Journal: PLOS Neglected Tropical Diseases

Article Title: The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation

doi: 10.1371/journal.pntd.0012146

Figure Lengend Snippet: Non-human VeroE6 cells were infected with MR766 MC or the chimeric MR766 MC virus with NS1 ZIKV-15555 (MR766 MC chimera) at an m.o.i. of 0.1. In ( A ), virus progeny production (PFU.mL -1 ) was determined by plaque-forming assay. In ( B ), intracellular viral RNA production was determined by RT-qPCR at 48 h p.i. I n ( C ), the percentage of ZIKV-infected cells based on FACS analysis using anti-E mAb 4G2. In ( D ), LDH activity were measured at 48h p.i and expressed as a percentage relative to mock-infected cells (control). Asterisks indicate that the differences between experimental samples are statistically significant, using an unpaired t test (**** p < 0.0001, ** p < 0.01, * p < 0.05).

Article Snippet: The purified mouse anti- pan flavivirus envelope E protein monoclonal antibody (mAb) 4G2 was provided by RD Biotech (Besançon, France).

Techniques: Infection, Virus, Quantitative RT-PCR, Activity Assay, Control

Journal: PLOS Neglected Tropical Diseases

Article Title: The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation

doi: 10.1371/journal.pntd.0012146

Figure Lengend Snippet: Human A549 cells were infected with MR766 MC or chimeric MR766 MC virus with NS1 CWA protein (MR766 MC chimera) at an m.o.i. of 1. In ( A ), virus progeny production (PFU.mL -1 ) was examined using a conventional plaque-forming assay. In ( B ), intracellular viral RNA production was determined by RT-qPCR at 48 h p.i. In ( C ), the percentage of ZIKV-infected cells based on FACS analysis using anti-E mAb 4G2. In ( D ), LDH activity was measured at 48h p.i and expressed as a percentage relative to mock-infected cells (control). Asterisks indicate that the differences between experimental samples are statistically significant, using an unpaired t test (*** p < 0.001, ** p < 0.01, * p < 0.05).

Article Snippet: The purified mouse anti- pan flavivirus envelope E protein monoclonal antibody (mAb) 4G2 was provided by RD Biotech (Besançon, France).

Techniques: Infection, Virus, Quantitative RT-PCR, Activity Assay, Control

Journal: PLOS Neglected Tropical Diseases

Article Title: The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation

doi: 10.1371/journal.pntd.0012146

Figure Lengend Snippet: A549 cells were infected with MR766 MC or chimeric MR766 MC virus with NS1 CWA protein (MR766 MC chimera) or mock-infected (no virus) for 24 h or 48 h at an m.o.i. of 1. In ( A ), visualization of intracellular rNS1 protein. Cells infected for 24h were stained with anti-E mAb 4G2 or anti-NS1 mAb 4G4 as primary antibody (green) for confocal immunofluorescence analysis. Nuclei were stained with DAPI (blue). The same magnification was used throughout. Scale bar, 25 μM. In ( B ), cell supernatant samples from three independent infections (exp. 1 to exp. 3) were analyzed in duplicates (A, B) by dot-blotting using mAb 4G4. The mean of signal intensity of each duplicate was determined using Image J software to estimate the relative amounts of secreted soluble NS1 protein. Results are the mean (± SEM) of three independent assays. Asterisks indicate that the differences between experimental samples are statistically significant, using an unpaired t test (** p < 0.01).

Article Snippet: The purified mouse anti- pan flavivirus envelope E protein monoclonal antibody (mAb) 4G2 was provided by RD Biotech (Besançon, France).

Techniques: Infection, Virus, Staining, Immunofluorescence, Software

Journal: PLOS Neglected Tropical Diseases

Article Title: The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation

doi: 10.1371/journal.pntd.0012146

Figure Lengend Snippet: A549 cells were infected with MR766 MC or the chimeric MR766 MC with NS1 ZIKV-15555 (MR766 MC chimera) at an m.o.i. of 1. In ( A ), the relative abundance of IFN-β and ISG mRNA was determined at 48 h p.i. by RT-qPCR. Housekeeping gene RPLPO36B4 mRNA served as an internal reference. The results are the mean (± S.D.) of three replicates. Asterisks indicate that the differences between MR766 MC and MR766 MC chimera for each cellular factor are statistically significant, using an unpaired t test (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). In ( B ), Immunoblot assay was performed on cell lysates using anti-ISG15 or anti-IFIT1 antibodies as indicated. Anti-E mAb 4G2 was used to detect ZIKV E protein. β-actin was detected as protein-loading control for lysate samples.

Article Snippet: The purified mouse anti- pan flavivirus envelope E protein monoclonal antibody (mAb) 4G2 was provided by RD Biotech (Besançon, France).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody

doi: 10.1038/srep16248

Figure Lengend Snippet: ( A ) Real-time interaction profiles of binding of native or heme-exposed human monoclonal IgG1, mAb21 to immobilized recombinant JEV E and EDIII proteins. The real-time interaction profiles obtained after injection of native mAb21, diluted to 500 nM are presented in the left panels. The binding profiles of heme-exposed mAb21 at 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 3.90 nM are presented on the right panels. The binding analyses were performed at 25 °C. The graphs show experimentally determined binding curves (black lines) and curves generated by globally fitting the data by BIA evaluation software (red line). The estimated kinetic parameters are presented on . ( B ) Arrhenius plots showing the natural logarithm values of association and dissociation rate constants of the heme-sensitive mAb21 obtained after interaction with JEV E (open circles) and JEV EDIII (filled circles) as a function of reciprocal values of temperature (in Kelvins). To generate these plots the kinetic rate constants were determined by global analysis of sensorgrams generated after evaluation of binding kinetics of the heme-exposed mAb21 with immobilized JEV proteins at varying temperatures (10, 15, 20, 25, 30, and 35 °C). Linear regression analyses were applied to obtain the slopes of the temperature dependency. ( C ) Association, dissociation and equilibrium thermodynamic parameters of binding of heme-exposed mAb21 to JEV E and EDIII. Changes in the enthalpy, entropy and free energy during different phases of the interaction of heme-exposed mAb21 with JEV E (white bars) and EDIII (black bars) are depicted. The changes in non-equilibrium thermodynamic parameters were evaluated by applying Eyring’s analyses on the data from Arrhenius plots.

Article Snippet: The recombinant envelope (E) protein (MBS143155) was originally cloned from JEV genotype III (Kamiyama strain) and obtained commercially from MyBioSource, San Diego, CA, USA.

Techniques: Binding Assay, Recombinant, Injection, Generated, Software