Journal: Nature medicine

Article Title: Identification of small molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

doi: 10.1038/nm.4184

Figure Lengend Snippet: Identification of PHA-690509 and Niclosamide as antiviral compounds. (a) Top, example western blot images of titration of PHA-690509 on SNB-19 cells for anti-ZIKV activity. Glioblastoma SNB-19 cells were treated with the indicated compound for 1 hour prior to inoculation with ZIKV FSS-13025, PRVABC59, or MR766 (MOI = 1) and cells were harvested 24 hours post infection for western blot analysis. Bottom, quantification of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). Insert shows chemical structure of PHA-690509. (b) Top, example western blot images of titration of Emricasan on SNB-19 cells for anti-ZIKV activity. Similar to (a). Values represent mean ± s.d. (n = 3 cultures; P > 0.1; One-way ANOVA for comparison with the DMSO treatment). (c) Top, chemical structure of Niclosamide and example western blot images of titration of Niclosamide on SNB-19 cells for anti-ZIKV activity using the PRVABC59 strain (MOI = 1). Bottom, quantification similar to (a). Values represent mean ±s.d. (n = 3 cultures; **P < 0.01; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (d) Summary of ELISA quantifications of secreted ZIKV-NS1 protein in the medium of infected cells upon treatment of different doses of Niclosamide (top) or PHA-690509 (bottom) for 24 hours. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.001; One-way ANOVA for comparison with the 0 μM group). (e) Summary of titration of Niclosamide and PHA-690509 on ZIKV viral production. SNB-19 cells were treated with each indicated compound at increasing concentrations for 1 hour prior to infection with PRVABC59 at MOI = 0.5. Cell culture supernatant was collected 24 hours post infection and infectious virions were quantified for focus-forming units (FFU) using Vero cells. Data represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating IC50, and the insets report the calculated IC50 value for each compound.

Article Snippet: The anti-ZIKV NS1 ELISA kit (ZKV-NS1-EK) was obtained from BioFront Technologies (Tallahassee, FL) and used according to the manufacturer’s protocol.

Techniques: Western Blot, Titration, Activity Assay, Infection, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nature medicine

Article Title: Identification of small molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

doi: 10.1038/nm.4184

Figure Lengend Snippet: Inhibition of ZIKV infection by Niclosamide and PHA-690509 at a post-entry step and blockade of ZIKV infection by additional CDKis. (a) Schematic illustration of time-of-addition experiment for Niclosamide and PHA-690509. (b) Top, example western blot images of SNB-19 cells treated with 10 μg/ml anti-AXL antibody, 2 μM Niclosamide, or 92 μM PHA-690509 for 1 hour prior to or 4 hours post infection with PRVABC59 (MOI = 1). Bottom, quantification of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; *P < 0.05; **P < 0.01; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (c) Inhibition of ZIKV infection in CDKi-treated cells. Left, example western blot images of SNB-19 cells treated with the indicated compound at 92 μM for one hour prior to infection with FSS13025 or PRVABC59 (MOI = 1). Right, quantification similar to (b). Values represent mean ± s.d. (n = 3 cultures; ***P < 0.01; One-way ANOVA for comparison with the DMSO treatment). (d) Effect of various CDKis on ZIKV production. Similar to Figure 2e. All data were normalized to 0 μM for each compound. Data represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating IC50 (listed on the right panel).

Article Snippet: The anti-ZIKV NS1 ELISA kit (ZKV-NS1-EK) was obtained from BioFront Technologies (Tallahassee, FL) and used according to the manufacturer’s protocol.

Techniques: Inhibition, Infection, Western Blot

Journal: Nature medicine

Article Title: Identification of small molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

doi: 10.1038/nm.4184

Figure Lengend Snippet: Niclosamide and PHA-690509 inhibit ZIKV infection in human astrocytes and forebrain-specific hNPCs. (a) Top, example immunostaining images of astrocytes treated with 2 μM Niclosamide, 92 μM PHA-690509, 9 μM Emricasan, or a combination of 92 μM PHA-690509 and 9 μM Emricasan for 1 hour prior to infection with PRVABC59 (MOI = 0.5). Cells were fixed 24 hours post infection and stained for ZIKVE (green) and DAPI (blue; Scale bar: 50 μm, applies to all image panels in a). Bottom, quantification of the percentage of ZIKV-infected astrocytes over DAPI. Values represent mean ± s.d. (n = 6 cultures; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (b) Top, example western blot images of hNPCs infected at a MOI of 0.1 and analyzed 48 hours post infection. Bottom, quantifications of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.01; One-way ANOVA for comparison with the DMSO treatment). (c) Virus production from compound treated iPSC-derived human astrocytes. Similar to Figure 2e. Data represent mean ± s.d. (n = 3 cultures). (d–e) Summary of additive effects of a combination of Emricasan and PHA-690509 on inhibiting increased caspase-3 activity in human astrocytes infected with FSS13025-ZIKV or MR766 ZIKV (d), and on improving cell viability as measured by ATP production (e). Similar to Figure 1a. Values represent mean ± s.d. (n = 3 cultures; *P < 0.05; **P < 0.01; One-way ANOVA).

Article Snippet: The anti-ZIKV NS1 ELISA kit (ZKV-NS1-EK) was obtained from BioFront Technologies (Tallahassee, FL) and used according to the manufacturer’s protocol.

Techniques: Infection, Immunostaining, Staining, Western Blot, Derivative Assay, Activity Assay

Journal: Sensors (Basel, Switzerland)

Article Title: Label-Free Electrochemical Biosensors for the Determination of Flaviviruses : Dengue, Zika, and Japanese Encephalitis

doi: 10.3390/s20164600

Figure Lengend Snippet: Survey of electrochemical label-free biosensors for Zika diagnostic.

Article Snippet: Microtiter plate precoated with ZIKV NS1/ Euroimmun anti-ZIKV IgM , Indirect ELISA , Antibody-HRP conjugates , DENV Ig M antibodies , 56% , West Nile virus , d, f , a, c , [ , ] .

Techniques: Diagnostic Assay, Virus, Polymer, Membrane, Indirect ELISA

Journal: Virology Journal

Article Title: Construction of an infectious clone of Zika virus stably expressing an EGFP marker in a eukaryotic expression system

doi: 10.1186/s12985-021-01622-z

Figure Lengend Snippet: Rescue of recombinant ZIKA–EGFP. a Detection of EGFP by electrophoresis. (1–5 lanes are generation p1-p5 of recombinant ZIKA-EGFP, respectively) b Fluorescence of Vero cells infected with the ZIKA–EGFP (generation P5) for 48 h (×200). c Western blot detection of E gene, NS1 gene, and EGFP gene levels in Vero cells infected with the ZIKA-WT, ZIKAi (generation P5), and ZIKA–EGFP (generation P5) for 48 h. Western blot analysis was repeated three times and representative result was exhibited. d Fluorescence of C6/36 cells infected with ZIKA–EGFP at different time points. e. The localization of EGFP and E protein for ZIKA-EGFP in Vero cells (×200)

Article Snippet: Anti-Zika E and NS1 antibody were obtained from GeneTex (San Antonio, TX, USA); anti-EGFP antibodies (Abcam, Cambridge, MA, USA); DAPI, Phycoerythrin (PE) labelled goat-anti-mouse antibody, anti-β-actin, anti-mouse and anti-rabbit secondary antibodies and enhanced chemiluminescence (ECL) Horseradish Peroxidase (HRP) substrate (Bioworld, Minneapolis, MN, USA); pEGFP-N1 vector, Infusion kit, TRIzol, PrimeScript RT reagent kit, BesstarTM qPCR Master Mix, TB GreenTM Premix (Takara, Dalian, China); pBAC11 (Addgene, Watertown, MA, USA); restriction enzymes Sfo I and Pac I, DH10B cells (New England Biolabs, Ipswich, MA, USA); Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA); 0.45-μm membrane filter, (Pall Corporation; Port Washington, NY, USA); polyvinylidene difluoride (PVDF) membrane (Millipore, MA,USA); bovine serum albumin (BSA), IPA lysis buffer (Genshare, Xian, China).

Techniques: Recombinant, Electrophoresis, Fluorescence, Infection, Western Blot

Journal: Viruses

Article Title: Construction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection

doi: 10.3390/v14040706

Figure Lengend Snippet: Analysis of stability of JEV-HA/NS2A/∆NS1’. The full-length gene sequence of JEV-HA/NS2A/∆NS1’ was amplified by RT-PCR from passage 6 and subjected to DNA sequencing. ( A ) Sequencing chromatogram of HA-tag insertion. ( B ) Sequencing chromatogram of A30P mutation. ( C , D ) BHK-21 cells were infected with the indicated recombinant JEV and subjected to western blots ( C ) and IFA analysis ( D ). Detection of HA-NS2A expression by IFA. HA-NS2A (green) and NS3 (red) were detected with anti-HA and anti-NS3 antibodies, respectively. The nuclei (blue) were stained with 4’,6-diamidino-2-phenylindole (DAPI).

Article Snippet: The commercial antibodies used in this study consisted of: mouse anti-HA monoclonal antibody (H9658, Sigma, St. Louis, MO, USA); rabbit anti-calnexin polyclonal antibody (C4731, Sigma); rabbit anti-Erlin-2 antibody (EPR8088, Abcam, Shanghai, China); mouse anti-β-actin monoclonal antibody (Proteintech Group, Chicago, IL, USA); rabbit anti-JEV C polyclonal antibody (GTX131368, GeneTex, St. Anthony, TX, USA); rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex); rabbit anti-JEV NS1 polyclonal antibody (GTX131370, GeneTex); mouse anti-JEV NS1 monoclonal antibody (GTX633820, GeneTex); rabbit anti-JEV NS2B polyclonal antibody (GTX125972, GeneTex); rabbit anti-JEV NS4A polyclonal antibody (GTX132028, GeneTex); and rabbit anti-JEV NS4B polyclonal antibody (GTX125865, GeneTex).

Techniques: Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Mutagenesis, Infection, Recombinant, Western Blot, Expressing, Staining

Journal: Viruses

Article Title: Construction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection

doi: 10.3390/v14040706

Figure Lengend Snippet: Replication of JEV-HA/NS2A/∆NS1’ in BHK-21 cells. ( A , B ) BHK-21 cells were infected with the indicated viruses at an MOI of 0.01. Viral titers in the supernatants and viral protein expression in the cells were examined at the indicated time points by TCID 50 assays ( A ) and western blot ( B ), respectively. ( C , D ) Plaque morphology of recombinant JEV. BHK-21 cell monolayers were infected with the indicated viruses for an analysis of plaque morphology. The plaques were stained with crystal violet at 5 dpi ( C ) and the plaque diameters were measured and plotted ( D ). The significant differences between the groups were tested by Student’s t -test.

Article Snippet: The commercial antibodies used in this study consisted of: mouse anti-HA monoclonal antibody (H9658, Sigma, St. Louis, MO, USA); rabbit anti-calnexin polyclonal antibody (C4731, Sigma); rabbit anti-Erlin-2 antibody (EPR8088, Abcam, Shanghai, China); mouse anti-β-actin monoclonal antibody (Proteintech Group, Chicago, IL, USA); rabbit anti-JEV C polyclonal antibody (GTX131368, GeneTex, St. Anthony, TX, USA); rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex); rabbit anti-JEV NS1 polyclonal antibody (GTX131370, GeneTex); mouse anti-JEV NS1 monoclonal antibody (GTX633820, GeneTex); rabbit anti-JEV NS2B polyclonal antibody (GTX125972, GeneTex); rabbit anti-JEV NS4A polyclonal antibody (GTX132028, GeneTex); and rabbit anti-JEV NS4B polyclonal antibody (GTX125865, GeneTex).

