Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: ZIKV and DENV2-infected A549 cells produce exosome-like particles associated with NS1 protein. ( A ) The NS1 protein of both ZIKV and DENV2 is found associated with the exosomal fraction obtained from the cell culture supernatants (CCS) of A549 infected cells. The tetraspanin CD63 detected on the dot blot of exosomal fractions provides insight into the quantity of exosomes extracted. ( B ) Western blot analysis of the exosome fraction, heated (HT) (+) or not (−), produced by DENV2 infected cells, show the presence of NS1 in dimeric form. A unique band with an apparent mass of ~48 kDa is observed after heating (+). ( C ) DLS analysis of exosomes produced by A549 cells during ZIKV (green) or DENV2 (blue) infection. On the y axis, the intensity gives the proportions of the different vesicle populations, depending on their size on the x axis. Both DENV2 and ZIKV infections appear to slightly reshape the distribution of vesicles.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: Infection, Cell Culture, Dot Blot, Western Blot, Produced

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: The overexpressed ZIKV-NS1 FLAG-tag and DENV2-NS1 V5-tag co-elute with exosome-like particles. ( A ) The recombinant ZIKV-NS1 FLAG-tag and DENV2-NS1 V5-tag , overexpressed in stable HEK cell lines, are present in the CCS, mainly as dimers. ( B ) The tetraspanins CD81 and CD63 are immunodetected in the exosome fractions of CCS from cells expressing the different secreted recombinant proteins. ( C ) When detecting the recombinant proteins in the CCS and exosomal fractions, only the NS1 proteins were detected in the exosomal fractions, and a similar signal was obtained whether or not the vesicles had been lysed. ( D ) SEAP activity was estimated by quantiblue assay. CCS and the flowthrough remaining from the exosome extraction, but not purified exosomes, exhibited SEAP activity. Ordinary one-way ANOVA test was performed using GraphPad Prism **** p < 0.0001, ns (not significant) ( E ) Western blot analysis of exosome protein extracts shows that ZIKV-NS1 FLAG-tag and DENV2-NS1 V5-tag are found associated with exosomes in their dimeric form.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: FLAG-tag, Recombinant, Expressing, Activity Assay, Purification, Western Blot

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: ZIKV-NS1 FLAG-tag is present in the exosome fraction. ( A ) Western blot analysis of HEK293-NS1 supernatant following ultrafiltration. Mouse anti-FLAG antibody was used to detect ZIKV-NS1 FLAG-tag , while the detection of albumin served as protein loading control. ( B ) Dot blot analysis of putative exosome fractions. After the ultrafiltration step, size exclusion chromatography was carried out on the >100 kDa fraction. Detection of ZIKV-NS1 FLAG-tag was achieved using a mouse anti-FLAG antibody, whereas CD81 was chosen as a marker for exosomes. ( C ) CCS containing CD63-positive EVs and secreted ZIKV-NS1 FLAG-tag (S1) were treated with PEG and centrifuged to obtain a pellet enriched in EVs. CD63 was found in the first pellet (P PEG) and after the second ultracentrifugation step (P2), validating the enrichment of exosomes which were also found associated with ZIKV-NS1 FLAG-tag . ( D ) ELISA capture of the exosomes in the supernatant of HEK293-ZIKV-NS1 FLAG-tag versus control. Exosomes were captured by an anti-CD81 antibody coated on Maxisorp ® plate. NS1 FLAG-tag protein was detected using a mouse anti-FLAG antibody. Ordinary one-way ANOVA test was performed using GraphPad Prism, ** p < 0.001. ( E ) DLS analysis of the exosomal fractions with the proportion of vesicles by size graph and the corresponding z-average-table .

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: FLAG-tag, Western Blot, Dot Blot, Size-exclusion Chromatography, Marker, Enzyme-linked Immunosorbent Assay

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: Overexpression of ZIKV and DENV2 NS1 lead to an increase in exosomes size.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: Over Expression, Standard Deviation

