Comparison of virus titre during infection in the presence or absence of cytokine-containing supernatants.

<p>Cytokine supernatants collected from primary human monocytes infected at an MOI of 0.1 with either TCRV or JUNV or mock infected at 4 days post-infection were subjected to ultracentifugation to remove virus particles. Macrophage cultures were then pretreated with the clarified supernatant of TCRV, JUNV or mock infected monocytes for 2 h prior to the addition of known amounts of (A) JUNV or (B) TCRV for infection. Samples were collected after 4 days and plaque assays performed to determine virus titres.</p

Comparison of virus growth and CPE formation during TCRV and JUNV infection of VeroE6 cells.

<p>(A) Virus growth during TCRV and JUNV infection. Sub-confluent VeroE6 cells were infected in a 6 well format with either TCRV or JUNV at an MOI of either 0.1 (upper panel) or 1 (lower panel). Cell culture supernatants were harvest immediately after infection and every 24 h thereafter for a period of 5 days. Virus titres in these supernatants were determined by plaque assay (B) CPE formation during TCRV and JUNV infection. VeroE6 cells were infected as described for (A) and CPE formation was monitored. Data are shown starting from the time of onset of CPE formation.</p

Cytokine production during exposure of primary human monocytes and macrophages to inactivated TCRV.

<p>Samples containing stock TCRV were inactivated by exposure to UV light for 60 min before being used to infect primary human monocytes at a dilution equivalent to an MOI of 0.1. Infection with untreated TCRV or mock infection of cells served as controls. Supernatants from the infected cells were analysed after 4 days using commercially available cytokine ELISA kits for IL-6 (left panel), IL-10 (center panel) and TNF-α (right panel) (R&D Systems) according to the manufacturer's instructions. The cytokine concentration for each of three donors (black box), as well as the mean for each group (black bar) is indicated for each treatment group: Untreated TCRV (Untr.), UV-inactivated TCRV (UV) or Mock (M).</p

Bioassay for IFN production during TCRV and JUNV infection of primary human monocytes.

<p>IFN-α/β activity in monocyte culture supernatants was detected using a biological assay for protection against vesicular stomatitis virus (VSV) infection and its associated cytopathic effects. For this assay, VeroE6 cells cultured in 12-well plates were treated with supernatants from monocytes infected with either JUNV or TCRV, supernatants from mock infected monocytes or dilutions of a commercial recombinant human IFN-β standard (50 ng/ml to 100 pg/ml). After 24 h the cells were washed and infected with VSV at an MOI of 0.001. Cells were then incubated at 37°C for 18 h before being examined for the formation of CPE.</p

Comparison of virus titre and CPE production during infection of primary human monocytes and macrophages.

<p>Growth kinetics of TCRV and JUNV in (A) primary human monocytes and (B) primary human macrophages. Primary human monocytes and macrophages were isolated by adherence of peripheral blood mononuclear cell fractions in 6-well plates with subsequent maturation for either 1 (monocyte) or 7 (macrophage) days. Subsequently, monocyte (upper panel) and macrophage (lower panel) populations were infected at an MOI of 0.1 with either TCRV or JUNV. Cell culture supernatants were harvest immediately after infection and every 24 h thereafter for a period of 5 days. Virus titres in these supernatants were determined by plaque assay. (C) Cytopathic effect during infection of primary human monocytes with TCRV and JUNV. The cells from which supernatants were collected for analysis in (A) were analysed by light microscopy for the formation of cytopathic effect in comparison to mock infected cells 4 days after infection.</p

Survival and weight loss in IFNAR^−/− mice.

<p>(<b>A</b>) <b>High dose (10³ ffu and 10⁴ ffu) infection.</b> IFNAR^−/− mice (n = 5–10) were infected via the intra-peritoneal route with either 10³ ffu or 10⁴ ffu per animal of recombinant (rZEBOV and rREBOV) or chimeric (rZEBOV-RGP and rREBOV-ZGP) Ebola viruses. Mouse-adapted ZEBOV (MA-ZEBOV) and wild-type Ebola viruses (wt-ZEBOV and wt-REBOV) served as controls. Animals were monitored for 14 days for survival (upper panel) and weight loss (lower panel) and observed for an additional 14 days to ensure no additional mortality occurred. Weights are shown as the mean values for each group along with bars indicating standard error values. (<b>B</b>) <b>Low dose (10 ffu) infection.</b> IFNAR^−/− mice (n = 10–15) were infected and monitored as indicated above, except that a dose of 10 ffu per animal was given.</p

The Ebola Virus Glycoprotein Contributes to but Is Not Sufficient for Virulence <em>In Vivo</em>

<div><p>Among the Ebola viruses most species cause severe hemorrhagic fever in humans; however, <em>Reston ebolavirus</em> (REBOV) has not been associated with human disease despite numerous documented infections. While the molecular basis for this difference remains unclear, <em>in vitro</em> evidence has suggested a role for the glycoprotein (GP) as a major filovirus pathogenicity factor, but direct evidence for such a role in the context of virus infection has been notably lacking. In order to assess the role of GP in EBOV virulence, we have developed a novel reverse genetics system for REBOV, which we report here. Together with a previously published full-length clone for <em>Zaire ebolavirus</em> (ZEBOV), this provides a unique possibility to directly investigate the role of an entire filovirus protein in pathogenesis. To this end we have generated recombinant ZEBOV (rZEBOV) and REBOV (rREBOV), as well as chimeric viruses in which the glycoproteins from these two virus species have been exchanged (rZEBOV-RGP and rREBOV-ZGP). All of these viruses could be rescued and the chimeras replicated with kinetics similar to their parent virus in tissue culture, indicating that the exchange of GP in these chimeric viruses is well tolerated. However, in a mouse model of infection rZEBOV-RGP demonstrated markedly decreased lethality and prolonged time to death when compared to rZEBOV, confirming that GP does indeed contribute to the full expression of virulence by ZEBOV. In contrast, rREBOV-ZGP did not show any signs of virulence, and was in fact slightly attenuated compared to rREBOV, demonstrating that GP alone is not sufficient to confer a lethal phenotype or exacerbate disease in this model. Thus, while these findings provide direct evidence that GP contributes to filovirus virulence <em>in vivo</em>, they also clearly indicate that other factors are needed for the acquisition of full virulence.</p> </div

Detection of virus in organs/tissues of IFNAR^−/− mice.

<p>(<b>A</b>) <b>Virus titration by TCID₅₀.</b> Homogenized liver and spleen samples, as well as blood samples, from animals (n = 3) infected with 10 ffu of either recombinant (rZEBOV and rREBOV) or chimeric (rZEBOV-RGP and rREBOV-ZGP) Ebola viruses were analysed at day 5 post-infection for viral load by calculating the tissue culture infectious dose (TCID₅₀) using the Reed and Muench method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002847#ppat.1002847-Reed1" target="_blank">[54]</a>. The values for each animal as well as the mean for each virus group are shown. (<b>B</b>) <b>Evaluation of virus infection in organs by immunohistochemistry.</b> The presence of viral antigen was detected in liver and spleen samples from infected animals by immunohistochemical straining using a cross-reactive anti-ZEBOV VP40 antibody.</p

Growth kinetics of wild-type, recombinant and chimeric Ebola viruses during infection in VeroE6 cells.

<p>VeroE6 cells were infected at an MOI = 0.1 with either recombinant REBOV (rREBOV), recombinant ZEBOV (rZEBOV), chimeric REBOV expressing the ZEBOV GP (rREBOV-ZGP), chimeric ZEBOV expressing the REBOV GP (rZEBOV-RGP), parental non-recombinant REBOV (wt-REBOV) or parental non-recombinant ZEBOV (wt-ZEBOV). Samples were collected at 0, 1, 2, 3, 4 and 7 days post-infection and titred based on focus-formation, which was visualized using either an anti-REBOV VP30 serum or an anti-ZEBOV serum. The mean values for each time point along with bars indicating standard error values are shown.</p

Reverse genetics for REBOV and rescue of chimeric Ebola viruses.

<p>(<b>A</b>) <b>Schematic diagram of the transcription cassette of the full-length REBOV cDNA plasmid.</b> The unique and rare restriction sites used to construct sub-genomic clones containing fractions of the REBOV genome, as well as to facilitate subsequent assembly of the full-length clone plasmid are indicated. Sites which have been knocked out through silent mutagenesis are shown crossed-out. An <i>XmaI</i> site inserted through silent mutagenesis is marked with an asterisk. A single silent point mutation in the GP ORF (G7252A) is also indicated, as are the T7 promoter (P_T7), T7 terminator (T_T7) and hepatitis delta virus ribozyme (Rib) sequences. (<b>B</b>) <b>Schematic diagram of recombinant and chimeric Ebola viruses.</b> The genomic composition of the recombinant parental REBOV (rREBOV) and ZEBOV (rZEBOV), as well as the chimeric REBOV expressing the ZEBOV GP (rREBOV-ZGP) and chimeric ZEBOV expressing the REBOV GP (rZEBOV-RGP) used in this study are illustrated. Dark grey indicates ORFs derived from REBOV while light grey indicates ORFs derived from ZEBOV. Untranslated and non-coding regions are shown in white and are derived from the respective parent virus. (<b>C</b>) <b>Analysis of the genetic composition of recombinant and chimeric Ebola viruses.</b> PCR fragments corresponding to the REBOV or ZEBOV nucleoprotein (NP) and glycoprotein (GP) were amplified using species-specific primer sets in order to identify the genetic composition of each of the recombinant parental (rREBOV and rZEBOV) and chimeric (rREBOV-ZGP and rZEBOV-RGP) viruses. Wild-type non-recombinant REBOV (strain Pennsylvania; wt-REBOV) and ZEBOV (strain Mayinga; wt-ZEBOV) served as controls. (<b>D</b>) <b>Analysis of the protein composition of recombinant and chimeric Ebola viruses.</b> Lysates from VeroE6 cells infected with each of the recombinant or chimeric Ebola viruses used in this study or the wild-type non-recombinant REBOV and ZEBOV controls were separated by SDS-PAGE and probed by Western blot for their VP40 and GP composition using specific antibodies. REBOV and ZEBOV VP40 (α-VP40) can be distinguished based on size, while the use of antibodies specific for ZEBOV GP (α-ZGP) or detecting both REBOV and ZEBOV (α-GP) were used to discriminate between REBOV and ZEBOV GP.</p