Journal: bioRxiv

Article Title: A novel zebrafish-based in vivo model of Zika virus infection unveils NS4A as a key viral determinant of neuropathogenesis

doi: 10.1101/2024.01.20.576493

Figure Lengend Snippet: (A) Schematic experimental design. Cell medium (mock), DENV viral particles (serotype 2, strain 16681s) or ZIKV viral particles (strain H/PF/2013) were microinjected in the zebrafish yolk at 2 hours post-fertilization (hpf). (B) Survival curve over 3 days post-fertilization (dpf) of mock-infected ( n =84), ZIKV-infected ( n =104), ZIKV-infected and treated with NITD008 ( n =94) and DENV-infected ( n =89) larvae ( N =3). (C) Representative pictures of microinjected larvae at 3 dpf. ZIKV infection induced both mild and severe developmental phenotypes. (D) Quantification of the proportion of larvae with the different phenotypes (No injection, n =64; Mock, n =54; ZIKV, n =67; ZIKV+NITD008, n =62; DENV, n =55. N =3). Data are shown as means ± SEM. *** P ≤ 0.001; ** P ≤ 0.01; 2-way ANOVA. (E) Head size at 3dpf of the larvae from (D). Data are shown as means ± SEM. **** P ≤ 0.0001; ns = not significant; one-way ANOVA. (F) ZIKV RNA levels in whole larvae at 3 dpf were determined using ddPCR. (G) Schematic representation of a zebrafish brain at 4 days post-fertilization. The forebrain is shown. Gray line represents the localization of the transverse sections. Th and PTh are areas of the diencephalon, a division of the forebrain. Dien= diencephalon; Tel= telencephalon; Th= thalamus; PTh= prethalamus; Ve= venticle. A= anterior; P= posterior; D= dorsal; V= ventral (H) Transverse brain cryo-sections of 4 days post fertilization mock-injected or ZIKV-injected larvae are shown. Sections were stained with anti-ZIKV E antibody (green) and analyzed by confocal microscopy. Nuclei were labeled with Hoechst. Scale bars = 50 μm. n represents the number of fish; N represents the number of independent experimental repeats.

Article Snippet: Four hours post-injection, fish were either treated with 100 μM NITD008 (Tocris Small Molecules) or 0.5% DMSO as control.

Techniques: Infection, Injection, Staining, Confocal Microscopy, Labeling

Journal: bioRxiv

Article Title: A novel zebrafish-based in vivo model of Zika virus infection unveils NS4A as a key viral determinant of neuropathogenesis

doi: 10.1101/2024.01.20.576493

Figure Lengend Snippet: (A) Schematic experimental setup and behavioral analysis. (B) Representative swimming tracks of control (mock), ZIKV-infected (ZIKV), and ZIKV-infected and NITD008-treated (ZIKV+NITD008) larvae at 4 days post-fertilization. (C) The distance moved by the larvae was assessed using the DanioVision device (mock, n =40; ZIKV, n =45; ZIKV+NITD008, n =30. N =3). Data are shown as median ± 95 CI. **** P ≤ 0.0001; Kruskal-Wallis test. (D) TUNEL staining at 2 days post-fertilization showing cell death in the developing brain following injection in zebrafish embryo. A = anterior; P = posterior. Scale bars = 50 μm (E) The number of TUNEL+ cells was quantified (Mock, n =15, N =3; ZIKV normal, n =20, N =3; ZIKV mild/severe, n =26, N =3; ZIKV+NITD008, n =14, N =2). Data are shown as median ± 95 CI. **** P ≤ 0.0001; Kruskal-Wallis test. n indicates the number of fish; N represents the number of independent experimental repeats.

Article Snippet: Four hours post-injection, fish were either treated with 100 μM NITD008 (Tocris Small Molecules) or 0.5% DMSO as control.

Techniques: Control, Infection, TUNEL Assay, Staining, Injection

Journal: Biochemistry

Article Title: Identification of Zika Virus Inhibitors Using Homology Modeling and Similarity-Based Screening to Target Glycoprotein E

doi: 10.1021/acs.biochem.0c00458

Figure Lengend Snippet: Compound cytotoxicity for 53 candidate compounds and positive control NITD008 (blue). Gray bars represent seven candidates with cytotoxicities of <1 (dashed horizontal bar). Compounds tested in triplicate at 25 μM.

Article Snippet: NITD008 (NR-36199) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), as part of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) program.

Techniques: Positive Control

Journal: Biochemistry

Article Title: Identification of Zika Virus Inhibitors Using Homology Modeling and Similarity-Based Screening to Target Glycoprotein E

doi: 10.1021/acs.biochem.0c00458

Figure Lengend Snippet: Cell viability results for 53 candidate compounds compared to the positive control NITD008 (blue). The data were normalized to ZIKV-infected cells. Values over 1 signifies inhibition of ZIKV-induced cell killing. Candidates 14–43 tested at 10 μM, all other candidates and the control NITD008 tested at 25 μM. Each group ordered by activity from high to low. Green bars represent candidates with mean activity of >1.

Article Snippet: NITD008 (NR-36199) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), as part of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) program.

Techniques: Positive Control, Infection, Inhibition, Control, Activity Assay

Journal: Biochemistry

Article Title: Identification of Zika Virus Inhibitors Using Homology Modeling and Similarity-Based Screening to Target Glycoprotein E

doi: 10.1021/acs.biochem.0c00458

Figure Lengend Snippet: TCID50 infectivity results for 53 candidate compounds compared to the positive control NITD008 (blue). Candidates 14 and 43 were tested at 10 μM; all of the other candidates and the NITD008 control were tested at 25 μM. Red bars represent candidates that inhibited infectivity to <0.6 (horizontal dotted line) in the TCID50 assay and >1.0 in the cell viability assay. Yellow bars represent candidates with TCID50 activity but not flagged as active in the cell viability assay. Candidates 5* and 52* did not yield results in the TCID50 assay.

Article Snippet: NITD008 (NR-36199) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), as part of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) program.

Techniques: Infection, Positive Control, Control, TCID50 Assay, Viability Assay, Activity Assay

Journal: Biochemistry

Article Title: Identification of Zika Virus Inhibitors Using Homology Modeling and Similarity-Based Screening to Target Glycoprotein E

doi: 10.1021/acs.biochem.0c00458

Figure Lengend Snippet: X – Y scatter plot defining activity from cell viability (>1.0 cutoff) and TCID50 (<0.6 cutoff) assays. Compounds colored red ( 8 , 15 , 30 , 36 , 41 , and 43 ) and blue (NITD008 control) were active in both assays. Compounds colored green ( 11 , 17 , 22 , and 27 ) or yellow ( 2 and 49 ) were active in one assay.

Article Snippet: NITD008 (NR-36199) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), as part of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) program.

Techniques: Activity Assay, Control