Journal: Immunology

Article Title: Non‐structural protein 1‐specific antibodies directed against Zika virus in humans mediate antibody‐dependent cellular cytotoxicity

doi: 10.1111/imm.13380

Figure Lengend Snippet: Isotype specificity of ZIKV‐immune sera. ELISA plates were coated with ZIKV NS1 (a, c) or ZIKV VLP (b, d), and a 1:100 dilution ( n = 28) (a, b) or serial dilutions ( n = 11) (c, d) of ZIKV‐immune sera were added to plates. Following incubation and washes, optimized concentrations of polyclonal secondary IgG, and monoclonal Abs against the hinge or Fc portion of IgG1, IgG2, IgG3 and IgG4 Abs were added to wells (a) and (b) and IgG1 hinge Abs to (c) and (d). OD values shown at 450 nm. OD values to ZIKV VLP and ZIKV NS1 using a 1:100 dilution of ZIKV‐naïve sera ( n = 4), <0·2 with polyclonal secondary IgG and <0·1 with IgG1 hinge, IgG1 Fc, IgG2, IgG3 and IgG4 secondary Abs. Bars represent mean with standard error of the mean of the OD values. * P < 0·05, *** P < 0·001 and **** P < 0·0001 were obtained using the non‐parametric Friedman test (three or more matched groups) with Dunn's multiple comparisons test

Article Snippet: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: Immunology

Article Title: Non‐structural protein 1‐specific antibodies directed against Zika virus in humans mediate antibody‐dependent cellular cytotoxicity

doi: 10.1111/imm.13380

Figure Lengend Snippet: Frequencies of ZIKV‐specific IgG and IgG1 B‐cell responses using fluorescently labelled ZIKV or ZIKV NS1. PBMCs from ZIKV‐immune and ZIKV‐naïve donors were stimulated in vitro for seven days with r848 + rIL‐2. FluoroSpot plates were coated with IgG and IgG1 capture Abs. (a) Images of wells containing media, total IgG, ZIKV and ZIKV NS1 IgG‐ or IgG1‐specific MBCs from one representative donor. (b) Frequencies of ZIKV (green) and NS1‐specific (red) ASCs per 10 6 input cells in MBC cultures from 12 ZIKV‐immune and three ZIKV‐naïve donors. Values are the mean of duplicate wells for each condition. The supernatants were evaluated for isotype‐specific Abs to (c) ZIKV VLPs and (D) ZIKV NS1. * P < 0·05, ** P < 0·01 and **** P < 0·0001 were obtained using the non‐parametric Mann‐Whitney or Friedman test (three or more matched groups) with Dunn's multiple comparisons test

Article Snippet: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

Techniques: In Vitro, MANN-WHITNEY

Journal: Immunology

Article Title: Non‐structural protein 1‐specific antibodies directed against Zika virus in humans mediate antibody‐dependent cellular cytotoxicity

doi: 10.1111/imm.13380

Figure Lengend Snippet: Opsonization and ADCC activity of a ZIKV NS1 monoclonal Ab. (a) K562 cells transfected with DC‐SIGN were infected with ZIKV (moi = 0·1 and 1), and 24 h later, the expression of E and NS1 was measured with monoclonal Abs 4G2 and B4 respectively. (b) Opsonization of ZIKV NS1‐transfected CEM‐NK R cells with a mAb to ZIKV NS1 from Native Antigen Company. CEM‐NK R cells transfected with ZIKV NS1 (c and d) or control CEM‐NK R cells (e and f) were opsonized with a ZIKV NS1 mAb (d and f), added to PBMC. A standard lymphocyte gate based upon light scatter properties followed by selection of singlet viable cells and dump channel exclusion (CD3 − T cells) defined the non‐T‐cell lymphocyte population, and Abs to CD16 and CD56 identified NK cells

Article Snippet: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

Techniques: Activity Assay, Transfection, Infection, Expressing, Selection

Journal: Immunology

Article Title: Non‐structural protein 1‐specific antibodies directed against Zika virus in humans mediate antibody‐dependent cellular cytotoxicity

doi: 10.1111/imm.13380

Figure Lengend Snippet: NK cell activation and degranulation in the presence of ZIKV‐immune sera. 1:100 dilution and 1:1600 dilution of polyclonal sera from ZIKV‐immune individuals were evaluated for their ability to mediate ADCC of ZIKV NS1 CEM‐NK R cells using intracellular cytokine assays. Shown are the frequencies of NK cells that (a) secrete IFN‐γ, (B) express CD107 or (C) express both CD107 and IFN‐γ in the presence of ZIKV‐immune sera. Background levels were subtracted

Article Snippet: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

Techniques: Activation Assay

Journal: Immunology

Article Title: Non‐structural protein 1‐specific antibodies directed against Zika virus in humans mediate antibody‐dependent cellular cytotoxicity

doi: 10.1111/imm.13380

Figure Lengend Snippet: Opsonization and target cell lysis using an NK‐TVA image cytometry assay. (a) Opsonization of ZIKV NS1 CEM‐NK R cells by ZIKV convalescent sera. (b) Representative images of wells acquired by an ImmunoSpot S5 Analyser of an NK‐TVA assay where ethanol, ZIKV NS1 or IgG Ab was added to the wells containing effector and target cells. (c) Using an optimized number of target cells, effector PBMC as a source of NK cells and different dilutions of naïve sera ( n = 5), positive control sera that opsonized ZIKV NS1 CEM‐NK R targets ( n = 5) and experimental sera ( n = 12), the loss of target cells at the single‐cell level was measured by the NK‐TVA image cytometry assay and calculated as a percentage of lysed target cells. * P < 0·05 and ** P < 0·01 were obtained using a Kruskal–Wallis test with Dunn's multiple comparisons test

