primary antibodies to ns5-zikv  (GeneTex)

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: Survival of AIR pups increases with decreasing amounts of virus and anti-IFNAR antibody administered to the dam. a Neonates born to AIR, AIR low and IgR treated, ZIKV infected pregnant mice (see Results section for different treatments) were followed for survival out to postnatal day 7 (P7) and the results are presented as Kaplan–Meier survival curves. Each curve indicates the survival of neonates from multiple dams treated with the indicated anti-IFNAR1/normal mouse IgG dose(s) and ZIKV infectious dose. The dash-dot line plotted on the Y-axis indicates median survival as determined by Kaplan–Meier. ** p < 0.01, **** p < 0.0001 indicate a significant increase in survival based on a Log-rank Mantel-Cox curve comparison test. All survival curves were compared relative to the AIR (red) curve. Whole-head H&E-stained sections of an b IgR and c AIR treated neonate at P5 are shown. Notice the diminished brain volume in the ( c , asterisks) AIR animal which corresponds to the only surviving AIR animal at P5 ( a , red line termination). The tissue disruption in the brainstem and cerebellum of ( b ) is due to tissue processing and cutting artifact and is not associated with virus-induced pathology

Article Snippet: Sections were then blocked at RT for 30 min (5% normal donkey serum, 0.01% TritonX100, 0.3 M Glycine in PBS) and primary antibodies against ZIKV NS5 (chicken, 1:1500, Aves Labs) and active Caspase 3 (rabbit, 1:250, Promega) or ionized calcium binding protein (rabbit, Iba1, 1:250, Dako) or glial fibrillary acidic protein (GFAP, rabbit, 1:250, Wako) were applied overnight at 4 °C.

Techniques: Virus, Infection, Comparison, Staining, Disruption

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV broadly infect neurons within the CNS of P7 AIR low neonates. P7 neonatal whole brain from naïve, IgR and AIR low pups was evaluated for ( a ) ZIKV RNA by qRT-PCR with virus-specific primers. Each symbol indicates an individual brain. The mean of the individually plotted data points for each group is represented by the horizontal black bars. The dotted line in ( a ) indicated the limit of detection for the assay. Data was analyzed by a One-way ANOVA (F 2, 20 = 12.56, p = 0.0003) with a Tukey Multiple Comparisons Test to determine significance between groups (***). Whole brain sections from b IgR and c AIR low neonates were immunohistochemically labeled for ZIKV NS5 antigen (green) and cell nuclei (grey, Hoechst) and visualized via fluorescence microscopy. Scale bar in ( c ) applies to ( b ). Cerebellum from ( d , h ) naïve, ( e , i ) IgR and ( f , j ) AIR low mice were dual chromogenic labeled for ( d – g ) Calbindin (yellow) and ZIKV NS2B (purple) or ( h – k ) NeuN (yellow) and ZIKV NS2B (purple) to demonstrate cellular organization and neuronal infection. The normal position and shape of the granular layer (GL), Purkinje cell layer (PCL), molecular layer (ML) and external germinal layer (EGL) are shown in a naïve mouse cerebellum labeled with ( d ) Calbindin and ( h ) NeuN at the P7 time point. Calbindin clearly labels Purkinje cell bodies in PCL ( d , lower left inset) and dendrites in the ML ( d , lower left inset, red arrow) and neurons associated with white matter (WM, d, lower right inset, red arrow) while NeuN labels neurons in the granular layer and maturing granular neurons as they emerge from the EGL to populate the GL. The black lines in ( e , i ) highlight the normal-appearing PCL and ML in an IgR mouse. These structures appear similar to those in the naïve mouse ( d , h , PCL and ML). In contrast, the black lines in ( f , j ) highlight the diminished PCL and ML in an AIR low mouse with the distance between each line having decreased. Likewise, notice the disorganization and sparsity of cells in the GL of IgR ( i ) and AIR low ( j ) mice, relative to the ordered GL in the naïve mouse ( h ). The asterisks in ( h – j ) highlight the maturing granular neurons that expression NeuN as they emerge from the EGL to populate the GL in a naïve, IgR and AIR low mouse respectively. The blue boxes in ( f ) and ( j ) corresponds to the higher magnification images in ( g ) and ( k ) respectively. The black arrows in ( g ) and ( k ) demonstrate ZIKV and neuronal marker dual-labeled infected neurons, which result in red-colored cells. In contrast, cerebellar neurons that did not label with Calbindin, but are ZIKV positive are labeled purple (green arrows in g ). The scale bar in ( d ) also applies to ( e , f , h and i )

Article Snippet: Sections were then blocked at RT for 30 min (5% normal donkey serum, 0.01% TritonX100, 0.3 M Glycine in PBS) and primary antibodies against ZIKV NS5 (chicken, 1:1500, Aves Labs) and active Caspase 3 (rabbit, 1:250, Promega) or ionized calcium binding protein (rabbit, Iba1, 1:250, Dako) or glial fibrillary acidic protein (GFAP, rabbit, 1:250, Wako) were applied overnight at 4 °C.

