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

Journal: Mediators of Inflammation

Article Title: Autophagy Contributes to Host Immunity and Protection against Zika Virus Infection via Type I IFN Signaling

doi: 10.1155/2020/9527147

Figure Lengend Snippet: (a) ZIKV-infected pregnant mouse model with different strains at 1 × 10 6 PFU per mice. The viral burden in neonatal mice was detected using PFU assay the first day after birth. N = 12, data expressed as means ± SD, ∗∗∗ p < 0.005. (b) Forebrain, midbrain, and hindbrain tissues of the neonatal mice infected with ZIKV-MR766 were sectioned for H&E staining. Arrows indicates the morphological changes. Scale bar = 100 μ m. (c) Hindbrain tissues were sectioned as above for immunostaining with CD11b, Ly6G, and other different antibodies to detect immune cells recruitment or infiltration in neonatal mice. Scale bar = 20 μ m. Data are shown as representative from 12 mice. (d) Statistic results of accumulated immune cells in each vision.

Article Snippet: The primary antibody to Zika Virus Envelope (E) Protein (D1-4G2-4-15) antibody was bought from Kerafast Boston, MA.

Techniques: Infection, Staining, Immunostaining

Journal: Mediators of Inflammation

Article Title: Autophagy Contributes to Host Immunity and Protection against Zika Virus Infection via Type I IFN Signaling

doi: 10.1155/2020/9527147

Figure Lengend Snippet: Phagocytes colocalized with ZIKV in brain. (a) The progression of ZIKV infection in neonatal mice in a time dependent manner. Iba1 and ZIKV were detected by immunofluorescence. PFU assay was performed by viral titer experiment. Scale bar = 20 μ m. (b) Hindbrain tissues were sectioned for immunostaining at 3 DPI with CD11b, Ly6G, and ZIKV E-protein antibodies, separately. (c) Colocalization of the cells (yellow) in the whole panoramic field was counted and compared, respectively. Scale bar = 2 mm. Data are representative from 3 independent experiment.

Article Snippet: The primary antibody to Zika Virus Envelope (E) Protein (D1-4G2-4-15) antibody was bought from Kerafast Boston, MA.

Techniques: Infection, Immunofluorescence, Immunostaining

Journal: Mediators of Inflammation

Article Title: Autophagy Contributes to Host Immunity and Protection against Zika Virus Infection via Type I IFN Signaling

doi: 10.1155/2020/9527147

Figure Lengend Snippet: (a, b) Hindbrain tissue was immunostained with ZIKV E-protein, LC3, p62, and LAMP1 antibodies. Data are representative of three experiments and expressed as means + SD. Scale bar = 20 μ m. One-way ANOVA (Tukey's post hoc), ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.005.

Article Snippet: The primary antibody to Zika Virus Envelope (E) Protein (D1-4G2-4-15) antibody was bought from Kerafast Boston, MA.

Techniques:

Journal: Mediators of Inflammation

Article Title: Autophagy Contributes to Host Immunity and Protection against Zika Virus Infection via Type I IFN Signaling

doi: 10.1155/2020/9527147

Figure Lengend Snippet: Autophagy promoted ZIKV clearance in RAW264.7 cells. (a) Raw264.7 cells were transfected with ctrl siRNA or Atg7 and Atg5 siRNAs. qRT-PCR was performed for knockdown efficiency test. Means + SD from triplicate. ∗ p < 0.05, ∗∗ p < 0.01. (b) Cells were infected with ZIKV for 1 hour, (MOI = 10 : 1). Before infection, the cells were also transfected with LC3-GFP and treated with Atg5 siRNA and Atg7 siRNA. Immunostaining was performed to detect LC3 puncta. (c) Puncta number in each cell was counted. Values are means ± SD from 20 cells/sample. One-way ANOVA; Tukey's post hoc test, ∗ p < 0.05. (d) RAW264.7 cells were infected with ZIKV for 1 hour (MOI = 10 : 1). Before infection, the cells were also treated with rapamycin and 3-MA; the rapamycin was used at (3 μ M, 12 hours); 3-MA was used at (3 mM, 3 hours). The number of internalized virus per cell after 1 h infection was counted by PFU assay. After infection for 12 h, the unbound virus was washed away and fresh medium was added. The clearance assay was performed as counting the number of internalizing virus per cell by PFU assay. (e) Before infection, RAW264.7 cells were treated with negative control siRNA, Atg5 siRNA, and Atg7 siRNA. The number of internalized virus per cell was counted as above. Data were representative of three experiment results. Means ± SD. One-way ANOVA; Tukey's post hoc test, ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The primary antibody to Zika Virus Envelope (E) Protein (D1-4G2-4-15) antibody was bought from Kerafast Boston, MA.

Techniques: Transfection, Quantitative RT-PCR, Infection, Immunostaining, Negative Control

Journal: Mediators of Inflammation

Article Title: Autophagy Contributes to Host Immunity and Protection against Zika Virus Infection via Type I IFN Signaling

doi: 10.1155/2020/9527147

Figure Lengend Snippet: (a) GSE97919 Zika data set. GSEA (gene set enrichment analysis) shows there was a positive correlation between ZIKV infection and IFN- α pathway activation. Enrichment score = 0.9344. (b) Different genes enriched by IFN- α activation upon ZIKV infection, and 70 IFN- α pathway genes were upregulated. (c) Analysis of interaction among IFN- α pathway, negative regulation of autophagy, and positive regulation of autophagy. (d) RAW264.7 cells were transfected with IFNAR1 siRNA, IFNAR2 siRNA, IFNGR1 CRISPR-Cas9, IFNGR2 siRNA, IFNLR1 siRNA, or IL10R2 siRNA, respectively. qRT-PCR was performed to test the knockdown or knockout efficiency. (e) Cells above were cultured and transfected with LC3-GFP, then infected with ZIKV, MOI = 10 : 1. LC3 fluoresce was observed using confocal microscopy. (f) LC3 puncta numbers in 100 cells of each sample were counted. Means ± SD. Data were representative of three experiment results.

Article Snippet: The primary antibody to Zika Virus Envelope (E) Protein (D1-4G2-4-15) antibody was bought from Kerafast Boston, MA.

Techniques: Infection, Activation Assay, Transfection, CRISPR, Quantitative RT-PCR, Knock-Out, Cell Culture, Confocal Microscopy