Article Snippet: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

Techniques: Lysis, Cytometry, Positive Control

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

Journal: Biosensors & Bioelectronics

Article Title: A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads

doi: 10.1016/j.bios.2021.113388

Figure Lengend Snippet: Operation of the MOnITOR chip for one-step indirect immunoassays. (a) In the ‘Preparation’ stage, the chip immediately started to operate. (b) Engineered bead lines were secured on the reaction zone. The scale bar represents 100 μm. (c) Red-dyed sample was loaded. Trapped gas bubbles eventually diffused through the PDMS matrix (blue arrows). Wasted volumes in the loading and dummy channels consume only 1.6% of the loaded sample. (d,e) Sequentially-loaded blue-dyed buffer flowed and filled the storage chamber. During storage chamber filling, 2nd Ab was rehydrated. Rehydrated 2nd Ab diffusion toward the buffer inlet was prevented by the delaying channel. (f) When loading sample with anti-ZIKV NS1 and anti-CHIKV E2 mAb, only the corresponding beads reacted. (g) Buffer continuously flowed toward the reaction zone by contouring the empty sample inlet. (h) Unbound 2nd Ab were washed by flowing buffer. An image of the reaction zone was acquired in this stage. The scale bar represents 1 cm. (i) In the case of (f), the transferred 2nd Ab only reacted with ZIKV, CHIKV, and + beads. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Anti-ZIKV NS1, anti-DENV NS1, anti-CHIKV E2, and anti-Mayaro virus (MAYV) E2 monoclonal antibodies (mAb) were also provided from BioNano Health Guard Research Center.

Techniques: Diffusion-based Assay

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: ZIKV NS4A disrupts mitochondrial dynamics and respiration profile, and induces mtROS production. (A) JEG-3 cells were transfected with mtDsRED-encoding plasmid. Next day, cells were mock-infected or infected with PRVABC59 (PRV) (MOI 10) and fixed at the indicated time points to visualize mitochondrial morphology using confocal microscopy. Scale bar = 10 μm. (B) ZIKV NS4A-induced mitochondrial fragmentation was visualized using JEG-3 cells transfected with mtDsRED and empty vector (EV) or FLAG-tagged ZIKV NS4A plasmids. Scale bar = 10 μm. CCCP-treated cells were included as a control for mitochondrial fragmentation. (C) Western blot analysis of proteins regulating mitochondrial dynamics in JEG-3 cells overexpressing EV, ZIKV NS4A or NS4B. Band intensities were quantified using ImageJ software and normalized against β-actin. (D) Real-time measurement of mitochondrial stress assay for EV or ZIKV NS4A-overexpressing cells was carried out to determine ZIKV NS4A-induced changes in oxygen consumption rate (OCR) following treatment with oligomycin (1.5 μM), FCCP (2μM) or rotenone + Antimycin A (0.5μM each) (E) Tetramethylrhodamine methyl ester (TMRM) assay of JEG-3 cells overexpressing increasing concentrations of ZIKV NS4A-encoding plasmid. NS4A-induced mitochondrial membrane depolarization was determined using fluorescence microplate reader. *p<0.05, **p<0.01, when compared to the control. (F) JEG-3 cells were transfected with different amount of ZIKV NS4A-encoding plasmid. Cells were treated with DMSO control (ctl), CCCP, or mito-TEMPO, an antioxidant targeted to mitochondria. Mitochondrial ROS (mtROS) was measured by MitoSox Red staining and mtROS levels were quantitatively determined using fluorescence analysis software (mean ± SD; n = 3).*p<0.05, ***p<0.001, when compared to the control.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Transfection, Plasmid Preparation, Infection, Confocal Microscopy, Control, Western Blot, Software, Membrane, Fluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: ZIKV NS4A induces mitophagy in human trophoblasts and neural progenitor cells. (A) JEG-3 cells were infected with mock- (m) or PRV (MOI 10) and stained with MitoTracker (green) (500 nM) and LysoTracker (red) (75 nM). Scale bar = 10 μm. Mitolysosomes were quantified by measuring percentage of cells with MitoTracker and LysoTracker colocalization (graph). The graph shows mean ± SD (n = 3). Statistical analysis; ***p<0.001, when compared to the mock. (B-D) HeLa cells expressing mtKeima (pH-dependent fluorescent protein) were infected with mock (m) or PRV (B) , mock (m), Brazil or MR766 (C) , or transfected with empty vector (EV), ZIKV NS4A or NS4B (D) were treated with CCCP (25 μM) for 2 (h) Green fluorescence of mtKeima reflects mitochondria in the cytosol (green), while red fluorescence of mtKeima reflects mitochondria in lysosomes (red). pH-dependent shift in excitation peaks were quantified to calculate % mitophagy (graph) using Zeiss ZEN software. *p<0.05, **p<0.01, and ***p<0.001, when compared to the mock or EV-transfected control. (E) MitoTracker Green and LysoTracker Red staining of mock- vs. PRV-infected human neural progenitor cells (hNPCs). Percentage of cells with MitoTracker and LysoTracker colocalization was calculated (graph). (F) TMRM staining of hNPCs transfected with varying concentrations of ZIKV NS4A-encoding plasmid. *p<0.05, ***p<0.001, when compared to the EV-transfected control.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Infection, Staining, Expressing, Transfection, Plasmid Preparation, Fluorescence, Software, Control