Techniques: Quantitative RT-PCR, Virus, Labeling, Fluorescence, Microscopy, Infection, Expressing, Marker

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV infection in the CNS of P7 AIR low neonates results in CNS structural abnormalities. H&E-stained whole brain sections from a IgR and b AIR low mice are shown as representative examples of morphometric measurements taken of cortical thickness (red line in a , b ) and cerebellar area (red polygon in a , b ). Actual recorded values for each section are shown. The scale bar in ( b ) applies to ( a ). Morphometric measurements from each hemisphere of 3 naïve, 10 IgR and 6 AIR low are shown for ( c ) cerebellar area and ( d ) cortical thickness. Each symbol represents the mean measurement from all sections for each hemisphere of each animal. The mean of the individually plotted data points for each group is represented by the horizontal black bars. A One-way ANOVA was used to compare cerebellar area ( c , F 2, 33 = 16.62, p < 0.0001) and cortical thickness ( d , F 2, 33 = 5.54, p = 0.0084) from samples from each group and a Tukey Multiple Comparisons Test was used to determine significance between groups. * p < 0.05, *** p < 0.001, **** p < 0.0001 indicate significance between groups

Article Snippet: Sections were then blocked at RT for 30 min (5% normal donkey serum, 0.01% TritonX100, 0.3 M Glycine in PBS) and primary antibodies against ZIKV NS5 (chicken, 1:1500, Aves Labs) and active Caspase 3 (rabbit, 1:250, Promega) or ionized calcium binding protein (rabbit, Iba1, 1:250, Dako) or glial fibrillary acidic protein (GFAP, rabbit, 1:250, Wako) were applied overnight at 4 °C.

Techniques: Infection, Staining

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV infection increases cell death in the CNS of P7 AIR low neonates primarily in the cerebellum, cortex and spinal cord. Representative H&E-stained sections from P7 ( a , c ) naïve and ( b , d ) AIR low cerebellum and spinal cord respectively. Degenerating cerebellar neurons ( b , black arrows) and axonal degeneration ( d , black arrows) were observed in AIR low mice. The scale bar in ( b ) applies to ( a ) and the bar in ( d ) applies to ( c ). Representative immunofluorescence labeled CNS sections from P7 ( e – g ) IgR and ( h – j ) AIR low pups demonstrate ZIKV NS5 (green) and active-Caspase 3 (magenta) positive cells in cortex, hippocampus and cerebellum. Nuclei are shown in grey. Images of active-Caspase 3 and nuclei staining only in AIR low mice are shown in ( k – m ). Red arrows and box insets in ( e – g ) indicate baseline active-Caspase 3 labeling in control animals. Red boxes in ( h – j ) correspond to insets in each specific brain region. Yellow arrows indicate ZIKV/active-Caspase 3 dual positive cells while orange arrows indicate active-Caspase 3 only cells. The scale bar in ( g ) applies to ( e – m )

Article Snippet: Sections were then blocked at RT for 30 min (5% normal donkey serum, 0.01% TritonX100, 0.3 M Glycine in PBS) and primary antibodies against ZIKV NS5 (chicken, 1:1500, Aves Labs) and active Caspase 3 (rabbit, 1:250, Promega) or ionized calcium binding protein (rabbit, Iba1, 1:250, Dako) or glial fibrillary acidic protein (GFAP, rabbit, 1:250, Wako) were applied overnight at 4 °C.

Techniques: Infection, Staining, Immunofluorescence, Labeling, Control

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV infection induces glial activation in the CNS of P7 AIR low neonates. Representative sections from P7 ( a , c , e and g ) IgR and ( b , d , f and h ) AIR low mice demonstrating ( a – d ) GFAP and ( e – h ) Iba1 immunofluorescence labeling in the cortex and cerebellum. Specific labeling from each antibody is shown in magenta and cell nuclei (Hoechst) are shown in grey for counterstain. Green boxes in ( g ) and ( h ) correspond to high magnification insets to demonstration cellular morphology. P7 neonatal whole brain from naïve, IgR and AIR low pups was evaluated for RNA expression of glial-specific genes including ( i ) Gfap , ( j ) Aif1 (Iba1) and ( k ) Gpr84 by qRT-PCR with specific primers. Each symbol indicates an individual brain. The mean of the individually plotted data points for each group is represented by the horizontal black bars. A One-way ANOVA was used to compare RNA expression of ( i ) Gfap (F 2, 20 = 44.50, p < 0.0001), ( j ) Aif1 (F 2, 20 = 76.88, p < 0.0001) and ( k ) Gpr84 (F 2, 20 = 103.2, p < 0.0001) from samples from each group and a Tukey Multiple Comparisons Test was used to determine significance between groups. **** p < 0.0001, indicates significance between groups

Article Snippet: Sections were then blocked at RT for 30 min (5% normal donkey serum, 0.01% TritonX100, 0.3 M Glycine in PBS) and primary antibodies against ZIKV NS5 (chicken, 1:1500, Aves Labs) and active Caspase 3 (rabbit, 1:250, Promega) or ionized calcium binding protein (rabbit, Iba1, 1:250, Dako) or glial fibrillary acidic protein (GFAP, rabbit, 1:250, Wako) were applied overnight at 4 °C.

