Article Figures & Data
Figures
- FIG 1
Primary structure of LLOV envelope GP. (A) Nucleotide and deduced amino acid sequences of LLOV GP. Deduced amino acid sequences are shown in boldface letters. Blue, green, and yellow lines represent the signal peptide, MLR, and transmembrane domain predicted by GENETYX Ver.10, NetOGlyc, and TopPred 0.01, respectively. Purple and red letters represent the putative editing and cleavage sites, respectively. (B) Comparison of EBOV and LLOV MLRs. Potential O-glycosylation sites were predicted by NetOGlyc, and MLRs were defined as the regions between the first and last amino acid residues showing a score above the threshold (0.5). Arrows indicate the cleavage sites. (C) Western blotting of filovirus GPs. Proteins in the lysate of HEK293T cells transfected with the plasmid expressing LLOV GP (lanes 1 and 2), EBOV GP (lanes 3 and 4), MARV GP (lanes 5 and 6), or empty vector (lanes 7 and 8) were separated by SDS-PAGE under nonreducing (lanes 1, 3, 5, and 7) or reducing (lanes 2, 4, 6, and 8) conditions.
- FIG 2
TEM of filovirus VLPs. Purified VLPs produced from 293T cells transfected with plasmids expressing LLOV (A, B, E, and F), EBOV (Zaire) (C and G), and MARV (Angola) (D and H) proteins were fixed and stained as described in Materials and Methods. For immuno-TEM (B and F), an anti-LLOV GP monoclonal antibody was used. Scale bars represent 500 nm (A to D) and 200 nm (E to H). Arrowheads indicate gold particles.
- FIG 3
SEM of LLOV VLPs. HEK293T cells transfected with pCAGGS expressing LLOV GP, VP40, and NP (A and B) or pCAGGS alone (C and D) were fixed at 48 h after transfection. Samples were observed with an S-4700 scanning electron microscope (Hitachi). Scale bars represent 5 μm (A and C) and 2 μm (B and D).
- FIG 4
Cross-reactivities of anti-GP sera among filoviruses in ELISA. Ten-fold serial dilutions of mouse antisera to EBOV (anti-Zaire), SUDV (anti-Sudan), TAFV (anti-Tai forest), BDBV (anti-Bundibugyo), RESTV (anti-Reston), LLOV (anti-Lloviu), and MARV (anti-Marburg) were tested for IgG reactivities to LLOV (A), EBOV (B), SUDV (C), TAFV (D), BDBV (E), RESTV (F), and MARV (G) GP antigens. Three mice were used for each virus, and averages and standard deviations are shown.
- FIG 5
Effects of chemical inhibitors on infectivities of pseudotyped VSVs. Vero E6 cells were pretreated with ammonium chloride (AMC) (A), monensin (MON) (B), and cathepsin B (CatB) (C) and L (CatL) (D) inhibitors for 30 min at 37°C. The treated cells were then infected with VSVΔG*-Zaire (Zaire), VSVΔG*-Angola (Angola), VSVΔG*-Lloviu (Lloviu), and VSVΔG*-G (VSV) appropriately diluted to yield 200 to 2,000 IUs/106 cells. At 20 h postinoculation, GFP-positive cells were counted using an IN Cell Analyzer 2000 (GE Healthcare). The percentages of infectivity were determined by setting the number of the untreated cells to 100%. Each experiment was performed three times, and averages and standard deviations are shown.
- FIG 6
Infectivities of pseudotyped VSVs in C-type lectin-expressing cells. K562 cells expressing hMGL or DC-SIGN were infected with VSVΔG*-Zaire, VSVΔG*-Reston, VSVΔG*-Lloviu, VSVΔG*-Angola, and VSVΔG*-Musoke. The infectivity of each pseudotyped VSV on K562 clones was determined by counting the number of GFP-positive cells using flow cytometry, and the percentages of infectivity in K562-hMGL and K562-DC-SIGN were determined by setting the number of the infected K562 cells as 100%. Each experiment was performed three times, and averages and standard deviations are shown. Statistical significance of the differences was determined by Student's t test (see P values in the text).
- FIG 7
ADE activities of antisera to EBOV and LLOV GPs. Ten-fold serially diluted mouse anti-EBOV (A) and anti-LLOV (B) sera were mixed with equal volumes of VSVΔG*-Zaire and VSVΔG*-Lloviu. Relative infectivity was determined by setting the number of infected K562 cells without antisera to 100%. Each experiment was performed three times, and averages and standard deviations are shown.
- FIG 8
Infectivities of pseudotyped VSVs in mammalian cell lines. VSVΔG*-Zaire, VSVΔG*-Reston, VSVΔG*-Lloviu, VSVΔG*-Angola, and VSVΔG*-Musoke were inoculated into several mammalian cell lines. (A) Infectious units (IUs) of each virus in different cell lines were determined by counting the number of GFP-expressing cells, and each IU value was standardized based on 106 copies of the VSV genome as determined by real-time RT-PCR. (B) Relative infectivities in bat cell lines are given by setting each IU value in Vero E6 cells to 1.0 (i.e., [IU in bat cells]/[IU in Vero E6 cells]). Each experiment was performed three times, and averages and standard deviations are shown. Infectivities of VSVΔG*-Angola and VSVΔG*-Musoke were under the limit of detection (*).
Tables
- TABLE 1
Origins of cell lines used in this study
Cell line Species Zoological name Organ Vero E6 African green monkey Chlorocebus sp. Kidney HEK293 Human Homo sapiens Kidney SK-L Pig Sus scrofa domesticus Kidney MDCK Dog Canis lupus familiaris Kidney BKT1 Greater horseshoe bata Rhinolophus ferrumequinum Kidney FBKT1 Yaeyama flying foxb Pteropus dasymallus yayeyamae Kidney YubFKT1 Eastern bent-winged batc Miniopterus fuliginosus Kidney IndFSPT1 Indian flying foxd Pteropus giganteus Spleen DemKT1 Leschenault's rousettee Rousettus leschenaulti Kidney ZFB11-97 Gambian epauletted fruit batf Epomophorus gambianus Kidney SuBK12-08 Schreiber's batg Miniopterus schreibersii Kidney