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: ZIKV and DV2 NS1 recombinant proteins bind to extracellular vesicles. ( A ) HEK 293 cells expressing ZIKV-NS1 Flag--tag protein were treated or not with GW4869 at 10 µM for 16 h in order to inhibit exosome’s biogenesis. The cell culture supernatant (CCS) was collected for exosomes extraction using the Exoeasy kit. A dot blot was performed using anti-Flag and then quantified using imageJ. The flow through (FT) is the remaining part of the first exosome extraction step, and the wash is the second flow through, considered as a negative control. The quantity of NS1 associated with exosomes was reduced when cells were treated with GW4869, thus eliminating the co-elution bias of NS1 protein with exosomes. Unpaired t-test was performed using GraphPad Prism, * p < 0.05. ( B ) CCS of HEK was collected 48 h post-platting and incubated with ZIKV-NS1 HIS-tag or DENV2-NS1 HIS-tag for 30 min and 4 h. CCS was then proceeded to PEG exosome precipitation. The negative control (CTL−) corresponds to panserin. The positive control (CTL+) corresponds to 0.2 ng of NS1 solubilized in panserin. Dot blots were performed using anti-His and anti-NS1 antibodies to detect ZIKV and DV2 NS1 proteins, respectively. NS1 of ZIKV and DV2 were found associated with EVs as early as 30 min post-incubation.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques: Recombinant, Expressing, FLAG-tag, Cell Culture, Dot Blot, Negative Control, Co-Elution Assay, Incubation, Positive Control

Journal: Viruses

Article Title: Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

doi: 10.3390/v15020364

Figure Lengend Snippet: Graphical overview of the association of ZIKV and DENV NS1 proteins with exosomes.

Article Snippet: Recombinant ZIKV-NS1 HIS-tag (strain Zika SPH2015) and DENV2-NS1 HIS-tag (Dengue type 2, strain New Guinea C) were from Sino Biological.

Techniques:

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: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Effect of human serum on ZIKV infectivity. ZIKV [1 x 10 6 PFU/mL] was incubated with active normal human serum (NHS) or 50% heat-inactivated NHS (hiNHS) for 1 h at 37°C. Thereafter, 10-fold dilutions of pre-incubated virus-serum mixtures were titrated on Vero cells. Plaques were visualized 4 days post infection using crystal violet staining. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (*** p < 0.001; ns indicates not significant). Mean data of at least 3 independent experiments are shown. The error bars represent the standard deviation.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Infection, Incubation, Virus, Staining, Comparison, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Time as a limiting factor on ZIKV infectivity. ZIKV [1 x 10 6 PFU/mL] was mixed with 50% active or heat-inactivated NHS and incubated for different time points (ranging from 1 min to 1 h) at 37°C. Virus-containing samples were then serially diluted and titrated on Vero cells. After 1 h incubation at 37°C, the cells were overlaid with agarose. Viral concentration was determined 4 days post infection using crystal violet staining. Mean data of 3 independent experiments are shown. The error bars represent the standard deviation.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Infection, Incubation, Virus, Concentration Assay, Staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: (A) EDTA inhibition confirms complement contribution to viral lysis. ZIKV [1 x 10 6 PFU/mL] was incubated with active or heat-inactivated human serum in the presence of increasing amounts of EDTA or Mg 2+ -EGTA. DMEM (not shown) and hiNHS were used as controls. (B) To prevent activation of the classical and lectin complement pathways, active or heat-inactivated human serum was pre-incubated with increasing amounts of C1 esterase inhibitor as indicated. (C) In the absence of C1q, most of the virus remained infectious whereas addition of purified C1q (70 μg/mL) restored the lytic effect on the virus. HiNHS was set to 100%. (D) Blocking of the lectin pathway by synthetic peptides (SFMI-1/2) did not rescue the virus. Combination of C1q-depleted serum and SFMI-1/2 served as additional control showing that the peptides had no effect on the infectivity of the virus. In all experiments, the viral titer was determined by plaque assays using Vero cells. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (** p < 0.005). All virus lysis experiments were conducted in duplicates and repeated three times. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Inhibition, Lysis, Incubation, Activation Assay, Virus, Purification, Blocking Assay, Control, Infection, Comparison

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Natural IgM blocking results in ZIKV rescue. Anti-human IgM blocking antibodies were incubated with 50% NHS or hiNHS for 30 min on ice before ZIKV was added. After incubation of 1 h at 37°C, the virus-serum mixture was serially diluted and titrated on Vero cells. After 1 h r incubation at 37°C, the cells were overlaid with agarose. Viral concentration was determined4 days post infection using crystal violet staining. All virus lysis experiments were conducted in triplicate, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Blocking Assay, Incubation, Virus, Concentration Assay, Infection, Staining, Lysis