Techniques: Infection, Activation Assay, Immunofluorescence, Labeling, RNA Expression, Quantitative RT-PCR

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: Prolonged ZIKV infection in AIR low mice results in sustained CNS structural abnormalities and glial activation leading to eventual clinical neurologic disease. a Neonates born to AIR low (blue line) and IgR (black line) treated, ZIKV infected, pregnant mice (see Results for specific mouse numbers) were followed for survival out to P14 and the results are presented as Kaplan–Meier survival curves. Each curve indicates the survival of neonates from multiple dams treated with the indicated anti-IFNAR1/normal mouse IgG dose(s) and ZIKV infectious dose. The dash-dot line plotted on the Y-axis indicates median survival as determined by Kaplan–Meier. **** p < 0.0001 indicate a significant decrease in survival based on a Log-rank Mantel-Cox curve comparison test where the AIR low curve was compared to the IgR. b P14 naïve, IgR and AIR low pups were evaluated for ZIKV RNA by qRT-PCR with virus-specific primers. The dotted line in ( b ) indicates the limit of detection for the assay. Morphometric measurements from each hemisphere of 3 naïve, 10 IgR and 3 AIR low mice that survived to P14 are shown for ( c ) cerebellar area and ( d ) cortical thickness. Each symbol represents the mean of 3 measured sections from each hemisphere of each animal. The mean of the individually plotted data points for each group is represented by the horizontal black bars. A One-way ANOVA was used to compare cerebellar area ( c , F 2, 28 = 9.64, p = 0.0007) and cortical thickness ( d , F 2, 28 = 1.384, p = 0.2673) from samples from each group and a Tukey Multiple Comparisons Test was used to determine significance between groups. ** p < 0.01, *** p < 0.001, indicate significance between groups. n.s. = not significant. Representative H&E-stained sections from P14 AIR low ( e ) cerebellum and ( f ) spinal cord demonstrate degenerating cerebellar neurons ( e , black arrows) and axonal degeneration ( f , black arrows). The scale bar in ( f ) applies to ( e ). Representative cerebellar sections from ( g ) IgR and ( h ) AIR low pups demonstrating ZIKV NS5 (green) and Iba1 (magenta) immunofluorescence labeling. The scale bar in ( h ) applies to ( g )

Article Snippet: Sections were then blocked at RT for 30 min (5% normal donkey serum, 0.01% TritonX100, 0.3 M Glycine in PBS) and primary antibodies against ZIKV NS5 (chicken, 1:1500, Aves Labs) and active Caspase 3 (rabbit, 1:250, Promega) or ionized calcium binding protein (rabbit, Iba1, 1:250, Dako) or glial fibrillary acidic protein (GFAP, rabbit, 1:250, Wako) were applied overnight at 4 °C.

Techniques: Infection, Activation Assay, Comparison, Quantitative RT-PCR, Virus, Staining, Immunofluorescence, Labeling

Journal: Viruses

Article Title: The Rescue and Characterization of Recombinant, Microcephaly-Associated Zika Viruses as Single-Round Infectious Particles

doi: 10.3390/v11111005

Figure Lengend Snippet: Analysis of viral proteins and genome synthesis in packaging cells transfected with the ZIKV Natal RGN replicon. ZIKV E, NS1, and NS5 expressions in mock cells (top), packaging cells (middle), and replicon-transfected packaging cells (bottom) were examined using the indicated primary antibodies and Alexa Fluor 546-conjugated secondary antibodies ( A ). Finally, cell imaging was conducted via immunofluorescence microscopy. In addition, relative copy numbers of sense (top) and antisense (bottom) genomes in the indicated cells were quantitated 72 h post-transfection using real-time PCR, and were then normalized to GAPDH mRNA ( B ). ***, p value < 0.001 compared with mock cells. Scale bar, 100 μm.

Article Snippet: To explore the expression of replicon-based EGFP and ZIKV reporter proteins, replicon-transfected cells were initially photographed using immunofluorescence microscopy; then, an immunofluorescence assay (IFA) was performed with primary antibodies against ZIKV NS1 and NS5 (GeneTex, Inc., Taiwan) and secondary AF546 goat anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Transfection, Imaging, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction