Journal: bioRxiv

Article Title: Highly multiplexed mRNA quantitation with CRISPR-Cas13

doi: 10.1101/2023.08.16.553527

Figure Lengend Snippet: a , Huh7 cells were infected with nine flaviviruses. Total RNA extracted from infected Huh7 cells was used as input for the qCARMEN ISG panel. b , Calculated fold-changes in expression for ISG panel genes across infected cells compared to naive cells. c , The distribution of fold-changes for each gene across viral infections and replicates. Boxplot whiskers extend to 1.5 times the interquartile range; outliers represented with diamond markers. d , Pairwise Spearman correlations of dysregulated ISGs in response to flavivirus infection. e , Gene expression vectors at each timepoint for all three IFNs plotted along two principal components to illustrate the trajectory of expression changes across core ISGs. PCA were transformations performed on flavivirus ISG signatures to overlay the expression profiles on top of IFN stimulation trajectories. Gold star represents 0-hour timepoint. Light to dark: 1, 2, 4, 8, 24, 48, 72-hour timepoints. f , Euclidean distance between flavivirus PCA projections and IFN timepoint vectors to characterize flavivirus ISG changes in the context of IFN responses.

Article Snippet: Pellets were subsequently incubated for one hour at room temperature with flavivirus group antigen antibody (clone 4G2, diluted 1:100, Novus Biologicals) for flavivirus-infected cells and murine-produced Clone 9E10 primary antibody specific for NS5A (kindly provided by Charles Rice at The Rockefeller University) diluted 1:8000 in FACS buffer (1% FBS (v/v) in DPBS) for HCV , .

Techniques: Infection, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Highly multiplexed mRNA quantitation with CRISPR-Cas13

doi: 10.1101/2023.08.16.553527

Figure Lengend Snippet: a , Percent of antigen-positive cells for HCV (2 MOIs). b , Percent of antigen-positive cells for flavivirus-infected cells. Error bars presented as SD. c , Infected cell populations for HCV samples. d , Infected cell populations for flavivirus samples.

Article Snippet: Pellets were subsequently incubated for one hour at room temperature with flavivirus group antigen antibody (clone 4G2, diluted 1:100, Novus Biologicals) for flavivirus-infected cells and murine-produced Clone 9E10 primary antibody specific for NS5A (kindly provided by Charles Rice at The Rockefeller University) diluted 1:8000 in FACS buffer (1% FBS (v/v) in DPBS) for HCV , .

Techniques: Infection

Journal: bioRxiv

Article Title: A mRNA-LNP vaccine against Dengue Virus elicits robust, serotype-specific immunity

doi: 10.1101/2021.01.05.425517

Figure Lengend Snippet: A) Schematic of the DENV genome and engineered mRNA construct. An mRNA encoding for the prM and ENV viral proteins was engineered with N-terminal signal peptide sequence, 5’ and 3’ untranslated regions (UTR) flanking the coding sequence, a 3’ poly-A tail, and a 5’ cap-1 structure. In vitro synthesized mRNA is encapsulated in a lipid nanoparticle for use in in vitro and in vivo experiments. B) 293T cells were transfected with the in vitro transcribed mRNA encoding for the wild-type sequence (WT), or a mutant version with amino acid substitutions in the fusion-loop epitope (ΔFL). Lysate was analyzed by western blot with the domain III specific 1A1D-2 monoclonal antibody and the fusion-loop specific 4G2 monoclonal antibody. C) Supernatant from transfected cells was purified and concentrated through ultracentrifugation and analyzed for VLPs by western blots with the 1A1D-2 monoclonal antibody or anti-GAPDH. Unpurified cell lysate from WT mRNA transfected cells is included as a control. Shown are representative blots. D) Electron microscopy image of VLPs from purified supernatant of transfected 293T cells showing homogenous shape and size of approximately 30nm.

Article Snippet: Membranes were blotted with envelope domain III specific 1A1D-2 (1:600) monoclonal antibody (CDC Arbovirus Reference Collection) or envelope fusion-loop specific 4G2 (3.33 mg/ml) (BEI Cat# NR-50327, Novus Biologicals Cat# NBP2-52709FR).

Techniques: Construct, Sequencing, In Vitro, Synthesized, In Vivo, Transfection, Mutagenesis, Western Blot, Purification, Control, Electron Microscopy

Journal: The American Journal of Pathology

Article Title: Hippo Signaling Pathway Has a Critical Role in Zika Virus Replication and in the Pathogenesis of Neuroinflammation

doi: 10.1016/j.ajpath.2019.12.005

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: Rabbit flavivirus group primary antibody (D1-4G2-4-15) , Novus Biologicals (Centennial, CO) , NBP2-52666.

Techniques: Produced, Purification, Virus

Journal: Journal of Neuroinflammation

Article Title: A central role for CCR2 in monocyte recruitment and blood–brain barrier disruption during Usutu virus encephalitis

doi: 10.1186/s12974-025-03435-1

Figure Lengend Snippet: USUV infection in the brain induces acute encephalitis in immunocompetent mice. a Changes in body weight of mock or USUV-infected (10 2 –10 4 PFU) mice. N = 6 per group. b Survival of C57BL/6J mice inoculated with vehicle (mock), heat-inactivated or infective USUV. N = 5 per group. c Infectious virus in the brain was measured at 6 dpi and is expressed as the log 10 -transformed 50% tissue culture infectious dose (TCID 50 ) per 100 mg of brain tissue. d Confocal images showing immunostaining of brain cryosections from USUV-infected mice (10 4 PFU) at 6 dpi, showing USUV infection (4G2, green), neurons (NeuN, magenta) and nuclei (Hoechst, blue). White square indicates the area zoomed. Scale bars = 1000 µm and 50 µm (zoom). e USUV infection is displayed as percentage of 4G2 stained area. f Representative images of microglia activation (Iba1, red) and nuclei (Hoechst, blue) in brain cryosections of mock and USUV-infected mice (10 4 PFU) at 6 dpi. Scale bars = 50 µm and 5 µm (zoom). g Quantification of microglia activation, displayed as % of Iba1 stained area in the cortex. h Representative images of immunostained USUV-infected cells (4G2, green) in the brain and microglia (Iba1, red). White squares indicate the zoomed areas. Scale bars = 20 μm and 5 μm. i Quantification of the percentage of infected cells that are associated with at least one microglial cell. j Levels of IL-6, CXCL1, CCL5, CXCL10, IFN-γ and CCL2 in full brain homogenates of mock and USUV-infected (10 2 –10 4 PFU) mice at 6 dpi. Protein levels are displayed in pg per 100 mg of brain tissue or OD450 value (CXCL10). See also Additional file : Fig. S1 for TNF-α levels. Data are shown as mean ± SEM. N.D. = not detected. Image quantifications were pooled from minimum 5 pictures per mouse brain. Each dot in the graphs represents a single mouse. p values were obtained with two-way ANOVA ( a ), Student’s t test ( e, g, j ) or one-way ANOVA followed by Dunnett’s multiple comparisons test ( j ). Significant differences compared to mock-infected mice are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We stained the sections with rabbit anti-Iba1 (Abcam), mouse or rabbit anti-flavivirus group antigen 4G2 (Novus Biologicals) and mouse anti-NeuN (Abcam).

Techniques: Infection, Virus, Transformation Assay, Immunostaining, Staining, Activation Assay