Fig 1.
Generation of lentiviral particles containing Ebola virus RNA.
A) Ebola virus genes nucleoprotein (NP, dark red), viral protein 35 (VP35, red), glycoprotein (GP, orange), viral protein 40 (VP40, light green) and the polymerase encoding gene L (dark green) were sequentially cloned into lentiviral vector pSF_lenti between the restriction sites SalI and BstEII. The main elements of the lentiviral vector used for the production of the viral RNA and incorporation within HIV-like particle are indicated. B) Particle size distribution of the 1:100 dilution in PBS of stock preparation of lentiviral particles containing Ebola virus RNA for np-vp35-gp genes. The graph represents the mean of 5 acquisitions (black line) ± standard deviation (red line). C) A representative image of the same preparation diluted 1:10 in PBS analysed by negative staining transmission electron microscopy. The average particle size was 116.02 nm (average of 9 fields).
Table 1.
Primers and probes sequences.
Fig 2.
Performance of the HIV-EBOV RNA preparations in qRT-PCR.
Representative amplification plot of two 10-fold serially diluted high titre LVV_NP-VP35-GP (A) and LVV_VP40-L (B) vials. Samples from two reconstituted vials per sample were run in duplicate and the mean values of the duplicates plotted as threshold cycle against fluorescence. The dilutions of the high titre LVV_NP-VP35-GP (C) and LVV_VP40-L (D) were also plotted against the dilution factor. Efficiencies of the reaction were calculated based on the slope of the regression line as 102% for np-target qRT-PCR and 110% for I-target qRT-PCR. The same samples, run without the reverse transcriptase reaction (RT negative), were detectable up to 10−2 dilution for LVV_NP-VP35-GP and 10−1 for LVV_VP40-L. Three 10-fold dilutions of the plasmids pSF-lenti-NP-VP35-GP and pSF-lenti-VP40-L were run in parallel and the values were the same between RT positive and RT negative qRT-PCR.
Table 2.
Evaluation of the HIV-EBOV RNA standard by quantitative RT-PCR.
Fig 3.
Efficiency of the quantitative RT-PCR targeting HIV LTR.
Viral RNA extracted from serial dilutions of WHO 3rd HIV-1 International standard (dilution factor 5, blue), LVV_NP-VP35-GP high (dilution factor 10, red) and LVV-VP40-L (dilution factor 10, green) were processed in duplicate in a quantitative RT-PCR using primers and probes annealing within the U5 region of the HIV-1 LTR. The graph shows a representative result of 3 independent experiments; the efficiencies of the reaction between samples were similar as represented by the slope of linear regression: HIV-1 IS = 3.122, LVV_NP-VP35-GP = 3.142, LVV_VP40-L = 3.110.
Table 3.
Examples of the HIV-EBOV RNA standard values using different technologies.
Table 4.
Stability of the freeze-dried preparations after two weeks of storage at different temperatures.