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Binding of C1q to recombinant ZIKV envelope (E) and NS1 proteins. ZIKV proteins or viral particles were coated to ELISA plates and incubated with decreasing amounts of C1q as indicated. Bound C1q was detected using a polyclonal anti-C1q antibody followed by a HRP-labeled goat anti-rabbit IgG and visualized by TMB. The absorbance was measured at 450 nm, using a Bio-Rad plate reader. Data show the mean of two experiments performed in duplicate.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Labeling

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Dissecting the role of the lectin and classical pathways on ZIKV derived from the human cell line A549. NHS (50%) reduced the viral titer for about one order of magnitude. Inhibition of the lectin pathway by a peptide mix of SFMI-1 and 2 had no effect. By contrast, C1q depletion rescued the virus and most of the virus remained infectious. Viral titer was determined by plaque assays using Vero cells. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (** p < 0.005). All virus lysis experiments were conducted in duplicates and repeated two times. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Derivative Assay, Inhibition, Virus, Comparison, Lysis

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Reduction of viral infectivity is linked to decreased RNA levels. After incubating ZIKV with active or heat-inactivated human serum, the viral RNA was digested by addition of RNases. Three hours after incubation at 37°C, the remaining genomic material was extracted and quantified by RT-PCR. The RNA copy number was calculated from the amount of RNA obtained by incubation of the virions with 50% hiNHS, which was set to 100%. Results are given as % of RNA loss. Data were analyzed with one-way-ANOVA with Bonferroni post-hoc comparison (**** p < 0.0001). All virus lysis experiments were conducted in triplicate. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Comparison, Virus, Lysis

Journal: Frontiers in Immunology

Article Title: Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex

doi: 10.3389/fimmu.2018.02177

Figure Lengend Snippet: Blocking the assembly of MAC leads to virus rescue. ZIKV [1 x 10 6 PFU/mL] was exposed to either 50% C9-depleted (C9dep) human serum or 50% heat-inactivated C9-depleted (hiC9dep) serum for 1 h at 37°C. As a control, the depleted serum was reconstituted with purified C9 protein, adjusted to its natural concentration in serum [60 μg/mL]. During incubation, the RNA of lysed ZIKV was digested by external RNase addition. Subsequently, the amount of complement lysis-resistant virions was determined by RT-PCR. The RNA copy number was calculated by incubation of the virions with 50% hiNHS, which was set to 100%. Results are given as % of RNA loss. All assays were performed in triplicate. The data represent mean values, and the error bars show standard deviations.

Article Snippet: To investigate the interaction of the complement component C1q with ZIKV-derived proteins, 5 μg/mL of recombinant ZIKV envelope or NS1 protein [MyBioSource, San Diego, USA] or 100 μL of ZIKV supernatant derived from C6/36 cells [containing 5 x 10 6 PFU/well] were coated in carbonate buffer (pH 9.6) on a 96-well flat-bottom plate (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C.

Techniques: Blocking Assay, Virus, Control, Purification, Concentration Assay, Incubation, Lysis, Reverse Transcription Polymerase Chain Reaction

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: Viruses

Article Title: Embryonic Stage of Congenital Zika Virus Infection Determines Fetal and Postnatal Outcomes in Mice