Techniques: Infection, Expressing, Western Blot, Recombinant, Staining

Journal: Viruses

Article Title: Construction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection

doi: 10.3390/v14040706

Figure Lengend Snippet: HA-NS2A localization to the ER. ( A ) BHK-21 cells were infected with JEV-HA/NS2A/∆NS1’ and harvested at 48 hpi for an isolation of the detergent-resistant membranes (DRM). The presence of the indicated proteins in the DRM was detected by western blots. ( B ) BHK-21 cells were infected with JEV-HA/NS2A/∆NS1’ and subjected to an immunofluorescence assay at 24 hpi. HA-NS2A (green) and the ER (red) were detected with anti-HA and anti-calnexin antibodies, respectively. The nuclei (blue) were stained with DAPI.

Article Snippet: The commercial antibodies used in this study consisted of: mouse anti-HA monoclonal antibody (H9658, Sigma, St. Louis, MO, USA); rabbit anti-calnexin polyclonal antibody (C4731, Sigma); rabbit anti-Erlin-2 antibody (EPR8088, Abcam, Shanghai, China); mouse anti-β-actin monoclonal antibody (Proteintech Group, Chicago, IL, USA); rabbit anti-JEV C polyclonal antibody (GTX131368, GeneTex, St. Anthony, TX, USA); rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex); rabbit anti-JEV NS1 polyclonal antibody (GTX131370, GeneTex); mouse anti-JEV NS1 monoclonal antibody (GTX633820, GeneTex); rabbit anti-JEV NS2B polyclonal antibody (GTX125972, GeneTex); rabbit anti-JEV NS4A polyclonal antibody (GTX132028, GeneTex); and rabbit anti-JEV NS4B polyclonal antibody (GTX125865, GeneTex).

Techniques: Infection, Isolation, Western Blot, Immunofluorescence, Staining

Journal: Viruses

Article Title: Construction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection

doi: 10.3390/v14040706

Figure Lengend Snippet: Interaction of HA-NS2A with viral proteins. BHK-21 cells were infected with JEV and harvested at 48 hpi for a co-immunoprecipitation assay. ( A ) The cell lysates were incubated with anti-HA antibodies and the immunoprecipitated proteins were detected with the indicated antibodies against different viral proteins. ( B ) The cell lysates were pre-treated with RNase A and the presence of NS1 and GAPDH RNAs was examined by RT-PCR. ( C ) The cells lysates were pre-treated with RNase A and subsequently subjected to co-immunoprecipitation with anti-HA antibodies. The immunoprecipitated proteins were detected by the indicated antibodies against different viral proteins.

Article Snippet: The commercial antibodies used in this study consisted of: mouse anti-HA monoclonal antibody (H9658, Sigma, St. Louis, MO, USA); rabbit anti-calnexin polyclonal antibody (C4731, Sigma); rabbit anti-Erlin-2 antibody (EPR8088, Abcam, Shanghai, China); mouse anti-β-actin monoclonal antibody (Proteintech Group, Chicago, IL, USA); rabbit anti-JEV C polyclonal antibody (GTX131368, GeneTex, St. Anthony, TX, USA); rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex); rabbit anti-JEV NS1 polyclonal antibody (GTX131370, GeneTex); mouse anti-JEV NS1 monoclonal antibody (GTX633820, GeneTex); rabbit anti-JEV NS2B polyclonal antibody (GTX125972, GeneTex); rabbit anti-JEV NS4A polyclonal antibody (GTX132028, GeneTex); and rabbit anti-JEV NS4B polyclonal antibody (GTX125865, GeneTex).

Techniques: Infection, Co-Immunoprecipitation Assay, Incubation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction