Figure 1.
Reverse genetics for REBOV and rescue of chimeric Ebola viruses.
(A) Schematic diagram of the transcription cassette of the full-length REBOV cDNA plasmid. 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 XmaI 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 (PT7), T7 terminator (TT7) and hepatitis delta virus ribozyme (Rib) sequences. (B) Schematic diagram of recombinant and chimeric Ebola viruses. 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. (C) Analysis of the genetic composition of recombinant and chimeric Ebola viruses. 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. (D) Analysis of the protein composition of recombinant and chimeric Ebola viruses. 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.
Figure 2.
Growth kinetics of wild-type, recombinant and chimeric Ebola viruses during infection in VeroE6 cells.
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.
Figure 3.
Survival and weight loss in IFNAR−/− mice.
(A) High dose (103 ffu and 104 ffu) infection. 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) Low dose (10 ffu) infection. IFNAR−/− mice (n = 10–15) were infected and monitored as indicated above, except that a dose of 10 ffu per animal was given.
Figure 4.
Detection of virus in organs/tissues of IFNAR−/− mice.
(A) Virus titration by TCID50. 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 [54]. The values for each animal as well as the mean for each virus group are shown. (B) Evaluation of virus infection in organs by immunohistochemistry. 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.
Figure 5.
Pathological evaluation of tissue samples from IFNAR−/− mice.
(A) Hematoxylin and eosin staining of tissues. Liver and spleens samples were harvested from animals (n = 3) infected with 10 ffu of either recombinant (rZEBOV and rREBOV) or chimeric (rZEBOV-RGP and rREBOV-ZGP) Ebola viruses. Samples were stained with hematoxylin and eosin prior to analysis of pathological changes. Areas of inflammatory cell infiltration are indicated with triangular arrow heads while areas of cellular necrosis are indicated with arrows. (B) Quantification of pathological changes present in tissues. Pathological changes in tissue samples were scored based on the degree of pathological change in comparison to mock infected animals (0 = normal; 1 = minimal change; 2 = mild change; 3 = moderate change; 4 = marked change; 5 = severe change). The values for each animal as well as the mean for each virus group are shown.