doi: 10.3390/v13091807

Figure Lengend Snippet: Fetal outcomes. ( A ) Percentages of each fetal outcome: fetuses that died in utero, were deformed, showed IUGR, or appeared normal at 6 days after congenital ZIKV infection. Survival of fetuses was confirmed by heartbeat or pulsation of the umbilical cord as observed under a microscope. The x -axis shows the embryonic days of ZIKV infection or 2MEM inoculation for uninfected controls. The ZIKV-infected group consisted of 10 fetuses from 1 dam infected at E6.5, 14 fetuses from 2 dams infected at E7.5, 22 fetuses from 3 dams infected at E8.5, 28 fetuses from 3 dams infected at E9.5, 21 fetuses from 2 dams infected at E10.5, 17 fetuses from 2 dams infected at E11.5, 13 fetuses from 2 dams infected at E12.5, and 7 fetuses from 1 dam infected at E13.5 or E14.5. The uninfected group consisted of 8 fetuses from 1 dam at E6.5 or E9.5 and 7 fetuses from 1 dam at E8.5 or E13.5. ( B ) Inverse correlation between IUGR prevalence at 6 dpi and infected embryonic days. The x -axis shows ZIKV-infected embryonic days. Significance was determined by Spearman’s correlation test. ( C ) Inverse correlation between the prevalence of deformed masses at 6 dpi and infected embryonic days. The x -axis shows ZIKV-infected embryonic days. Significance was determined by Spearman’s correlation test. ( D ) Fetal CRL at 6 dpi. Dams were infected with ZIKV or inoculated with 2MEM (uninfected) at the indicated embryonic days. Individual dams are indicated on the x -axis; each square represents one fetus. Vertical dashed gray lines separate litters from each dam. If the fetal heads were indistinguishable from the body, their CRL was considered as zero (below the detection limit). Significance was determined by t -test or Kolmogorov–Smirnov test. ( E ) Fetal head length at 6 dpi. Data are from the same fetuses as described for panel C. If the fetal heads were indistinguishable from the body, their head length was considered as zero (below the detection limit). Significance was determined by t -test or Kolmogorov–Smirnov test. ( F ) Fetal weights at 6 dpi. Dams were infected with ZIKV or inoculated with 2MEM (uninfected) at the indicated embryonic days. Individual dams are indicated on the x -axis; each square represents one fetus. Vertical dashed gray lines separate litters from each dam. Significance was determined by t -test. ( G ) Percentages of each fetal outcome at 2 or 4 dpi. Dams were infected with ZIKV at E9.5 or E13.5, and fetuses were visually inspected at 2 or 4 dpi. The data include 18 fetuses from 2 litters at 2 dpi at E9.5, 10 fetuses from 1 litter at 4 dpi at E9.5, 4 fetuses from 1 litter at 2 dpi at E13.5, or 75 fetuses from 8 litters at 4 dpi at E13.5. ( H ) The fetus with intracranial hemorrhage at 4 dpi at E13.5. Scale bar = 1 cm. ( I ) The fetus with ocular malformation and an apparently normal littermate. Scale bar = 1 cm.

Article Snippet: IHC was performed using an anti-ZIKV NS1 antibody (C01886G, Meridian Bioscience, Cincinnati, OH, USA) as the primary antibody [ , ].

Techniques: In Utero, Infection, Microscopy

Journal: Viruses

Article Title: Embryonic Stage of Congenital Zika Virus Infection Determines Fetal and Postnatal Outcomes in Mice

doi: 10.3390/v13091807

Figure Lengend Snippet: Viral titers and histological findings in fetal tissues or placentas. ( A ) Viral titers in fetal whole bodies, placentas, and deformed masses at 6 dpi. Dams were infected with ZIKV at the indicated embryonic days. Individual dams are indicated on the x -axis. Vertical dashed gray lines separate litters from each dam. Symbols represent individual fetus, placenta, or deformed mass. Limit of detection was 0.83 log 10 CCID 50 /g as indicated by the horizontal dashed line. ( B ) Viral titers in fetal heads, placentas, and deformed masses at 2 or 4 dpi, as described for panel A. Percentage of fetuses that were infected for each dam at 2 or 4 dpi is indicated. Kolmogorov–Smirnov test or t -test was used for statistical analysis. ( C ) Lack of correlation between virus titers in placentas and fetal heads at 2 dpi and 4 dpi as determined by Pearson or Spearman’s correlation test. ( D ) Placental weights at 6 dpi. Dams were infected with ZIKV or inoculated with 2MEM (uninfected) at the indicated embryonic days. Individual dams are indicated on the x -axis; each square represents one placenta. Significance was determined by t -test or Kolmogorov–Smirnov test. ( E ) H&E staining of placentas at 6 dpi; dams were infected at E7.5. Representative image of placentas from 2 dams. ( F ) As described for panel E at higher magnification. ( G ) IHC of placenta at 6 dpi at E7.5 using anti-ZIKV NS1 antibody. Positive staining (brown) was detected in decidual cells. Representative image of placentas from 2 dams. ( H ) As described for panel G at higher magnification. ( I – L ) H&E staining and IHC of placentas at 6 dpi; dams were infected at E13.5; otherwise as described for E–H. ( M ) H&E staining of placentas from uninfected dams. Representative image of placentas from 2 dams. ( N ) As described for panel M at higher magnification. ( O ) IHC of placenta from uninfected dams using anti-ZIKV NS1 antibody. Representative image of placentas from 2 dams. Scale bars; 500 µm ( E , I , M ), 100 μm ( F – H , J – L , N , O ).

Article Snippet: IHC was performed using an anti-ZIKV NS1 antibody (C01886G, Meridian Bioscience, Cincinnati, OH, USA) as the primary antibody [ , ].

