficiency of vectors bearing HAL.

<p>Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated surface proteins of FLUAV or batFLUAV were treated with trypsin before inoculation of MDCK and EpoNi/22.1 cells. At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase. The combined data from three independent experiments (quadruplicate samples) with separate pseudotype preparations are shown. Transduction efficiencies were normalized against pseudotypes harboring only HA or HAL (set as 1) and are given as x-fold changes on a logarithmic scale. Error bars indicate standard error of the mean. A two-tailed, paired student’s t-test was used to test statistical significance (* = p < 0.05).</p

HAL of batFLUAV does not require sialic acids for host cell entry.

<p>EpoNi/22.1 cells were incubated for 1.5 h in the absence (black bars) or presence (white and grey bars) of increasing concentrations of exogenous sialidase and were subsequently inoculated with trypsin-treated vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated viral glycoproteins. At 1 h post inoculation, the inoculum was removed, the cells were washed and further incubated for 18–20 h with fresh medium before transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase. Transduction of sialidase-treated cells is shown relative to that measured for mock-treated cells (on a linear scale), which was set at 100%. The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in two independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance of differences measured for sialidase- versus mock-treated samples (* = p < 0.05).</p

Cell lines used to study batFLUAV tropism.

<p>Cell lines used to study batFLUAV tropism.</p