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: Drp1-mediated mitochondrial fission & PINK1 are important for ZIKV replication. (A) Western blot analysis of JEG-3 transfected with ctl (siCtl) vs. PINK1-specific siRNA (siPINK1) to confirm successful knockdown. Band intensities were quantified using ImageJ. (B) ZIKV vRNA levels were quantified using qRT-PCR in siCtl- vs. siPINK1-transfected cells infected with PRV. **p<0.01, when compared to the siCtl-treated cells (C, D) JEG-3 cells transfected with control siRNA (siCtl), Drp1-specific siRNA (siDrp1) (C) or MFN2-specific siRNA (siMFN2) (D) were infected with PRV (MOI 10). ZIKV vRNA expression levels were quantified with qRT-PCR. (E, F) Mdivi-1 was added to JEG-3 cells at various concentrations and cells were infected with PRV (MOI 10). Mdivi-1-induced downregulation of mitochondrial fission was confirmed via suppression of FIS1 mRNA levels (E) . Mdivi-1 treatment at various concentrations suppressed ZIKV vRNA gene expression levels, as quantified by qRT-PCR (F) (mean ± SD; n = 3). *p<0.05, **p<0.01, and ***p<0.001, when compared to the control. Relative expression levels were normalized against GAPDH.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Western Blot, Transfection, Knockdown, Quantitative RT-PCR, Infection, Control, Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: ZIKV NS4A upregulates mitophagy receptor expression, and BNIP3 and NIX modulate ZIKV replication. (A) Western blot analysis of mitophagy receptor expression levels in cytosolic (cyto) and mitochondrial (mito) fractions obtained from mock- vs. PRV-infected JEG-3 cells. Band intensities were quantified using ImageJ and normalized against TOM70. (B, C) JEG-3 cells were transfected with siCtl, siBNIP3 (B) or siNIX (C) and infected with PRV (MOI 10). BNIP3 and NIX mRNA levels were determined by qRT-PCR. Data represented as mean ± SD. **p<0.01, when compared to the siCtl-transfected cells. Relative expression levels were normalized against GAPDH. (D, F) qRT-PCR analysis of BNIP3 or NIX siRNA-transfected JEG-3 cells infected with PRV (MOI 10). Relative viral transcription levels (D) and IFN-β, ISG15 , and OAS1 mRNA levels (F) were determined. Relative expression levels were normalized against GAPDH. (E) TCID 50 assay of Vero cells infected with supernatants collected at 48 hpi from siBNIP3- or siNIX-transfected JEG-3 cells infected with PRV (MOI 10). Data represented as mean ± SD. *p<0.05, **p<0.01, and ***p<0.001, when compared to the siCtl-treated PRV-infected cells.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Expressing, Western Blot, Infection, Transfection, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: ZIKV NS4A reduces mitochondrial mass & blocks MAVS oligomerization to suppress IFN response. (A, B) Genomic DNA isolated to determine relative mitochondrial DNA (mtDNA) present in JEG-3 cells infected with mock (m), PRV (MOI 10) or treated with CCCP (25 μM, 2 h) (A) or transfected with empty vector (EV), ZIKV NS4A, NS4B or treated with CCCP (25 μM, 2 h) (B) . Relative mtDNA amount was normalized against 18S rRNA. *p<0.05, **p<0.01, and ***p<0.001, when compared to the m or EV control. (C) Western blot analysis of MAVS oligomerization and expression levels in JEG-3 cells transfected with MAVS in combination with increasing concentrations of ZIKV NS4A. (D) JEG-3 cells overexpressing IFN-β or ISRE reporter plasmids were transfected with EV, ZIKV NS4A or NS4B and treated with poly(I:C) (20 μg/mL, 2h). Luciferase reporter assay is shown (mean ± SD). (E, F) Luciferase assay of HEK293T cells transfected with IFN-β or ISRE reporter plasmids in combination with ZIKV NS4A and one of the following RLR signaling effectors (MDA5, RIG-I, MAVS, TBK1, IRF3). Data represented as mean ± SD. *p<0.05, **p<0.01, and ***p<0.001, when compared to the EV control.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Isolation, Infection, Transfection, Plasmid Preparation, Control, Western Blot, Expressing, Luciferase, Reporter Assay

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: Small extracellular vesicles (sEV) proteomics profiling revealed presence of mitochondrial components. (A) Schematic representation of the experimental procedures taken to isolate, purify, and characterize sEVs released from mock- vs. PRV-infected JEG-3 cells. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis was performed to characterize the contents of sEVs from PRV-infected JEG-3 cells (B) Nanoparticle tracking analysis of sEVs secreted by mock- or PRV-infected JEG3 cells is shown. (C) Western blot analysis of sEVs confirm the presence of ZIKV NS1 and sEV markers (CD9, CD63, TSG101, and ALIX). Endoplasmic reticulum localized calnexin was used as a negative marker. (D) Transmission electron microscopy of sEVs. Scale bar = 100 nm. (E) Pie chart of functional Gene Ontology (GO) terms associated with differentially expressed proteins detected within sEVs (PRV- vs. mock-infected; p < 0.05, fold change > 2). (F) Heatmap of top expressed proteins that were upregulated in PRV-infected sEVs. Two biological replicates per samples were included for further analysis. (G) LC–MS/MS-based proteomics data were validated by Western blot analysis. Snx6, Snx9, LAMP1, TOM70, IRF3, p62, and G3BP1 were upregulated in PRV-infected sEVs compared with mock sEVs. (H) Number of protein-encoding genes of the detected proteins were quantified according to their subcellular localization. Proteins that can localize to the mitochondria are shown in gray.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Infection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Marker, Transmission Assay, Electron Microscopy, Functional Assay

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: MDV components (Snx6 and Snx9) regulate ZIKV replication & infectivity. (A) Heatmap of upregulated sEV proteins that can localize to the endosome. Snx6 and 9 were chosen for further analysis. (B) Western blot analysis of JEG-3 cells transfected with Snx6- or Snx9-specific siRNAs and infected with mock or PRV (MOI 10). Snx6 and Snx9 protein levels were determined and quantified below the blot. (C) qRT-PCR quantification of ZIKV vRNA transcription levels in siSnx6- and/or siSnx9-transfected JEG-3 cells infected with PRVABC59. Expression levels were quantified relative to siCtl-transfected JEG-3 cells infected with PRV. (D) TCID 50 assay of Vero cells infected with supernatants collected at 48 hpi from siSnx6- and/or siSnx9-transfected JEG-3 cells infected with PRV. (E) siSnx6- and/or siSnx9-transfected JEG-3 cells were infected with PRV for 24h. Relative ISG15 mRNA levels were quantified relative to siCtl-transfected JEG-3 cells infected with PRV. (F) Cytosolic mtDNA amount was quantified with qRT-PCR. (+) = JEG-3 cells transfected with siSnx6 and siSnx9 in combination. Data represented as mean ± SD (n=3). *p<0.05, **p<0.01, and ***p<0.001, when compared to the siCtl-transfected cells infected with PRV.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Infection, Western Blot, Transfection, Quantitative RT-PCR, Expressing

Journal: Frontiers in Immunology

Article Title: Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells

doi: 10.3389/fimmu.2023.1203645

Figure Lengend Snippet: ZIKV NS4A dysregulates mitochondrial dynamics and quality control mechanisms to suppress host antiviral response in trophoblast cells. ZIKV NS4A triggers changes in mitophagy, and MAVS-mediated antiviral immunity to evade the host innate immune response and facilitate viral infection, while ZIKV modulation of MDV components, such as Snx6 and Snx9, may affect ZIKV replication and infectivity.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: ZIKV NS1 (Genentech, CA, USA); MFN1, OPA1, phospho-Drp1 (Ser616), Drp1, LC3, p62, cleaved/total Caspase 3, cleaved/total PARP, PINK1, Parkin, BNIP3, NIX (Cell Signaling Technology, MA, USA); GFP, IRF3, Tom20, G3BP1, LAMP1 (Santa Cruz biotechnology, San Diego, CA, USA)); TOM70, Snx6, Snx9 (Proteintech, IL, USA); FLAG tag (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), myc, β-actin (Abcepta, CA, USA); MAVS/VISA (Bethyl laboratories, MA, USA).

Techniques: Control, Infection

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: Post‐translational modification of ZIKV viral proteins. A) SH‐sy5y cells were infected with ZIKV (MR766) at a multiplicity of infection (MOI) of 1, and cell samples were collected 48 h post‐infection. Mass spectrometry was employed to analyze the acetylation, methylation, phosphorylation, and ubiquitination patterns of viral proteins. B and C) 293T cells were transfected with plasmids encoding various ZIKV proteins tagged with Flag (NS1‐Flag, NS3‐Flag, NS5‐Flag, PrM/E‐Flag, NS2A‐Flag, NS2B‐Flag, NS4A‐Flag, NS4B‐Flag, or C‐Flag). B) In another set of experiments, these proteins were co‐transfected with a plasmid expressing ubiquitin tagged with HA (Ub‐HA). C) After 24 h, cells were treated with MG132 (5 µM) for 4 h. Cell lysates were collected, and immunoprecipitation with Flag antibody‐coupled magnetic beads was performed. The ubiquitination levels of viral proteins were then analyzed by Western Blot. The presented data are representative of three independent experiments.

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Modification, Infection, Mass Spectrometry, Methylation, Phospho-proteomics, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Magnetic Beads, Western Blot

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: E3 ubiquitin ligase WWP2 interacts with NS1. A) NS1‐Flag was transfected into 293T cells, followed by ZIKV infection (MOI = 0.5) 24 h later. Immunoprecipitation of NS1‐Flag was performed with Flag antibody‐coupled magnetic beads 24 h post‐infection to analyze the ubiquitin‐associated enzymes interacting with NS1‐Flag protein using mass spectrometry. B–E) WWP2‐Myc was co‐transfected with NS1‐Flag expression plasmid in 293T cells. After 24 h, immunoprecipitation of NS1‐Flag was performed, and WWP2‐Myc protein was detected by Western Blot. B) In another set of experiments, immunoprecipitation of WWP2‐Myc was carried out, and NS1‐Flag protein was detected by Western Blot. C) In addition, 293T cells were infected with ZIKV (MOI = 1), and after 48 h, endogenous WWP2 was immunoprecipitated. ZIKV NS1 protein was then detected by Western Blot D), and endogenous WWP2 protein was detected by immunoprecipitation of ZIKV NS1 and Western Blot E). F) 293T cells were infected with ZIKV(MOI = 0.5) for 48 h. The intracellular localization of the endogenous WWP2 and NS1 proteins was observed using laser confocal imaging. G and H) Each of the WWP2 truncates was co‐transfected with NS1‐Flag in 293T cells, and the cells were collected after 24 h. Immunoprecipitation with NS1‐Flag was performed, and Western Blot detected the expression of Myc‐tagged truncated proteins. I) Molecular docking prediction results for WWP2‐WW and ZIKV NS1 proteins. The data presented are representative of three independent experiments.

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Ubiquitin Proteomics, Transfection, Infection, Immunoprecipitation, Magnetic Beads, Mass Spectrometry, Expressing, Plasmid Preparation, Western Blot, Imaging

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 ubiquitinates NS1 and leads to NS1 degradation. A and B) NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) were co‐transfected in 293T cells, and the protein levels of NS1‐Flag were detected by Western Blot 48 h later. C and D) NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) were co‐transfected in 293T cells, and the mRNA levels of NS1 were detected by qRT‐PCR 48 h later. E) NS1‐Flag and WWP2‐Myc were co‐transfected in 293T cells, which were then treated with MG132 (5 µM, 4 h) 24 h later. The protein levels of NS1‐Flag were detected by the Western Blot method. F) NS1‐Flag and WWP2‐Myc were co‐transfected in 293T cells. 24 h later, cells were treated with Chloroquine (10 µM, 6 h). The protein levels of NS1‐Flag were detected by the Western Blot. G and H) NS1‐Flag and shWWP2/WWP2‐Myc (1 µg) were co‐transfected in 293T cells and treated with MG132 (5 µM) for 4 h after 48 h. The ubiquitination level of NS1‐Flag protein was detected by the Western Blot method after immunoprecipitation of NS1‐Flag. I and J) NS1‐Flag and WWP2‐Myc (WT or C838A) plasmids were co‐transfected in 293T cells. After 24 h, immunoprecipitation of NS1‐Flag was performed, and the ubiquitination levels of NS1‐Flag protein were detected by Western Blot (F). The protein levels of NS1‐Flag were detected by Western Blot (G). K) NS1‐Flag, purified WWP2 (or WWP2‐C838A), E1 (Hdm2), and E2 (UbcH5a) were incubated for 1 h in the presence of ATP. The in vitro ubiquitination level of NS1 was analyzed by Western Blot. The data presented are representative of three independent experiments. ns, non‐significant (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Immunoprecipitation, Purification, Incubation, In Vitro

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 expression was upregulated during ZIKV infection. A) SH‐sy5y or 293T cells were infected with ZIKV (MR766) (MOI = 0.5). WWP2 levels was analyzed by qRT‐PCR and Western Blot. B) THP‐1 or 293T cells were treated with IFN‐α (500 U ml −1 ) for 6 h, and WWP2 levels were determined using qRT‐PCR and Western Blot. C) WWP2 expression was up‐regulated during ZIKV infection based on GEO databases. Data are representative of 3 independent experiments and presented as mean ± SD. ** P < 0.01, and *** P < 0.001, **** P < 0.0001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 restricts ZIKV infection. A–C) SH‐sy5y (A)/U3A (B) cells were infected with lentivirus overexpressing or knocking down WWP2 (MOI = 10). Subsequently, cells were infected with ZIKV (MOI = 0.5) 48 h later. Viral mRNA levels in the cells were detected 24 h later using qRT‐PCR. Viral load in the supernatant was visualized by TCID50, and the infectious viral load in U3A supernatants was determined by plaque assay (C). D) WWP2‐Myc (WT or C838A) plasmid was transfected into 293T cells and infected with ZIKV (MOI = 0.5) after 24 h. Cellular RNA was extracted at 24 and 48 h, and the viral RNA levels were analyzed by qRT‐PCR. The viral supernatant titer after 48 h was determined by TCID50. E‐H) Ifnar1 −/− mice (6 weeks old, 12 mice per group) were injected with 5 × 10 7 PFU shmWWP2 lentivirus via the tail‐vein route; 7 days later, mice were injected intraperitoneally with 10 7 PFU ZIKV. Hemocytes and serum were collected on days 3 and 5. Blood cell RNA was extracted, and qRT‐PCR was used to detect the RNA content of ZIKV and shmWWP2 in the cells E). Viral titers in the serum of mice on day 5 were detected by TCID50 F). Infectious virus in the serum of mice on day 5 was detected by the plaque assay G). The status and survival of mice were recorded by daily observation (* P < 0.05, Log‐rank test) (H). Data are representative of 3 independent experiments and presented as mean ± SD. ns, non‐significant, * P < 0.05, ** P < 0.01, and *** P < 0.001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Infection, Quantitative RT-PCR, Plaque Assay, Plasmid Preparation, Transfection, Injection, Virus

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 ubiquitinates amino acids K265 and K284 of ZIKV NS1. A and B) WWP2‐Myc was co‐transfected with NS1‐WT or its mutants in 293T cells for 24 h. After 24 h, the cells were treated with MG132 (5 µM) for 4 h. NS1‐Flag was immunoprecipitated, and the ubiquitylation level of NS1 proteins was detected by Western Blot A). The protein level of NS1 was detected by Western Blot B). C) NS1‐WT or its mutants were transfected into 293T cells and treated with actinomycin ketone CHX (50 µM) for 0, 2, 4, and 8 h after 24 h. NS1 protein levels were detected by Western Blot. D) Secondary mass spectrometry analysis of ubiquitinations at positions K265 and K284 of NS1. E) WWP2‐Myc, NS1‐Flag, and ubiquitin molecule mutant plasmids (K6, K11, K27, K29, K33, K48, and K63) were co‐transfected into 293T cells. After 24 h, the cells were treated with MG132 (5 µM, 4 h), and NS1‐Flag was immunoprecipitated. NS1 proteins were detected by Western Blot method, and the ubiquitination level was assessed. F and G) WWP2‐Myc or shWWP2 (1 µg), NS1‐Flag, were co‐transfected into 293T cells. After 24 h, the cells were treated with MG132 (5 µM, 4 h), and NS1‐Flag was immunoprecipitated. NS1 proteins were detected by Western Blot, and the ubiquitination types of NS1 were detected using K48 and K63 antibodies. H and I) WWP2‐Myc, NS1‐Flag individual point mutants, and ubiquitin molecule mutant K48/K63‐HA were co‐transfected into 293T cells. After 24 h, the cells were treated with MG132 (5 µM, 4 h), and immunoprecipitated with NS1‐Flag. The level of ubiquitination of NS1 proteins was detected by Western Blot. Data are representative of 3 independent experiments.

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Transfection, Immunoprecipitation, Western Blot, Mass Spectrometry, Ubiquitin Proteomics, Mutagenesis

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: Amino acid mutation at position NS1 K265, K284 alters ZIKV virulence. A and B) Schematic diagram of the ZIKV packaging process (A): The full‐length plasmid of the 2016 GZ01 strain was used as a template. The full‐length plasmid of the NS1 point‐mutated K265R, K284R, and K265/284R viral genomes was obtained by targeted mutagenesis. The full‐length plasmid was transfected with RNA into BHK21 cells after in vitro transcription, and the viral supernatant was collected after culture to obtain the WT viruses and mutant viruses (B). C–E) WT, K265R, K284R, and K265/284R viruses were packaged with the same mass of RNA, and the titer of the viral particles was detected by TCID50 (C). 293T cells were infected with the same titer of the mutant viruses (MOI = 1), and the intracellular viral load was detected by qRT‐PCR 48 h later (D). The same titer of mutant viruses was used to infect 293T cells (MOI = 1), and the level of ZIKV NS1 in the supernatant was detected by ELISA after 72 h (E). F and G) 293T (F) and SH‐sy5y (G) cells were infected with lentiviruses knocking down the expression of WWP2, and then infected with WT and mutant viruses after 48 h. Cells were collected after 48 h to extract the RNA, and the viral load in the cells was detected by qRT‐PCR. H) Ifnar1 −/− mice were infected with 10 7 PFU viruses (WT, K265R, K284R, and K265/284R), and viral loads were detected by qRT‐PCR on day 5 after infection. I) A search of the Virus Sequence Library ( https://nextstrain.org ) revealed the existence of a naturally occurring strain of ZIKV NS1 mutated at amino acid positions 265/284. J) Model for regulation of ZIKV NS1 by WWP2. Data are representative of 3 independent experiments and presented as mean ± SD. ns, non‐significant, * P < 0.05, ** P < 0.01, and *** P < 0.001, **** P < 0.0001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Mutagenesis, Plasmid Preparation, Transfection, In Vitro, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Virus, Sequencing

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: WWP2 is a broad‐spectrum arthropod‐borne flavivirus suppressor. A) Conservation of site 284 of the arthropod‐borne flavivirus NS1 protein. B–D) After overexpression or knockdown of WWP2 in 293T (B) and U3A (C) cells, the cells were infected with JEV (MOI = 0.5), and cellular RNA was extracted after 24 and 48 h. The viral content of the cells was detected by qRT‐PCR; the amount of infectious viruses in the supernatant of U3A was detected by plaque assay (D). E–G) Using WT and Wwp 2 −/− mice, 10 7 PFU JEV (SA14) was injected intraperitoneally, and hemocytes and serum were collected by orbital blood sampling on days 3 and 5, respectively. Blood cell RNA was extracted, and the amount of JEV in the cells was detected using qRT‐PCR (E); the viral titer in the serum of mice on day 5 was detected by TCID50 (F); the survival of mice was observed and recorded daily (* P<0.05, Log‐rank test) (G). H) JEV NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) plasmids were co‐transfected in 293T cells and treated with MG132 (5 µM) for 4 h after 48 h. NS1‐Flag was immunoprecipitated, and ubiquitination of JEV NS1‐Flag protein was detected by Western Blot. I) JEV NS1‐Flag and WWP2‐Myc/shWWP2 (1 µg) plasmids were co‐transfected in 293T cells, and the cells were collected after 48 h. NS1 protein levels were detected by Western Blot. J) JEV NS1‐Flag and WWP2‐Myc were co‐transfected in 293T cells, which were treated with MG132 (5 µM) for 4 h after 24 h. JEV NS1 protein levels were detected by Western Blot. K‐N) Cells were infected with LGTV after overexpression or knockdown of WWP2 in 293T (K) and U3A cells (M) (MOI = 1), and cellular RNA was extracted after 48 h. The viral RNA load in the cells was detected by using qRT‐PCR; etch‐a‐sketch assay was performed to detect the amount of infectious virus in the supernatants of U3A cells (N). Data are representative of 3 independent experiments and presented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Over Expression, Knockdown, Infection, Quantitative RT-PCR, Plaque Assay, Injection, Sampling, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Virus

Journal: Advanced Science

Article Title: Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors

doi: 10.1002/advs.202408024

Figure Lengend Snippet: Ubiquitination of NS1 by WWP2 homologs in mosquitoes promotes ZIKV infection of mosquitoes. A) ZIKV NS1‐Flag was transfected in C6/36 cells and treated with MG132 (5 µM) for 4 h after 24 h. Cells were collected and immunoprecipitated with Flag antibody‐coupled magnetic beads, and the ubiquitination level of viral proteins was detected by Western Blot. B) The E3 ligase Su(dx), which is highly homologous to human WWP2, is present in Aedes albopictus. (WWP2 GenBank: U96114.2; Su(dx) GenBank: XM_01 969 6185.2) C) NS1‐Flag was co‐transfected with Su(dx)‐His expression plasmid in C6/36 cells and infected with ZIKV (MR766) (MOI = 0.5) 24 h later for 24 h. NS1‐Flag was immunoprecipitated, and Su(dx)‐His protein was detected by Western Blot. D) NS1‐Flag and different doses of Su(dx) were co‐transfected in C6/36 cells, and NS1‐Flag protein levels were detected by Western Blot 24 h later. E) Co‐transfected siSu(dx) (50 nM) with NS1‐Flag (1 µg) in C6/36 cells, treated with MG132 (5 µM) for 4 h after 24 h. Immunoprecipitation of NS1‐Flag was performed, and the ubiquitination level of NS1 protein was detected by Western Blot method. F) Su(dx)‐Flag (1 µg) was transfected into C6/36 cells. After 24 h, the cells were infected with WT, K265R, K284R and K265, 284R viruses (MOI = 0.5), respectively. 48 h later, the cells were treated with MG132 (5 µM, 4 h) and NS1 was immunoprecipitated. NS1 protein was detected by Western Blot and its ubiquitination level was determined. G) Transfection of siSu(dx) (50 nM) in C6/36 cells was followed by infection with ZIKV (MOI = 1) after 48 h. Viral mRNA levels in the cells, as well as Su(dx) knockdown efficiency, were detected after 24 h using qRT‐PCR. H) Su(dx)‐His was transfected in C6/36 cells, infected with ZIKV (MOI = 1) 24 h later, and the viral mRNA level as well as the efficiency of Su(dx) overexpression was detected in the cells 48 h later using qRT‐PCR. I) NSC2805 (10 µM, 4 h) treated C6/36 cells were infected with ZIKV and viral RNA levels were detected by qRT‐PCR at 24 h J) Aedes aegypti mosquitoes were divided into two groups, the experimental group was injected with Su(dx) dsRNA, and the control group was injected with Luc dsRNA. 100 PFU of MR766 strain virus was injected into each mosquito. The viral mRNA level and Su(dx) knockdown efficiency in mosquitoes were detected by qRT‐PCR on day 7 after infection. K) Recombinant viruses (WT, K265R, K284R, and K265/284R) of the same titer were injected into the thoracic cavity of Aedes aegypti mosquitoes (50 PFU of virus per mosquito), and viral loads in the mosquitoes were detected by qRT‐PCR on day 7 after infection. Data are representative of 3 independent experiments and presented as mean ± SD. ns, non‐significant, * P < 0.05, ** P < 0.01, and *** P < 0.001, **** P < 0.0001 (Student's t‐test).