Techniques: Infection, Virus, Staining

Journal: Viruses

Article Title: Embryonic Stage of Congenital Zika Virus Infection Determines Fetal and Postnatal Outcomes in Mice

doi: 10.3390/v13091807

Figure Lengend Snippet: Postnatal outcomes. ( A ) Survival of offspring. Data are from 8 offspring from 1 dam infected at E8.5, 16 offspring from 3 dams infected at E9.5, 12 offspring from 2 dams infected at E10.5, 14 offspring from 2 dams infected at E11.5, 75 offspring from 9 dams infected at E12.5, 20 offspring from 3 dams infected at E13.5, 25 offspring from 4 dams infected at E14.5, and 19 offspring from 3 uninfected dams. Comparison of Kaplan–Meier survival curves between groups was performed by log-rank analysis. ( B ) Weight of offspring at P3. Individual litters are indicated on the x -axis; each square represents a single offspring. Vertical dashed gray lines separate each litter, which was infected at the indicated embryonic days. The pale green shaded area represents 2SD below the mean body weight of uninfected offspring. ( C ) Weight gain of each litter. The pale green shaded area represents 2SD below the mean body weight of uninfected offspring. Data consist of 6 survived offspring out of 8 offspring (6/8) for litter 1 (L1) infected at E10.5, 2/7 for L1 infected at E11.5, 3/5 for L1 infected at E12.5, 2/4 for L2 infected at E12.5, 7/7 for L3 infected at E12.5, 7/7 for L1 infected at E13.5, and 3/5 for L2 infected at E13.5. The uninfected group consisted of 18 offspring from 3 litters. Statistical analyses were performed by repeated-measure ANOVA. ( D ) Head circumference of offspring at P3. Individual litters are indicated on the x -axis; each square represents a single offspring. Vertical dashed gray lines separate each litter, which was infected at the indicated embryonic days. The pale green shaded area represents 2SD below the mean head circumference of uninfected offspring. Head circumference was calculated by multiplying the head diameter by Pi (3.14). ( E ) Growth of head circumference of each litter. Data are from the same litters as described for panel C. The pale green shaded area represents 2SD below the mean head circumference of uninfected offspring. Asterisks show the value 2SD below that of the uninfected mean. ( F ) Weight of offspring born to ZIKV-infected or uninfected dams at P7 or P8. Individual litters are indicated on the x -axis; each square represents a single offspring. Vertical dashed gray lines separate each litter, which was infected at E12.5 or uninfected. Dorsal view of two small offspring infected at E12.5 compared with each littermate. ( G ) SHIRPA scores of 4-week-old mice born to ZIKV-infected dams at E12.5. The horizontal axis shows the score. Each bar represents one mouse. Statistical analyses were performed by Kolmogorov–Smirnov test or t -test: * p < 0.05; ** p < 0.01. Longer lines along the y -axis separate litters.

Article Snippet: IHC was performed using an anti-ZIKV NS1 antibody (C01886G, Meridian Bioscience, Cincinnati, OH, USA) as the primary antibody [ , ].

Techniques: Infection, Comparison

Journal: Viruses

Article Title: Embryonic Stage of Congenital Zika Virus Infection Determines Fetal and Postnatal Outcomes in Mice

doi: 10.3390/v13091807

Figure Lengend Snippet: Neutralizing antibodies in dams protect the offspring from vertical ZIKV infection. ( A ) Experimental timeline of each group. ( B ) ZIKV-specific neutralizing antibody titers of each dam. Limit of detection was 1 in 10 dilutions as indicated by the horizontal dashed line. Titer was determined by PRNT 50 assays. Kolmogorov–Smirnov test was used for statistical analysis. ( C ) Survival of offspring. Comparisons for Group E versus either Group A or Group C, p < 0.0001. Comparisons of Kaplan–Meier survival curves between the different groups were performed by log-rank analyses. The data are from 51 offspring from 7 litters for Group A, 21 offspring from 3 litters for Group B, 35 offspring from 4 litters for Group C, 6 offspring from 1 litter for Group D, 67 offspring from 10 litters for Group E, and 60 offspring from 7 litters for the uninfected group. ( D ) Correlation between neutralizing antibody titers of each dam and the percent survival of each litter in Groups A, C, and E. Significance was determined by Spearman’s correlation test.

Article Snippet: IHC was performed using an anti-ZIKV NS1 antibody (C01886G, Meridian Bioscience, Cincinnati, OH, USA) as the primary antibody [ , ].

Techniques: Infection