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Results: 13

1.
Fig 6

Fig 6. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

Viral titers in supernatants of DC infected with the indicated viruses (mean ± SE based on triplicate samples). For some values, the error bars cannot be seen due to their small size.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
2.
Fig 7

Fig 7. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

Formation of homotypic DC clusters (day 2 postinfection). Disabling any of the four EBOV IRADs by a single point mutation each results in effective formation of homotypic clusters by infected DC.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
3.
Fig 11

Fig 11. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

CCL19-driven migration of infected DC. Shown are the numbers of DC infected with the indicated viruses that migrated to the lower chamber during 3 h. The experiment was performed three times, with DC from each donor shown with the same symbol for each treatment and the mean values indicated by horizontal bars. The 100% level in DC infected by wt EBOV is shown by the dashed line. A statistically significant difference between cells infected with EBOV/VP35-R312A and wt EBOV is indicated by the asterisk (P = 0.002).

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
4.
Fig 1

Fig 1. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

DC are not readily infected by EBOV. (A) Bright-field (left and middle) and UV (right) microscopy of Vero-E6 cells (day 2; top) or DC (day 3; bottom) infected or mock infected with wt EBOV. (B) Flow cytometry analysis of expression of EGFP by infected DC on days 1 to 5 after infection with wt EBOV. The experiment with DC was repeated with cells derived from blood from two different donors, which resulted in essentially similar results.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
5.
Fig 10

Fig 10. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

Disabling any of the individual EBOV IRADs by a single point mutation each results in effective secretion of chemokines and cytokines by infected DC. The levels of CXCL chemokines (row 1 from top), CCL chemokines (row 2), and cytokines, including IFNs (rows 3 and 4), in supernatants of DC infected with the VP24 and VP35 EBOV mutants, HPIV3 and HPIV3/ΔF-HN/EboGP, normalized to that for DC infected with wt EBOV. The 100% levels in DC infected by wt EBOV are shown by the red lines. Shown are results of two independent experiments, in which DC from subject 1 or DC from subject 2 were used.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
6.
Fig 2

Fig 2. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

GP of EBOV, not whole EBOV particles, induces the formation of homotypic clusters. (A) Schematic representation of the viral particles used in the study. The EBOV proteins and RNA are depicted in red and pink, respectively, and those of HPIV3 in blue and light blue, respectively; the inserted EGFP gene is depicted in green. (B) Western blot analysis of GP expressed by wt EBOV (lanes 2) or by HPIV3/ΔF-HN/EboGP (lanes 3) on days 1 and 2 postinfection. Lanes 1, protein standards; lanes 4, mock infection. (C) Formation of homotypic clusters of DC infected with HPIV3/ΔF-HN/GP, but not EBOV or HPIV3, on day 2 postinfection.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
7.
Fig 9

Fig 9. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

The mutated EBOVs induce maturation regardless of the level of infection. (A) CD86 expression in EGFP+ (green histograms) and EGFP (black histograms) DC. (B) Expression of the three maturation markers in EGFP+ and EGFP DC infected with the four mutants and normalized to the levels of the markers in DC infected with wt EBOV (100%). The log10 MFI values ± SE for EGFP+ DC infected with wt EBOV were 3.1 ± 0.3, 3.4 ± 0.1, and 3.1 ± 0.2 for the three markers of maturation, and the respective values for EGFP DC were 3.5 ± 0.2, 3.7 ± 0.1, and 2.9 ± 0.2. Values for each donor are indicated by the same symbol in each plot, and the mean values are indicated by horizontal bars. No statistical difference between EGFP+ and EGFP DC was observed for any of the three markers of activation for any virus.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
8.
Fig 13

Fig 13. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

Individual IRADs act synergistically to disable the adaptive immune response during EBOV infections. (A) Summary of the effects of EBOV GP and IRADs located in VP24 and VP35 on DC maturation. ND, not determined; ++++, a very strong effect; +++, a strong effect; ++, a moderate effect; +, a weak effect; −, no effect. (B) Hypothetical model of the effects of individual IRADs located in the VP35 protein on the adaptive immune response. While disabling of any of the IRADs results in effective DC maturation sufficient for the induction of a T cell response (left), the synergistic effect of at least two individual IRADs of wt EBOV effectively blocks DC maturation and the induction of a T cell response (right).

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
9.
Fig 3

Fig 3. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

GP of EBOV induces maturation of DC. (A) Representative flow cytometry data on the expression of maturation markers CD54, CD80, and CD86; the MFI values are shown in the upper right corner. (B) MFI of CD54, CD80, and CD86 normalized to the level of expression induced by wt EBOV in DC from the same donor (100%, indicated by the red horizontal lines). The log10 MFI values ± standard errors (SE) for DC infected with wt EBOV were 3.5 ± 0.2, 3.7 ± 0.1, and 3.0 ± 0.2, respectively, for the three markers of maturation. The values for each donor are indicated by symbols, and the mean values are indicated by horizontal bars. Statistical significance for each treatment compared to wt EBOV: *, P < 0.05; **, P < 0.001.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
10.
Fig 8

Fig 8. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

Disabling any of the individual EBOV IRADs by a single point mutation each results in effective maturation of infected DC. (A) Expression of maturation markers CD54 and CD86 by DC infected with the indicated viruses; the MFI values are shown in the upper right corner. (B) MFI of CD54, CD80, and CD86 normalized to the level of expression induced by wt EBOV in DC from the same donor (100%, indicated by the red horizontal lines). The log10 MFI values ± SE for DC infected with wt EBOV were 3.5 ± 0.1, 3.5 ± 0.1, and 2.7 ± 0.2 for the three markers of maturation. Values for each donor are indicated by the same symbol in each plot, and the mean values are indicated by horizontal bars. Statistical significance of values for each individual mutant or LPS treatment compared to wt EBOV: *, P < 0.05; **, P < 0.01. Note that most of the DC samples were also included in the experiment shown in Fig. 3.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
11.
Fig 12

Fig 12. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

EBOV exposure without infection is not sufficient for DC maturation. The indicated viruses were subjected to 1-h-long incubation at 56°C, overnight incubation at 4°C in tubes, or overnight incubation at 4°C in ELISA plates. The next day, the wells of ELISA plates containing the viruses were washed, and DC were added. Alternatively, DC were added to wells containing the viruses treated in tubes or fresh, untreated viruses. At 40 h, expression of CD86 was measured by flow cytometry. The CD86 MFI values were normalized to the level of expression in mock-infected DC (100%), indicated by the dashed line. Values for each donor are indicated by symbols, and the mean values are indicated by horizontal bars. The P values of the difference between the level of CD86 in DC exposed to EBOV/VP35-R312A preincubated in a plate at 4°C versus the same virus preincubated at 4°C in a tube (*) or untreated virus (**) are 0.02 and 0.007, respectively.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
12.
Fig 4

Fig 4. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

(A) Generation of the recombinant viruses. Recombinant EBOVs were generated that express EGFP and carry amino acid mutations in GP, IRADs located in the VP35 and VP24 proteins, or the RNA-editing site to disable the transcription of sGP and to restore the open reading frame encoding the transcription of the full-length GP1/2 without the requirement for transcriptional editing. The enlarged GP gene is shown in blue, with the functional elements of the protein indicated in yellow, and the EGFP gene is shown in light green. The transcriptional gene start and gene stop signals are shown as green and red bars, respectively, and the introduced mutations are indicated with red arrows. (B) Viral titers in supernatants of Vero cells infected with the indicated viruses (mean ± SE based on triplicate samples). For some values, the error bars cannot be seen due to their small size.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.
13.
Fig 5

Fig 5. From: The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains.

Disabling of IRADs alters EBOV infectivity for DC. (A) Bright-field (top) and UV (bottom) microscopy of the same field on day 3 postinfection of DC with the indicated viruses. DC infected with EBOV/VP24-K142A demonstrated greater intensity of EGFP than those infected with wt EBOV, while no EGFP-positive cells were visible among DC infected with the three VP35 mutants. (B) Flow cytometry analysis of EGFP expression on day 2 after infection of DC with the viruses indicated in panel A. Percentages of EGFP+ cells and MFI are shown. The experiment was performed three times. (C) Percentages (left) and MFI (right) of EGFP+ DC on days 1 to 5 postinfection for wt EBOV and the two mutants. The experiment was performed two times. Note that DC from different donors were used in the experiments shown in panels A, B, and C, which had somewhat different levels of susceptibility to the viruses.

Ndongala M. Lubaki, et al. J Virol. 2013 July;87(13):7471-7485.

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