Article Snippet: The following antibodies were used in this study: GAPDH Mouse Antibody (Proteintech, Cat # 60004‐I‐Ig), DYKDDDDK Tag Mouse Antibody (ABclonal, Cat # AE005), anti‐rabbit IgG HRP‐linked antibody (CST, Cat # 7074), HRP goat anti‐mouse IgG (BioLegend, Cat # 405 306), HA‐tag Rabbit Polyclonal Antibody (CST, Cat # 3724), Ubiquitin Antibody, (Santa Cruz, Cat # sc‐8017), K63‐linkage Specific Polyubiquitin (D7A11) Rabbit mAb (CST, Cat # 12 930), K48‐linkage Specific Polyubiquitin (D9D5) Rabbit mAb (CST, Cat # 12 805), CACYBP Polyclonal Antibody (Abclonal, Cat # A8757), TRIM4 Polyclonal Antibody (Abclonal, Cat # A15922), WWP2 Polyclonal Antibody (Abclonal, Cat # 12197‐1‐AP), Myc‐tag Rabbit Polyclonal Antibody (Proteintech, Cat # I6286‐I‐AP), Goat Anti‐Mouse IgG Antibody (H+L), DyLight 488 (SeraCare, Cat # 5230‐0391), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 647) (abcam, Cat # ab150083), GST Tag Antibody (ABGENT), ZIKV virus NS1 protein antibody (GeneTex, Cat # GTX133307, GTX634158).

Techniques: Ubiquitin Proteomics, Infection, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Expressing, Plasmid Preparation, Knockdown, Quantitative RT-PCR, Over Expression, Injection, Control, Virus, Recombinant

Journal: Viruses

Article Title: Seroprevalence of Zika Virus in Amphawa District, Thailand, after the 2016 Pandemic

doi: 10.3390/v14030476

Figure Lengend Snippet: Schematic diagram of the study design and procedures. Zika virus (ZIKV) and dengue virus nonstructural protein 1 antibodies were examined at enrollment (Day 0) and after 6 and 12 months. Urine ZIKV RT-PCR was performed for volunteers who visited a hospital with acute febrile illness.

Article Snippet: A solution containing 50 µL of serum (1:10 dilution) or the ZIKV NS1-specific antibody ZKA35 (Ab1036-10.0) (Absolute Antibody, Oxford, UK) as a positive control at 5 μg/mL prepared in PBS was immediately mixed.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Viruses

Article Title: Seroprevalence of Zika Virus in Amphawa District, Thailand, after the 2016 Pandemic

doi: 10.3390/v14030476

Figure Lengend Snippet: The number (%) of subjects who had Zika virus nonstructural protein 1 seropositivity by age group and visit.

Article Snippet: A solution containing 50 µL of serum (1:10 dilution) or the ZIKV NS1-specific antibody ZKA35 (Ab1036-10.0) (Absolute Antibody, Oxford, UK) as a positive control at 5 μg/mL prepared in PBS was immediately mixed.

Techniques:

Journal: Viruses

Article Title: Seroprevalence of Zika Virus in Amphawa District, Thailand, after the 2016 Pandemic

doi: 10.3390/v14030476

Figure Lengend Snippet: The results of the Zika virus nonstructural protein 1 (NS1) blockade-of-binding (BOB) ELISA and the neutralizing antibodies against Zika virus and dengue virus serotypes detected by PRNT50 among the 12 participants with definite seroconversion.

Article Snippet: A solution containing 50 µL of serum (1:10 dilution) or the ZIKV NS1-specific antibody ZKA35 (Ab1036-10.0) (Absolute Antibody, Oxford, UK) as a positive control at 5 μg/mL prepared in PBS was immediately mixed.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Science translational medicine

Article Title: Rapid Antigen Tests for Dengue Virus Serotypes and Zika Virus in Patient Serum

doi: 10.1126/scitranslmed.aan1589

Figure Lengend Snippet: (A) Table listing mAb names, mAb immunochromatography applications, mAb linear epitope sequences and starting amino acid positions, and NS1 domain positions. (B) Comparison of amino acid similarity based on analysis of NS1 protein sequences from the following viruses: DENV1- Strain Singapore/S275/1990, accession number P33478; DENV2 -Strain NGC, accession number AAA42941; DENV3- Philippines/H87/1956, accession number AAA99437; DENV4- Singapore/8976/1995, accession number AAV31422; Zika virus, accession number KU497555.1. Amino acid sequences were compared using Color Align Conservation http://www.bioinformatics.org/sms2/color_align_cons.html to enhance the output of sequence alignment program. Residues that are identical among the sequences are boxed. Linear peptide epitopes (B) are italicized and indicated in color on the figure, with the key to the right of the figure.

Article Snippet: Monoclonal antibody production strategy DENV and ZIKV anti-NS1 monoclonal antibodies were produced in mice under contract (Covance, Inc), following an approved animal care protocol.

Techniques: Comparison, Virus, Sequencing

Journal: Science translational medicine

Article Title: Rapid Antigen Tests for Dengue Virus Serotypes and Zika Virus in Patient Serum

doi: 10.1126/scitranslmed.aan1589

Figure Lengend Snippet: (A-B) Images of rapid test strips. Strip numbers refer to the DENV serotype NS1 (1–4), pan-dengue (P; all four DENV serotype NS1 proteins), or ZIKV virus NS1 (Z) detected. Recombinant NS1 proteins, indicated with a lower case “r’ preceding the virus name, were prepared at 500 ng/ml, and the strips were run using 50 μl of solution. Strip #1 (detects DENV serotype 1): mAb pair 912/271; Strip #2 (detects DENV serotype 2): mAb pair 243/1; Strip #3 (detects DENV serotype 3): mAb pair 411/55; Strip #4 (detects DENV serotype 4): mAb pair 626/55; Strip P (“pan dengue”; detects all four DENV serotypes): mAbs 271–243-411–626/323; Strip Z (detects ZIKV): mAbs 130/110. The test proteins run on the strips are recombinant DENV NS1, serotypes 1–4 (rDENV1-rDENV4), as well as recombinant NS1 proteins from ZIKV (rZIKV), West Nile Virus (rWNV), Yellow Fever Virus (rYFV), Japanese encephalitis virus (rJEV), and Tick Borne encephalitis virus (rTBEV). C = control, NS1 = detection site for specific NS1 protein. (C-E) Limits of detection for viral NS1 proteins using the serotype-specific (SSp) DENV strips 1–4 (C), the pan-dengue strip (D), and the ZIKV virus strip (E). The limits of detection, representing three independent determinations, are recorded on the figures. Each point (C-E) is presented as the mean and standard deviation. (F) NS1-containing supernatants from Vero cells infected with DENV serotypes 1–4 (Vs DENV1–4) or ZIKV virus (Vs ZIKV-U (Uganda) or ZIKV-B (Brazil)) were chromatographed on strips 1–4, pan-dengue (P), or ZIKV (Z) NS1 strips. The arrows indicate the strips with positive NS1 signals. Horizontal test lines (panel F) result from applying antibodies to the nitrocellulose using a mechanical striper device; the circular dot signals result from applying antibodies to the nitrocellulose using a standard pipettor.

Article Snippet: Monoclonal antibody production strategy DENV and ZIKV anti-NS1 monoclonal antibodies were produced in mice under contract (Covance, Inc), following an approved animal care protocol.

Techniques: Stripping Membranes, Virus, Recombinant, Control, Standard Deviation, Infection

Journal: Science translational medicine

Article Title: Rapid Antigen Tests for Dengue Virus Serotypes and Zika Virus in Patient Serum

doi: 10.1126/scitranslmed.aan1589

Figure Lengend Snippet: (A) Map showing the endemic virus regions where the rapid tests were deployed to analyze patient serum samples. The areas of the circles correlate with the numbers of samples analyzed. The blue colors, faint to dark, represent DENV serotypes 1–4. ZIKV is indicated in orange color. (B) ELISA results showing the amounts of DENV (left) and ZIKV (right) NS1 found in patient serum and supernatants from infected cell cultures. Lanes 1 and 6 are supernatants from Vero cells infected with DENV; lanes 2 and 7 are supernatants from Vero cells infected with ZIKV. Lanes 3 and 8 are PCR-negative sera; lanes 4 and 9 are sera from PCR-positive DENV patients. Lanes 5 and 10 are sera from PCR-positive ZIKV patients. (C) Images of rapid test analysis of DENV NS1 serotypes 1–4 and ZIKV NS1 on serotype specific strips 1–4, as well as pan-dengue (P) and ZIKV (Z); the upward arrows mark positive tests, and θ is serum from an uninfected patient. (D-G) Quantification of rapid test results. Dipstick tests were run with PCR-confirmed DENV sera or ELISA-validated ZIKV serum (panel C), and the resulting signals were quantified and expressed as box plots. Statistical significance, based on one one-way ANOVA, is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001. (H) Statistical significance, based on an unpaired T-test, is presented as * P < 0.05. Box and whiskers plots: the black ✕ represent the maximum and minimum measured normalized intensity values, while the small square box ☐ represents the mean value, and the larger box represents the 25–75% range of the data. Individual colored points represented individual patient samples measured. (I-J) Images of rapid tests showing that DENV and ZIKV NS1 tests do not cross-react. (I) Supernatants from Vero cells infected with DENV serotype 4 were chromatographed on DENV serotype strips 1–4, on the pan-dengue strip (P), and on the ZIKV NS1 strip (Z). (J) Supernatants from Vero cells infected with ZIKV virus were chromatographed on DENV serotype strips 1–4, on the pan-dengue strip (P), and on the ZIKV NS1 strip (Z). (K) Images of rapid tests showing ZIKV NS1 is detected in serum samples concentrated 5X, but ZIKV virus NS1 is not detected in concentrated urine. Three sets of paired serum and urine samples were concentrated 5X by filter centrifugation and chromatographed on the ZIKV dipsticks. S: serum; U: urine. (I-K) The red boxes and vertical black lines serve as fiducial markers for image recognition and processing. Upward arrows indicate positive tests, using the serum samples. (L-N) Quantification of NS1 protein in supernatants of Vero cells infected separately with three DENV4 patient isolates (L) or three ZIKV patient isolates (M), or five paired serum/urine patient samples (N). Fig. 3L-N: One-way ANOVA was used to calculate statistical significance of the dengue and Zika tests: p<0.05, p<0.01, and p<.0.001 are indicated as *, **, and ***, respectively. Box and whiskers plots: the black ✕ represent the maximum and minimum measured normalized intensity values, while the black ☐ represents the mean value, and the larger box represents the 25–75% range of the data. Individual colored points represented individual patient samples measured.

Article Snippet: Monoclonal antibody production strategy DENV and ZIKV anti-NS1 monoclonal antibodies were produced in mice under contract (Covance, Inc), following an approved animal care protocol.

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Infection, Stripping Membranes, Centrifugation

Journal: Science translational medicine

Article Title: Rapid Antigen Tests for Dengue Virus Serotypes and Zika Virus in Patient Serum

doi: 10.1126/scitranslmed.aan1589

Figure Lengend Snippet: (A) ROC curve analysis of the patient sample data collected for dengue serotypes 1–4, Dengue Pan, and Zika virus. TPR: true positive rate (sensitivity); FPR: false positive rate (1-specificity). (B) Table listing numerical values of the sensitivity and specificity results.

Article Snippet: Monoclonal antibody production strategy DENV and ZIKV anti-NS1 monoclonal antibodies were produced in mice under contract (Covance, Inc), following an approved animal care protocol.

Techniques: Virus